Advances in Immunology Volume 68

ADVANCES IN

Immunology VOLUME 68

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ADVANCES IN

Immunology EDITED BY

FRANK J . DIXON The Scripps Research Institute La Jolla, California ASSOCIATE EDITORS

Frederick Alt K. Frank Austen Tadamitsu Kishimoto Fritz Melchers Jonathan W. Uhr

VOLUME 68

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CONTENTS

ix

CONTRIBUTORS

Posttranscriptional Regulation of mRNAs Important in T Cell Function JAMES

S. MALTER

I. Introduction 11. 111. IV. V. VI. VII.

Measurement of mRNA Decay Rates Measurement of Translation mRNAs Regulated by Posttranscriptional Control cis Elements tran.s Factors Concluding Remarks References

1 1 3 4 18 29 36 37

Molecular and Cellular Mechanisms of T Lymphocyte Apoptosis JOSEF

M. PENNINCER A N D GUIDO KROEMER

I. Introduction 11. Degradation Phase of Apoptosis 111. Effector Phase of Apoptosis IV. Initiation Phase of Apoptosis V. Conclusions References

51 54 65 89 122 124

Prenylation of Ras GTPase Superfamily Proteins and Their Function in lmmunobiology

ROBERTB. LOBELL I. Introduction 11. The Ras Superfamily Members 111. The GTPase Cycle IV. Downstream Signaling Effectors: Ras and the Rho/Rac Connection V. Rho/Rac Effectors VI. Prenylation of the Ras Superfamily Members VII. Preriylation and Processing of CaaX Substrates V

145 145 147 148 150 150 152

vi

CONTENTS

VIII. IX. X. XI. XII. XIII. XIV. XV. XVI. XVII. XVIII. XIX. XX. XXI. XXII.

CaaX Prenyltransferases CaaX Protease and Carboxymethyltransferase Rab GGTase-I1 Role of Prenylation in Membrane Binding and in Protein-Protein Interactions Role of Ras GTPase Family Members in Immunobiology: The Ras Pathway The Rho/Rac Pathway and Leukocyte Function Regulation of the Neutrophil NADPH Oxidase by Rac and Rap Re lation of Phos holi ase D by RhoA Ro e of C-Termina Met ylation of Prenylated Proteins in NADPH Oxidase Regulation and Other Leukocyte Functions Role of Rab Proteins in Membrane Transport in Leukocytes Regulation of Vesicular Transport by Rho Proteins Other Prenylated Proteins Prenyltransferase Inhibitors Effects of Prenylation Inhibitors on Leukocyte Function Conclusion References

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152 157 158 162 166 168 169 171 171 172 174 174 175 178 179 180

Generation and TAP-Mediated Transport of Peptides for Major Histocompatibility Complex Class I Molecules

FRANK MOMBURG A N D GUNTER J. HAMMERLING I. Introduction 11. TAP as the Principal Peptide Supplier for MHC Class I Molecules enic Peptides from Endogenous Antigens 111. Generation of Class I Molecules in the ER VII. VIII. IX. X. XI. XII. XIII. XIV. XV.

TAP as a Member of the ABC Transporter Superfamily Structure of TAP Molecules In Vitro Assays for Pe tide Binding and Transport by TAP Substrate S ecificity o Peptide Transport Biochemic Characteristics of Peptide Transport Linking TAP Structure and Function Export of Peptides from the ER Involvement of TAP in Diseases Concluding Remarks References

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191 192 199 205 209 211 213 214 215 218 228 231 233 234 239 240

Adoptive Tumor Immunity Mediated by Lymphocytes Bearing Modified Antigen-Specific Receptors

THOMAS BROCKER AND KLAUSKARJALAINEN I. Ado tive Tumor Therapy 11. Sing e-Chain Fv Receptors 111. New Approaches References

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CONTENTS

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Membrane Molecules as Differentiation Antigens of Murine Macrophages

ANDREWJ. MCKNIGHTAND SIAMON GORDON I. Introduction 11. Differentiation Antigens Expressed by Murine Monocytes and Macro hages 111. Use of Di erentiation Antigens to Characterize Macrophages in Situ and in Vitro IV. Conclusion References

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271 271 298 303 305

Major Histocompatibility Complex-DirectedSusceptibility to Rheumatoid Arthritis

GERALD T. NEPOM

I. Introduction 11. Mechanisms to Account for the Association of the Shared Epitope with RA 111. Clinicd Applications References

3 15 318 326 327

Immunological Treatment of Autoimmune Diseases

J. R. KALDEN,F. C. BREEDVELD, H. BURKHARDT, A N D G. R. BURMESTER

I. Introduction 11. Cytokines and Anticytokine-Related Treatment Principles in Autoimmune Diseases 111. Anti-CD4 mAb in the Treatment of Autoimmune Diseases IV. Monoclonal Antibody Treatment against Cell Surface Antigen of T Cells (with the Exception of Anti-CD4) V. Immunological Treatment Principles in Animal Models of Autoimmune Disease References INDEX OF RECENT VOLUMES CONTENTS

333 337 347 353 365 397 419 43 1

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CONTRIBUTORS

Nuinhers in parentheses indicate the pages on which the authors’ contributions begin

F. C. Breedveld (333),Department of Rheumatology, Leiden University Hospital, Leiden, The Netherlands Thomas Brocker (257), Basel Institute for Immunology, CH-4005 Basel, Switzerland H. Burkhardt (333),Department of Internal Medicine 111 and Institute for Clinical Immunology, University Hospital Erlangen-Nurnberg, Germany G. R. Burmester (333),Department of Internal Medicine 111, Medical Faculty of the Humboldt University, Berlin, Germany Siamon Gordon (271), Sir William Dunn School of Pathology, University of Oxford, Oxford OX1 3RE, United Kingdom Gunter J. Hammerling (191), Department of Molecular Immunology, German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany J. R. Kalden (333),Department of Internal Medicine 111 and Institute for Clinical Immunology, University Hospital Erlangen-Nurnberg, Germany Klaus Karjalainen (257), Basel Institute for Immunology, CH-4005 Basel, Switzerland Guido Kroemer (51),CNRS-UPR 420, F-94801 Villejuif, France Robert B. Lobell (145), Merck Research Laboratories, Department of Cancer Research, Merck and Company, Inc., West Point, Pennsylvania 19486 James S. Malter (I),Department of Pathology and Laboratory Medicine, University of Wisconsin Hospital and Clinic, Madison, Wisconsin 53792 Andrew J. McKnight (271), Sir William Dunn School of Pathology, University of Oxford, Oxford OX1 3RE, United Kingdom Frank Momburg (191), Department of Molecular Immunology, German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany Gerald T. Nepom (315),Virginia Mason Research Center and Department of Immunology, University of Washington School of Medicine, Seattle, Washington 98101 Josef M. Penninger (51),The Amgen Institute, Ontario Cancer Institute, and Departments of Medical Biophysics and Immunology, University of Toronto, Toronto, Ontario M5G 2C1, Canada ix

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ADVANCES IN IMMUNOLOGY, VOI.. hH

Posttranscriptional Regulation of mRNAs Important in T Cell Function JAMES S. MALTER Deparhnent of Parhobgy and laboratory Medicine, University of Wsconsin Hospifal and Clinic, Madison, Wisconsin 53792

I. Introduction

The development and activation of T lymphocytes involves substantial and complex alterations in gene expression. In this way, T cells can prepare for or react to changes in their environment. A great deal of attention has focused on understanding the transcriptional events that underlie T cell gene expression during differentiation or activation. Relatively little is known as to the extent of or mechanisms of posttranscriptional gene regulation under these conditions. Despite the infancy of this field, it is clear that a variety of critical genes are dominantly regulated at the level of posttranscriptional control. It is the intent of this review to discuss how posttranscriptional gene regulation, especially alterations in mRNA stability, contributes to the ultimate phenotype of a T lymphocyte. In some cases, I will extrapolate from other cell systems to T cells where insufficient data are available but likely applicable. Posttranscriptional regulation is often employed by cells or tissues that must respond quickly to changes in their environment with corresponding changes in gene expression. For most genes, transcription takes several hours to initiate and in the case of extremely long mRNAs (such as dystrophin) may take many hours to complete. Posttranscriptional control mechanisms, however, can be evoked in seconds to minutes, permitting cells much more control over mRNA and protein abundance and function. Therefore, posttranscriptional regulation affords greater speed in responding to stimuli. Second, such regulation is extremely versatile. As will be described later, cells can selectively regulate the stability, quantity, and translatability of individual mRNAs. Thus, modulation of a subclass of target mRNAs can be accomplished without affecting other cell functions. These features mandate the existence of regulatory systems capable of discriminating among different mRNAs. II. Measurement of mRNA Decay Rates

A variety of methods have been developed for the measurement of how rapidly mRNAs decay within cells. The rate of mRNA decay is equal to 1

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the production rdte (transcription) minus net accumulation. Therefore, in the absence of ongoing transcription, the rate of mRNA decay equals the change in mRNA amount over time. Thus, all methods for the measurement of inRNA decay must quench ongoing transcription or mask its presence. Current methods include pulse chase, transcriptional blockade, and a variety of inducible promoter systems. The measurement of decay of abundant mRNAs can be accomplished by puke chase (Alterman et at., 1984; Levis and Penman, 1977). Radiolabeled ribonucleotides are added to the culture supernatant and allowed to be incorporated into elongating transcripts. After a time adequate for radiolabeling of nascent mRNAs, a vast excess of unlabeled nucleotides are added to chase the radiolabeled homolog. This effectively produces a wave or pulse of radiolabeled mRNAs whose abundance can be followed over time. Although this method is effective for the measurement of high-abundance mRNAs such as globin, its application is quite limited. In addition to being insufficiently sensitive for low-abundance mRNAs, it remains unclear whether the instantaneous dilution of radiolabeled nucleotide occurs upon addition of unlabeled homolog. However, this methodology does not perturb the cells under investigation and, thus, where usable, remains the gold standard. Alternative methodologies have been developed to measure the decay rates of less abundant mRNAs. The most commonly employed use drugs that block transcription. Under such conditions, the amount of target mRNA remaining over time (as determined by Northern blot or RNAse protection) equals the decay rate. Transcriptional blockade can be accomplished with RNA polymerase I1 inhibitors, such as actinomycin D (Act-D) (Singer and Penman, 1972), cr-arnanitin (Chen et al., 1993), or the adenosine analog 5,6-dichloro-l-/3-~-ribofuranosyldencimidazole (DRB) (Sehgal et aZ., 1975). Act-D blocks transcription by intercalating between DNA stands, whereas DRB appears to function by interfering with kinases necessary for RNA polymerase I1 activity. The effective concentrations of these agents vary for different cell types, necessitating control experiments demonstrating that transcription has been inhibited. It is worth emphasizing that transcriptional blockage with these agents is global. Therefore, the measurement of decay of a particular mRNA occurs in the context of complete shutdown of the transcriptional apparatus. In addition, the effects of these drugs on other metabolic pathways are poorly characterized. Recent data have demonstrated that c-fos (Shyu et al., 1989; Chen, C. Y. et al., 1995), erythropoietin (Goldberget al., 1991), transferrin receptor (Seiser et al., 1995), and granulocyte macrophage-colony stimulating factor (GM-CSF) (Chen et al., 1993) mRNAs were stabilized in cells treated with Act-D. We have recently evaluated the effect of Act-D on

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3

the decay of capped, polyadenylated GM-CSF mRNA transfected into resting lymphocytes (Rajagopalan and Malter, 1996). GM-CSF mRNA was precipitated onto gold beads and accelerated into resting lymphocytes by particle-mediated gene transfer. After cells were washed to remove external, exogenous mRNAs, decay was assessed in the presence or absence of Act-D. When present, Act-D rapidly stabilized exogenous GM-CSF m R N A by greater than 10-fold. We also observed that the synthesis and secretion of GM-CSF protein was inhibited for approximately 2 hr when compared to untreated cells, although treated cells caught up by 10 hr. Therefore, Act-D has a profound effect on the mRNA decay machinery, as well as the translational apparatus. It remains unknown at this time if DRB induces similar effects. In order to avoid problems associated with the use of metabolic poisons, like Act-D or DRB, several inducible promoter systems have been developed. One of the most popular utilizes the c-fos promoter (Kabnick and Housman, 1988; Shyu et al., 1989). The presence of a serum response element within the promoter permits transient activation in response to serum. Transcription is self-limited, lasting approximately 15-30 min (Greenberg and Ziff, 1984; Kruijer et al., 1983), which produces a pulse of mRNA whose decay can be monitored by standard techniques. Functionally, this is equivalent to pulse chase but with a major advantage that the cDNA for any low-abundance mRNA can be used. However, in order to induce a brief pulse of transcription, cells harboring the transgene must undergo serum starvation for at least 12 and often 24 hr ( S h p et ul., 1989). Under such conditions, protein synthesis, gene transcription, and mRNA decay are reduced. For the study of c-fos mRNA, which is normally induced by serum, such issues are probably irrelevant. However, insufficient data exist to determine the appropriateness of these manipulations to study the decay of other mRNAs. Recently, other regulatable promoters have been employed for producing a controlled pulse of mRNA. These include the metallothionine metal response element (Hurta et al., 1993) as well as those activated by antibiotics such as tetracycline (Eldredge et al., 1995; Grossen and Bujard, 1992). The former is rather leaky, difficult to control, and requires cell culture in high concentrations of &valent metals. However, the tetracycline response promoters are gaining widespread use because these antibiotics have no apparent effects on eukaryotic cell metabolism, can be rapidly shut down, and are sufficiently active to produce a large bolus of niRNA on demand. 111. Measurement of Translation

The measurement of mRNA translation in intact cells is somewhat less problematic than mRNA decay. Clearly, the production of a previously

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JAMES S. MALTER

absent protein either in cell pellet or supernatant is a direct and irrefutable sign that translation has occurred. Protein can be detected by a variety of means including Western blotting, ELISA, or bioassay against a responsive cell line. It is important to point out that the rapid appearance of protein in a cell supernatant may reflect mobilization of preformed stores rather than de novo synthesis. Incubating the cells under study with 3sS-labeled amino acids with subsequent incorporation provides incontrovertible evidence of de novo protein synthesis. Another commonly used technique to evaluate translation is polysome profiling. Intracellularly, mRNAs can be partitioned to the soluble, ribonuclear protein fraction (mRNP) or attached to single or multiple ribosomes (so-calledpolysomes). Movement of an mRNA from soluble mRNPs to polysomes often precedes enhanced translation. Such changes often occur in response to changes in environmental conditions or cell activation. Translation can also be enhanced by cytoplasmic elongation of the 3’ polyadenylate tail. Well-described examples include vasopression mRNA (Carranza et al., 1988; Waller et al., 1993) in response to dehydration or water restriction. Finally, enhanced translation is often associated with increased activity of proteins important in translation initiation (Boa1 et al., 1993), which is the rate-limiting step. For example, on cell activation, eukaryotic initiation factor 4e (eIF4e or cap-binding protein) is phosphorylated, which increases its activity (Ma0 et al., 1992). Translation can be globally arrested by a variety of agents of which cycloheximide (CHX) is the most widely used. This drug freezes polypeptide elongation. Cycloheximide has profound effects on mRNA abundance by inhibiting mRNA decay. Many cytokine and protooncogene mRNAs, including GM-CSF (Rajagopalan and Malter, 1996), IL-2 (Shaw et al., 1988), IL-1 (Gorespe et al., 1993),cfos (Wilson and Treisman, 1988), and c-myc (Reed et al., 1987),are stabilized by CHX suggesting decay requires ongoing protein synthesis or the replenishment of a labile protein that destabilizes these m RNAs. IV. mRNAs Regulated by PosiiranscripiionalControl

The ability to rapidly modulate cytoplasmic mRNA quantity and translatability is critical for T lymphocytes to effectively respond to environmental change. Such change might be the binding of cell surface cytokine, interactions between the T cell receptor and specific antigen, differentiation, or in vitro manipulation with a variety of activating agents, including phorbol ester, ionophore, or lipopolysaccharide. During the past 10 years, our understanding and appreciation of posttranscriptional regulation has grown immensely. A large number of T lymphocyte and other cell mRNAs are

POS'ITRANSCRIPTIONAL REGULATION OF mRNAs

5

dominantly controlled by alterations in their stability and/or translatability rather than by transcriptional control. In the next sections, I will identify and discuss mRNAs under such regulation. A. PROTOONCOGENES The ability to increase or decrease protooncogene levels is integral to appropriate progression through the cell cycle, proliferative response, and apoptosis. Generally, mRNAs coding for protooncogenes are exquisitely unstable in resting T lymphocytes, due in large measure to the presence of cis-acting destabilizing domains. The best characterized of these is the AUUUA motif (see Section V). As cells move from Gointo GI,protooncogene mRNAs accumulate dramatically (Reed et al., 1987). The accumulation of c-myc mRNA occurred rapidly in normal B and T lymphocytes stimulated with phorbol ester, ionophore, or phytohemagglutinin (Reed et al., 1987). After stimulation with PHA, steady-state levels of c-myc mRNA increased 20- to 40-fold within 1 hr with only modest increases (3- to 5-fold) in the rate of transcription. It was also noted that c-myc mRNA stabilization was cycloheximide sensitive. C-fos mRNA is expressed rapidly and transiently following stimulation with growth factors, membrane depolarizing agents, neurotransmitters, and phorbol esters (Greenberg and Ziff, 1984). Transient expression of cfos mRNA was due to rapid decay mediated by a 3' untranslated region (UTR) AU-rich element (Wilson and Treisman, 1988) and a coding region instability determinant (Shyu et al., 1989). In fibroblasts the half-life of serum-induced c-fos mRNA was approximately 9 min, but can be indefinitely prolonged by the addtion of protein synthesis inhibitors such as cycloheximide (Rahmsdorf et al., 1987). In addition to cycloheximide, phorbol ester superinduced c-fos mRNA by approximately 10-fold,whereas calcium ionophore (A23187) had little, if any, effect (Shigeoka and Yang, 1990). In macrophages, c-fos mRNA can be induced by phorbol ester, lipopolysaccharide(LPS),or calcium ionophore. These effects were antagonized by interferon-y ( IFN-.)I),which enhanced c-fos mRNA degradation without affecting its transcription rate (Radzioch and Varesio, 1991).Others have shown that c-myc mRNA can also be downregulated by interferon-y (Harel-Bellan et al., 1988). Because interferon-y typically induced the expression of a variety of genes, c-fos and c-myc mRNA downregulation appears somewhat atypical. Pokeweed mitogen (PWM) or anti-CD3 antibodies significantly increased c-jun mRNA in T lymphocytes (Chauhan et al., 1993). The elevation of c-jun mRNA was maximal after 15-30 rnin of exposure of T cells to PWM. Although nuclear run on assays demonstrated enhanced transcription, c-jun mRNA was also stabilized. Cycloheximide treatment had no

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JAMES S. MALTER

effect on c-jun mRNA induction by PWM. Others have demonstrated that cycloheximide superinduced cjun in 3T3 fibroblasts (Lamph et al., 1988). They also observed upregulation of c j u n mRNA after phorbol ester treatment, although specific effects on mRNA stability were not assessed. The expression of the serine-threonine kinase pim-1 mRNA has been investigated in mitogen-treated ovine lymphocytes (Wingett et al., 1991). After a 4-hr stimulation with Con A and phorbol ester, a fourfold induction of pim-1 mRNA was observed. By 17 hr post stimulation, pim-1 mRNA had decreased by 50%. pim-1 mRNA half-life was 80 min at 4 hr poststimulation and fell to 35 min after 17 hr. In addition, cycloheximide superinduced the stability of pim-1 mRNA consistent with its function on other AUUUA-containing, protooncogene mRNAs. pim-1 mRNA is expressed as an alternatively spliced, 2.4-kb transcript in germ cells that lack AUUUA motifs. The decay of this mRNA was substantially slower than the AUUUAcontaining full-length message, consistent with a destabilizing role of the adenosine-uridine (AU)-rich element (ARE). IL-2 caused a transient increase in pim-1 mRNA in the IL-2-dependent murine CTLL-2 line (Dautry et al., 1988). The protooncogene c-kit encodes a transmembrane tyrosine kinase receptor whose ligand is the recently described stem cell factor. IL-3, IL4,and GM-CSF downregulated the expression of c-kit in murine myeloid mast cell progenitor cells (Sillaber et al., 1991). In normal CD34 bone marrow progenitor cells, phorbol ester downregulated steady-state levels of c-kit transcripts and stem cell factor receptor surface expression (Asano et al., 1993). The primary effect of phorbol ester (TPA) was to accelerate the decay of c-kit mRNA by fourfold, which was concentration dependent with a maximum at 10 mM. These effects were antagonized by cycloheximide. Transforming growth factor /3 (TGF-/3)treatment of murine hematopoetic progenitor cell lines also destabilized c-kit mRNA. Within 2 hr of TGF-P treatment, c-kit transcripts decayed with an accelerated half-life of approximately 1 hr. The decrease in c-kit mRNA was associated with a decrease in cell surface receptor expression (Dubois et al., 1994).As can be seen from the previous examples, cell activation with a variety of agonists often modulated the stability of rotooncogene and cell cycle progression factors. The tyrosine kinase p56k! ' was transiently downregulated upon T cell receptor/CD3 complex engagement (Paillard and Vaquero, 1991). Northern blotting and nuclear run-off assays revealed low basal transcription and accelerated decay, which combined to reduce steady-state p56Ick mRNA levels. This effect was antagonized by cycloheximide, which superinduced ~ 5 6 mRNA "~ levels by enhancing its stability. Thus, p56IckmRNA was downregulated by signal transduction pathways that usually increased lymphokine mRNA levels.

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Glucocorticoids induced Go/G1arrest of lymphoid cells by decreasing the abundance of cyclin D3. When dexamethasone was added to P1798 murine T lymphoma cells, cyclin D3 mRNA was reduced by 50% within 2 hr and 80% within 4 hr (Reisman and Thompson, 1995). The effects of glucocorticoids were reversible, however, with a return to control levels within 2 hr after their removal. Interestingly, there was no change in the transcription rate of cyclin D3 within 6 hr after the addition of glucocorticoids suggesting posttranscriptional regulation. Measurement of the decay rate of cyclin D3 showed a half-life of 8 hr in mid-log phase cells that was reduced to less than 1 hr after glucocorticoid treatment. These effects were antagonized by actinomycin D. Other cyclin mRNAs show considerable variation of expression throughout the cell cycle. Cyclin B1 peaks in G2/M with minimal levels in GI. BI mRNA can be induced transiently in HeLa cells after y-irradiation coincident with the development of a G2 block. Measurement of cyclin B, mRNA stability during different phases of the cell cycle revealed that the half-life varies from 1.1 hr in GI to 8 hr in S and 13 hr at the GJM interphase. Irradiation decreased the stability of cyclin B1 mRNA through an unknown mechanism (Maity et al., 1995). Similar regulation of the halflife of p53 mRNA was observed after the activation of peripheral blood mononuclear cells with a combination of phytohemagghtinin (PHA) and TPA (Voelkerding et al., 1995). At the GO/GIinterface, p53 mRNA was rapidly degraded with a half-life of 1 hr. Cycloheximide treatment superinduced p53 levels by stabilizing the mRNA. Cells driven into the cell cycle showed progressive stabilization of p53 mRNA to a half-life of 6 hr after 24 hr of stimulation. The combination of PHA and TPA was a more potent stabilizer than were TPA or PHA alone. This common theme of mRNA accumulation and stabilization on transit through the cell cycle also applies to mRNAs encoding c-rel (Gruniont and Gerondakis, 1990), B-myb (Reiss et ul., 1991), rufl (Colotta et al., 1991), the transcription factor spi-1 (PU.l) (Hensold et al., 1996), and the protein tyrosine phosphatase PTP-S (Rajendrakamar et al., 1993). In all cases, dramatic increases in mRNA levels were driven by enhanced mRNA stability.These effects were manifest at different points of the cell cycle corresponding to physiological requirements for these particular proteins. In most cases, cycloheximide antagonized regulated stability and typically increased steady-state mRNA levels. The mechanism of cycloheximide action remains obscure but has been ascribed to a requirement for a labile protein integral to mRNA destabilization, Conversely, cycloheximide and other protein synthesis inhibitors may directly interfere with polysome based mRNA decay. An additional level of regulation has been demonstrated for protooncogenes coded for by alternatively spliced mRNAs. Both c-re1 (Grumont and

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Gerondakis, 1990) and the human splicing factor PR264SC35 are coded for by multiple mRNAs which differ in their 3' untranslated regions. When measured simultaneously, these mRNAs exhibited dramatically different half-lives (Sureau and Perbal, 1994).

B. CYTOKINES Posttranscriptional gene regulation plays a critical role in the regulated expression of cytokines upon activation or differentiation. In many cases, increased cytokine mRNA stability preceded cytokine elaboration and constituted the dominant means by which T cells enhanced the production of these critical molecules. Although this field remains relatively new and certainly far less intensively studied than transcriptional regulation, the number of cytokines controlled through alterations in mRNA stability is striking. GM-CSF is a hematopoietic growth factor produced by a variety of cells, including T and B cells, monocytes, endothelial cells, and fibroblasts. A variety of activating agents, such as antigen, plant lectins (PHA, PWM, and Con A), mitogenic anti-cell surface antibodies, and phorbol esters (TPA), induced GM-CSF mRNA in T cells (Granelli-Piperno et al., 1984; Shaw and Kamen, 1986; Lindsten et al., 1989; Thorens et al., 1987). Thorens et al. (1987) demonstrated that mouse peritoneal macrophages can be induced to accumulate GM-CSF mRNA and release GM-CSF by inflammatory agents, phagocytosis, or adherence to substrates coated with fibronectin. GM-CSF mRNA accuinulation was blocked by dexamethasone and IFN-y. After activation, GM-CSF mRNA transcription rates were unchanged with accumulation of message entirely dependent on enhanced cytoplasmic stability. Shaw and Kamen (1986) and Caput et al. (1986) identified the presence of multiple AUUUA motifs in the 3' untranslated regon of GM-CSF. They demonstrated that GM-CSF mRNAs intrinsic instability in resting cells was due to these repeats, which could confer instability on a chimeric mRNA such as globin. However, Thorens et al. (1987) showed that c-sis, which codes for the chain of platelet-derived growth factor (PDGF), was differentially regulated from GM-CSF after macrophage adherence or LPS treatment. c-sis also contains AU multimers in the 3' untranslated region suggesting this class of unstable mRNAs may not be coordinately regulated at all times. After these initial observations, a number of groups have confirmed and extended them to better characterize the kinetics, types of activating agents, and responsible cis-acting elements. Lindsten et al. (1989) demonstrated that GM-CSF, IL-2, IFN-y, and tumor necrosis factor-a (TNF-a) mRNAs displayed differential regulation depending on how T cells were activated. Antibodies directed against the T cell receptor/CD3 complex induced

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upregulation of cytokine transcription but had no affect on cytoplasmic mRNA stability. However, when anti-CD28 and anti-T cell receptor antibodies were jointly employed, cytokine mRNAs were stabilized and markedly accumulated. Under such conditions, cytokine transcription rates were not enhanced. Interestingly, c-fos and c-myc mRNAs remained unstable despite activation through CD28. These data suggested that mRNA stability could be induced through specific signaling pathways. Just as mRNA stabilization appears necessary for cytoplasmic accumulation and gene expression, reinstitution of rapid decay must occur to quench cytohne production. Treatment of mitogen-activated peripheral blood lymphocytes as well as immortalized T lymphocyte lines with vitamin D3 downregulated GM-CSF mRNA by50 and 90% at 6 and 48 hr of exposure, respectively (Toebler et nl., 1988). The effects of vitamin D3 required protein synthesis as they were antagonized by cycloheximide. Nuclear runoff assays demonstrated that GM-CSF gene transcription was unchanged, but that mRNA decay was accelerated by approximately 10-fold. The immunosuppressive drugs FK506 and rapamycin selectively enhanced the degradation of GM-CSF and IL-2 mRNAs (Hanke et al., 1992). These drugs downregulated the IL-2 and GM-CSF promoters as well as enhanced the degradation of these mRNAs. Interestingly, neither the stability of IL2 receptor nor GAPDH mRNAs were affected by FK506 or rapamycin. These data suggested that commonly employed immunosuppressants may partially exert their effects by destabilizing cytokine mRNAs. Similar effects have been observed in activated lymphocytes treated with glucocorticoids or dexamethasone (Fessler et al., 1996). Finally, GM-CSF mRNA can be downregulated by cytokine treatment. In long-term bone marrow cultures stimulated with IL-1, TNF-a, or endotoxin, IFN-a inhibited the expression of GM-CSF mRNA (Gollner et al., 1995).The effects of IFN-a were dose and time dependent with maximal inhibition at 500 U/ml, which started approximately 90 min posttreatment. Transfection of GM-CSF promoter fragments revealed IFN-a had no affect on GM-CSF transcription but accelerated GM-CSF mRNA decay. Irradiation of cells induces the stress response associated with cytokine production. Hachiya et al. (1994) showed that irradiated fibroblasts increased their production of GM-CSF mRNA and protein. Irradiation was capable of potentiating the effects of phorbol ester stimulation. Removal of IL-1 bioactivity partially effaced the GM-CSF response suggesting IL1 contributed to the irradiation effect. Although runoff analysis revealed that the rate of transcription was increased, stability studies showed GMCSF mRNA half-life increased greater than fivefold in irradiated cells. These data suggested that GM-CSF can be upregulated through nonprotein kinase pathways.

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In addition to the previously described effects of phorbol ester, antigen, mitogens, and irradiation, calcium flux can also trigger GM-CSF expression. Treatment of EL-4 thymoma cells (Iwai et al., 1993) with the calcium ionophore A23187 induced a 12-fold increase in GM-CSF mRNA stability. These effects were mediated by the AUUUA boxes in the 3’ end of GM-CSF, although an upstream region approximately 160 bases away also contributed. IFN-y, like GM-CSF mRNA, can be superinduced by the treatment of cells with inhibitors of translation as well as low-dose y-irradiation (Lebendiker et al., 1987). Increased IFN-y mRNA levels could not be accounted for by increased transcription or decreased mRNA turnover. These data are in contradistinction to those of Lindsten et al. (1989), who demonstrated IFN-.)I was stabilized when T cells were activated with a combination of anti-CD28 and anti-CD3 antibodies. Peritoneal macrophages treated with LPS and IFN-y showed increased levels of IFN-P mRNA (Gessani et al., 1991). Measurement of IFN-/3 transcription failed to show an enhancement after LPS and IFN-.)I treatment despite a 20fold increase in steady-state mRNA levels. These authors attributed IFN-/3 accumulation under these conditions to enhanced stability. The recently described IL-12/NK stimulating factor has potent effects on human T cells. Addition of neutralizing anti-IL-12 antibody to PHAstimulated peripheral blood mononuclear cells markedly reduced both IFN-y protein and mRNA levels. Conversely, treatment of purified T cells with PHA and recombinant IL-12 increased IFN-y mRNA stability and protein production, whereas IL-2 mRNA levels were unaffected (Nagy et al., 1994).Interestingly, accessory signaling through CD40 synergized with IL-12 to upregulate the production of IFN-y as well as a variety of other TH1 and TH2 cytokines (Ping et al., 1996). These agents increased both the protein and the mRNA levels; however, determination of IFN-y mRNA half-life was not done. Purified human T lymphocytes activated by CD38 ligation also secreted a variety of cytokines, including IL-6, GM-CSF, IFN-y, and IL-10 (Ausiello et al., 1996). IL-7 stimulation of T lymphocytes synergized with anti-CD3 or anti-CD3/anti-CD28 to induce IFN-y and IL-4 mRNA expression (Borger et al., 1996a). At optimal concentrations, IL-7 (5 ng/ml) increased IFN-y mRNA levels by &fold and IL-4 mRNA by 3-fold. These effects could not be blocked by anti-IL-12 antibody. IL-7 induced the stabilization of both IFN-y and IL-4 mRNAs as well as enhancing their transcription. The regulated production of IL-la and -P has been intensively studied. In human fibroblasts and fibrosarcoma cells, IL-la and -p are constituitively transcribed but fail to accumulate. These data are consistent with a rapid decay rate, which has been estimated at approximately 20 min.

POS’ITRANSCRIPTIONAL REGULATION OF mRNA\

11

Treatment of fibroblasts with TNF-a, cycloheximide, or phorbol ester stabilized IL-1 mRNAs (Gorospe et al., 1993). Similar protein kinase C (PKOdependent IL-1 mRNA stabilization has been observed in human peripheral blood monocytes (Smith et al., 1991).However, downregulation of PKC by prolonged phorbol ester treatment followed by LPS stimulation revealed IL-la and -p mRNA levels can be regulated by an alternative signal transduction pathway. Stimulation of neutrophils with a combination of IL-1 and TNF-a upregulated IL-IP mRNA and protein (Marucha et al., 1991). These treatments induced large increases (30- to 90-fold) in the transcription rates of the IL-@ gene along with a modest (3- to 5-fold) increase in IL-1 mRNA stability. Retinoic acid enhanced IL-1 mRNA levels by altering the processing of precursor transcripts (Jarrous and Kaempfer, 1994). Under these conditions, IL-10 mRNA stability was not changed. Based on chimeric globin mRNAs containing the 3’ UTR of IL1, the 3’ AUUUA motifs were responsible for rapid IL-1 decay in resting cells (Kern et al., 1997). However, an LPS response element could not be mapped to the AU-rich determinant, suggesting another region of the 3’ UTR participated in this effect. Along with TNF-a, IL-1, and GM-CSF, IL-2 mRNA regulation has been intensively studied. Shaw et al. (1988) showed that IL-2 mRNA has a half-life of 1 hr in Jurkat cells or resting PBLs. IL-2 mRNA degradation was sensitive to cycloheximide as well as to actinomycin D. Lindsten et al. (1989) demonstrated that IL-2, GM-CSF, IFN-7, and TNF-a mRNAs were stabilized in normal T cells by mitogenic combinations of anti-CD28 and anti-CD3 antibodies. IL-2 mRNA accumulation can also be upregulated by treatment with PHA and TPA (Nordmann et al., 1989).Cyclosporin completely blocked IL-2 transcription but had no apparent effect on IL-2 mRNA decay. Garlesi and Mastro (1992) showed that pretreatment with phorbol ester for 10 hr blocked a response to subsequent mitogenic challenge with Con A or Con A plus TPA. Under such conditions, IL-2 mRNA accumulation was inhibited, which could be partially counteracted by cycloheximide. Conversely, mitogenic treatment with Con A plus TPA in the absence of pretreatment with TPA caused augmented IL-2 mRNA accumulation. These data suggested that protein kinase C activation induced IL-2 mRNA stabilization and accumulation (Dill et al., 1994). These authors noted that different mitogens differentially stabilized IL-2 mRNA. A combination of phorbol ester plus Con A appeared maximal and increased IL-2 inRNA levels by >20-fold and the half-life by >5-fold. Phorbol ester plus ionophore increased IL-2 mRNA by >100-fold and the half-life by 10-fold. In addtion to mitogens, IL-2 mRNA was stabilized by the treatment of Jurkat cells with substance P (Calvo, 1994).

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JAMES S . MALTER

Interestingly, substance P-mediated stabilization was abolished by concominant treatment with cyclosporin A, actinomycin D, or cycloheximide. Laff et al. (1995) evaluated the kinetics of IL-2 mRNA production and stabilization in mouse T cells stimulated through the CD28 and T cell receptors. CD28 signaling increased IL-2 mRNA levels by 20-fold compared to T cell receptor signaling alone. In the absence of CD28 costimulation, IL-2 mRNA rapidly decreased secondary to accelerated IL-2 mRNA decay, However, CD28 costimulation also involved nuclear effects as the levels of unspliced IL-2 mRNA were increased, Gerez et al. (1995) have shown that mitogenic induction of IL-2 gene expression also involved nuclear accumulation of precursor IL-2 mRNAs. The net production of mature message from precursor was greatly facilitated, causing a superinduction of cytoplasmic mRNA. The CD28 costimulatory pathway can be blocked by treatment of T cells with glucocorticoids (Fessler et al., 1996). No changes in transcription rate were observed in the presence of dexamethasone, demonstrating that the IL-2 mRNA levels must be controlled by posttranscriptional mechanisms. In addition to glucocorticoids, FK506 and rapamycin selectively enhanced the degradation of IL-2 mRNA (Hanke et al., 1992). Although it was not demonstrated, these data suggested that other AUUUA-containing cytokine mRNAs may be similarly affected. Destabilization was antagonized by okadaic acid, demonstrating the importance of phosphorylation in FK506-mediated degradation of IL-2 message. TNF-a is a critical cytokine produced by many cells of the hematopoietic and lymphoid systems. It has wide-ranging systemic effects and plays a central role in the pathophysiology of cachexia, inflammation, and septic shock. At the cellular level, TNF stimulated the production of cytokine by lymphocytes and macrophages. Sung et al. (1988) showed that TNF-a mRNA was stabilized in T cell lines and normal peripheral blood T lymphocytes by costimulation with phorbol ester and anti-CD3 antibody. Phorbol ester can substitute for CD28 stimulation in providing necessary signals for TNF-a mRNA stabilization. Protein kinase C inhibitors destabilized TNF-a mRNA in virus-infected astrocytes (Lieberman et al., 1990).These data supported a role for kinase-mediated regulatory systems in the control of TNF-a mRNA stability. Using a reversibly bound, protein kinase C activator, Sung et al. (1991) confirmed that TNF-a mRNA was stabilized through PKC-mediated events that could be antagonized by removing the agonist or adding the protein kinase inhibitor H-7. TNF-a mRNA can also be induced by ionizing y-irradiation (Weill et al., 1996). Because these effects were observed within 30 min, it is likely they involved some degree of posttranscriptional control. A variety of cytokines and drugs also modulated TNF-a mRNA stability. Treatment of murine macrophages with IL-4 plus LPS enhanced the degradation of TNF-a message, which was not observed

POSTTRANSCRIPTIONAL REGULATION OF m R N A s

13

after IFN-.)I plus LPS (Suk and Erickson, 1996). Thalidomide accelerated the decay of TNF-a message (Moreira et ul., 1993). The effect of this drug appeared specific because other LPS-induced monocyte cytokines were unaffected. In addition to the previously discussed mRNAs, a number of other cytokines are also controlled at the posttranscriptional level. IL-10 is produced spontaneously by monocytes and B cells but not by T cells of healthy donors. Cycloheximide treatment of normal T cells superinduced IL-10 inRNA levels through mRNA stabilization (Stordeur et al., 1995). IL-4 mRNA can be induced in resting T lymphocytes by Con A (Dokter et al., 1994). Treatment of Con A-stimulated T cells with IL-7 enhanced IL-4 inRNA levels by increasing its stability. Antibodies against IL-1 and TNF-a had no effect on the IL-7-induced enhancement of IL-4 mRNA suggesting a direct effect of IL-7. IL-4 mRNA was induced through antiCD3 or anti-CD3 plus anti-CD28 treatment of human T lymphocytes (Borger et al., 1996b). Treatment of stimulated cells with CAMP as well as prostaglandin E2 blocked mitogen-induced IL-4 mRNA accumulation. These effects were dominantly transcriptional when Con A was used to activate T cells, whereas the modulation of IL-4 inRNA stability was the dominant feature in CD3KD28-activated T lymphocytes. Interestingly, TPA plus calcium ionophore-induced IL-4 mRNA expression was insensitive to the effects of CAMPand prostaglandin E2.Treatment of lymphocytes with cross-linked TCR with vasoactive intestinal peptide inhibited IL-4 production at the posttranscriptional level (Wanget al., 1996).These effects were antagonized by recombinant IL-2. IL-3 is transiently produced by T lymphocytes stimulated with mitogen or antigen. Stimulation of mast cells with calcium ionophore or phorbol ester stabilized IL-3 rnRNA without affecting its transcription rate ( Wodnar-Filipowicz and Moroni, 1990). These effects disappeared when stimulating agonists were removed, demonstrating a necessity for ongoing signal transduction. In activated human T cells, IL-7 stabilized IL-3 and GM-CSF rnRNAs (Dokter et al., 1993). The IL-7-mediated effect was independent of protein synthesis and had no effect on the transcription rate. Enhanced mRNA levels led to 4-fold increases in IL-3 and GM-CSF protein secretion. In addition to blocking IL-2 transcription, cyclosporin A destabilized IL-3 mRNA (Nair et al., 1994). Interestingly, IL-4 and IL6 transcripts that were coexpressed with IL-3 mRNA were not affected by cyclosporin. All three cytokine rnRNAs contain 3’ AUUUUA motifs, suggesting selective regulation of IL-3 must occur through an additional mechanism. IL-6 has recently been shown to be regulated at a posttranscriptional level. Kuo et nl. (1996) showed that IL-lP added to c-kit ligana IL-10-stimulated mast cells prolonged the half-life of IL-6 m R N A by

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JAMES S. MALTEH

approximately 4 hr and induced a 50-fold increase in the level of IL-6 protein. IL-11 can be stabilized in phorbol ester-stimulated primate bone marrow stromal cells, which were susceptible to protein synthesis inhibition (Yang et al., 1996). Chimeric transcripts demonstrated that the 5’ UTR coding region and 3’ UTR contributed to regulated IL-11 mRNA decay. IL-8 mRNA was stabilized by prolonged (24-hr) treatment of human T cells with LPS (Villarete and Remick, 1996). Vascular endothelial growth factor (VEGF) is expressed by CD3-positive peripheral blood T cells. This potent endothelial cell mitogen and angiogenic factor can be induced in CD3-positive T cells by hypoxia (Freeman et al., 1995). Levy et al. (1996) demonstrated that hypoxia increased VEGF mRNA stability from 43 to 106 min. Several hypoxia-induced proteins were identified that bound specifically to 3’ UTR VEGF mRNA sequences and whose activity increased with message stabilization. Other members of the TNF-a family, including lyinphotoxin and lymphotoxin /3, have shown variable levels of posttranscriptional regulation in activated murine T cell clones (Millit and Ruddle, 1994).In anti-CD3-activated T cells, lymphotoxin and lymphotoxin fi mRNAs were stabilized. However, these two mRNAs showed differential sensitivity to cycloheximide, with lymphotoxin superinduced but lymphotoxin /3 unaffected. Other monocyte/macrophage-derived chemokine mRNAs, including HILDNLIF, GRO-a, and GRO-/3, can be synergistically induced with a combination of phorbol ester, LPS, and vitamin D3 (Anegon et al., 1991; Iida and Grotendorst, 1990). These mRNAs can be superinduced with cycloheximide and appear to be stabilized by mitogens. Finally, colony stimulating factor-1 (CSF-1) can be induced by phorbol ester, TNF-a, or cycloheximide (Koeffler et al., 1988). The half-life of GCSF mRNA was increased 16-fold in cells cultured with TNF-a, TPA, or cycloheximide. C. CELLSURFACERECEPTORS Just as cytokines are regulated by alterations in inRNA stability, cell surface cytokine receptors often show similar regulation. When T cell clones were anergized with high concentrations of peptide in the presence of antigen presenting cells (APCs) cell surface CD28 was decreased. This was accompanied by accelerated CD28 mRNA decay (Lake et al., 1993). The early lymphocyte-activation antigen, CD69, was rapidly induced during lymphoid activation. This molecule can transmit stimulatory signals in T and B lymphocytes, NK cells, and platelets. In phorbol ester-activated T lymphocytes, CD69 mRNA declined rapidly with a half-life of less than 60 min. The 3’ UTR of this mRNA contains AUUUA motifs that, when fused to a previously stable globin transcript, conferred instability (Santis et al., 1995). In the S1A T lymphoma cell line, polyunsaturated lipids

POSTTRANSCRIPTIONAL REGULATION OF inKNA\

15

enhanced Thy-1 inRNA and protein levels (Deglon et nl., 199Fj).Increased Thy-1 mRNA was entirely due to enhanced stability and appeared to be mediating by the coding region alone. IL-2 receptor-a (IL-2Ra) chain was upregulated in T lymphocytes activated with combinations of antiCD3 or anti-CD2 plus anti-CD28 antibodies. The costimulatory effect of dual-receptor ligature resulted in enhanced stability of IL-2Ra mRNA (Cerdan et a l , 1995). Combined treatment of human inonmytes with IL-2 and IFN-y has also been show to stabilize IL-2Ry mRNA (Bosco et al., 1994). CD7 is a 40-kDa member of the immunoglobulin superfamily expressed earIy in T cell development. Ligand binding to CD7 can deliver a costimulatory signal with CD3-mediated activation. Treatment of peripheral blood T cells with a nonmitogenic ionophore increased CD7 transcription without altering CD7 mRNA stability.After stimulation with mitogenic doses of ionomycin, PHA, and anti-CD3 antibody, CD7 mRNA stabilitywas enhanced (Ware and Elaynes, 1993). IL-4 receptor mRNA accumulated in human T cells after activation with Con A, TPA, calcium ionophone, or a combination of these agents (Dokter et nZ., 1992). Mitogens increased IL-4 receptor mRNA stability by two or threefold, which could be further enhanced by treatment with IL-4. The CSF-1 mRNAs can be upregulated in phorbol ester-stimulated monocytes. These effects were antagonized by cotreatment with dexamethasone and cyclosporin, which destabilized CSF-1 mRNA (Chambers et al., 1993). IL-6 treatment of the human monocytic line THP-1 stabilized IFN-y receptor inRNAs (Sancau et al., 1992).These data were in contradistinction to a dominantly transcriptional effect mediated by TNF-a treatment alone. mRNAs coding for the CD45 isoforms were controlled through alterations in stability (Deans et nl., 1992). After T cell activation high-molecular-mass isofornis of CD45 were preferentially expressed for approximately 2 days followed by rapid downregulation with increased expression of a low-molecular-weight isoform, CD45 KO. The switch from high- to low-molecular-mass isoforms appeared to be controlled by rapid mRNA degradation sensitive to cycloheximide. Specific ligation of a cytokine to its cell surface receptor has also been shown to alter receptor mRNA stability. IL-1 downregulated cell surface expression and mRNA levels of the IL-1 receptor type 1 (Ye et nE., 1992). When 2-15% of the IL-1 surface receptor was occupied, IL-1 receptor mRNA stability was reduced from 6 to 1 hr. These effects were blocked by cycloheximide suggesting de izovo protein synthesis may be necessary for decreased RNA stability. The IL-6 receptor is encoded by two distinct mRNAs of different lengths that vary only in the 3' untranslated region (Bowman et al., 1990). The longer mRNA contains multiple AUUUA motifs, suggesting that IL-6 receptor expression can be controlled by mitogens as well as alternative splicing to vary steady-stable levels. Expression

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JAMES S . MALTER

of the transferrin receptor is controlled by 3’ untranslated region iron response elements (Teixeira and Kuhn, 1991). In addition to iron levels, IL-2 treatment of the murine T cell line B6.1 induced transferrin receptor mRNA by 50-fold when added to arrested, IL-2-deprived cells (Seiser et al., 1993). In these cells, IL-2 increased the binding activity of the iron response element-binding protein, which prevented transferrin receptor mRNA degradation. CD2 is an important T cell surface receptor capable of transmitting proliferative signals. The stimulation of human peripheral blood mononuclear cells with PHA, anti-CD3 monoclonal antibody, and TPA rapidly increased CD2 mRNA levels by stabilizing CD2 mRNA by 4-fold (Malter et al., 1988). The upregulation of CD23 by IL-4 can be antagonized by IFN-.)I (Lee et al., 1993). The inhibitory action of IFN-.)I required new protein synthesis and occurred by decreasing the stability of CD23 mRNAs. D. OTHERmRNAs REGULATED BY ACTIVATION A well-known phenomenon associated with T cell activation is enhanced mRNA translation, which can increase by approximately 10-fold (Boa1 et al., 1993). Resting T lymphocytes express low levels of critical initiation factors, including eIF-2a, 4E, and 4A mRNAs, compared to proliferating T cells. Activation resulted in a rapid, 20- to 50-fold increase in the level of these three mRNAs (Ma0 et al., 1992). New protein synthesis was not required for increased initiation factor mRNA level, nor could transcription upregulation account for these changes. Therefore, eIF-4 mRNA stability was likely enhanced during T cell activation. For eIF-2a, the expression of alternatively spliced mRNAs with different 3’ UTRs may account for some of this effect. The 1.6- and 4.2-kb transcripts differed in their stability with the larger message more stable in activated cells (Miyamoto et al., 1996). The regulated expression of molecules critical for normal immune response, antigen recognition, and humoral immunity are partially or fully controlled by posttranscriptional regulatory pathways. CD3 is a multisubunit assembly associated with the T cell receptor. The CD36 gene produces three distinct, mature mRNAs of 0.7, 1.5, and 2.5 kb (Wilkinson et al., 1989).Cycloheximide treatment increased the expression of all three CD36 transcripts, which along with variably spliced mRNAs that differ only in the 3’ untranslated region, suggested a component of posttranscriptional control. Accumulation of the TCRa gene can be induced by phorbol ester, calcium ionophore, or protein synthesis inhibitors ( Wilkinson and Macleod, 1988). Treatment with multiple agonists increased the level of TCRa message that could not be suppressed by cyclosporin. HLA class I1 expression was also regulated at a posttranscriptional level in human T cells.

POSTTRANSCRIPTIONAL REGULATlON OF rnRNAs

17

Although resting T cells do not express detectable, cell surface class 11, activation with PHA and TPA caused a rapid appearance (Caplen et al., 1992).Northern blotting revealed constitutive expression of class I1 mRNAs that could be superinduced with cycloheximide. Del Pozzo and Guardiola (1996) showed that in vitro, HLA class I1 mRNAs associated with polysomes derived from cells treated with puromycin or cycloheximide were more rapidly degraded than those in the absence of protein synthesis inhibitors. Therefore, it appeared that ongoing translation was required for the stabilization of HLA class I1 mRNAs. Class I expression may also be partially controlled by regulated stability. Compared to HLA-A and -B, HLA-C showed low levels of cell surface expression. Northern blotting revealed that HLA-C mRNA was expressed at lower levels than HLA-B mRNA and that this difference resulted from faster degradation of the HLA-C mRNA (McCutcheon et al., 1995).A 3’ UTR domain approximately 600 bp downstream of the stop codon appeared responsible for this effect. In mature B cells, steady-state immunoglobulin mRNA levels were increased by approximately 50-fold over earlier B cell progenitors. Early stage lymphomas degraded p inRNA in approximately 2 hr, which was a 9-fold increase in stability in hybridomas (Genoviese and Milcarek, 1990). Cox and Emtage (1989) demonstrated a 6-fold stabilization of p mRNA as B cells differentiated into plasma cells. Enhanced stability was almost sufficient to account for the differences in steady-state mRNA levels between the two cell lines. Similar data have been shown by Reed et al. (1994). E. DIFFERENTIATION Very little information is available concerning alterations in mRNA stability in the developing thymus. In the microenvironment of the thymus, cytokines are probably carefully and precisely regulated to ensure the appropriate differentiation of T cells. Le et al. (1991) have shown that thymic epithelial cells produce IL-1 and IL-6. Primary cultures of normal human thymic epithelial cells treated with EGF or TGF-a increased IL-1 and IL-6 mRNA levels. In both cases, transcription rates were unchanged, but IL-1 and IL-6 mRNAs were stabilized. Because both EGF and TGFa can induce tyrosine phosphorylation, enhanced cytokine mRNA stability was likely dependent on tyrosine kinase cascades. In the most complete study to date, Takahama and Singer (1992) evaluated the regulation of CD4 and CD8 mRNAs as thymocytes transited from dual- to single-positive cells. T cell receptor cross-linlang induced such differentiation, which was entirely dependent on enhance degradation of CD4 or CD8 mRNAs. Interestingly, the VDJ recombinase gene, RAG-1, is also tightly regulated at the posttranscriptional level (Neale et al., 1992). RAG-1 and RAG-2 are

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JAMES S. MALTER

both expressed only in immature lymphocytes. Treatment of immature lymphocytes with phorbol ester caused a rapid elimination of RAG-1 and RAG-2 mRNA. In addition to blocking the transcription of RAG-1, phorbol ester accelerated the decay of RAG-1 mRNAs by more than twofold. V. cis Elements

Given the vast number of mRNAs that coexist in any cell at any point in its life cycle, mechanisms must exist by which the cellular machinery can discriminate one mRNA from another. The ability to localize mRNAs to distinct subcellular fractions, selectively degrade or stabilize, as well as load them onto polyribosomes at different times under different conditions requires elaborate regulatory capacity. This regulatory machinery appears to require both cis-acting elements embedded within mRNAs and trans factors capable of interacting with these cis elements. Cis elements can be produced by unique, contiguous nucleotide sequences, secondary structure such as stem loops, or higher folding between distant regions separated by as many as several thousand bases. The diversity of structural elements reflects the intrinsic ability of mRNAs to fold, bringing nearby or distant regions close together for the creation of regulatory sites. The location of cis elements can be predicted by the alignment of mRNA sequences coding for a single or a family of genes from divergent species. The alignment of mouse, rat, and human mRNAs often reveals unexpected homologies, especially outside of the coding regions. The conservation of 5’ or 3’ UTRs strongly suggests the presence of a regulatory element. For example, cytokine mRNAs typically show substantial homology throughout their entire length. Although the coding regions would be expected to share substantial homology at the nucleotide level, the 5’ and 3’ UTRs can be >90% identical (Shaw and Kamen, 1986). In general, conservation in the 5‘ UTR often implies the presence of translational control elements, whereas that in the 3’ UTR typically suggests domains important in mRNA stability or localization. In some cases, these domains may interact as well. The previous discussion is not meant to imply that all mRNA cis elements reside in untranslated regions. Recently, cfos (Kabnick and Housman, 1988; Shyu et al., 1989; Schiavi et al., 1994; Shyu et al., 1991; Wellington et al., 1993) and c-myc (Bernstein et al., 1992; Prokipeak et al., 1994) mRNAs have been shown to contain a destabilizing coding region element. Such domains are difficult to identify by homology search but tend to become apparent after other potential elements have been experimentally manipulated without loss of the expected phenotype.

POSTI'RANSCRIPTIONAL REGULATION OF mRNAs

19

A. ADENOSINE-URIDINE-RICH ELEMENTS The putative identification of regulatory domains by homology search necessitates experimental demonstration of their functionality. In 1986, Shaw and Kamen, coincident with Caput et al. (1986), identified a conserved nucleotide sequence consisting of repeated, tandem AUUU boxes within the 3' UTR of mRNAs encoding inflammatory mediators, cytokines, and protooncogenes. They were usually organized as AUUUAUUUA repeats varying in number from several to eight (Shaw and Kamen, 1986). These domains showed remarkable conservation across species lines and appeared restricted to cytokine and protooncogene mRNAs. The repeated AUUUA motifs (also known as Shaw-Kamen boxes, AU-rich elements, or AREs) failed to demonstrate obvious relationships to either the stop codon or the poly A tail. In addition, some mRNAs, such as IFN-.)I, contained two or three AUUUA tandem pentamers separated by 20-50 dissimilar bases followed by another cluster of AUUUA repeats. GM-CSF mRNA, on the other hand, showed much tighter packing of these domains. Measurement of GM-CSF mRNA decay in a T cell line after transcriptional blockade with Act-D revealed very rapid degradation with a half-life (&) of approximately 45 min (Shaw and Kamen, 1986). TNF-a mRNA decay showed similar kinetics (Caput et al., 1986). P-Globin, however, was supremely stable, with a calculated ti > 15 hr (Shaw and Kamen, 1986). Therefore, AREs destabilize mRNAs that contain them. Mutagenesis of the AUUUA repeats present in GM-CSF, TNF-a, or cfos mRNAs dramatically reduced the decay rate of the mutant mRNAs (Shaw and Kamen, 1986; Rajagopalan and Maker, 1996; Iwai et al., 1993). Conversely, chimeric globin mRNAs fused to the AUUUA motifs derived from GM-CSF or c-jios greatly accelerated the decay of this previously stable transcript from 17 hr to approximately 45 min (Shaw and Kamen, 1986). In addition, protein synthesis was required for the rapid decay of wild-type GM-CSF or chimeric globin GM-CSF mRNAs because it was prevented by cycloheximide (Shaw and Kamen, 1986). Finally, labile mRNA decay was coupled to PKC-regulated events because TPA treatment of T lymphocytes or fibroblasts stabilized most, but not all, AU-containing mRNAs (Shaw and Kamen, 1986; Koeffler et nl., 1988). Thus, these pioneering studies demonstrated a link between mRNA turnover mediated through AU motifs, translation, and PKC-mediated signal transduction pathways. These data showed that regulatory pathways that modulated transcriptional events coordinately controlled posttranscriptional events. Database searches have revealed many mRNAs with single reiterations of the AUUUA motif. P-Globin mRNA, for example, contains a single motif. Given the known stability of globin mRNA, these data suggested that

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JAMES S . MALTER

single reiterations of the AUUUA motif were unlikely to be a destabilizing element. Because multiple, often tandem reiterations of AUUUA were found in rapidly degraded mRNAs, this suggested that a higher order or reiterative structure containing multimers of this sequence might be the true element. Recent work (Zubiaga et al., 1995; Lagnado et al., 1994) has conclusively shown that the nonamer UUAUUUA (UIA) (U/A) is the true destabilizer formed by reiterations of the AUUUA motif or a single motif in a U-rich context. Interestingly, somewhat greater instability was conferred by reiterations of this nonamer as found in GM-CSF, IL-2, and TNFa mRNAs. Reiterations of AUUUA (AUUUAAUUUA)are not destabilizing (Lagnado et al., 1994) suggesting that multiple interior purines interfere with the dominantly U-rich element. Whether this destabilizing element assumes higher order structure remains unknown. Computer-assisted folding has failed to demonstrate a stable stem-loop structure and ribonuclease mapping has yet to be reported. Thus, current data suggest that the AUUUA motifs function as a primary sequence. Additional work has demonstrated that the 3’ untranslated location of the AU motif is not coincidental. P-Globin GM-CSF chimeras showed identical stability to wild-type P-globin if (i) the mRNA could not be translated, (ii) the AUUUA motifs were disrupted by guanosine or cytosine substitutions, and (iii) the stop codon was mutated, allowing polyribosomes to be translated into or through the 3’ untranslated region (Savant-Bhonsale and Cleveland, 1992). Untranslatable, chimeric mRNAs remained associated with cytoplasmic mRNPs rather than polyribosomes. This was in marked contrast to wildtype GM-CSF mRNAs containing functional stop codons that were associated with very large (>20 S) translation-dependent destabilizing complexes. Finally, ARE-containing mRNAs remained unstable only when fully translated. Inhibition of ribosome translocation as the result of the insertion of a stable stem-loop structure in the 5’ UTR prevented AREmediated destabilization (Aharon and Schneider, 1993). Somewhat unexpectedly, a stable stem loop within the 3’ UTR upstream of the ARE also blocked rapid decay (Curatola et aZ., 1995). These data suggested that ARE-mediated decay likely involves ribosomeassociated, translation-dependent decay factors that require particular topography only achieved under conditions of normal translation. These could include alterations in secondary or tertiary structure assumed by the 3’ untranslated region or possibly the juxtaposition of particular protein factors such as a ribonuclease to the mRNA target. However, Chen, C. Y. et aZ. (1995) showed that P-globin c-fos chimeras were rapidly degraded in the absence of translation. When chimeric mRNAs containing a c-fos ARE and a 5’ iron response element to permit regulated translational

POSTTRANSCRIPTIONAL REGULATION OF mRNAs

21

initiation were evaluated, the chimeric message also decayed rapidly in the absence of translation (Koeller et al., 1991).Finally, there have been recent reports that GM-CSF inRNA decay can occur in the absence of ongoing translation (Chen, C. Y. et al., 1995). Whether these discrepancies reflect true differences between the cfos ARE and GM-CSF remains unresolved. Other possible explanations include the types of cells used and the presence of metabolic poisons, such as actinomycin-D, used for the measurement of mRNA decay. Although the use of chimeric mRNAs has been invaluable to demonstrate the function of cis elements, such studies must be interpreted cautiously. For example, the Yj’ UTR or coding region may contain additional ancillary information necessary for appropriate regulation. The demonstration of coding region destabilizing elements in cfos (Shyu et al., 1989) and c-myc (Bernstein et al., 1992) mRNAs demonstrates that such regions should not be ignored. Finally, long-range interactions within the 3’ UTR of the insulin-like growth factor I1 mRNA (Meinsma et al., 1992; Scheper et al., 1995) show how distant sequences can assemble to form a functional destabilizing domain. Finally, chimeric mRNAs containing AREs are usually nonresponsive to the effects of phorbol ester or other agents known to stabilize their wildtype counterparts (Akashi et al., 1994). Therefore, it is highly likely that the AREs, although active as dominant destabilizers, may lose some functionality without additional ancillary information provided by nearby or distant sequences within the body of the mRNA. Given the paradigms of GM-CSF, IL-2, or TNF-a mRNAs, how do the AREs direct rapid decay? The biochemistry of this process remains largely unknown. Clearly, once initiated, decay is extremely rapid. Only on rare occasions have intermediates in this process been identified (Stoeckle, 1992), suggesting that once the process begins it occurs with extreme speed. Based on the requirement for translation, mRNA decay likely occurs on a polysome or in close association with it. Wilson and Treisman (1988) were first to demonstrate that c-fos mRNA decay was initiated by poly A tail shortening. They observed progressive loss of the polyadenylate tail before disappearance (with presumed cleavage) of the coding region. Shortly after the deadenylation to approximately 30 A residues, the mRNA body disappeared as assessed by Northern blotting. These data have been confirmed by many other investigators and suggested that deadenylation was the first step in labile mRNA decay. When the A tail reached a critical length of approximately 30 residues, destruction of the mRNA body was triggered. Such destruction could be the result of progressive and continued 3’ to 5’ exonuclease action or of internal endonuclease digestion. Using an in witro system to study c-myc mRNA decay, Brewer and Ross (1988) identified an mRNA decay intermediate whose termini was near to or at

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the AUUUA motifs. These data were interpreted to suggest that c-nzyc mRNA was cleaved by an endoribonuclease that may recognize the AU motifs. Several putative ribonucleases potentially specific for AUUUAcontaining mRNAs have been partially or completely purified (Astrom et al., 1992; Huaet al., 1993;Wennborget al., 1995; Caruccio and Ross, 1994).

B. APPROACHES TO IDENTIFYING NEWcis ELEMENTS Although the AU-rich element remains among the most intensively studied mRNA instability determinants, it is by no means the only one. As discussed herein, there appear to be dozens, and perhaps hundreds of mRNAs that are regulated partially or dominantly at the posttranscriptional level. Typically, nuclear runoffs fail to account for changes in mRNA abundance, suggesting alterations in mRNA stability may be operative. Because many mRNAs regulated at this level lack AU-rich elements, there must be additional domains with distinctive sequences, structures, and shapes capable of mediating regulated and selective decay. Clearly, the presence of shared elements among coregulated mRNAs provides a powerful means for coordinated control. Thus, both experimental data and common sense suggest that a plethora of distinctive cis elements exist. Identification of novel cis elements can be performed in a variety of ways. As mentioned previously, homology searches between divergent species can reveal unexpected conservation. This approach was used to identify the AUUUA motifs in cytokine and protooncogene mRNAs (Shaw and Kamen, 1986; Caput et al., 1986). Second, the availability of “natural experiments” can point to the presence of a previously undefined element. For example, IL-2 (Henics et al., 1994) and IL-3 (Mayo et al., 1995; Algate and McCubrey, 1993) mRNAs accumulate in MLA-144 and FL5.12 cell lines, respectively. Sequencing these mRNAs revealed the insertion of retroviral long terminal repeats (LTRs) into the 3’ UTRs. The LTRs disrupted the endogenous AUUUA motifs causing message stabilization. In some cases, the coopting of cellular protooncogenes into retroviruses has been associated with the loss of similar regulatory domains. v-fos lacks the terminal 3’ untranslated region of its cellular homolog c-fos. When measured, the decay rate of v-fos mRNA is far slower than that of c-fos, contributing to its accumulation and transforming activity (Rahmsdorf et al., 1987). Finally, the identification of protein binding sites may pinpoint the location of regulatory domains. We have used this approach to identify an instability determinant present in the amyloid protein precursor (APP) mRNA (Zaidi and Malter, 1994; Zaidi et al., 1994). In this case, a large 3’ untranslated region (1.2 kb) made classical mutagenesis approaches impractical. Therefore, we produced radiolabeled APP mRNA and used

POSTTRANSCRIPTIONAL REGULATION OF inRNAs

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it for mobility shift assays with cytoplasmic lysates from neuronal cell lines. APP mRNA was previously shown to be abnormally stable in these lines. We were able to identify multiple mRNA-protein interactions that ultimately were mapped to a 29-base domain approximately 200 bases from the stop codon. After mutagenizing this domain, APP mRNA was stabilized, demonstrating the functional significance of the protein binding site (Zaidi et al., 1994).

C. OTHERcis ELEMENTS Because many posttranscriptionally regulated mRNAs lack AU repeats, additional instability determinants clearly exist. There is no a priori reason why such domains need be AU rich. In this section, I will discuss some of the better defined mRNA instability elements found in mRNAs expressed by T lymphocytes. In many cases, such regulation has not been demonstrated in T cells but is likely. APP mRNAs are expressed by T cells as well as by most other nucleated human cells. At least five alternatively spliced APP mRNAs exist that code for amyloid precursor protein from which P-amyloid is proteolytically derived (Beyreuther et al., 1991). Overproduction of 0-amyloid is likely pivotal for the development of Alzheimer’s disease. In addition to overproduction of APP and 0-amyloid, a subset of patients also overexpress APP mRNA (Jacobsen et al., 1991). Elevated levels could be the result of enhanced transcription or decreased m RNA degradation or both. Therefore, we examined if APP mRNA was regulated at the posttranscriptional level by measuring its half-life in resting and activated peripheral blood mononuclear cells. In resting cells, APP mRNA decayed with a half-life of approximately 4 hr, which could be increased to >12 hr after cell activation with a mitogenic combination of phorbol ester and phytohemagglutinin (Zaidi et al., 1994). These data suggested that APP mRNA contained a domain through which its stability was controlled. By the use of protein binding and mutagenesis, a 29-base region approximately 200 bases from the stop codon was implicated in APP mRNA regulation (Zaidi and Malter, 1994).This region is highly conserved between murine and human APP mRNAs with 26 of 29 bases in common. Homology search has failed to reveal any additional mRNAs containing like sequences. At the nucleotide level, the regon is 61% AU but not organized into AUUUA motifs. Computer modeling revealed the presence of a potential stem-loop structure, although ribonuclease mapping has yet to be performed. Insertion of guanosine in place of adenosine and cytosine in place of uracil completely ablated the functionality of this domain, rendering APP mRNA constitutively stable (Zaidi and Malter, 1994).

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The coordinated regulation of intracellular iron metabolism occurs at both transcriptional and posttranscriptional levels. The iron response element (IRE) is a stem-loop structure common to several mRNAs whose levels vary with the iron status of the cell. The IRE is highly conserved from oocytes to mammals (Harford et al., 1994; Klausner et al., 1993). In particular, a single IRE is found in the 5‘ untranslated region of ferritin mRNA, whereas five copies are present in the 3’ untranslated region of transferrin mRNA. IREs have also been described in several other mRNAs that code for proteins involved in iron metabolism, such as ALA synthase. Based on computer folding, this sequence forms a stable stem loop (Harford et d.,1994; Klausner et d.,1993). The length of the stem appears variable, but there remains an absolute requirement for a conserved cytosine residue on the 5’ end of the stem, 5 nucleotides from the loop (Harford et al., 1994; Klausner et al., 1993). The loop itself is highly conserved and composed of 6 nucleotides with a conserved cytosine residue at the 5’ end. When this element is present in the 5’ UTR of ferritin, it confers translational regulation. When present in the 3’ UTR the IREs modulate transferrin receptor mRNA stability. The position of the element in respect to the 5’ cap in ferritin mRNA is critical for its function (Gray and Hentze, 1994). As it is moved toward the start codon, it becomes progressively less active, suggesting that it can inhibit the assembly of ribosomal components but not block the movement of a completely assembled ribosome. [For a more complete discussion of the IRE, please see one of the many recent reviews (Hentze and Kuhn, 1996)l. Ribonucleotide reductase is a highly regulated, rate-limiting enzyme responsible for the reduction of ribonucleotides to their corresponding deoxyribonucleotides (Wright et al., 1987). The enzyme is composed of dissimilar heterodimers, referred to as R1 and Rz, that are encoded by different genes. R1 is itself a homodimer with an aggregate molecular weight of 170 kDa that contains substrate and allosteric binding sites, whereas Rz is also a homodimer that binds iron (Kabnick and Housman, 1988; Verma and Sassone-Corsi, 1987). Appropriate enzymatic activity requires both R, and R2 subunits, whose levels change during proliferation (Weber, 1983) as well as after treatment with TGF-P1 (Hurta et al., 1991) or TPA (Choy et al., 1989). After stimulation with phorbol ester or TGF-P, both R, and Rz mRNA levels increase due to elevated stability of the coding mRNA. Through careful mutagenesis as well as the construction of chloramphenicol acetyltransferase R1 and Rz mRNA chimeras, Wright and coworkers (Chen et al., 1994) have conclusively demonstrated that regulated R1 mRNA stability was determined by a 49-nucleotide region located at the distal portion of the 3‘ UTR. In unstimulated cells, removal of this domain minimally destabilized R, mRNA, but its loss prevented

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25

phorbol ester-mediated stabilization.Therefore, the 49-nucleotide element mediates TPA-induced R1 stability but appears dispensable for rapid R1 mRNA decay in resting cells. Like R,, R2 mRNA levels are controlled by regulated stability. R2 mRNA contains an 83-nucleotide element located in the mid-3’ untranslated region that appears to function as a TGF-P1 response element (Amara et al., 1996a). In the presence of TGF-P, R2 mRNA was stabilized. Interestingly, the R1 and R2 3’ UTR cis elements are dissimilar and without sequence homology to other known stability elements. Functional mapping of these large domains has recently been reported (Amara et al., 1996b). The inevitable cellular production of superoxide and hydrogen peroxide as a result of oxygen-based metabolism requires abundant antioxidant enzymes (superoxide dismutase, catalase, and glutathione peroxidase; Fridovich and Freeman, 1986). In newborns, exposure of the lungs to hyperoxia results in increased antioxidant enzyme activity, which appears to be mediated by changes in catalase mRNA stability (Clerch et al., 1991). The response element has been putatively mapped to a conserved 240-base domain sequence within the 3’ UTR of catalase. The 3’ UTR contains two elements, one of which is a 36-base, stem-loop structure and the other a CA dinucleotide repeat. These domains together appear both necessary and sufficient for the specific binding of a cytoplasmic, catalase-specific, RNA-binding protein (Clerch, 1995). Despite the identification of an RNAbinding protein, it is yet to be shown (by deletion studies) that this domain confers regulated stability to catalase mRNA. Insulin-like growth factor-I1 ( IGF-I1) is a 67-amino-acid polypeptide structurally related to IGF-I and insulin (Rinderknecht and Humbel, 1978). IGF-I1 mRNA undergoes developmental and tissue-specific regulation by differential activation of four promoters and alternative splicing (de PagterHolthuizen et al., 1988; Van Dijk et al., 1991). In addition to full-length IGF-I1 mRNA, an uncapped 1.8-kb, 3’ cleavage product with an intact poly A tail has been identified (Meinsma et al., 1991). The cleavage of IGF-I1 mRNA is directed by 3’ UTR sequence elements separated by approximately 2 kb (Scheper et al., 1995). Each element is approximately 300 nucleotides long with the distal element encompassing the cleavage site. The downstream element was necessary for cleavage, whereas the upstream element controlled the rate at which cleavage occurred. When introduced into a chimeric mRNA (P-globin), these two elements directed cleavage of the chimera, suggesting they are necessary and sufficient. These interacting domains are to date the most widely separated and clearly demonstrate how distant regions can cooperate to form a functional element.

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The existence of extremely stable mRNAs could be accounted for by the absence of destabilizing elements or the inclusion of domains that retard mRNA decay. It appears that P- and y-globin mRNAs contain stabilizing domains. Globin mRNAs are produced in a tissue-restricted pattern by erythroid cells and eiythroid precursors. Liebhaber’s group has demonstrated the presence of distinct stability determinants within the 3‘ untranslated region of both a- and P-globin (Weiss and Liebhaber, 1995; Russell and Liebhaber, 1996). The a-globin domain appears to be composed of three cytosine-rich regions in which base substitutions cause message destabilization through a translationally independent mechanism (Weiss and Liebhaber, 1995).When ribosomes were permitted to translate into the 3’ UTR by mutagenesis of the stop codon, the stability element was nonfunctional. These data have been interpreted to imply that the aglobin determinant must be spatially organized and can be disrupted by ribosomal read-through or targeted mutation. The P-globin element showed similar sensitivity to ribosomal read-through, demonstrating that an unperturbed 3’ untranslated region was also critical for its normal function. However, unlike a-globin, antiterminated P-globin mRNA was unstable irrespective of whether it was translated (Russell and Liebhaber, 1996). The stability of P-globin may be further modulated by 5’ untranslated region sequences that, when mutated, also influence mRNA stability (Ho et al., 1996). Effector T cells release cytolytic enzymes such as perforin, a 70-kDa protein with cytolytic activity. Interestingly, cytolytic cells downregulate perforin and esterase mRNAs upon exposure to targets (Bajpaiet al., 1991). This decrease in perforin inRNA content was caused by accelerated mRNA decay that was unaffected by protein synthesis blockade by cycloheximide (Goebel et al., 1996),suggesting cotranslational degradation was not operative. Mutagenesis as well as the production of reporter chimeric mRNAs revealed perforin mRNA instability determinants were present in the coding region rather than the 3’ UTR. Based on the inability of small fragments of the coding region to confer regulation to the chimera, the authors concluded that the element consists of multiple domains that likely interact to form a functional unit. Interestingly, NK cells treated with IL-2 and IL-12 show enhanced stability of perforin mRNAs (Salcedo et al., 1993). Whether regulated perforin mRNA stability is solely dependent on the coding region determinant remains to be shown. Intracellular adhesion molecule-1 (ICAM-1; CD54) is one of several cell surface molecules belonging to the immunoglobulin superfamily. ICAM-1 serves as a ligand for Pzintegrins, lymphocyte function-associated antigen1 (LFA-1; CD-11A/CD-18) (Diamond et al., 1991), and MAC-1 (CDllB/ CD-18), which is critical for a variety of immune functions including T

POSTTRANSCRIPTIONAL REGULATION OF mRNAs

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cell-mediated killing, T-helper responses, as well as leukocyte trafficking and adherence to vascular endothelium and epidermal cells (Springer, 1990). ICAM-1 is inducible on a variety of cells but is dramatically upregulated at sites of inflammation (Springer, 1990). Many inflammatory mediators, such as IL-l, IFN-7, and synthetic mimetics such as phorbol esters, increase ICAM-1 expression (Carlos and Harlan, 1994). Sequence analysis revealed ICAM-1 mRNA contains multiple reiterations of AUUUA motifs that appear responsible for rapid decay (tt < 1 hr) in unstimulated monocytic cell lines (Ohh et al., 1994). Consistent with the role of AUUUA motifs in the posttranscriptional regulation of ICAM- 1, cycloheximide, phorbol ester, and IFN-y all stabilized ICAM-1 mRNA (Ohh et al., 1994). Mutagenesis revealed that the AUUUA motifs function as destabilizers in resting cells and appear to act as the TPA response element. However, IFN-7 stabilized ICAM mRNA from which the AUUUA motifs had been deleted (Ohh and Takei, 1994). The IFN-.)I response element was subsequently mapped to an 87-nucleotide region upstream of the AUdestabilizing motifs. The mRNA coding for protooncogenes cfus and c-myc is regulated at both transcriptional and posttranscriptional levels. The immediate-early response gene, c-fos, is dramatically upregulated (50- to 100-fold) during the transition of fibroblasts from Goto S phase (Muller et al., 1984). Similar upregulation has been observed when cells are exposed to ultraviolet light (Angelet al., 1986). Finally, stimulation of semm-deprived fibroblasts with serum or PDGF rapidly upregulated cfus mRNA transcription, which returned to prestimulation levels within 1 hr (Greenberg and Ziff, 1984). Because cfos mRNA accumulation largely mirrored transcriptional activity, it was clear that cfos mRNA must be exquisitely unstable, with an estimated half-life of between 5 and 20 min (Greenberg and Ziff, 1984). Sequence analysis of the 3' UTR of c-fos mRNA revealed the presence of three AUUUA motifs within a U-rich 67-base region. This regon was highly conserved between murine and human cfus genes, which differed by only two nucleotides (Van Straaten et al., 1983). The removal of this domain greatly enhanced the transforming potential of c-fos by stabilizing cfos mRNA and increasing the amounts of Fos protein. Of note, the viral homolog offos (vfus) lacks the 67-base destabilizing domain (Meijlink et al., 1985). Extensive mutagenesis of the 3' UTR has revealed the presence of two destabilizing domains (Chen, C. Y. et al., 1994). Domain 1is located within the proximal 49 nucleotides of the AU-rich element and contains 3 AUUUA motifs. This domain can function independently as an mRNA destabilizer. Domain 2 is a 20-nucleotide U-rich sequence 3' to the AU motifs that in and of itself cannot function as a destabilizer. However, this region enhanced the destabilizing activity of domain 1 and appeared to

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buffer potentially stabilizing mutations within domain 1. These data are consistent with a two-step mechanism for c-fos mRNA decay, with deadenylation followed by body cleavage, and are consistent with data showing that the initial steps in c-fos mRNA decay involved shortening of the polyadenylate tail prior to body cleavage (Wilson and Treisman, 1988). Despite the presence of the potent AU-rich element as a 3’ untranslated region destabilizer, c-fos mRNA contains a second domain within the protein coding region that appears to function independently as an instability determinant (Shyu et al., 1989). When inserted into P-globin mRNA, this region destabilized the chimeric message. As c-fos mRNA accumulated when translation was inhibited, deadenylation and decay appeared coupled to translation. The coding region determinant within c-fos mRNA encodes the nucleic acid binding and heterodimerization domains. This region is purine rich and approximately 60 bases in length. When inserted into a heterologous mRNA in frame, the resultant chimeric message was partially destabilized suggested the participation of additional coding region or 3’ UTR determinants. Finally, the coding region determinant was insensitive to actinomycin-D, which has been shown to impede the functionality of the AU-rich element. The c-myc protooncogene encodes proteins involved in transcriptional regulation and possibly DNA replication (Luscher and Eisenman, 1990). c-myc mRNA is exquisitely unstable and normally decays with a t 4 of approximately 15 min. Like c-fos mRNA, it contains two AU-rich regions preceding dual polyadenylation signals. Each of these domains is approximately 50 bases long and contains at least one or more AUUUA pentamers. Both lymphomas and myelomas have been identified with translocations causing the loss of the 3’ UTR of c-myc mRNA. In these cells c-myc RNA levels were elevated through message stabilization (Hollis et al., 1988). However, the translocations added additional heterologous sequences to both the 5‘ and the 3‘ ends of c-myc mRNA. Thus, the additional sequences may have altered c-myc mRNA decay. Indeed, 5’ UTR-truncated myc transcripts derived from fusions with immunoglobulin sequences were more stable than the wild type (Eick et al., 1985), demonstrating that additional heterologous sequences stabilized c-myc mRNA rather than the deleted 5’ UTR contained an instability determinant. Some investigators have been unable to show that the deletion of the AU-rich domains enhanced c-myc mRNA stability (Laird-Offringa et d.,1991). These authors also showed that complete translation was not necessary for rapid decay of c-myc mRNA. These observations suggested that an additional mRNA instability determinant may be present within c-myc mRNA. Recently, Wisdom and Lee (1991) demonstrated that the protein coding region confers instability on c-myc mRNA. Herrick and Ross (1994) demonstrated

POSTTRANSCRIPTIONAL REGULATION OF mRNAs

29

that this region can destabilize the normally stable p-globin mRNA when fused in frame. Interestingly, the coding region determinant coded for the nucleic acid binding and heterodimer-forming regions of c-myc protein -but was nonfunctional in the presence of actinomycin-D (Wisdom and Lee, 1991). In addition, depending on the cell type used for analysis, the coding region determinant may be silent (Yeilding et al., 1996). Thus, depending on the transcriptional blockers employed as well as the cell types used, the c-myc instability determinants may or may not be active. VI. hcrns Factors

Given the large number of both cis elements and mRNAs that contain them, cellular mechanisms must exist for the identification and regulation of specific RNAs. It seems inconceivable that ribonucleases, in and of themselves, can adequately discriminate between stable and unstable mRNAs. Therefore, many investigators have proposed the existence of cytoplasmic proteins capable of interacting with cis elements. Such proteins could function as destabilizers either by recruiting ribonucleases to a particular mRNA or by containing ribonuclease activity themselves. Conversely, proteins could act as stabilizers by directly interacting with mRNA targets and blocking ribonuclease recognition. A more complex model can also be envisioned whereby trans factors interact with mRNA outside of a putative cis element. Under such conditions, folding or conformation of the mRNA may be changed, obscuring or potentially increasing the presentation of a ribonuclease recognition site. Such binding need not be close to the cis-acting element but rather could be hundreds or even thousands of nucleotides away. The presence of RNA-protein interactions can be detected by the use of mobility shift assays or filter hybridization (Northwestern blotting). The most commonly employed assay has been the electrophoretic mobility shift assay. This technique is very similar to the DNA mobility shift assays commonly employed to detect DNA-binding proteins. Radiolabeled RNA is prepared in vitro and incubated with cytoplasmic extracts derived from the tissue or cell of interest. Assay conditions are critical, with most investigators favoring near-physiologic pH and relatively low ionic strength buffers. Nonspecific competitors, such as tRNA, poly-I, poly-C, poly-G, or heparin, are also included to decrease nonspecific interactions. After brief incubation at room temperature (5-30 min), ribonuclease (T-1 or A) is typically added to destroy any free RNA probe as well as portions of bound RNA that are not protected by the interacting protein. Samples can then immediately be electrophoresed on nondenaturing acvlamide gels, usually under low ionic strength conditions (0.25-0.5 X TBE) or ultraviolet light

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cross-linked and electrophoresed on denaturing SDS acrylamide gels (SDS-PAGE). Controls typically include the untreated RNA ligand and the RNA ligand treated with ribonuclease in the absence of cytoplasmic protein. Using a combination of native and SDS-PAGE, we were able to identify several distinct mRNA-binding proteins that interacted with the AUUUA motifs (Malter, 1989; Gillis and Malter, 1991), the APP 29-base element (Zaidi et al., 1994), and an erythropoietin mRNA destabilizer (Rondon et al., 1991). It is important to remember that the observed mass of the complex represents a combination of both protected RNA and protein, making definitive calculation of the protein’s molecular weight approximate. In addition, the differences in buffer and irrelevant competitors (tRNA vs. heparin vs. poly-I : poly-C) can lead to vastly different results using identical cell lysates. Northwestern blotting has been infrequently employed due to difficulties in achieving native folding of immobilized RNA-binding proteins. We observed a loss of binding specificity when nucleolin and hnRNP C proteins were immobilized on membranes (Zaidi and Malter, 1994). Others, however, have been successful in employing Northwestern blotting for the direct cloning of mRNA-binding proteins from cDNA libraries expressed in Escherichia coli (Qian and Wilusz, 1994). Thus, most investigators begin the search for trans factors interacting with their mRNA of interest by mobility shift assays. Using this approach, many RNA-binding proteins have been identified and their target sequences determined. In only a few cases have these proteins been cloned, but it is likely that within the next few years many more cDNAs will be obtained.

A. AU-SPECIFIC RNA-BINDING PROTEINS Due to the large number and critical nature of mRNAs containing AUUUA motifs, many investigators began to search for the existence of specific binding proteins. As previously discussed, such proteins could stabilize or destabilize AUUUA-containing mRNAs. Functionality might be determined by their affinity for RNA and their subcellular location, intracellular concentration, and activity. The first definitive report of an AU-specific mRNA-binding protein was made by Malter (1989). RNA mobility shift assays were performed with cytoplasmic lysates derived from log phase Jurkat cells (J32,T cell leukemia) and an 80-base radiolabeled, in uitro transcript containing four tandem repeats of the AUUUA motif. Cytoplasmic lysates were incubated in the presence of radiolabeled ARE containing RNA for 10 min in low ionic strength buffer at physiologic pH with a vast excess of nonspecific tRNA competitor. After 10 min, reaction mixtures were treated with RNase A or T-1 to cleave unprotected RNA, followed by nondenaturing polyacrylamide gel electrophoresis or ultraviolet cross-linking and SDS-PAGE.

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31

Using these two approaches, a dominant RNA-protein complex with a molecular mass of approximately 36 kDa was identified. This complex could be specifically competed by unlabeled AUUUA-containing RNA but not by irrelevant competitors. Based on binding specificity, this protein was dubbed the AU-binding factor or AUBF. Finally, it was demonstrated that AUBF must have very high affinity for AUUUA RNAs because complex formation was nearly instantaneous in solution phase. Interestingly, very short RNAs of less than 30 nucleotides, but containing multiple AU repeats, failed to interact with AUBF (Gillis and Malter, 1991). This suggested that secondary or higher order structures were involved in presenting the primary sequence to AUBF. Computer folding of AU-containing mRNAs has not revealed a thermodynamically preferred structure. This is not to say that such a structure is not adopted by the AU-rich RNA in solution, however. In addition, RNA ligands with less than three AUUUA repeats failed to interact with AUBF. Therefore, AUBF would not interact with mRNAs such as globin that contain a single AUUUA motif. Mutagenesis of the AUUUA repeats dramatically reduced the ability of AUBF to bind. Especially deleterious were conversions of the middle U to G (binding decreased by more than 95%) or U to C (binding decreased by approximately 80%) (Gillis and Malter, 1991). These data suggested that AUBF recognition was highly sequence specific. If the ribonuclease machinery that normally recognized and rapidly degraded AU-containing mRNAs has similar specificity, mRNAs with mutations in the AU-rich regions would escape ribonuclease surveillance and likely be long-lived. Indeed, transformed cell lines containing viral insertions that disrupt the AU-rich 3' UTRs of IL-2 (Chen et al., 1985)or IL-3 (Algateand McCubrey, 1993; Hirsch et al., 1993) create cytokine mRNAs with 5- to 10-fold greater half-lives than normal. Truncation of the c-fos mRNA has also been reported to stabilize this message and facilitate cellular transformation (Meijlink et al., 1985). Because cytokine mRNAs are stabilized in lymphoid cells by activation with phorbol ester (Shaw and Kamen, 1986), PHA (Shaw and Kamen, 1986), LPS (Thorens et al., 1987), ionophore ( Wodnar-Filipowicz and Moroni, 1990), cytokines ( Wodnar-Filipowicz and Moroni, 1990), or antigen (Takahama and Singer, 1992), changes in binding protein quantity or activity would provide important insight into their function. AUBF was inactive in resting lymphocytes (Malter and Hong, 1991). After treatment with PHA, TPA, or ionophone, activity was rapidly upregulated and maintained for at least 8 hr. Even in the presence of actinomycin-D and cycloheximide, similar upregulation of AUBF activity was observed. This suggested that preformed AUBF existed in an inactive state that, through posttranslational modification, acquired binding activity (Malter and Hong, 1991).

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Other nucleic acid-binding proteins including NF-KB can be similarly regulated (Kopp and Ghosh, 1995). Because phorbol ester and ionophore stimulated AUBF activity, PKC was likely involved in modulating the mRNA decay machinery. PKC or a kinase downstream of PKC might phosphorylate AUBF, causing alterations in binding activity. Such data would be consistent with the observed upregulation of AUBF activity in the context of both transcriptional and protein synthesis inhibition (Malter and Hong, 1991). This was tested directly by treating active cytoplasmic lysates with potato acid phosphatase (PAP), with subsequent measurement of AUBF activity. Under such conditions, PAP completely ablated AUBF activity, suggesting a requirement for phosphorylation. In addition to the PKC pathway, activation with 8-bromoCAMPalso increased AUBF activity (Stephens et al., 1992).Thus, it appears that multiple phosphorylation cascades can modulate AU-specific binding proteins. An interesting observation first made with the bacteriophage R17 RNAbinding protein was sensitivity to oxidants (Starzyk et aZ., 1982). A similar requirement has been noted for the iron response element-binding protein (Hentze et d.,1989) suggesting this might be a generalized phenomenon. Treatment of cytosolic lysates with oxidants such as diamide or N ethylmaleimide (NEM) completely ablated AUBF activity (Malter and Hong, 1991). After diamide oxidation, incubation with reducing agents such as 2-mercaptoethanol fully restored binding activity. These data suggested that redox changes within the cytosol, along with phosphorylation, regulated AUBF activity. Redox sensitivity suggested the presence of critical sulfhydryl groups that participated directly or indirectly in RNA binding. Reduced sulfhydrylscould be present in the active site or stabilize AUBF’s secondary or tertiary structure by chelating metals. The so-called zinc finger proteins chelate zinc through a coordination complex formed by cysteine and histidines. In order to differentiate between these two options, AUBF-AUUUA RNA complexes were treated with NEM. If cysteines were directly bound to the RNA ligand, they would be unavailable for modification by NEM and complexes would be maintained. Conversely, if sulfhydrylswere chelating metal ions at a site distant from that interacting with AUUUA RNA, NEM would block complex formation. Because NEM treatment inhibited preformed AUBF-AUUUA RNA complexes, it was likely that critical cysteine residues were distant from the RNA binding site and probably interacted with divalent metals. After extensive dialysis of AUBF-containing cytoplasmic lysates against EDTA and EGTA, a variety of divalent or trivalent metals were added back prior to binding activity assays (Malter et al., 1990). Only magnesium and calcium ions were able to reconstitute binding activity suggesting that

POSTTRANSCRIPTIONAL RECUJATION OF mRNAs

33

these metals likely interacted with AUBF and are important for its function. Based on these data, the authors proposed that AUBF was regulated by reversible phosphorylation as well as by reductiodoxidation. Given the absence of AUBF activity in resting lymphocytes and its rapid upregulation after phorbol ester, ionophore, or CAMPactivation, conditions that enhanced the stability of AUUUA mRNAs, it was proposed that AUBF might function as an AUUUA rnRNA stabilizer (Malter and Hong, 1991). In order to test this hypothesis, polysomes were isolated from mitogenactivated peripheral blood mononuclear cells and used to determine GMCSF mRNA decay in vitro (Rajagopalan and Malter, 1994). Under such conditions, GM-CSF mRNA decayed with a half-life of approximately 90 min. When polysomes were depleted of AUBF by RNA affinity chromatography, GM-CSF inRNA decay was accelerated fivefold. Although it is possible that proteins in addition to AUBF were removed by affinity chromatography, these observations support the proposal that polysomebased proteins specific for the AUUUA determinants of cytokine and potentially protooncogene messages can stabilize them. Direct confirmation of this conclusion must await cloning and expression of recombinant AUBF.

B. OTHERAUUUA-BINDING PROTEINS Polysomes from log phase tumor cells have long been used as an in vitro mRNA decay system (Brewer and Ross, 1988). Because in vitro decay on polysomes can discriminate between AU- and non-AU-containing mRNAs, they are a logical location for potential trans factors involved in regulated decay. As described previously, AUBF has been localized to polysomes (Rajagopalan and Maker, 1994). Brewer showed the presence of a destabilizing activity in the S130 soluble fraction obtained after the centrifugation of polysomes through sucrose gradients (Brewer and Ross, 1988). Using density gradient centrifugation, this activity was isolated, cloned, and denoted Auf-1 (Brewer, 1991). Auf-1 consisted of 37- and 40-kDa isoforms. Cloning of the 37-kDa isoform revealed two nonidentical RNA recognition motifs (Burd and Dreyfuss, 1994). The larger isoform appeared to contain an additional 19 amino acids located N terminal to the first RNA recognition motif (Ehrenman et al., 1994). In vitro binding assays with both cellular and recombinant Auf-1 showed high-affinity association constants in the low nanomolar range (DeMaria and Brewer, 1996). Indeed, DeMaria has shown that the affinity of Auf-1 to different ARES reflects the potency of ARE as an mRNA destabilizer. These data have been interpreted to support a role for Auf-1 in ARE-mediated decay. In addition to c-fos and c-myc mRNAs, Auf-1 interacts with GM-CSF, glucose transporter-1, and p-adrenergic receptor mRNAs (Pende et al., 1996).

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Because Auf-1 activity increased as AU-containing mRNA decay accelerated, it has been proposed to function as a destabilizer. Although the mechanism is unknown, Auf-1 may alter the secondary structure of target mRNAs, directly recruiting ribonucleases or in some other way making target mRNAs more susceptible to decay. Auf-1 has not been shown to possess intrinsic ribonuclease activity. Despite these associations, there is yet to be an unambiguous demonstration, either in uitro or in uiuo, of the functionality of Auf-1. Shortly after the description of AUBF, Bohjanen and coworkers (1991, 1992) identified a series of proteins termed AU-A, AU-B, and AU-C that bind to mRNA with AUUUA multimers. AU-A resembled AUBF as well as a similar protein described by Vakalopoulou et al. (1991) and migrated as a 34-kDa protein when associated with RNA. AU-A was primarily localized to the nucleus although it was also detected in the cytoplasm. This protein was capable of interacting with the 3’ untranslated region of GMCSF, IL-2, TNF-a, and c-myc mRNAs. A second RNA-binding protein, called AU-B, was not present in unstimulated T lymphocytes but was rapidly induced following engagement of the TCR-CD3 complex (Bohjanen et al., 1991).This protein has a predicted mass of 30-kDa and interacts with the AU-containing mRNAs listed previously with the exception of cmyc. AU-B appeared localized exclusively to the cytoplasm. Based on protease mapping, AU-A and AU-B are likely distinct proteins. A third activity, denoted AU-C, appeared structural related to AU-B. Both AU-B and AU-C required three or more AUUUA repeats for efficient and highaffinity binding and, like AUBF, were intolerant of mutations within the AUUUA recognition motif (Bohjanen et aZ., 1992). Because AU-A was constituitively present and tolerated wider variation of sequence than AU-B or AU-C, it has been proposed that AU-A may play a more general role in the metabolism of U-rich or AU-rich mRNAs. The upregulation of AU-B/AU-C upon CD3-TCR activation, along with their narrow sequence specificity, suggested that they may participate in the regulation of cytoplasmic inRNA decay. However, because TCR-CD3 activation is insufficient to stabilize GM-CSF, IL-2, and INF-.)I(Lindsten et aZ., 1989), conditions that upregulated AU-B and AU-C, it remains unclear what additional regulatory steps might be involved. Several other groups have described AU binding activities with molecular inasses of 30-50 kDa. These include a 32-kDa AU binding factor described by Vakalopoulou et ul. (1991) and Myer et nl. (1992), a group of four Urich sequence-binding proteins (You et al., 1992), and a group of three proteins designated A, B, and C that are capable of interacting with the TNF-a AU-rich element (He1et aE., 1996).The 32-kDa protein described

I'OS'ITRAN SCHIPTION A L HEGU LATION OF

111 R NA\

35

by the Steitz lab has recently been cloned and been designated hnKNP 0 (Meyer and Steitz, 1995). In addition to the novel activities described previously, it has recently become apparent that abundant nuclear proteins, such as heterogenous nuclear ribonuclear proteins A and C (hnKNP A and C), can be found in the cytoplasm, associated with polysomes and based on mobility shift assays, and able to interact with AU-containing mRNAs (Hamilton et nl., 1993). These data suggest that RNA-binding proteins may have distinctive functions depending on subcellular location. Changes in phosphorylation or other posttranslational modification rnay enhance their transit to the cytosol, where they can participate in labile rnRNA decay.

C OTHERCYTOKINE A N D PROTOONUI 3' non-coding sequence in an initial step in degradation of groa mRNA and is regulated by IL-1. N ~ i c l ~Acid i c Res. 20, 1123-1127. Stordeur, P., Schandene, L., Durez, P.. Gerard, C., Goldnian, M., and Velu, T. (1995). Spontaneous and cyclohexiinide induced interleukin-I 0 inRNA expression in human mononilclear cells. Mol. Zmmunol. 32, 233-239. Suk, K., and Erickson, K. (1996). Differential replation of tumor necrosis factor a nrRNA degradation and macrophages by IL-4 and interferon y . Inmunology 87, 551-558. Sung, S., Bjorndahl,J.. Wang, C., Kao, H., and Fu, S. (1988).Production of turnor necrosis factor by human T-cell lines and peripheral blood T-lymphocytes stimulated by phorbol niyristate acetate and anti CD3 antibody. J . Exp. Med. 167, 937. Sung, S., Waiters, J., Hudson, J.. and Gimball, J. (1991).Tumor necrosis factor a mRNA accumulation in human milo-nionocytic cell lines: Role of transcriptional regulation by DNA seqiience motifs and inHNA staltilization.J. ZtwnmoZ. 147, 2047-2054. Sureau, A,, and Perbal, B. (1994). Several mRNAs with variable 3' untranslated regions and different stahility, The human PR264 SC35 splicing factor. Proc. Natl. Acad. Sci. USA 91,932-936. Takahama, Y., and Singer, A. (1992).Post-transcriptional regulation of early T-cell development by T-cell receptor signals. Science 258, 1456- 1462. Teixeira, S., and Kuhn, L. (1991). Post-transcriptional regulation of the transferrin receptor and 4F2 antigen heavy chain rnRNA during growth activation of spleen cells. Ettr. J . Biochetii. 202, 819-826.

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Thorens, B., Mermod, J.-J., and Vassalli, P. (1987). Phagocytosis and inflammatory stimuli induced GM-CSF mRNA in macrophages through post-transcriptional regulation. Cell 48,671-679. Toebler, A,, Miller, C., Norman, A., and Koeffler, H. (1988). 1,25-Dihydroxyvitamin D3 modulates the expression of a lymphokine (granulocyte-macrophage colony stimulating factor) post-transcriptionally.J. Clin. Invest. 81, 1819-1823. Vakalopoulou, E., Schaack, J., and Shenk, T. (1991). A 32-kilodalton protein binds to AUrich domains in the 3' untranslated regions of rapidly degraded mRNAs. Mol. Cell. Bid. 11(6),3355-3364. van Dijk, M. A., van Schaik, F. M., Bootsma, H. J., Holthuizen, P., and Sussenbach, J. S. (1991). Initial characterization of the four promoters of the human insulin-like growth factor I1 gene. Mol. Cell. Endocrinol. 81(1-3), 81-94. van Straaten, F., Muller, R., Curran, T., Van Beveren, C., and Verma, I. M. (1983).Complete nucleotide sequence of a human c-onc gene: Deduced amino acid sequence of the human c-fos protein. Proc. Natl. Acad. Sci. USA 80(11), 3183-3187. Verma, I. M., and Sassone-Corsi, P. (1987). Proto-oncogene fos: Complex but versatile regulation. Cell 51(4), 513-514. Villarete, L., and Remick, D. (1996). Transcriptional and post-transcriptional upregulation of interleukin-8. Am. I. Puthol. 149, 1685-1693. Voelkerding, K., Steffen, D., Zaidi, S., and Maker, J. (1995). Post transcriptional regulation of the P53 tumor suppressor gene during growth-induction of human mononuclear cells. Oncogene 10,515-521. Waller, S. J., Carter, D. A,, Ang, H.-L., Ho, M.-Y., Zeng, Q., and Murphy, D. (1993). Regulation of the extent of polyadenylation of vasopressin and growth hormone mRNAs in response to physiologic stimuli. Reg. Peptides 45, 37-41. Wang, H., Xin, Z., Tang, H., and Ganea, D. (1996). Vasoactive intestinal peptide inhibits IL-4 production in murine T-cells by a post-transcriptional mechanism. J. Immunol. 156, 3243-3253. Ware, R., and Haynes, B. (1993). T-cell CD7 mRNA expression is regulated by both transcriptional and post-transcriptional mechanisms. Int. Immunol. 5, 179-187. Weber, G. (1983). Biochemical strategy of cancer cells and the design of chemotherapy: G. H. A. Clowes Memorial Lecture. Cancer Res. 43(8), 3466-3492. Weill, D., Gay, F., Tovey, M., and Chouaib, S. (1996). Induction of tumor necrosis factor (Y expression in human T-lymphocytes following ionizing y irradiation. J Interferon Cytokine Res. 16, 395-402. Weiss, I. M., and Liebhaber, S. A. (1995). Erythroid cell-specific mRNA stability elements in the a 2-globin 3' nontranslated region. Mol. Cell. B i d . 15(5),2457-2465. Wellington, C. L., Greenberg, M. E., and Belasco, J. G. (1993).The destabilizing elements in the coding region of c-fos mRNA are recognized as RNA. Mol. Cell. Biol. 13(8),50345042. Wennborg, A., Sohlberg, B., Angere, D., Klein, G., and Von Cabain, A. (1995). A human RNase E-like activity that cleaves RNA sequences involved in mRNA stability control. Proc. Natl. Acad. Sci. USA 92,7322-7326. Wilkinson, M., and Macleod, C. (1988). Complex regulation of the T-cell receptor (Y gene: Three different modes of triggering induction. Eur. J. Immunol. 18, 873-879. Wilkinson, M., Ceorgopoulos, K., Terhorst, C., and Macleod, C. (1989). The CD3S gene encodes multiple transcripts regulated by transcriptional and post-transcriptional mechanisms. Eur. J. Immunol. 19,2355-2360. Wilson, T., and Treisman, R. (1988). Removal of poly(A) and consequent degradation of c$os mRNA facilitated by 3' AU-rich sequences. Nature 336, 396-399.

POS’ITRANSCRIPTIONAL REGULATION OF inRNAs

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Wingett, D., Reeves, R., and Magnuson, N. (1991). Stability changes in Pim-1 protooncogene mRNA after mitogen stimulation of normal lymphocytes. J. Immunol. 147, 3653-3659. Wisdom, R., and Lee, W. (1991). The protein-coding region of c-myc inRNA contains a sequence that specifiesrapid mRNA turnover and induction by protein synthesis inhibitors. Genes Dev. 5(2), 232-243. Wodnar-Filipowicz,A., and Moroni, C. (1990). Regulation of interleukin-3 mRNA expression in mast cells occurs at the post-transcriptional level and is mediated by calcium ions. Proc. Natl. Acad. Sci. USA 87, 777-781. Wright, J. A,, Alam, T. G., McClarty, G. A,, Tagger, A. Y., andThelander, L. (1987).Altered expression of ribonucleotide reductase and role of M2 gene amplification in hydroxyurearesistant hamster, mouse, rat and human cell lines. Sorn. Cell Mol. Genet. 13(2),155-165. Yang, L., Steussy, C., Fuhrer, D., Hamilton, J., and Yang, Y.-C. (1996). Interleukin-11 mRNA stabilization in phorbol ester stimulated primate bone marrow stromal cells. Mol. Cell. Biol. 16, 3300-3307. Ye, K., Koch, K., Clark, B., and Dinarello, C. (1992). Interleukin-1 downregulates gene and surface expression of interleukin-1 receptor type 1by destabilizing its mRNA, whereas interleukin-2 increases it expression. Immunology 75, 427-434. Yeilding, N. M., Rehman, M. T., and Lee, W. M. (1996). Identification of sequences in cmyc mRNA that regulate its steady-state levels. Mol. Cell. B i d . 16(7), 3511-3522. You, Y., Chen, C. Y., and Shyu, A. B. (1992). U-rich sequence-binding proteins (URBPs) interacting with a 20-nucleotide U-rich sequence in the 3’ untranslated region of c-fos mRNA may be involved in the first step of c-fos mRNA degradation. Mol. Cell. B i d . 12(7),2931-2940. Zaidi, S. H., and Malter, J. S. (1994).Amyloid precursor protein mRNA stability is controlled by a 29-base element in the 3’-untranslated region.J. BioZ. Chern. 269(39), 24007-24013. Zaidi, S. H., Denman, R., and Malter, J. S. (1994). Multiple proteins interact at a unique cis-element in the 3’-untranslated region of ainyloid precursor protein mRNA. J. Biol. Chem. 269(39), 24000-24006. Zubiaga, A. M., Belasco, J. G., and Greenberg, M. E. (1995). The nonamer UUAUUUAUU is the key AU-rich sequence motif that mediates inRNA degradation. Mol. Cell. B i d . 15(4), 2219-2230. This chapter was accepted for publication on July 2, 1997.

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A J N A N L E 5 IN IMMLINOLOL\

VOI

hX

Molecular and Cellular Mechanisms of T lymphocyte Apoptosis JOSEF M. PENNINGER’ AND GUIDO KROEMERt ‘The Amgen lnrtiiufe, Ontario Comer Institute, and Deporhnenk of Medical Biophysics and Immunology, Universily of Toronto, Toronto, Onterio M5G 2C1, Canado; ond tCNRS-UPR 420, F-9480 1 Villejuif, France

I. Introduciion

According to the French philosopher Albert Camus, the primordial question of philosophy is whether life is worth living (1).It appears that this existential problem also affects various cell types in the multicellular organism. Apoptosis is now generally conceived as a strictly regulated (“programmed”) device for the removal of superfluous, aged, or darnaged cells within the immune system as well as in all other organ systems. It is fundamental for development, throughout embryogenesis, organ metamorphosis and organogenesis, including synaptic interactions of neurons, and repertoire selection of T lymphocytes. The process of apoptosis has attracted much attention in immunology, given that lymphocytes are continuously at risk of comitting suicide during positive and negative selection processes. In addition, acquired or genetically determined dysregulations of apoptosis have a major pathogenic impact, underlining the physiological importance of apoptosis control for the normal immune function. Enhanced resistance to apoptosis induction can lead to the persistance of self-reactive or mutated cells, which in nonnal circumstances would be eliminated to avoid the development of autoimmune diseases, lymphocyte hyperplasias (lymphadenopathy + splenornegaly due to lymphocyte acculeukemia). In mulation), or lymphocyte-derived tumors (lymphomas contrast, an enhanced susceptibility to apoptosis can cause a numeric or functional immunodeficiency. An ever-increasing number of spontaneous or experimentallygenerated genetic models of irninunopathologyare linked to dysregulations of apoptosis (Table I). Froin an immunologist’s point of view, another interesting facet of apoptosis concerns the handling of autoantigens. It is now generally agreed on that apoptosis is a physiological means of cell removal in which dying cells undergo subtle changes in membrane physicochemistry that trigger their recognition and phagocytic removal by normal adjacent cells. Classical apoptosis de facto precludes the release of cellular contents into the interstitiurn, thereby avoiding secondary inflammatory or autoimmune responses. Perturbation of apoptotic cell removal thus might play an important role in the initiation and/ or perpetuation of autoaggressive diseases. Nonetheless, this review will

+

52

JOSEF M. PENNINGER AND GUIDO KROEMER

TABLE I EXAMPLES OF H O M O L O G O U S R E C O M B I N A T I O N OF GENES AFFECTINGLYMPHOCV~E ApopTosIs IN THE MOUSE Knockout

Phenotype

Fds/CD%-’-

Lympoproliferative syndrome, liver hyperplasia, autoimmunity Defect in deletion of peripheral CD8+ T cells Defect in negative selection, large thymus Immunodeficiency, polycystic kidneys, hair hypopigmentation, and other defects Impaired lympocyte maturation, massive neuronal apoptosis Lymphoid hyperplasia, hypospermia Enhanced apoptosis in response to CD3 and Fas embryonic lethal

TNF-R1-lCD30-/BcI-2-’Bc1-X-IBtWSEKF

Reference 578 266, 579 256 418,419 420 580 380

focus on the genetic and molecular mechanisms of T lymphocyte apoptosis rather than discussing the death of antigen presenting or target cells. The modem era of apoptosis research has been initiated by the discovery that glucocorticoid-treated thymocytes undergo characteristic morphological changes involving shrinkage and chromatin condensation, accompanied by a characteristic DNA fragmentation into mono- and oligomers of -200 bp. This finding gave rise to the obvious speculation that endonuclease-mediated DNA fragmentation was the decisive event of the cell death process (2), and one of the first models of apoptosis regulation suggested that an augmentation of cytosolic and nuclear Ca2+concentrations would function as second messenger linking the initial trigger (glucocorticoid ligation) to the crucial biochemical event of (Ca2+-dependent) endonuclease activation (3). This simplistic model constitutes a good example how scientists tend to order isolated pieces of existing knowledge to construe theories explaining complex systems. In the meantime, it has been discovered that apoptosis-triggering stimuli can use an ever-increasing number of distinct signal transduction modules, that Ca2+ elevation is not decisive for apoptosis to occur in most models, and that endonuclease activation is a late event that actually is dispensable for apoptosis. Moreover, multiple additional elements of apoptosis regulation have been discovered during the past decade and have led to the refinement of the actual cell death researcher’s worldview. This review constitutes another attempt of ordering the known pieces of the apoptotic puzzle. According to current understanding, the process of apoptosis can be subdivided into at least three different phases (Fig. 1) (4,5). During

53

MECHANISMS OF APOPTOSIS

Inducer

Inducer

Inducer

central executioner

manifestations

Fie. 1. Schematic view of the three phases of apoptosis. During the initiation phase, different inducers trigger disparate pathways that finally trigger the central executioner. These pathways are “private” in the sense that they depend on the initial apoptosis trigger. The common phase of apoptosis has two distinct phases; the effector phase, during which the central executioner is activated and which is subject to regulatory mechanisms, and the degradation phase (beyond the point of no return), during which apoptosis becomes manifest at the levels of morphology and biochemical catabolism.

the initiation phase, cells receive apoptosis-triggering stimuli. Such death inducers include ligation of certain receptors [Fas/APO-l/CD95, tumor necrosis factor receptor (TNF-R), transforming growth factor receptor (TGF-R), etc.] or, in the case of obligate growth factor receptors, the absence of receptor occupancy. In addition, interventions on second messenger systems (Ca”, ceramide, kinases, etc.), suboptimal growth conditions (shortage of essential nutrients and hypoxia), mild physical damage (radiotherapy), and numerous toxins (reactive oxygen species, chemotherapy, and toxins strict0 sensu) can induce apoptosis. Virus infection and attack by cytotoxic T cells or NK cells is another way of apoptosis induction. Nonspecific or receptor-mediated death induction involves a stimulusdependent (“private”) biochemical pathway, and it is only after this initiation phase that common pathways come into action. It is generally assumed

54

[OSEF M. PENNINGER AND GUIDO KROEMER

that the execution phase of apoptosis defines the “decision to die” at the “point of no return” of the apoptotic cascade. During the execution phase the “central executioner of apoptosis” is activated. It is at this level that the different private pathways converge into one (or few) common pathway(s) and that cellular processes (redox potentials and expression levels of oncogene products including Bcl-2-related proteins) still have a decisive regulatory function. Once the cell has been irreversibly committed to death, the different manifestations classically associated with apoptosis such as DNA fragmentation become detectable. This degradation phase is similar in all cell types. It is characterized by the action of catabolic enzymes, mostly specific proteases (caspases) and endonucleases, within the limits of a near-to-intact plasma membrane. Thus, the cell actively contributes to its removal in a “suicidal”fashion and undergoes stereotyped biochemical and ultrastructural alterations. This review will focus on the various phases of apoptosis initiation, execution, and degradation of lymphocytes. First, we will elaborate on the common aspects of the late phase of the death process, which actually produces the phenotypic appearance of apoptosis. Second, we will discuss the actual conception of the effector stage, during which the cell activates the central executioner. Third, we will detail upstream (private) signal transduction pathways that regulate resistance or susceptibilty to death, as well as those proapoptotic signals that activate the central executioner. Thus, this review aims at integrating common principles of apoptosis executiom‘degradation with particular pathways of death control prevailing in lymphocyte (path0)physiology. II. Degradation Phase of Apoptosis

The degradation phase of apoptosis is probably very similar in all cell types. Electron microscopic features of apoptosis include a variety of changes in cellular ultrastructure. These changes are most prominent at the level of chromatin and nuclear structure. At the level of the nucleus, easily recognizable changes include condensation of chromatin that appears divided into compact and diffuse areas, progressive chromatin condensation that eventually involves all the nucleus with homogeneously electron-dense areas, reduction in nuclear volume (pyknosis), destruction of the nuclear envelope with disappearance of nuclear pores from the nuclear envelope that surrounds the compact areas of chromatin, and fragmentation of the nucleus (karyorrhexis). In contrast, morphological changes in the cytoplasma are less impressive: rounding up of the cell, blebbing of the plasma membrane, increased vacuolization, reduction in cell volume, and later cytolysis with signs of secondary necrosis (i.e., necrosis after apoptosis).

MECHANISMS OF APOPTOSIS

55

Organelle ultrastructure appears to be roughly preserved in cells that have undergone full-blown nuclear apoptosis. One important feature of apoptosis concerns the plasma membrane, which remains near-to-intact until late stages of the process. Thus, in contrast to primary necrosis (i.e., necrosis without apoptosis), apoptotic cells do not release their content and may form membrane-surrounded apoptotic bodies that contain organelles, nuclear fragments, and parts of the cytosol. Today’s overwhelming consensus is that these obvious changes in cellular structure occur well after that cells have “decided” to undergo cell death, during the so-called degradation phase of apoptosis. Obviously, the apoptotic process is accompanied by major changes in cellular biochemistry involving the activation of catabolic enzymes, mostly proteases and nucleases. In addition, major changes in energy metabolism, redox potentials, and ion homeostasis occur during the apoptotic degradation phase. Some particularly striking features of apoptotic degradation phase will be discussed in this section. However, this review will not cover all aspects of apoptotic degradation.

A. ENDONUCLEASES ACTIVATEDDURINC: APOPTOSIS During apoptosis, DNA fragmentation occurs via the activation of nucleases that generate either large fragments 2 5 0 kbp of DNA or monoand oligomers of 180-200 bp corresponding to the length of the nucleosome. This latter fragmentation, which is due to inteniucleosomal DNA cleavage, is generally referred to as oligonucleosomal DNA fragmentation. Apparently, different enzyme systems are involved in both types of DNA fragmentation, as suggested by the existence of cell lines that undergo high-molecular-weight DNA fragmentation without oligonucleosomal DNA fragmentation (6). Moreover, ZnZt typically inhibits only the oligonucleosomal type of DNA fragmentation (7).Typically, DNA froin apoptotic cells demonstrates double-strand breaks with single-base 3’ overhangs as well as blunt ends. It appears that single-base 3’ overhangs, as they are generated by some but not all nucleases, are seen in apoptotic but not in necrotic cell death (8).DNases that generate this type of cleavage include DNase I and cyclophilins A, B, and C (9,lO).Both DNase I and cyclophilin C induce 250 kbp DNA fragmentation but not oligonucleosomal DNA fragmentation ( 11). Nucleosomal-size DNA fragmentation may involve nuclear, -30- and -97-kDa endonucleases, as well as an -65-kDa cytoplasmic endonuclease, which are all inhibited by Zn2+(12, 13). Endonuclease G, which is also found in the mitochondria1 matrix, has recently been isolated from thymocyte nuclei (14). However, it appears that mitochondrial DNA is not degraded during apoptosis (15).

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JOSEF M. PENNINGER AND GUIDO KROEMER

Endonucleases contained in the nucleus may be activated in uitro by a number of different stimuli includng increases in Ca” or Mg2+ (16) concentrations, low pH (17),proteases such as trypsin (18),mitochondrial apoptosis-inducing factor (AIF) (19),and a heterodimeric (40/45 kDa) socalled DNA fragmentation factor (DFF) that is activated by caspase-3 (20). In addition caspase-mediated cleavage of actin may abolish actin’s inhibitory action on DNAse I (21). The relationship between DNA fragmentation and chromatin condensation is not clear. According to one report (22), chromatin condensation of rat thymocytes treated with lysates from Fas/APO-l-stimulated Jurkat cells requires ATP, whereas chromatin cleavage into 250-kbp and oligonucleosomal fragments does not, suggesting that both changes can be dissociated. That this is the case is also suggested by experiments involving Schizosaccharomyces pombe in which overexpression of proapoptotic genes from mammals (bax)or Caenorhabditis elegans (Ced-4)induces chromatin condensation without major DNA fragmentation (23, 24). Based on the fact that certain endoiiuclease inhibitors, such as Zn2+and aurintricarboxylic acid, can inhibit apoptosis in some models, it has been inferred that endonucleases are responsible for cell death. This is certainly not correct because some cell types and cell lines undergo apoptosis without endonuclease activation (6, 25) and because anucleate cells can be stimulated to undergo cell death (26, 27). Instead, it appears that Zn2+, which has pleiotropic effects on multiple enzyme systems, acts on yet undefined upstream events of the apoptotic cascade (28). This is also true for aurintricarboxylic acid, which inhibits, among other enzymes, calpain (29) and topoisomerase I1 (30). Thus, endonucleases probably are not central to the apoptotic process and rather fulfill the function of “cleaning up after death” (31).To our knowledge, the only case in which endonucleases may constitute a prime mechanism of cell death is provided by certain Mycoplasma species. Mycoplasma penetrans may cause cell death via the hrect action of pathogen-encoded endonucleases (32, 33). This type of cell death lacks certain features of apoptosis such as chromatin condensation and early disruption of mitochondrial functions (32). Advanced endonuclease activation occurs only at a late stage of the apoptotic process. Thus, after injection of glucocorticoidsin viuo, no major DNA fragmentation can be seen among lymphoid cells, even in conditions in which the cellularity of thymocytes and splenocytes is strongly declining (34). In situ, almost all thymocytes showing DNA fragmentation are localized within other cells (35),indicating that heterophagic recognition of apoptotic cells occurs before DNA fragmentation begins.

MECHANISMS OF APOF’TOSIS

57

B. CASPASES Overwhelming evidence suggests the involvement of specific cysteine proteases cleaving after aspartic acid (“caspases”),which catalyze a highly selective pattern of protein degradation, in apoptosis (36-40). At least 14 different caspases exist in humans. All caspases are synthesized as proenzymes (zymogens),which are proteolytically processed to form active heterodimeric enzymes. The cleavage sites for proteolytic maturation of procaspases are themselves cleaved by caspases, suggesting that caspases may engage in a cascade of proteolytic activation and amplification steps, much as this is known for the complement system. Despite a notable similarity in structure, different members of the caspase family possess distinctive activation requirements, substrate specificities, and inhibitory profiles (Table 11). Some, but not all, caspases are endowed with the capacity of autoactivation.Thus, for instance, caspase-1 cleaves procaspase1. Moreover, caspases can activate others following an ordered sequence. Thus, caspase-10 cleaves caspase-6, -7, and -8, which in turn do not cleave caspase-10, suggesting that different caspases may act at distinct levels of the apoptotic cascade. Gene knockout experiments have demonstrated an essential role for capase-1 in Fas/Apo-l/CD95-induced apoptosis (41, 42) as well as a role of caspase-3 in the regulation of neuronal apoptosis (43). However, these knockouts have no major impact on apoptosis in general. Thymocytes from either caspase-1 or caspase-3 knockout mice undergo normal glucocorticoid-induced apoptosis (41, 43), a finding that is widely interpreted to mean that caspases can function in a redundant fashion. The following findings underline the role of caspases in the apoptotic degradation phase: Addition of caspases to cell-free extracts is sufficient to trigger nuclear apoptosis. To obtain this effect, caspases have to trigger endonuclease activators contained in the cytosol(44-46). One such endonuclease activator is DFF, a heterodimeric protein that is activated by the proteolyic action of caspase-3 (20). Induction of apoptosis is accompanied by activation of caspases, which can be measured using fluorogenic substrates such as a fluorogenic substrate containing the cleavage site WAD [4-(4’-dimethylaminophenylazo) benzoic-YVADAPV-5-[-2-aminoethyl)-amino] naphtalene-l-sulfonic acid] or DEVD (Ac-DEVD-amino-4-methylcoumarin) (45). The activation of caspases cleaving DEVD appears to be a general phenomenon, whereas activation of YVAD-cleaving caspases is less frequent (47, 48). In apoptotic cells, some but not all caspases are found in the activated, proteolytically cleaved form. This applies in particular to caspase-3. Moreover,

TABLE I1 PKINCXPAL FEATURES OF CASPASES 1-10 No.

cn a3

Synonyms (special signs)

Optimal substrate (inhibitors)

Activators

1

ICE

WEHD [WAD.cink] [crmA]

2

ICH-1 Nedd-2 CPP32 Yama Apopain

DEVD

Granzyme B

DEVD [ DEVD.cmk]

Granzyine B Cytochroine c Caspase-6, -7, -10

3

Substrates Prointerleulan-lp Interferon-y-inducing factor a-Spectrin PITSLRE kinases Procaspase-1, -2, -3 PARP Procaspase-2 Sterol regulatory element binding protein Huntingtin DNA-dependent protein kinase a-Spectrin Protein kinase Cy (PKC-y) Actin B Fodrin Gas-2 Heteronuclear ribonucleoproteins C PITSLRE hnases PARP Retinoblastoina protein (Rb) U l 70 kDa PAK2 Procaspase-3, -6

5

Tx ICH-2 ICErel-I1 ICErel-111

6

TY Mch-2

4

I

8

in

9

Ls

10

Mch-3 ICE-LAP3 CMH-1 Mch-5 FLICE Mach- 1 [ FADD domain] ICE-LAP6 Mch-6 Mch-4 [FADD doinain]

WEHD

WEHD (IN/L)EXD

Caspase-10 Caspase-3

DEVD

Caspase-10

(IN/L)EXD [crniA]

Caspase-10 DISC

(IN/L)EXD

Granzyme B

(IN/L)EXD [crmA]

DISC

PARP Laniin A Heteronuclear riboniicleoproteins C PARP Sterol replator). element binding protein All known caspases

All known caspases

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JOSEF M. PENNINCER AND GUIDO KROEMER

a number of caspase substrates found in the nucleus [lamin, poly(ADPribose) polymerase (PARP), U1-70 kDa, etc.] and in the cytoplasma (e.g., fodrin) are constantly cleaved in apoptosis, at the same site of the primary sequence as that recognized by caspases. The subcellular localization of caspases has not been investigated in detail. It appears that some caspases are localized in the cytosol (procaspase-3 and -9) (49), whereas active interleukin-fl converting enzyme (ICE) distributes to the plasma membrane (50). It is unclear how caspases gain access to the nucleus to cleave nuclear substrates including PARP, U1-70 kDa, and the nuclear lamins. Inhibitors acting on a wide range of caspases, such as the baculovirus protein p35 (51-53), N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (z-VAD.fmk) (54-62), its truncated analog Boc-Asp(0Me)fluoromethyl ketone (B-D.fmk) (59), or acetyl-Tyr-Val-Ala-Asp-chloromethylketone (Ac-DEVD.cmk) (63), inhibit the acquisition of apoptotic morphology in a wide range of experimental models. According to one report, some cell lines (e.g., CTLL-2 cells) respond poorly to Z-VAD.fmkmediated inhibition of apoptosis but do respond to B-D.fmk (59).However, the overall consensus is that inhibition of caspases by z-VAD.fmk can fully prevent several aspects of classical apoptosis including endonuclease activation. The previous findings have suggested that caspase activation and apoptosis are near to synonymous. This idea has received apparent support by the fact that at least two procaspases (caspase-8 and -10) possess long N-terminal prodomains with homology to the so-called FADD/MORTl molecule, allowing its interaction with and activation by the Fas/APO-l/ CD95 or TNF-R complexes (64-66). Moreover, numerous natural inhibitors of apoptosis are inhibitors of caspases. Such caspase inhibitors are encoded by baculovirus (p35) (51),cowpox virus (crmA) (67), Kaposi’s sarcoma-associated human herpesvirus-8, or human molluscipoxvirus (68). Caspases are also inhibited by nitric oxide (NO), which acts via specific S-nitrosylation of cysteine residues important for proteolytic function (69). Multiple different proteins can be digested by caspases (Table 11).Some of these proteins have been considered to have a particularly important role in causing cell death. Thus, the “death substrate” PARP has been thought to be particularly important because cleavage of PARP causes enzymatic activation with consequent NAD depletion. However, cells from PARP knockout mice undergo apoptosis with normal kinetics (70). Actin cleavage has been thought to be important for the changes in overall cell morphology. However, actin is not cleaved in all cell types undergoing

MECHANISMS OF APOPTOSIS

61

apoptosis (71). The role of lamins, which are cleaved by a number of caspases in the apoptotic process, has also been overestimated. Thus, transfection of cells with caspase-resistant lainin B only retards the process of nuclear apoptosis (72). Functionally, more important caspase substrates may be DFF (20),which can stimulate endonucleases, and PAK2. Transfection of cells with caspase-3-resistant PAK2 abolishes formation of apoptotic bodies although it leaves unaffected endonuclease activation and phosphatidylserine exposure (73). Full inhibiton of caspase activation with consequent absence of protein degradation and endonuclease activation does not always rescue cells from death. In a series of different models, z-VAD.fmk does not prevent membrane blebbing, does not maintain the clonogenic potential of cells, and ultimately does not prevent cytolysis (60-62, 74) (Fig. 2), suggesting that, at least in some models of apoptosis, the activation of caspases, although necessary for acquisition of apoptotic morphology, occurs after the decision to die has been made. It thus appears that the activation of both endonucleases and “downstream caspases” occurs after the point of no return of the apoptotic process. Only in certain circumstances do caspases act in signal transduction pathways that may be linked to apoptosis induction. This applies, for instance, to caspase-8, which links the Fas/APO-l/CD95 receptor to induction of a number of kinases (35,38,46, and 54 kDa) including the stress-responsive mitogen-activated protein kinase p38/HOG (75, 76) and the dephosphorylation of retinoblastoma protein (77). Similarly, a crmA-inhibitable caspase links TNF-R-mediated signaling to ceramide generation (78). In synthesis, it appears that caspase activation is an obligatory corrolary of the apoptotic mode of cell death. In several cases, caspase activation links signal transduction pathways to apoptosis induction. However, caspases are not (or not always) involved in the initiation and effector stages of apoptosis. Moreover, cell death can occur in the absence of caspase activation.

C. OTHERPROTEASES In addition to caspases, a number of different proteases have been implicated in the apoptotic process. Putative apoptosis-triggering proteases include serine proteases, calpains, and proteasomes. Evidence for the involvement of these enzymes is based on studies using inhibitors that can prevent apoptosis induction in some models of cell death. These data are difficult to interpret because supposedly specific inhibitors of a protease can act on other enzyme systems. For instance, “calpain inhibitor I” is a relatively efficient inhibitor of proteasomes. Probably, most of these noncaspase proteases are only involved in some particular types of apoptosis induction, Thus, proteasome inhibitors such as lactacystin and MG132

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JOSEF M . PENNlNGEH AND GUlDO KROEMER

FIG. 2. Caspase inliibition can inhibit apoptosis without preventing cell death. Mouse thyinocytes were stiimilated for 12 hr with etoposide (2 p M ) ,either in the absence (A) or in the presence (B) of z-VAD.fink (100 p M ) . Note the typical apoptotic nuclear morphology (condensed, homogeneously electron-dense nuclei) that is visible with etoposide alone (A). This morphology is found in a cell that has undergone secondary necrosis ( i t . , necrosis after apoptosis). In the presence of etoposide plus z-VAD.fink (B) cells undergo cytolysis without acquiring an apoptotic morphology (i.e., primary necrosis). Similar results are obtained when, instead of etoposide, glucocorticoids are used as apoptosis inducers. For details see Ref. (62).

protect thymocytes against a number of different apoptosis inducers (glucocorticoids, etoposide, etc.) (79), have no effect on camptothecin-induced apoptosis of HL60 cells (SO), and induce apoptosis in proliferating cell lines (81, 82). How proteasome-mediated degradation of ubiquinated proteins can regulate apoptosis is not understood. Similarly to proteasome inhibitors, inhibitors of calpain and serine proteases prevent cell death in

MECIIANISMS OF APOF'TOSIS

63

some but not all systems of apoptosis induction (83, 84). The inhibitors of calpain (benzyloxycarbonyl-Leu-Leu-Tyrdiazomethylketone and acetylLeu-Leu-Nle aldehyde) and serine proteases (3,4-dicliloroisocoumariiiand tosyl-Phe chloromethylketone) may actually induce apoptosis (85). Altogether, these data suggest that proteasomes, calpains, and serine proteases are not involved in the common phase of the apoptotic process; these enzymes rather participate in private pathways upstream of the common effector phase. In addition to these upstream effectors, some iioncaspase proteases may be involved in the degradation phase. Thus, inhibition of the nuclear scaffold-associated serine protease by AAPFcmk prevents lamin breakdown (86).A number of different serine protease inhibitors have been shown to inhibit oligonucleosoinal DNA fragmentation without affecting

64

JOSEF M. PENNINGEH AND CUlDO K R O E M E R

formation of 250-kDa fragments and chromatin condensation (83). Inhibition of this serine protease does not prevent cell death, as might be expected. Currently, the molecular nature of these downstream proteases remains elusive.

D.

OTHER FEATUHES OF TIIE

DEGRADATION PHASE

1 . Cell Membrane Changes In normal intact cells, lipids contained in the plasma membrane are distributed in a rigorously asymmetric fashion. Phosphatidylserine and phosphatidylethanolamine are only found on the inner side of the plasma membrane. Apoptosis is accompanied by a loss of membrane asymmetry with consequent “flipping out” of phosphatidylserine and phosphatidylethanolamine, which become detectable on the surface of apoptotic cells (87, 88). This is a general feature of apoptosis regardless of the initiating stimulus (89-91). Phosphatidylserine exposure is prevented by caspase inhibition and occurs in anucleate cells (61, 92, 93), indicating that is independent of the nucleus but requires caspase activation. Chelation of extracellular (but not intracellular) Ca2+has a partial inhibitory effect (94). The exact mechanisms of phosphatidylserine exposure are elusive. It might involve the inhibition of an ATP-dependent aminophospholipid translocase (“flippase,” which would counteract the entropy of the outer membrane) (95) or activation of a Ca2+-activated“scramblase” (which would actively perturb the plasma membrane) (96-98). Alternatively, it might be due to the caspase-mediated derangement of the cytoskeleton with fodrin cleavage, thereby disrupting the anchoring of phosphatidyl serine residues on the internal side of the plasma membrane (99). The exposure of phosphatidylserine on the cell surface typically occurs before the membrane becomes permeable to vital dyes such as ethidium bromide or trypan blue. It appears, however, that some changes in plasma membrane permeability occur relatively early (100) and possibly facilitate the outflow of glutathione (101) and potassium (102). Phosphatidylserine exposure is a functionally important event because phagocytes possess receptors for phosphatidylserine ( 103).Thus, surface exposure of phosphatidylserine facilitates the recognition and removal of apoptotic cells. Phosphatidylserine also causes activation of procoagulant enzymes (104).

2. Redor Status Apoptosis is generally associated with major changes in the redox status (105). Such changes include a loss of nonoxidized glutathione (106, 107), which is extruded from the cell (101, 108) and/or may be oxidized during the process of apoptosis. Hyperproduction and/or reduced detoxification of reactive oxygen species is generally found in cells undergoing apoptosis

MECHANISMS OF APOPTOSIS

65

(109). Accordingly, membrane lipids tend to be oxidized in apoptotic cells (110). 3. Ion Fluxes During apoptosis, changes in subcellular Ca’+ distribution have been investigated in detail. It appears that during the late degradation phase, cytosolic Ca2+levels tend to increase to supraphysiologicallevels (>1 p~ ) (107), whereas K+ levels decrease (102). These changes may have some functional impact because they can participate in the activation of endonucleases. Whether they correspond to a general dysfunction of plasma membranes or a specific dysregulation of specific ion transporters is not clear. In conclusion, it appears that most if not all structures of the cell are severely perturbed during the apoptosis degradation phase. Thus, catabolic enzymes, mostly proteases and nucleases, become activated and the overall entropy increases, causing a loss of membrane asymmetry and later a disruption of membrane barrier function. Because direct inhibition of proteases and nucleases or interventions on other manifestations of the degradation phase (e.g., oxidative processes or increases in cytosolic Ca2+ levels) fail to preserve the cell’s integrity, with the exception of some special cases, these changes are not (or not always) decisive for the cell’s fate and rather become manifest after the cell has decided to die. The nature of this decision process-the effector phase of apoptosis-will be discussed in the next section. 111. Effector Phase of Apoptosis

As pointed out in the Introduction, the common phases of apoptosis, which includes the effector phase (regulated) and the degradation phase (beyond regulation), are likely to be the same in all cell types. Therefore, this section will discuss data obtained in different experimental systems, without any specific consideration of lymphocyte physiology.

A.

THE

“CENTRAL EXECUTIONER”: A THEORETICAL CONCEPT

Because apoptosis is induced by a myriad of different inducers (5, 111) but demonstrates a stereotyped pattern of morphological and biochemical changes, irrespective of the cell type and the initial trigger, several investigators have postulated the existence of a so-called central executioner (112) or “death machine” (38), colloqually also referred to as “great integrator” or “apostat.” Activation of the hypothetical central executioner during the effector stage would allow the decision to die to be made and to streamline

fifi

JOSEF M PENNINGEK AND GUIDO KIiOEMER

the many private pathways of apoptosis into one common pathway. The nature of this hypothetical entity has long remained elusive. What would we expect from the central executioner, based on functional considerations? In other terms, what criteria should a change in cellular biochemistry fulfill so that it may be considered a part of the central executioner?

Chronological criterion: For obvious reasons, the central executioner should become activated before alterations classically associated with the degradation phase occur (full-blown activation of the caspase cascade with lamin degradation, endonuclease activation, phosphatidylserine e.xposure on the plasma membrane, etc.) (Fig. 3A). Functional criterion: When apoptosis is induced, activation of the central executioner and of the degradation phase should be undissociable (Fig.3B). Criterion of conuei-gence: The central executioner should function as the great integrator. In other words, it should sense multiple proapoptotic signal transduction cascades as well as damage pathways, all of which should converge onto the central executioner (Fig. 3C). Criterion of coordination: Triggering of the central executioner should suffice to induce the entire spectrum of apopotic changes at the levels of the nucleus, the cytoplasrna, and the plasma membrane. In other words, the central executioner, once activated, should have pleiotropic effects on several organelle systems (Fig. 3D). Criterion of universulity: Because apoptosis follows the same end stage pathway in all cell types, the central executioner should be the same in lymphocytes and in all nonlymphoid cells. Similarly, the central execution should constitute a critical event of apoptosis that is independent of the cell death-initation stimulus (Fig. 3E). Criterion of vitality: All cells can be driven into apoptosis. This applies to primary cells from various tissues, to tumor cells, as well as to transformed

FIG.3. A heptalog of criteria that should be fulfilled by the hypothetical central executioner. (A). Criterion of chronology: The central executioner should be activated before the different facets of apoptotic degradation become apparent. (B). Functional criterion: During naturally occurring apoptosis, activation of the executioner and the manifestation of apoptotic degradation should be undissociable. (C). Criterion of convergence: Very different damage pathways (symbolized by lightnings) or receptor-mediated signal transduction pathways should converge on the central executioner. (D). Criterion of coordination: Activation of the central executioner should have pleiotropic effects on different organelle systems, thereby causing the full spectrum of apoptosis-linked changes. (E).Criterion of universality: The central execution should be the same in different cell types and in different models of apoptosis induction. (F).Criterion of vitality: The central executioner (or its components) should exert functions vital for normal cell survival. ( G ) .Criterion of the switch: The central executioner should be a self-amplifying device that is either in the on or in the off position.

A Criterion of chronology

B

Functional criterion

C

Criterion of convergence D

I

E

Criterion of universality

F

Criterion of vitality

G

Crit. of coordination

I

central executioner

I

Criterion of the switch

Growth factor withdr. Ceramide etc. Pro-oxidants (ROS, NO) Ischemidreperfusion Excitotoxiq calcium Metabolic toxins, etc. mnifertations olapoptosis

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JOSEF M. PENNINGER AND GUIDO KROEMER

cell lines that have been cultured during decades in vitro (113).Based on this premise, it may be speculated that the structures necessary for apoptotic cell death are also indispensable for normal cell survival; otherwise, mutated cells that are completely resistant to apoptosis induction would have arisen during in vivo or in vitro selection for tumor growth (Fig. 3F). Criterion ofthe switch: Cell death is an all-or-nothing process. There is no such thing as a “half-dead’ or “half-alive” cell. Accordingly, the central executioner should be activated in an all-or-nothing fashion. In other biological systems such irreversible ordoff decision processes are obtained by positive feedback loops that lock the cell in a committed state. [This applies, for instance, to differentiation processes involving transcription factors that stimulate their own transcription ( 114)].Accordingly, the central executioner should have self-amplifying properties so that it can behave like a switch that is either in the on or in the off position (Fig. 3G).

B. A FEWERRONEOUS PROPOSALS ON THE NATUREOF THE CENTRAL EXECUTIONER Since the rediscovery of apoptosis by Andrew Wyllie and colleagues (115),numerous studies have claimed the discovery of the universal mechanism of apoptosis. Most if not all of these theories have been invalidated. Unfortunately, it takes a long time for the scientific community to dismiss wrong hypotheses. Thus, the recent literature on apoptosis is still plagued with a number of erroneous a priori ideas on apoptosis that have been en vogue during short periods. Here we will discuss some of these incorrect assumptions on the general mechanism of apoptosis (Table 111).

1. “Killer Genes” and “Death Programs”? Based on the observation that inhibition of transcription and translation can prevent apoptosis in several models, it has been widely assumed that apoptosis would require the expression of killer genes or the realization of genetic death programs. This idea was apparently supported by the observation that numerous genes regulate apoptosis susceptibility. Stimulated by the hypothetical existence of so-called killer genes, hundreds of groups have attempted to isolate genes specifically expressed during apoptosis using differential display and subtracted libraries. The final result of these efforts has been extremely deceiving. No universal killer gene has been discovered in mammalian cells. The few genes that have been implicated in death induction are only involved in particular induction pathways but are dispensable for apoptosis induction in most systems. Thus, for instance, nur77, whose expression is involved in T cell receptor (TCR)-mediated thymocyte deletion, is not required for glucocorticoid-

69

MECHAR‘ISMS OF APOPTOSIS

EmovEous ASSEHTIONSO N

THE

TABLE I11 NAIUIIE01;T H E CENTRAI. EXECUTIONER OF APOITOSIS

Assertion

Invalidation

Apoptosis requires the expression of “killer genes” or “death programs” Apoptosis is an abortive cell cycle or mitotic catastrophe

Apoptosis c m Iw induc.ed in all cells in tlw presence of cyclohexiinide (113) Apoptosis can he induced dnring any pliase of the cell cycle [reviewed by Ref.

Apoptosis is a nuclear process

Cytoplasts (anucleate cells) can uudergo receptor-tnedi~itrdcell death (26, 27) Apoptosis can be induced in/by the al)sence o f oxygen, in a Bcl-2-regiilatetl fhshion (135, 1%) Ca” depletion can induce apoptosis ( 143); nuclear apoptosis can be induced in Ca”-free inedia (147, 179, 581) I n some inodels. acidification inhibits cell death (150) Bax and Bak overexpression intluces death in the presence of a caspase inhibitor (60, 61); Bcl-XI,overexpression can resciie cells i n which caspases have cleaved PARP (162) 111 some cases, necrosis is inhibited by the apoptosis regulator Bcl-2 (167, 169); classical apoptosis inducers provoke necrosis i n the presence of caspase inhibitors (Tuhlr IV) or in conditions of ATP depletion (171, 582) Cell-free systenis of apoptosis require mitochondria or mitochondria1 products (19. 147, 179)

(511 Apoptosis requires the action of reactive oxygen species Apoptosis requires the elevation of Ca’+ i n sonie subcellular conipartnient Apoptosis requires cytosolic acidification Apoptosis always requires the action of caspses

Apoptosis is fundainentally different froin necrosis

Apoptosis does not involve initochondria

induced thymocyte apoptosis (116, 117). In the meantime, it has become clear that protein neosynthesis is not required for apoptosis induction in most systems (118, 119). On the contraiy, it appears that all primary cells can be driven into apoptosis by a combination of the kinase inhibitor staurosporine A and the protein synthesis inhibitor cyclohexiinide (58, 113).This iinplies that all proteins and probably all nonprotein structures necessary for the acquisition of the apoptotic phenotype are constitutively expressed. 2. Apoptosis as an “Aliortizje Cell Ciple” of “Mitotic Catastrophe”? A few gene products that regulate apoptosis susceptibility (e.g., p53, Bcl-2, Bax, c-Myc, E2F-1) have marked effects on cell cycle control (120-

70

JOSEF M. PENNINGER A N D GUlDO KHOEMER

127).In addition, in some special cases, apoptosis induction has been linked to a determined phase of the cell cycle (128).These findings suggested that apoptosis might constitute a sort of abortive cell cycle. Apoptosis and mitosis share some characteristics, such as cytoskeletal changes, rounding up of the cell, nuclear envelope breakdown, and chromatin condensation, thus favoring the speculation that programmed cell death (PCD) might represent an aberrant cell cycle, involving out-of-phase expression in postmitotic cells of mitotic activities with lethal consequence; PCD would be a mitotic catastrophe (129). Nonetheless, it appears that the link between apoptosis and cell cycle, if such a link truly exists, must be loose. Although in some special cases apoptosis induction is linked to a perturbation of cell cycle advancement, in principle apoptosis can be induced both in resting and in proliferating cells, during any phase of the cell cycle ( 5 ) . Recent studies have also shown that disassembly of nuclei is radically different in mitosis and apoptosis. In mitosis, p34'd"-mediated phosphorylation of lamins results in their reversible depolymerization, whereas in apoptosis lamin is irreversibly degraded by proteases f 130).Thus, apoptosis is not an aborted mitosis.

3. The Nucleus CIS a Prime Target of the Apoptotic Process? Because changes in nuclear morphology are the most spectacular ultrastructural features of apoptosis, it has been assumed for a long time that the nucleus would be a prime target of the apoptotic effector phase. This idea was strengthened by the fact that endonuclease-mediated DNA cleavage was the first biochemical alteration specifically associated with apoptosis. In the meantime, several independent groups have reported that cytoplasts (anucleate cells) can undergo receptor-induced regulated cell death that shares several features with normal apoptosis: regulation by Bcl-2 (26,27),caspase activation, phosphatidylserine exposure (92,131), and disruption of the mitochondria1 transmembrane potential (131, 132). Apoptosis-like death of cytoplasts has been induced by a variety of stimuli including growth factor withdrawal, cross-linking of Fas, staurosporine, ceramide, and granzyme B (26, 27, 92, 131-133). This indicates that the nucleus cannot be part of the central executioner and rather constitutes a downstream target of the degradation phase.

4. Reactive Oxygen Species as Universal Apoptosis Effector,s? Most events of apoptosis are accompanied by dramatic changes in the cellular redox balance: depletiodoxidation of glutathione ( 101, 106, 107), hyperproduction of reactive oxygen species (109, 110), and oxidation of cellular constituents including lipids (110). Moreover, in many (but not all) cases, antioxidants prevent apoptosis, whereas prooxidants induce or facilitate apoptosis ( 105).Although these findings suggested that apoptosis

MECHANISMS OF APOPTOSIS

71

might be an oxidation process, it has been shown that apoptosis induced by a variety of different stimuli (staurosporine, growth factor withdrawal, and anti-Fas) can be induced in cells kept under anaerobic conditions, in the manifest absence of reactive oxygen species (ROS) (134, 135), and that hypoxia can be an apoptosis-triggering condition (136). Only in some pathways (e.g., in glucocorticoid-induced apoptosis) does the formation of ROS appear to be essential for apoptosis induction (137). Thus, redox processes constitute one way of regulating apoptosis but cannot be central to the apopoptotic process.

5. Ca2+:An All-Explaining Cation? Increases in cytosolic Ca2+can induce apoptosis, and chelation of intracellular calcium prevents apoptosis induction in a number of models (138). This also applies to TCFUCD3- or glucocorticoid receptor-triggered thymocyte apoptosis, and Ca" has also been proposed to be the factor responsible for nuclear endonuclease activation (139, 140). Although Ca2+may serve as a proapoptotic second messenger, several examples have been reported in which apoptosis is induced in or by the absence of extracellular Ca2t (141-143) or in the presence of intracellular Ca2' chelators such as BAPTAAM (144). Although cytosolic Ca2+tends to increase to nonphysiological levels ($10 W M ) during the late stage of apoptosis (107) and Ca2+can activate endonucleases (140, 145), many cell-free systems of apoptosis do work in the presence of CaZt chelators (20, 45, 146, 147). Thus, Ca2+ elevations are unlikely to be essential parts of the effector or degradation phases. 6. Cytosolic Acidijication: The Panacea?

The degradation phase of apoptosis is accompanied by major changes in ion homeostasis including a cytosolic acidification (148). Attempts have been undertaken to reduce the death/life decision to a question of pH regulation (149). Nonetheless, in certain cell types, e.g., thymocytes, cytosolic acidification actually inhibits apoptosis (150).Altogether, the present data suggest that changes in ion fluxes and compartmentalization are a byproduct of apoptotic degeneration downstream of protease activation rather than essential elements of the process (151, 152).

7. Caspases: Decisive for Cell Death? During the past few years it has been widely assumed that caspases might constitute the central executioner (36-38, 112). This is suggested by numerous observations: Caspases are constantly activated during apoptosis (see Section 11).

72

[OSEF M. PENNINGER A N D CUIDO KROEMER

Transfection-enforced overexpression of caspases causes apoptosis, at least in several models (153-156). Addition of caspases to cell extracts can induce nuclear apoptosis in uitro (45, 46). Inhibition of caspase activation by broad-spectrum inhibitors precludes acquisition of the apoptotic phenotype (60, 61, 135). Several apoptosis-inducing pathways, including those activated by ligation of TNF-R p55, Fas/APO-l/CD95 (64,65),or granzyme B (157, 158), are directly coupled to caspase activation. Caspases can engage in a cascade of sequential (and sometimes mutual) activation, suggesting that they can participate in (aut0)amplification processes (36, 37, 40, 158). Nonetheless, a few facts argue against the obligatory participation of caspases in the central executioner: Caspase can exert other functions than those involved in apoptosis. Thus, caspase-1 is necessary for the processing of interleukin-lp (159) and interferon-y-inducing factor (160). Caspase-3 activation can be observed in the absence of apoptosis (161),and the caspase-mediated digestion of the nuclear substrate PARP has been observed in cells that maintain their clonogenic potential (162). In several systems, inhibition of caspase activation using the broadspectrum inhibitor z-VAD.fmk prevents acquisition of the apoptotic phenotype but does not inhibit cell death. This applies to a number of different models (Table IV), suggesting that caspase activation is decisive for the degradation phase but dispensable for the effector phase of apoptosis. In synthesis, caspases are not (or not always) involved in the effector phase of cell death. 8. Apoptosis in Opposition to Necrosis? Conventional textbook knowledge insists on the opposition between apoptosis and necrosis. However, several facts weaken the idea that apoptosis and necrosis involve fundamentally different mechanisms:

After apoptosis, cells undergo necrosis. The same toxin can induce apoptosis (at low doses) and primary necrosis (at high concentrations) (111, 163). Many pathologies labeled as “necrotic” are now known to involve apoptosis. This applies to myocardial infarction, cerebral apopleloj, and excitotoxin-induced neuronal cell death (164-166).

73

MECHANISMS OF APOPTOSIS

TABLE IV MODELSOF APOPTOSISI N WHICH THE CASPASE INHIBITOR z-VAD.fmk FAILSTO INHIBITCYTOLYSIS Inducers of Apoptosis Overexpression of bax

'

c-myc overexpression and serum withdrawal or overexpression of bak Protonophore, protoporphyrine IX, dexamethasone, etoposide, or nitric oxide CTL granule exocytosis

Effect of z-VAD.fmk Disruption of the mitochondrial transmembrane potential, followed by death without DNA fragmentation Cytoplasmic blebbing for hours, followed by cytolysis without DNA fragmentation Disruption of mitochondrial transmembrane potential; retarded nonapoptotic cytolysis in the absence of caspase or endonuclease activation (see Fig. 2) Inhibition of nuclear apoptosis without prevention of target cell Iysis

Reference 60

61

62, 216

74

Bcl-2 overexpression can inhibit apoptosis and, at least in some cases, necrosis (167-169). Inhibition of caspases can induce a switch from the apoptotic to the necrotic mode of cell death, as discussed previously (60-62, 74). Manipulations of the ATP level can influence the choice between the two modes of cell death. Thus, in cells in which the ATP level is lowered, for instance, by withdrawal of glycolytic substrates and inhibitors of mitochondrial ATP generation, stimuli that conventionally induce apoptosis (e.g., Fas- cross-linking) cause necrosis (170, 171). Altogether, these recent findings imply that apoptosis and at least some examples of necrosis share a common effector pathway. 9. Lack of Mitochondria1 Involvement in Apoptosis? Several studies have reported that cells lacking mitochondria1 DNA could undergo full-blown apoptosis (172, 173). Although these data were correctly interpreted by the authors of these studies, many investigators have short-circuited them to assume that cells without mitochondria would retain the capacity of undergoing apoptosis and that therefore mitochondria would not be important for the apoptosis effector stage. Nonetheless, cells without mitochondrial DNA do possess morphologically normal mitochondria that just lack some components of the respiratory chain complexes I, 111, IV,and V encoded by the mitochondrial genome and thus are respira-

74

JOSEF M . P E N N I N C E R A N D GUIDO KROEMER

tion deficient. Therefore, the assumption that mitochondria are not relevant to apoptosis, as based on the aforementioned experiments, is certainly incorrect [for review see Ref. (174)]. Indeed, numerous recent finding suggest that mitochondria do have a central role for the apoptotic process. C. THECENTRAL EXECUTIONER OF APOPTOSIS:A MITOCHONDRIAL HYPOTHESIS Mitochondria undergo major changes in their structure/function early during apoptosis [for review see Refs. (163)and (175)-( 177)].Two different major changes in membrane permeability have been observed. On the one hand, the electrochemical gradient built up on the mitochondrial inner membrane dissipates during apoptosis (109, 178). On the other hand, proteins that normally are sequestered in mitochondria are released through the outer mitochondrial membrane. Such proteins include cytochrome c (179-181) and a so-called apoptosis inducing factor (19, 147). These two proteins both activate caspases and trigger nuclear apoptosis in cell-free systems. The exact molecular mechanisms and the cause/effect relationship between the increase in inner and outer mitochondrial membrane permeability are a matter of debate. We have advanced the hypothesis that opening of the so-called “mitochondrial permeability transition pore” (also called “mitochondrial megachannel”), which is formed by apposition of proteins within the contact site of the inner and outer membranes, might be closely linked to both the dissipation of the inner transmembrane potential (A?,,) and the cytochrome c release (163, 175-177). Importantly enough, it appears that proteins of the Bcl-2 family, many of which are selectively enriched in the mitochondrial innedouter membrane contact site, regulate apoptosis via affecting the mitochondrial permeability transition (177).In most experimental systems, Bcl-2 must be present in the mitochondrial membrane to inhibit apoptosis (182,183).Overexpression of the apoptosis-inhibitory genes bcl-2 and bcZ-X, prevents the A!Pm collapse and/or the release of apoptogenic activities (cytochrome c, and AIF) (Table V). This result has been observed in intact cells as well as in isolated mitochondria (19, 132, 147, 162, 177, 178, 180, 181, 184) (Fig, 4). Conversely, proapoptotic members of the Bcl-2 family such as Bax favor the loss of mitochondrial function (60). Interestingly, several members of the Bcl-2 family have been shown to have a channel-like function, when incorporated into artificial membranes (185, 186). Whether this function is related to their regulatory effects on mitochondria remains unknown, It thus appears that mitochondrial membrane permeability is a prime target of apoptosis regulation by Bcl-2-like proteins. Bcl-2 has been speculated for a long time to act on the apoptotic effector stage (4,187-189). Does this mean that the central executioner involves the mitochondrion?

75

MECHANISMS OF APOPTOSIS

TABLE V A ~ ~ I ~ T O S I S - ~ N REGIMES D U ~ : I NI N~ :WHICH BCI-2 PRESERVES MITOCHONUHIAL, FUNC:TION

Cell Type

Inducers of Apoptosis

Effect of Bcl-2 on Mitochondria

Reference ~

Thymocytes or T cells

T cytoplasts B cells

PC12 cells

Fibroblasts HL60 cells

Glucocorticoids, ceratnide ter-Butylhydroperoxide Protoporphyrine IX Etoposide (VP-1G) y-Irradiation, doxorubicin Cytosine arabirioside inClCCP (protoiiophore 1 Cerainide Surface IgM cross-linking Cyclosporin A, etoposide ( VP- 16) y-Irradiation, doxorubicin Cytosine arabinoside, adriamycin ter-Butylhydroperoxide Ceramide, senim withdrawal Cyanide, rotenone Antiinycin A. etoposide (VP-16) Calcium ionophore p53 Etoposide, staurosporine

Stabilizes AT,,,

Partial A*,,, stabilization Stabilizes AT,,, Stabilizes AT,,,

109, 203 19 199 132 132 132 19 131 132 132 132 132 Unpublished Unpublished

Stabilizes AT,,,

Stabilizes A q , , , Prevents cytoehrome c release and ATn,

184 184 184 S83 180

In the following paragraphs we will examine the question of whether changes in mitochondrial membrane permeability fulfill the seven criteria of the central executioner that we have previously discussed. 1, Chronological Criterion If the mitochondrial membrane change constitutes part of the central executioner, it should occur before the degradation phase of apoptosis becomes manifest. The mitochondrial transmembrane potential (A?,") results from the asymmetric distribution of protons on both sides of the inner mitochondrial membrane, giving rise to a chemical (pH) and electric gradient that is essential for mitochondrial function (190). The inner side of the inner mitochondrial membrane is negatively charged. As a consequence, cationic lipophilic fluorochromes, such as rhodamine 123, 3,3'dihexyloxacarbocyanine iodide [DiOC6(3)], chloromethyl-X-rosamine (CMXRos),or 5,5',6,6'-tetrachloro-l, 1',3,3'-tetraethylbenzimidazolcarbo-

76

JOSEF M. P E N N I N G E R AND GUIDO KROEMER

FIG.4. Bcl-2 effects on cells, cytoplasts,and mitochondria. Transfection-enforced overexpression of Bcl-2 prevents rnitochondrial changes associated with apoptosis in intact cells, cytoplasts, and isolated mitochondria. This indicates that the antiapoptotic effect of Bcl-2 does not require the nucleus and that Bcl-2 affects mitochondrial function due to its local presence within the mitochondrial membrane.

cyanine iodide (JC-l), distribute to the mitochondrial matrix as a function of the Nernst equation, correlating with the A",,, (Fig. 5 ) . Using a cytofluorometer or a confocal microscope, these dyes can be employed to measure variations in the AqInona per mitochondrion or per cell basis. Cells induced to undergo apoptosis manifest an early reduction in the incorporation of A*",-sensitive dyes, indicating a disruption of the A*,, . This A q I ncollapse can be detected in many different cell types including lymphocytes, irrespective of the apoptosis-inducing stimulus (19, 60, 109, 131, 132, 147, 162, 174, 175, 178, 191-201). Upon induction of apoptosis, the A",, disruption is also found in cells lacking mitochondrial DNA (which have a normal steady-state A",)) (174, 196). It addition, it appears that early during apoptosis, mitochondrial intermembrane proteins such as cytochrome c leak out into the cytosol (179-181). Whether this loss of the outer mitochondrial membrane function is a cause or a consequence of, or without any relationship to, the disruption of the inner membrane is still unknown. In a number of different models, the A*,, disruption becomes manifest before cells aberrantly expose phosphatidylserine (PS) on the outer cell membrane leaflet, fragment nuclear DNA, hyperproduce ROS, or manifest a massive dysre ulation in ion homeostasis (107, 109, 131, 178, 193, 202, 203). Only (but not AWmhlgh)cells contain proteolyticilly activated caspase-3, indicating that the activation of caspase-3 is probably secondary to the A",,, disruption (203).Cells that have reduced their A",,, are irreversibly committed to undergo death, even when the apoptosis-inducing trigger is withdrawn (109). Thus, the A",, collapse marks the point of no return

77

MECHAEU'ISMS OF APOPTOSIS

DiOC6(3) (3,3'dihexyloxacarbocyanlne iodide)

HE

ethidium

hydroethidine (= dihydroethidium)

distribution into mitochondrial matrix

cationic lipophilic

hydrophilic fluorescent

lipophilic non-fluorescent

CMXRos (chloromethyl-X-rosamine) R

R

thiol-

conjugstion

cnsi + w - ~ p t i e

cnm-psptie

lipophilic cationic

JC-1

fiiable

NAO (nonyl acridine orange)

(5,5',6,6'-letrrchloro-1,1',3'3,3'-

tetraahylbsndmidazolca~ocyanine iodide)

cn3

Cn3

I Cn,

I CHZ

R-cc-mi, I

w-cwcn I

n

HCDCOR'

I

n,c-~po,~n,C.fflH,o-Po~~cn~ I

OH

Cardiolipin (diphosphatidylglycerol) cationic, lipophilic green monomer red aggregate

distribution into mitochondrial matrix

F I ~5.. Structures of five fluorochromes allowing for the assessment of apoptosisassociated mitochondrial alterations. DiOC6(3),CMXRos, and JC-1 are lipophilic cationic fluorochromes that distribute through intracellular membranes as a function of the Each of these dyes has different properties: DiOC6(3) is nonfixable, CMXRos reacts with thiol to produce aldehyde-fixable thiolesters, and JC-1 can be used as a ratiometric probe because it emits two fluorescence wavelengths, depending on its concentration. HE and NAO incorporate into cells independently from the A",,,. Both are nonfluorescent and react with superoxide anion or nonoxidized cardiolipin, respectively, to form fluorescent products. The fluorescence produced by HE (i.e.,the conversion HE + Eth) is proportional to the time of incubation and the generation of reactive oxygen species. The fluorescence produced by NAO is directly proportional to the cell's content in nonoxidized carcholipin. Changes in HE and NAO fluorescence are found at a later stage of apoptosis than changes in the incorporation of AY,,,-sensitivedyes. For details see Ref. (589).

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JOSEF M . PENNINCER AND GUIDO KROEMER

of apoptosis but precedes all common signs of the apoptotic degradation phase: nuclear apoptosis, PS exposure on the membrane, activation of CPP32-related caspases, Ca2+influx, K+ loss, and cell shrinkage. To understand the mechanism by which cells undergoing apoptosis lose their ATl,,, experiments have been performed in which cells were first labeled with Aq,,,-sensitive fluorochromes and then purified in a fluorocytometer, based on their AT,,,. In appropriate conditions, this procedure allows for the purification of cells with low ATl,, values and a still normal DNA content and morphology (= preapoptotic cells) or, alternatively, of cells with a high ATIl,that will lose their AT,,, upon a short-term (30-120 min) culture period (109, 178,204).A!Pl,1" (but not ATmh'gh) cells undergo oligonucleosomal DNA fragmentation upon short-term (3060 min) culture at 37°C. The AT],, loss of Aq)Rh cells is prevented by three inhibitors of the mitochondrial PT pore: cyclosporin A (CsA), the nonimmunosuppressive CsA derivative N-methyl-Val-4-CsA, and bongkrekic acid (178, 198, 204). These data indicate that the permeability transition (PT) accounts for the AT,,, collapse observed during early apoptosis. According to one report, cytochrome c release can occur before the AT,,, collapse (which is indicative of irreversible opening of the inner membrane PT pore) (180). In contrast, opening of the PT pore in isolated mitochondria causes the release of cytochrome c (205), during both the initial (reversible) and the advanced (irreversible) stages of megachannel opening (G. Kroemer, unpublished results). As a possibility, an inital "flickering" of the PT pore (that would not cause a manifest Aq,disruption) may lead to cytochrome c release in cells before the AWm dissipates. The relationship between cytochrome c release and PT requires further clarification.

2. Functional Criterion The PT-mediated A*,,, reductions should be undissociable from the subsequent degradation phase, if this mitochondrial change formed part of the central executioner. Apparently, the Aql,l reduction and later nuclear apoptosis are closely linked with each other. Very different inducers of apoptosis provoke the same sequence of events (mitochondrial followed by nuclear alterations). Moreover, whenever an inhibitor interrupts the cascade of signals leading to apoptosis, both the mitochondrial and the nuclear changes are abolished. This is illustrated in Table VI for thymocytes but also applies to other cell types, including primary peripheral lymphocytes, T cell hybridoma cells, lymphoma cells, as well as nonlymphoid cell types. In one particular case, in thymocyte cell death induced by CD99 cross-linking, cell death occurs in the absence of full-blown nuclear apop-

79

MECHANISMS OF APOPTOSIS

TABLE VI APOPTOW I N D U C INC, REC.IMLS IN THYMOCYTES THATCAU5.E A AT,,,DI~RKJFTIO~ PHEC E I ~ NCn c M A R DNA FRAGMENTATIO~ A N D THEIRI N H I B I T I O ~ Inducer of Apoptosis Gliicocorticoids

DNA diunage (etoposide or y-irradiation )

Anti-Fas antibody

TNF-a, cerainide Thapsigargine Negative selection or the absence of positive selection Menadione (ROS generator) ter-Butyhydroperoxide

NO Diamide

Protopoi-phyrine IX

Inhibitor of Apoptosis

Reference

RU38486 (receptor blockade) 192, 194, 198 Actinomycin D (transcription 131 inhibitor) Cycloheximide (translation inhibitor) 131 TLCK (trypsin inhibitor) 198 Lactacystin, MG132 (proteasome 163 inhibitors) N-acetylcysteine (GSH precursor) 178 Catalase ( H 2 0 2detoxifying enzyme) 107 Bongkrekic acid (ANT ligand) 198 Monochlorobiinan (thiol reactive) 200 Chloromethyl-X-rosamine (thiol 200 reactive) p 5 3 null mutation 131, 198 Actinomycin D (transcription 131 inhibitor) Cyclohexiniide (translation inhibitor) 131 TLCK (trypsin inhibitor) 198 Lactacystin. MC,132 (proteasoirie 163 inhibitors) Bongkrekic acid (ANT ligand) 198 Monochlorobiinane (thiol reactive) 200 Cliloroiiiethyl-X-rosaniine(thiol 200 reactive) Ar-WAD.cmk (caspase-1inhibitor) 131 Unpublished Cyclosporin A 584 194

Monochlorobinian (thiol reactive) Ch loromethyl -X- rosain ine ( t h iol reactive) Cyclospoiin A Bongkrehc acid Monochlorobiman (thiol reactive) Chloroniethyl-X-rosaiiiine(thiol reactive) Bongkrekic acid (ANT ligand)

194 200 200 216 216 200 200 199

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JOSEF M. PENNINGER AND GUIDO KROEMER

tosis, but the AT,,, disruption precedes phosphatidylserine exposure (206). In most experimental systems, it appears that PT is both sufficient and necessary for nuclear apoptosis to occur. Induction of PT by agents acting on the PT pore complex causes apoptosis, and inhibition of PT by pharmacological agents of overexpression of Bcl-2 prevents apoptosis (62, 132, 147, 198-200). PT inducers that also induce apoptosis include inhibitors of the respiratory chain, protonophores, and ligands of the peripheral benzodiazepin receptor. Pharmacological PT inhibitors with antiapoptotic effects include cyclophilin ligands (cyclosporin A, N-methyl-4-Valcyclosporin A) and bongkrekic acid, a ligand of the adenine nucleotide translocator (62, 147, 198-200).

3. Criterion of Convergence The mitochondrion should be capable of integrating very different proapoptotic signal transduction and damage pathways. In this context it appears important that the PT pore is a dynamic multiprotein complex located at the contact site between the inner and the outer mitochondrial membranes, one of the neuralgic sites of metabolic coordination between the cytosol, the intermembrane space, and the mitochondrial matrix. Although the exact composition of the PT pore complex is not known, it is currently thought to involve cytosolic proteins (hexokinase), outer membrane proteins (peripheral benzodiazepin receptor; porin, also called voltage-dependent anion channel), intermembrane proteins (creatine kinase), at least one inner membrane protein (the adenine nucleotide translocator; ANT), and at least one matrix protein (cyclophilin D) (207-211). Speculatively, it may also interact with proteins of additional multiprotein complexes, namely, the Tim (transporter of the inner membrane) complex, the Tom (transporter of the outer membrane) complex (212), and the Bcl2 complex (177) (Table VII). Irrespective of its exact composition, the PT pore complex contains multiple targets for pharmacologd interventions and is regulated by numerous endogenous physiological effectors (Table VIII). Such effectors include ions (divalent cations, mainly Ca2+and Mg2+),protons, the AT,,,, the concentration of adenine nucleotides (ADP and ATP) (208, 209), the pyrimidine redox state (NAD vs. NADH,; NADP vs. NADPH2),the thiol redox state (controlled by glutathione) (213), reactive oxygen species and nitric oxide (214-216), the concentrations of lipoids (lipid acids, acyl-CoA, and perhaps ceramide and derivatives) (208,209,217),the concentrations of some peptides (amphipathic peptides and perhaps signal sequences of peptides targeting proteins to the mitochondrial import machinery) (218, 219), and changes in the composition or function of the Bcl-2 complex (109,147,177).As a general rule, it appears that any major change in energy

MOLECULESIN

THE

TABLE VII PT PORECOMPLEX AS TARGETS FOR ENDOGENOUS EFFECTORS AND PHARMACOLOGICAL AGENTS Nomd Funchon

Molecule (Topology) ~

Adenine nucleotide translocator (ANT) (inner membrane)

Matrix thiols (within the inahix side of the ANT?)

Peripheral henzodiazepin receptor (PBR) (outer membrane) Porin (outer membrane) Cyclophilin D (matrix)

Ca%ensitive sites Tim23 (and other proteins of the Tim and Tom wmplexes?) Hexoldnase (cytosol) Creatine kinas? (intermembrane)

Modulators and Role in PT

Helerence

~

ADP and ATP inhibit PT Bongkreldc acid (binds to matrix site): favors m-state and inhibits PT Atractyloside (hinds to intermembrane site): favors c-state and induces PT Thiol oxidation (e.g., phenylarsenine oxide) and disnlfide Thiol sensor for redox potentials (in quilibnum with bridge formation (e.g., diamide) favor FT glutathione) Thiol derivatimtiun hy monochlorohiman or chloromethyl-Xrnsamine prevents FT Protoporphyin N:induces PT Reeptor for endozepin (= CoA-binding protein) PK11195, FGIN 1-27. chlorndiazepam: facilitate PT Voltage Voltage-dependent anion channel, ATP transport Cyclosprine A: inhibits interaction with inner membrane Peptidyl prolyl isomerase (chaperone function) N-methyl-Val-4-cyclosporine A (nonimmunosuppressive) acts like cyclosporine A Low pB: prevents interaction with inner membrane Calcium favors PT Sensor for divalent cations Transport of proteins through the outer (Tom) or iniier (Tim) Signal peptides of proteins targeted to the mitochondrial matrix inhibit a FT pore-like channel nutochondrial membranes Facilitates or regulates IT? Phosphorylates hexasacchandes (mainly glucose) while hydrolysing ATP Inhibits FT? Transfers phosphate from creatine phosphate to ADP or from ATP to creatine

ATPIADP antiport

210.230

200. 213

223, 233 21 1 586

208, 209 219 211 211

TABLE VIII FUNCTIONS OF THE PERMEABILITY THANSITION PORE Function Voltage sensor Thiol sensor Sensor of pyridine oxidation Matrix pH sensor

Principles of Modulation

Example

Low ATl,, induces PT High AT,,, inhibits PT Oxidation of a critical matrix dithiol (in the ANT?; regulated by GSH) induces PT Oxidation of NAD(P)H2 favors PT (in equilibrium with GSH oxidation)

Anoxia, respiratory inhibitors induce PT Hyperpolarization (nigericin) inhibits PT Prooxidants and thiol cross-linking induce PT Prevention of thiol cross-hnlang prevents PT NAD(P)H2prevents PT Antioxidants prevent PT Prooxidants favor PT Akalinization (pH = 7.3)of matrix favors PT Neutral or acidic matrix pH inhibits PT Increase in matrix CaZ+induces PT M$+ and Zn2i prevent PT Extra ATP (glycolytic substrates) prevents PT Oligomycin (F, ATPase inhibitor) favors PT Caspase-1 induces PT Calpain-like enzyme may favor PT Palmitate and stearate induce PT Carnitine prevents PT Mastoparan induces PT Peptide ligands of Tim 23 inhibit PT?

ADPIATP sensor

Reversible histidine protonation (of cycophilin D?) prevents PT Ca2+induces PT Other divalent cations inhibit PT ADP (and ATP) inhibits FT '

Protease sensor?

Direct action of proteases on outer membrane proteins

Lipid acid sensor?

Long chain lipid acids induce PT

Peptide sensor?

Amphipathic peptides induce PT

Cation sensor

Note. For references and details consult main text.

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83

balance (the absence of oxygen, depletion of ATP and ADP, depletion of NAD(P)H?, or disruption of tlie A*,,,) or in redox balance (oxidation/ depletion of nonoxidized glutathione or NAD(P)H2and hyperproduction of reactive oxygen species) can provoke PT. This implies that PT functions as an integrator of stress responses and that major damage of cells will invariably cause PT. In addition, a number of signal transduction pathways triggered via intracellular or cell surface receptors can result in PT (Fig. 6). Thus, PT is facilitated by a nuniber of second messengers: increases in cytosolic Ca2+ concentration (220-222), ceramide (109,203,217,223),and some caspases such as caspase-1 (but not caspase-3) (203). Because PT is regulated by the Bcl-2 complex, changes in tlie stoicliioinetry of this complex (e.g., enhanced synthesis of tlie Bcl-2 antagonist Bax) or signal transduction pathways culminating in posttraiislational modifications of the Bcl-2 complex can also facilitate PT. The phosphorylation of Bcl-2 or Bcl-2 honiologs

I

Changesinthe

1

Redox catastrophe

Slgnai transductlon Bioenergetic catastrophe

Fic:. 6. Inducers of perineability transition. Different signal transduction pathways can promote the activation of caspnses, increases in cytosolic Ca” levels, and nitric oxide, ainphipatliic peptides, or lipid mediators (e.g..ceraniide) provoke F’T. In addition, hyperexpression of the Bcl-2 antagonist Bax or posttranslational effects affecting the function of the Rcl-2 complex can induce F’T. Major changes in the cellular redox and energy balance also trigger PT. Note that PT is a s e l f - q M ) i n g process (hvo-headed arrows). Certain PT inducers (increases in cytosolic (=a‘+. reactive oxygen species, etc.) may be involved in “private” patliways of apoptosis indiictioii and tliiis function as facultative PT inducers. yet constitute constant by-products of the apoptotic process (striped boxes). This explains why several conseqnences of PT are constant by-products of :ipoptosis, hut itre not necessary for apoptosis to occur in all instances.

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JOSEF M . PENNINGER AND GUIDO KROEMER

(e.g., Bad), as well as the subcellular distribution of Bcl-2-related proteins (e.g., Bad) or Bcl-2-associated proteins (e.g., Bag-1 and Raf-1), is regulated by growth factor receptors (177, 224, 225), thus suggesting how growth factor withdrawal can trigger PT via changes in the composition of the PT-regulatory Bcl-2 complex (177). Speculatively, an additional pathway that may converge at the level of mitochondria may involve the Src-like kinase substrate HS-1, a proapoptotic molecule (226) that interacts with the mitochondrial outer membrane protein Hax-1 (227). The functional significance of pathways leading to the formation of a mitochondrial p38 neoantigen (228) in apoptotic cells still remains elusive. Altogether these findings suggest that, in addition to integrating various damage responses, PT can be triggered via receptor-connected pathways. It thus may constitute the crossroad of both nonspecific damage responses and responses mediated via specific receptors. 4. Criterion of Coordination The central executioner should be capable of coordinating the different manifestations of apoptosis at the levels of the nucleus, the cytoplasm, and the plasma membrane. Hence, the question is whether and how the disruption of mitochondrial membrane integrity may provoke the entire spectrum of apoptotic degradation. Does PT coordinate the apoptotic degradation phase? Pharmacological inhibition of PT prevents all manifestations of apoptosis, at the levels of the nucleus, the plasma membrane, and the cytoplasma (198, 200), emphasizing that PT can indeed function as a central coordinating event. How does PT cause cell death? In normal circumstances, the inner mitochondrial membrane is near-to-impermeant. This feature is required for building up and mantaining the inner transmembrane potential ( A q m ) .Opening of the PT pore allows for the diffusion of solutes with a MW of I 1 5 0 kDa, according to gross estimations based on the use of polyethylen glycol polymers (208, 209). Prolonged opening of the PT pore causes the dissipation of the A q mwith consequent loss of mitochondrial RNA and protein synthesis, cessation of the import of most proteins synthesized in the cytosol, release of Ca2+and glutathione from the mitochondrial matrix, uncoupling of oxidative phosphorylation with cessation of ATP synthesis, oxidation of NAD(P)H2and glutathione, and hyperproduction of superoxide anion on the uncoupled repiratory chain (208) (Fig. 6). Accordingly, cells undergoing apoptosis manifest an arrest of mitochondrial biogenesis, at both the transcriptional and translational levels (191, 229), and perturbations in mitochondrial electron transport (230). Multiparameter fluorescence analyses revealed that the preapoptotic A*,,, collapse is closely linked to major changes in cellular redox potentials, namely, NAD(P)H2 depletion (192), GSH depletiodoxidation (107), and

MECHANISMS OF APOPTOSiS

85

later increases in superoxide anion generation (109) and massive cytosolic Ca2+elevations (107). Superoxide generation is reduced in several models by rotenone, an inhibitor of the respiratory chain complex I, suggesting that it is formed on the uncoupled respiratory chain (109, 193). The bioenergetic and redox changes of PTs themselves are sufficient to cause cell death by necrosis (220, 223, 231-234). How does PT trigger apoptosis? PT allows for the release of proteins that are usually confined to the mitochondria1 compartment. Thus, PT causes the release of cytochrome c from the intermembrane space into the cytosol(205).The protein precursor of cytochrome c (apocytochrome c) is synthesized in the cytosol and transported into the intermembrane space, where the heme lyase attaches a heme group to generate holocytochrome c (235). Holocytochrome c (but not its precursor apocytochrome c, still lacking a heme group) can interact with other yet unknown cytosolic factors to activate caspase-3 (179-181). Caspase-3 then can activate the DFF, which in turn acts to activate nucleases (20). In addition to cytochrome c, mitochondria undergoing PT release AIF, a protein of approximately 50 kDa that suffices to cause nuclear apoptosis and activation of caspase-3 in cell-free and cytosol-free systems (19, 203). As also true for cytochrome c, this activity appears to be ubiquitous and preformed. Exhaustive studies have identified an inhibitor of AIF: z-VAD.fmk (19). This protease inhibitor abolishes all activities of AIF on the nucleus. z-VAD.fink is also a universal inhibitor of nuclear apoptosis occurring in intact cells, irrespective of the cell type (54-62), thus emphasizing the possible in vivo relevance of AIF. In summary, mitochondria contain several proteins endowed with the capacity of stimulating at least some facets of the apoptotic program in cell-free systems. Currently, it appears that at least two biochemical pathways may link mitochondria to nuclear apoptosis (AIF; cyctochrome c + factor X + caspase 3 + DFF). It is not known which among these pathways prevails in vivo or whether both pathways are complementary. If PT can stimulate both primary necrosis and apoptosis, what does make the difference? As a possibility, the bioenergetic and redox catastrophe ensuing PT (which would induce necrosis) and the activation of catabolic enzymes (caspases and nucleases) might compete among each other in a sort of race. Cells would only die from primary necrosis when apoptogenic proteases fail to come into action, either because they are inhibited (e.g., by addition of z-VAD.fmk or by natural compounds such as NO) (60-62, 69) or because the time frame of the process is too rapid to allow for protease activation. This view of cell death would be compatible with the observation that many substances induce apoptosis at low doses (when PT is induced smoothly, perhaps first in a fraction of mitochondria, and cells can activate proteases) but necrosis at higher doses (when PT is caused

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JOSEF M. PENNINGER AND GUIDO K H O E M E R

abruptly and cells lyse before proteases come into action). Finally, it would explain how maintenance of cytosolic ATP levels can influence the decision between the two modes of apoptosis induction, with high ATP levels favoring apoptosis and low ATP levels favoring necrosis (170,171),whereas inhibition of proteases or manipulations that reduce cellular ATP levels favor necrosis over apoptosis (Fig. 7). It may be noteworthy that, in contrast to other cell types such as neurons and myocardiocytes, lymphocytes undergo apoptosis rather than necrosis in response to most death-inducing regimens (111).This may be due to the fact that ATP generation in lymphocytes mainly relies on glycolysis.

5. Criterion of Uniuersality The central executiorer should be operative in all cells and in all events of apoptosis. Molecules such as the ANT [which exchanges matrix ATP for cellular ADP on the inner membrane (236)] or porin [which allows for the exchange of ATP on the outer membrane (237)] are essential for oxidative phosphorylation and thus must be expressed in all living cells. For

I

mitochondria1 permeability transition

i

it of redox potentials and bioenergetic catastroph

I

primaKy necrosis (without apoptosis)

[necrosis) secondary necrosis

FIG7 . Hypothetical model of the apoptosis/necrosisdichotomy. Disruption of initochondrial function caused by mitochondria1 permeability transition can cause necrosis via its consequences on redox and enerQ metabolism. Alternatively, the liberation of apoptogenic mitochondria1 proteins, such as cytoclirorne c and AIF, causes the proteolytic activation of different cytosolic caspase precursors, as well as the activation of DNA fragmentation factor (DFF) via caspase-3. Caspases, DFF, and AIF can act on the nucleus (and on cytoplasmic stmctures) to cause apoptosis. Depending on the pathway that prevails, necrosis oc'ciirs before apoptosis (primary necrosis) or after apoptosis (secondary necrosis).

MECHANISMS OF APOPTOSIS

87

obvious reasons, the apoptogenic factor cytochrome c, which participates in the respiratory chain, also must be present in all respiring cells. AIF-like activity has also been detected in many different tissues (19), suggesting that it may be ubiquitous. Although mitochondrial PT pores have been classically studied in hepatocytes (208, 209), it appears that mitochondria of very different cell types possess cyclosporin A-sensitive PT pores: heart muscle cells (238),neurons (211,221,222,239),kidney cells (240),lymphocytes (175), and fibrosarcoid cells (223).This suggests that PT pores are ubiquitous. Moreover, it appears that mitochondrial membrane changes affecting the outer and/or inner membrane occur in all cases of apoptosis investigated thus far (175, 176, 241).

6. Criterion of Vitality The central executioner or the compounds that compose it must have some function(s) that is essential for normal cell survival. Some if not all PT pore components are essential for cellular metabolisms, and this also applies to cytochrorne c. However, it is not clear whether the PT functions in physiological situations not related to apoptosis. According to Brdiczka and co-workers (211), the PT pore might be important for the handling of ATP and metabolic control. This idea is based on the fact that many of the proteins involved in formatiodregulation of the PT pore specifically interact with ATP and ADP: porin, hexokinase, and the ANT. Bernardi and Petronilli (209) propose an alternative physiological role for PT. Short spikes of PT may be involved in the periodic outflow of calcium from the mitochondria1 matrix, and this would be necessary to avoid excessive calcium accumulation in mitochondria. Kinnally and colleagues (212, 242) suggest that the PT pore might be identical with a mitochondrial multiple conductance channel modulated by peptides responsible for targeting mitochondrial precursor proteins. This would imply that the PT pore participates in the import of nuclear gene products into mitochondria. If any one among these hypotheses was confirmed, PT would be essential for normal mitochondrial function.

7. Criterion ($the Switch The central executioner should function as a switch that is either off or on and thus should be endowed with the capacity of self-amplification. Many of the metabolic consequences of PTs themselves trigger PT (Table IX). Two types of self-amplification can be conceived, one at the level of the organelle and the other at the level of the cell. At the level of single disruption, which mitochondrion, the immediate consequence of PT, in turn Favors PT, is likely to lock the organelle, once the is below a critical threshold, in an irreversible stage of damage. It appears that PT

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JOSEF M. PENNINGER AND GUIDO KROEMER

TABLE IX SELF-AMPLIFYING FEATURES OF MITOCHONDRIAL PERMEABILITY TRANSITION Inducer of PT

AT,,,reduction facilitates PT (587) Calcium causes PT (208, 588) Thiol oxidation of matrix proteins facilitates PT (213) Oxidation of NAD(P)Hz favors PT (213) Reactive oxygen species induce FT (208) Proteases can cause PT (198) AIF induces PT (203)

Consequence of PT PT causes AT,,, disruption (208) PT causes outflow of matrix calcium and ATP depletion, thereby disrupting calcium homeostasis (208) PT causes depletion of nonoxidized glutathione, thereby favoring protein thiol oxidation (208) PT results in NAD(P)H2oxidation (192) PT causes hypergeneration of superoxide anion on the uncoupled respiratory chain (109, 191) PT results in protease activation (19, 179) PT causes AIF release (19)

is accompanied, at the local level, by a loss of mitochondrial matrix glutathione and ADP/ATP (which extrude through the PT pore), two conditions that facilitate PT (208, 243). In addition, local oxidation of mitochondrial compounds, such as cardiolipin (109, 244) and the PT-induced extrusion of cytochrome c (205,245), are likely to interfere with respiratory function, thereby preventing recovery of the mitochondrion that has undergone PT. Several among the consequences of PT affect the whole cell and thus could act on other mitochondria than those undergoing PT via a sort of domino effect. These changes include oxidative changes in the redox potential (107, log), increases in cytosolic free CaZt concentrations (probably due to Ca" influx through the plasma membrane) (107), and caspase activation (203), which all induce PT (208, 243; J. Penninger and G. Kioemer, unpublished data). AIF released from mitochondria undergoing PT can also induce PT (203).Thus, PT initially occurring in some mitochondria of a cell is likely to affect the metabolism in a way that causes PT in all remaining mitochondria. The capacity of self-amplification renders PT an attractive candidate to constitute the (or a) death switch. In conclusion, it appears that mitochondrial PT (or a closely associated mitochondrial process) fulfills most if not all criteria of the central executioner. As a word of caution, it should be mentioned that additional processes including caspase activation (37, 38, 112) have been suggested to form part of the executioner. It is possible that the central executioner involves an intricate interplay between mitochondrial membrane alterations, caspase activation, changes in redox metabolism, and ion fluxes rather than the mitochondrion alone. Be that as it is, it appears plausible that mitochondria exert a decisive role in the effector stage of apoptosis.

MECHANISMS OF APOPTOSIS

89

IV. initiation Phase of Apoptosis

The preceding sections of this review dealt with the common pathway of apoptosis. Here, we will describe private pathways; that is, pathways that operate in response to determined signal and that thus are activated in a cell type- and trigger-specific fashion. Whenever possible, we will concentrate on pathways operating in T cells and in their precursors. Cell fate decisions in developing and mature T cells depend on signal transduction via the antigen-specific TCR and additional receptors in a complex interplay. Thus, the same TCR can signal for survival or cell death depending on the affinity for the ligand and second modulatory signals. Here, we will concentrate on signal transduction pathways that link surface receptors to the death effector machinery. In particular, the role of signal transduction via TNF-R superfamily proteins, stress kinases, tyrosine kinase-based receptors, NFKB, PI3'K, and protein kinase B in the decision between apoptosis and survival will be discussed. A. THYMIC T CELLDIFFERENTIATION AND CLONAL SELECTION: GENERAL PRINCIPLES T ceU development within the thymus is a well-defined process during which precursor thymocytes divide, rearrange, and express TCR. Thymocytes undergo two selective processes: positive selection and negative selection. Mechanisms governing positive and negative T cell selection are critically dependent on physical interactions between the antigen-specific TCRs on developing thymocytes and major histocompatibility complex molecules (MHC) expressed on thymic stromal cells (246-248). Recognition of MHC class I molecules by the TCR commits thymocytes to the CD8' lineage, whereas interactions with MHC class I1 molecules determines the generation of CD4+ T lymphocytes (Fig. 8) (249). Recognition of self-MHC molecules expressed on thymic epithelial cells by immature thymocytes and subsequent differentiation into CD4+or CD8' T cells are the basis for positive selection (246-248, 250). Thus, positive selection generates a T cell repertoire that is restricted to self-MHC. Positive selection mechanisms must trigger a survival signal in developing thymocytes, which subsequently migrate from the thymus to commence their life as mature peripheral T cells. Thymocytes expressing TCRs that recognize self-antigens on bone marrow-derived cells with high affinity/avidity are clonally deleted, leading to the removal of T cells that express T cell receptors with potentially harmful self-reactivity (248-251). The outcome of both positive and negative selection events is directed by the specificity of the T cell receptor expressed on the developing thymocyte (Fig. 8) (250). Developing thymo-

90

JOSEF M . PENNINGER AND GUIDO KROEMER

Bone Marrow precursers (adult) Fetal liver (embryo)

Thymic cortex

CD4-CDB-

RAG-1IRAG-2 Rearrange TCRa locus

NeProllferatlon TCRap.int

RAG-l/RAG-2 Rearrange TCRa locus

I T cell selection

~

TCRaphbh

TCRaPl9h

Switch off RAG-11RAG-2

Thymic medulla FIG.8. Outline of thymocyte differentiation. Positive, negative, and default selection occur at the CD4+CD8’TCRcr/3’”ghstage of differentiation and depend on the affinity of the TCR for the peptide/MHC ligand and on specific second signals provided by thymic strornal cells. The second signals for positive and negative selection appear to be biochemically distinct, and these signals are mediated by distinct cell populations of the thymic microenvironment. The signals involved in selection and development of the second T lymphocyte lineage expressing y8 TCRs are still elusive and involve the signal transduction via the protein tyrosine kinases ~ 5 6 ’and ‘ ~ SYK and the protein tyrosine phosphatase CD45.

cytes that express a TCR with low affinity for self-peptides in association with MHC molecules die via neglect (nonselection default). It appears that more than 80% of developing thymocytes undergo death by default

MECHANISMS OF APOPTOSIS

91

due to expression of a low-affinity TCR (35). Apoptotic cell death thus ensures the survival of only those T cells that express a self-MHC-restricted and self-tolerant TCR. Although default selection and negative selection pathways are fundamentally different, both pathways trigger morphological and biochemical changes typical for programmed cell death, indicative of a common death effector pathway. Thus, thymocytes undergoing apoptosis in response to a variety of different stimuli manifest the key features of the effector stage, including a disruption of the mitochondria1 transmeinbrane potential (A?,,,) (131, 194, 200), and the typical characteristics of the degradation phase, including caspase and endonuclease activation and phosphatidylserine exposure (5, 111). In contrast, a number of regulators of apoptosis have a restricted effect on thymocyte apoptosis, depending on the apoptosis trigger. Thus, transgene-enforced overexpression of bcl-2 prevents glucocorticoid or irradiation-induced thymocyte apoptosis but has little if any effect on negative selection of thyinocytes (252,253). Similarly, knockout of the tumor suppressor gene p53 only affects apoptosis induced by genotoxic stress but fails to interfere with selection-induced apoptosis or glucocorticoid-mediated thymocyte apoptosis (254, 255). In contrast, a member of the TNF-R family, CD30, appears to be necessary for TCW CD3-triggered thymocyte apoptosis but does not influence the induction of cell death by damage pathways (256). These examples illustrate the existence of multiple pathways of apoptosis induction that may or may not be influenced by ~ ~ 5bcl-2, 3 , or members of the TNF-R family. B HOMEOSTASIS OF MATURE T CELLSGENERAL PRINCIPLES Activation of mature T cells induces the switch from high- to lowmolecular-weight CD45 isoforms. The activation-induced transition from CD45RB"g'l'' to CD45RO""Rh' and the transition from CD45RA+ to CD45RO' T cells correlates with a decrease in Bcl-2 expression and IL-2 synthesis, whereas the expression of Fas, the Hodgkin's lymphoma antigen CD30, and the low- and high-molecular-weight TNF-Rp55 and TNF-Rp75 is increased in primed T cells (Fig. 9) (257, 258). In addition, IL-2 and fibroblasts can prevent apoptotic cell death, presumably by enhancing Bcl2 expression or by affecting cycle progression, respectively. These data imply that activation of T cells generates lymphocytes that are prone to apoptosis, unless these cells are rescued by IL-2 or reside in a specialized environment (257-261). Thus, activated lymphocytes undergo programmed cell death by default in the absence of rescue signals mediated by cytokines or cell-cell contacts. This mechanism maintains leukocyte homeostasis in vivo and may explain why memory T cells, which often display a CD45 phenotype similar to primed T cells (CD45ROt), may

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JOSEF M. PENNINGER AND GUIDO K R O E M E R

FIG.9. Proposed correlation between activation and programmed cell death in mature peripheral T cells. Regulated expression of Bcl-2, Fas, CD30, and both TNF receptors confers susceptibility or resistence to apoptosis and survival signals, depending on the state of activation. Besides cell death due to the absence of continuous stimulation by peptides or cytokines, it appears that apoptosis of activated T cells is mainly triggered through ligation of Fas, TNF-Rp55, and TNF-Rp75.

depend on continuous stimulation or the presence of antigen in vivo (257, 258). Antigen-dependent peripheral deletion of peripheral T cells is mainly mediated via the death receptors Fas, TNF-Rp55, and TNF-Rp75, and perhaps additional molecules of the TNF-Rp55 family (262-266). Although in determined circumstances T cell death can be induced by starvation from IL-2, this lymphokine can also facilitate T cell apoptosis. Thus, pretreatment with IL-2 renders T cells susceptible to TCR-induced apoptosis, perhaps by dysregulated activation of the cell cycle (267) or by influencing Fas/Fas-L signaling (268). Based on these data, a scenario emerges in which antigen recognition by naive peripheral T cells triggers IL-2 production, proliferation, and functional differentiation (Fig. 9). Dysregulated activation of the antigen receptor during cell cycle progression may lead to programmed cell death, a mechanism that would maintain clonal tolerance through inactivation of bystander T cells that have been unspecifically activated by IL-2. Only cells that receive signals in the correct

MECHANISMS OF APOPTOSIS

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temporal order and within the proper spatial confinements are allowed to expand and differentiate into effector lymphocytes. After completion of the effector functions, for instance, after clearance of a virus infection, primed T cells undergo programmed cell death to maintain leukocyte homeostasis, with the exception of a few T cells that are rescued by unspecific stimuli such as IL-2 signaling or by the persistance of antigen. This model also implies that activation of self-reactive T cells that are frequently found in mice and humans can only occur in rather exceptional circumstances: in the context of a temporally and/or spatially dysregulated activation of both the TCR and additional signals (259, 260, 269).

SIGNALS TRIGGERED VIA THE TCWCD3 C APOPTOSISREGULATORY RECEPTORCOMPLEX Specific recognition of antigens by T cells is mediated by the TCWCD3 receptor complex and nonspecific accessory molecules such as CD4 and CD8 glycoproteins. Antigen receptor-induced activation of T cells initiates a cascade of signal transduction molecules that results in transcriptional activation or repression of a variety of genes involved in T cell activation and development (Fig. 10) (270-272). These newly transcribed genes can encode for surface receptors, transcnption factors, or cytokines that in concert regulate T cell proliferation and coordinate the immunological response (273). Crucial players for TCWCD3-mediated signal transduction include the Src-family protein tyrosine kinases (PTK) p59'p and p56Ick, which physically interact with the TCWCD3 complex and CD4CD8 accessory molecules (271,274-276); the cytoplasmatic PTK ZAP70, which associates with the phosphorylated CD35 chain via SH2 domains (277); the guanine nucleotide exchange factor p9Sav(278, 279); the ring finger containing protooncogene ~ 1 2 0 '(280); ~ ' p50'jk,which negatively regulates the catalytic function of Src-family kinases (281);protein tyrosine phosphatases (PTPase) such as CD45 (271); and probably other molecules (270, 272, 273). After an initial wave of tyrosine phosphorylation and tyrosine dephosphorylation events, phospholipase C-yl (PLC-yl)is activated and mediates hydrolysis of phosphatidyl inositol into diacylglycerol (DAG) and inositol 1,4,5-triphosphate (InsP3) (Fig. 11). DAG leads to activation of protein kinase C (PKC) and InsP3 mediates an increase in the concentration of cytoplasmically free Ca2+through binding and opening of InsP3-regulated receptors in the endoplasmatic reticulum (282). Besides PKC and PLCy l , avariety of other signal transducing molecules, such the protooncogenes p21rd7and p74' rd, mitogen-activated serinekhreonine kinases (MAPK), or the stress-activated protein serinelthreonine kinases (SAPK, also termed Jun-N-terminal kinases or JNKs) are activated (Fig. 10) (270-273, 283,

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FIG.10. Schematic model of antigen receptor-mediated signal transduction. Other signaling molecules that are phosphorylated on tyrosine residues after TCR activation are CD3l, ZAP70, SPL76, SLAPl30, the molecular adapter p120"c"', Ras-CAP, the regulatory p85 subunit of the P13' kinase, or the cyoskeletal proteins ezrin, talin, and vinculin (not shown).

284). Antigen receptor-induced signal transduction is organized in cascades and ultimately results in the activation of transcription factors including c-fos, c-jun, or NF-AT (273). TCFUCD3 signaling is intrinsically complex because its final outcome is influenced by multiple parameters: concentration, affinity, and avidity of the ligand; chronology of receptor ligation; activation stage; differentiation stage; cell type; and simultanous ligation of other receptors. In addttion, many of the consequences of TCWCDS signaling will affect the expression level of other receptors (e.g., Fas/Apo-l/CD95 and IL-2Ra) or ligands (e.g., Fas-L and IL-2), thereby having indirect effects on the cell's fate via cross talk with additional receptors and signal transduction cascades.

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FIG.11. Model for antigen receptor-indiiced activation of PLC-yl and PKC. Stimulation of TCWCD3 molecules and accessory receptors triggers the rapid activation of tyrosine kinases, in particular p56''k and p59'". which then leads to tyrosine phosphorylation and subsequent activation of PLC-yl. Interactions between PLC-yl and PTKs are mediated via SH2 domains present in PLC-yl. Active PLC-yl translocates to the inetnhwne and hydrolysis inembrane-hound phosphatidyl inositol (PIP2) into diacylglycerol (DAG) and inositol 1,4,5-triphosphate (InsP3). InsP3 triggers the release of Ca" stored in the endoplasmatic reticnlum through activation of' InsP.3-sensitive receptors { InsP3-H). Increased oytoplasmaticillyfree Ci2+probably mediates phosphorylation of InsP3 to generate inositol 1,3,4,S-tetraphosphate (InsP4), which triggers the opening of Ca2+channels located at the p h n a metnhrane. Elevated Ca2+levels in concert with DAG activate the serine/threonine kinase PKC, wliich translocates to the membrane. To coinpensate for the increase of cytosolic Ca", K' channels are opened that pump K t from the cytoplasm into the extracellular spaces. Speculatively, this might explain the fact that K' channel blockers such as the hormone somatostatin can inhibit T cell activation and proliferation.

1. TCR Signtiling and Thyinocyte Selection: The Impact of lntmcellular Ca2+and InsP3 Receptors TCR-dependent interactions between immature thymocytes and thymic stromal cells presenting peptide antigens or superantigens trigger an activation signal that results in functional changes of the developing T cell and expression of activation markers (271,285-287). Antigen-specific activation of thymocytes can mediate signals that lead to either positive T cell selection

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or activation-induced programmed cell death (261, 288, 289). In vitro and in vivo cross-linking of the TCWCD3 complex expressed on immature CD4'CDS' double-positive thymocytes can induce TCR-mediated signal transduction and increase the concentration of cytoplasmatically free Ca2' (Fig. 11) (285, 286). In addition, the catalytic activity of two Srcrelated PTKs, ~ 5 6 ' and ' ~ p59'y", is increased in transgenic TCRaP T cells undergoing positive selection (290), and activation of thymocytes can be blocked with PTK inhibitors (291). Thus, similar to mature T cells, antigen receptor-mediated signaling in developing thymocytes is associated with Ca2+mobilization and the activation of tyrosine kinases (270, 271, 272). Whereas antigen receptor stimulation preferentially leads to proliferation of peripheral, mature T lymphocytes, activation of the TCWCD3 signaling cascade in immature thymocytes leads to programmed cell death by "poptosis (292). Thymocytes undergoing negative selection and programmed cell death have been shown to contain elevated levels of Ca2+ in the cytoplasm (139,291). Moreover, increased Ca" levels correlate with negative selection, and antigen receptor-triggered clonal deletion of thymocytes can be inhibited using CaZt-chelating agents (293). Interestingly, cyclosporin A, which blocks activation-induced cell death (294) by inliibition of the Ca2+-dependentphosphatase calcineurin (295), does not block negative selection of developing thymocytes (296)but rather blocks positive T cell selection (297). Increased expression of the type 3 inositol 1,3,4-triphosphate receptor ( InsP3R3), an InsP3-gated Ca2+-release channel in the endoplasmatic reticulum (Fig. 11) (282), has been linked to antigen receptor and glucocorticoid-induced apoptosis in T and B lymphocytes (298). Antigen receptor-mediated activation induces physical interaction between the TCR-associated protein tyrosine kinase ~ 5 9 and ~ ' InsP3 receptors in T cells, and InsP3R can be tyrosine phosphorylated and activated by p5gfP (299). Moreover, it has recently been shown that type 1 inositol 1,3,4triphosphate receptor-deficient Jurkat cell lines are protected from antiTCWCD3, anti-Fas, and dexamethasone-induced apoptosis and that induction of apoptosis in Jurkat cells is dependent on opening of intracellular InsP3R-gated Ca2+channels (300). This suggests the possibility that increases in cytosolic Ca2+levels secondary to the action of InsP3R-gated Ca" channels may be a major signal transduction pathway contributing to the induction of apoptosis. Obviously, Ca2+is a pleiotropic second messenger, and the link between increases of cytosolic CaZt concentrations and apoptosis may be complex. In a number of experiinental systems of cell death, Ca2+elevation is necessary (and sometimes sufficient) to induce opening of the mitochondrial megachannel

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(220, 221, 239, 301). Indeed, Ca2+has been known for a long time to induce or to fkilitate mitochondrial PT in isolated mitochondria (208, 209). Thus, Ca" may provide a direct link between receptor triggering and activation of the mitochondrial executioner. Whether this also applies to T cells remains to be elucidated.

2. Protein Tyrosine Kinases: p56lCkand p59fy" Elevation of Cap+levels after TCR ligation in mature T cells depends on upstream events including the activation of PTKs (282, 299). The Srcfamily protein tyrosine kinases ~ 5 6 'and ' ~ p59fp are associated with the cell membrane through a myristoylated glycine residue and mediate very early events in antigen receptor-induced signal transduction because the transmembrane TCWCD3 complex does not possess any intrinsic tyrosine kinase activity (270-272, 274-276). The PTK ~ 5 6 ' interacts '~ noncovalently with cysteine residues in the cytoplasmic region of both CD4 and CD8 molecules (274-276), whereas p59fv"can directly associate with the TCW CD3 complex (302). Negative selection of thymocytes expressing an MHC class II-restricted TCR is not prevented by PTK inhibitors (291),indicating that PTK-mediated signal transduction may not be required for the induction of thymocyte apoptosis. However, PTK inhibitors can block programmed cell death in T cell hybridomas (303), and activation-induced cell death of proliferating peripheral T cells correlates with alterations of tyrosine phosphorylation (304). In addition, cross-linkingof CD4 molecules using anti-CD4 antibodies (305) or HIV gpl20 protein (306) renders mature T cells susceptible to apoptosis. Moreover, transgenic overexpression of CD4 molecules impairs positive and negative T cell selection of CD8' thymocytes expressing the transgenic H-Y TCR presumably by sequestrating ~ 5 6 ' (307). '~ Because CD4 molecules are noncovalently associated with the Src-family kinase ~ 5 6 ' "which ~ is critically involved in TCR-mediated signal transduction (274-276, 308), it appears possible that ~ 5 6 ' 'and ~ similar kinases may be involved in activation-induced cell death. Recently, it has been demonstated in ~ 5 6 ' " (308, ~ - 309) or p59fy" (310, 311)-deficient mice that these PTKs relay TCWCD3-mediated signals to the apoptotic machinery. In a model of superantigen-driven cell death, ~ 5 6 ' is ' ~not required for the deletion of CD4' cells but influences the death of CD8' T cells (312). Because superantigens are presented by MHC class I1 molecules and thus are more efficient stimulators of CD4' than CD8' thymocytes due to differences in affinity, ~ 5 6 appears "~ to be an important signal transduction molecule involved in the deletion of superantigen-reactive CD8' T cells and perhaps in CD4' thymocytes expressing a low-affinity TCR on the cell surface. CD4' thymocytes expressing TCRs with higher affinity for ligand can, however, undergo nega-

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tive selection in the absence of ~ 5 6 ’(312). ‘ ~ This interpretation is corroborated by results in CD4-’- mice (313)and CD4-’-CD8-’- double-knockout mice (314) in which cloiial deletion occurs in thymocytes expressing TCRVP chains with high affinity for superantigens but not in thyinocytes expressing low-affinity TCRVP chains. Altogether, these data are consistent with a model in which quantitative differences in the TCWligand interaction and/or coreceptor signaling determine the requirement for ~ 5 6 during “ ~ positive or negative selection events. Besides the Src kinases ~ 5 6 ’ ‘and ~ p59fy”, it has recently been demonstrated that the CD3c-associated protein tyrosine kiiiase ZAP70 (315),and the protooncogene product p95”“ ( J. Penninger, unpublished data), an SH2 and SH3 domain-containing guanine nucleotide exchange factor for the small Ras-family proteins Racl, CDC42, and RhoA (316), may have a direct role in TCR-mediated apoptosis in thymocytes. Thus, a number of different tyrosine kinases are essential signal transduction modules linking the TCR to downstream effectors of the apoptotic cascade. Currently, it appears unlikely, however, that p56ILkand p59fy” are direct activators of the central executioner. Thus, addition of these molecules to a cell-free system of apoptosis fails to trigger the apoptosis machinery (317),whereas the addition of another molecule possessing a Src homology 2 domain, Crk, can accelerate apoptosis in such a system (318).

3. The CD45 Protein Tymsirie Phosphatase in the Regulation of T Cell Apoptosis Signal transduction by the antigen receptor in T cells depends on the balance between protein tyrosine kinases and protein tyrosine phosphatases (PTPases) (271). Antigen receptor-induced signal transduction in both T and B lymphocytes requires expression of the transmembrane receptor PTPase CD45 (319-322). The intracellular CD45 PTPase domain dephosphorylates negative regulatory tyrosine residues of ~ 5 6 and “ ~ p59‘”’ (but not of ~6,”“)(271, 322). CD45 is expressed on the cell surface of all nucleated hematopoietic cells and their precursors. CD45 is expressed in various isoforms with a molecular weight ranging from 180 to 235 kDa. These isoforms arise from alternative splicing of variable exons (exons 4-7) that encode sequences at the amino-terminal domain. Almost all sites for N- and 0-linked glycosylation are located in a serinehhreonine-rich region corresponding to exons 3-8 of the CD45 molecule. Changes in the expression of the variable exons modify the molecular architecture of CD45, as well as the amount of negatively charged sugar residues of the extracellular domain. Expression of distinct CD45 isoforms also correlates with different hematopoietic cell lineages and depends on the state of differentiation (271, 322). Thus, all B lymphocytes express the high-molecular-weight

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isoform of 220 kDa (also termed B220), which includes all CD45 exons. By contrast, immature CD4+CD8' thymocytes mainly express low-inolecularweight CD45 isoforms, whereas mature CD4' or CD8+ thymocytes and peripheral CD4' or CD8+T cells can express multiple isoforms (323-326). Expression of different CD45 isoforms changes during T cell activation. For instance, naive T cells switch from high-niolecular-weight to lowmolecular-weight CD45 isoforins upon stimulation (327,328). Beside exon switching, CD45 glycosylation depends on the cell type. T and B cells have different patterns of CD45 glycosylation due to differences in enzymatic glycosyltransferase activities within the Golgi apparatus (271, 322). Although the biological function of CD45 isoforms might be different, TCRmediated signal transduction can be restored in CD45-defective cell lines with chimeric molecules containing only the intracellular CD45 PTPase domain (329, 330). Thus, it appears that expression of cytoplasmic CD45 PTPase domain alone is necessary and sufficient for TCWCD3-mediated signal transduction to occur in uitro. Similar to the activation event in peripheral T cells, alternative splicing of CD45 exons changes upon receptor ligation in thymocytes undergoing positive and negative selection (325).Immature CD4'CD8+ thymocytes express lower-molecular-weight CD45 isoforms, whereas mature CD4t or CD8+ T cells express higher-inolecular-weightCD45 isoforms due to exon switching (325,331).Alterations in CD45 isoform expression could change the enzymatic PTPase activityvia interactions with distinct ligands or association with different coreceptors on the cell surface (271,322).Alternatively, different CD45 exon switching may be required for T cell migration from the thymus and homing to distinct peripheral organs (309) or could simply correlate with the generation of mature unprimed T cells (271,322). Mice lacking the alternatively spliced CD45 exon 6 (321) or the CD45 exon 9 (332) exhibit a block in T cell development at a late stage of development, namely, at the transition of immature CD4+CD8' doublepositive to mature CD4' or CD8' single-positive thymocytes (321, 332). Accordingly, it appears that CD45 can participate in the control of positive selection of thymocytes expressing transgenic TCRaP heterodimers (309, 333).Moreover, the PTPase CD45 is crucial for the induction of immature thymocyte apoptosis by superantigen (309, 312, 321), anti-CD3 crosslinking (332), and antigenic peptides ( J. Penninger, unpublished data). Transgenic overexpression of the low-molecular-weight CD45 isoform, CD45R0, in mice can augment antigen receptor-mediated cell death and negative selection of thymocytes expressing the autoreactive H-Y antigenspecific ap TCR (334). Interestingly, the endogenous lectin, galectin-1, which is produced by thymic epithelial cells (335), can induce apoptosis of developing thymocytes and activated human T cells (336). Because

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galectin-l-induced apoptosis depends on the expression of the lowmolecular-weight CD45RO isoform on T cells (336),differential expression of CD45 isoforms may determine the interaction with receptors expressed on cells of the thymic microenvironment, thereby influencing the fate of thymocytes. Because CD45 isoforms can regulate the function of adhesion receptors on thymocytes (337) and peripheral lymphocytes (338),it is possible that different CD45 isoforms mediate physical interactions of thymocytes with different thymic microenvironments. Deregulated positioning of thymocytes may trigger cell death due to temporally or spatially “wrong” interactions with thymic stromal cells. Apoptosis as a control mechanism for cell positioning has been recently termed “anoikis” (homelessness) and appears to be controlled by integrin receptors (339). Thus, activation of integrin receptors (VLA-5) with fibronectin can induce apoptosis in hematopoietic cells (340) and VLA-4 and VLA-5 expression is differentially regulated on developing thymocytes (341). CD45 can control the activity of integrin receptors (337) and different CD45 isoforms preferentially associate with the integrin receptor LFA-1(342). These findings suggest another possibility of how CD45 may influence life and death in the thymus. A wrong CD45 isoform is expressed on abnormal T cells accumulating in mice homozygous for the MRL-lpr or gld mutations, namely, mutation of the Fas surface receptor (343) or the Fas ligand (344), respectively. Such mice develop severe lymphadenopathy and systemic autoimmune disease (345, 346). The lymphadenopathy is caused by the accumulation of TCRaPtCD4-CD8-HSA+ T cells expressing B220, i.e., a glycosylated isoform of CD45 normally expressed on the surface of B cells. Similar to systemic autoimmunity in Iprlgld mice, “disproportional” expression of certain CD45 isoforms on T cell subsets has been implicated in the pathogenesis of various autoimmune diseases such as diabetes (347) or experimental allergic encephalomyelitis (348). It is conceivable that signal transduction by different CD45 isoforms may be directly or indirectly involved in the regulation of apoptotic cell death or induction of autoimmunity. To directly test whether CD45 exon switching and expression of different CD45 isoforms are required for T cell development and thymocyte selection, CD45-’- mice have been reconstituted with transgenes encoding the low- or the high-molecular-weight isoforms of CD45, CD45R0, and CD45ABC, respectively (334,349). Both CD45 isoforms can restore maturation of TCRaPt thymocytes, indicating that the CD45 PTPase activity alone is sufficient to generate a positive selecting signal, in accord with the fact that the extracellular CD45 domain is dispensable for antigen receptor-mediated signal transduction in cell lines (329,330). Interestingly, the main signal transduction defect in CD45-’- thymocytes is inappropriate

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activation of PLC-71 and opening of Ca2+channels, implying that Ca2+fluxes might relay TCWCD3-triggered PTK and PTPase signaling to the apoptotic rnachinery during thymocyte selection ( J. Penninger, unpublished data). D STRESSKINASES During the development of all inulticellular organisms, cell Fate decisions determine whether cells undergo proliferation and differentiation or apoptosis. Distinct and evolutionarily conserved signal transduction cascades mediate survival or death in response to developmental programs and environmental triggers. Multiple stimuli for differentiation and cell growth activate the MAPK, aIso known as the extracellular signal-regulated kinases ERKl and ERK2 (350-353), which translocate to the nucleus and regulate the activity of transcription factors (354). MAPKs are activated by the phosphorylation of a threonine and a tyrosine residue by the dual-specificity MAPK kinases MEKl and MEK2, which relay Ras and Raf signal transduction to MAPKs (Fig. 12) (355-358). A parallel signaling cascade leads to the activation of SAPWJNK (Fig. 12) (357,359).SAPKs/JNKs are activated in response to a variety of cellular stresses such as changes in osmolarity and metabolism, DNA damage, heat shock, ischemia, shear stress, inflammatory cytokines such as TNF and IL1,or ceraniide (360-368). When activated, SAPKs/JNKs phosphorylate cJun, thereby activating the transcriptional complex AP-1 (369). SAPKs/ JNKs are activated by the phosphorylation of tyrosine and threonine residues in a reaction that is catalyzed by the dual-specificity kinase SEKl (also known as MKK4 and JNKK) (359,370-372). SEKl/MKK4 transmits signals from upstream activators such as Rac-1, CDC42, PAK65, MEKK1, ASK1, HPK1, or MLK3, to SAPK activation (Fig. 12) (360, 361, 371, 373-377). Although SEKl is structurally related to MEKl and MEKB, MEKs do not activate SAPKsIJNKs and, conversely, SEKl does not activate ERKs. This implies that parallel and independent signaling cascades exist for MAPK and SAPWJNK activation (378,379),thus introducing a dichotomy into hnase cascades. 1. Distinct Stress Kinase Pathways .for D i f f rent Types of Cellular Stress Genetic data in SEK1-deficient embryonic stein (ES) cell clones (380) and results using chromatographically fractionated extracts (381,382) indicate that SEK1-dependent and SEK1-independent intracellular signaling pathways for SAPWJNK activation exist, and that different types of stress trigger distinct signaling pathways for SAPWJNK activation. Thus, SEKl is the critical activator of SAPKdJNKs in response to the protein synthesis inhibitor anisomycin and heat shock, whereas SAPK activation in response

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MAPK

SAPK

hwosina klnasa Receptor

p38/HOG

1 Iachernlaraperfuslon,Heat shock, Irrrdlatlon, UV, hyparosrnolarlty

GrbUSOS Pyla

Vav

\r Racl, CDC42 HPKl

Raf-1

Tpl-2

MEKKl

MLK3

SEKllMKK4 SEWKK7

ASK1

?

MKK3 MKK6

+

ERK2

p9ORsk GSK-3

Fos

Elk-1

c-Jun

ATF2

Mef

kinase2

FIG. 12. Signal transduction pathways for MAPK, SAPK, and p38/HOG induction. Various cross talks exists between these pathways, and distinct pathways have multiple upstream regulators. For example, Ras can activate Racl/CDC42via PI3’ kinase (not shown). Theoretically, distinct upstream activators might relay signals from different stresses or receptors. This is, however, complicated by the fact that, for example, MLK3 can activate SAPK and p38/HOG, whereas HPKl is a direct activator of MLK3, but HPKl can only activate SAPK. Thus, by analogy with yeast signaling in reponse to osmolarity changes, a hypothetical mammalian scaffolding protein has been proposed to spatially confine and channel similar signal transduction pathways to distinct downstream effectors such as SAPK or p3WHOG. The downstream molecules that relay Pyk2 signaling to MAPK and SAPK activation are not known. Only few downstream molecules are known to be substrates of MAPK, SAPK, and/or p38/HOG. A forth parallel signaling pathway that involves MKK5 + ERK5 has been omitted from the diagram. HPK1, hematopoietic protein kinase1; GCK, germinal center kinase; ASK1, apoptosis-inducing kinase-1; MLK3, mixed-lineage kinase-3: SOS, son of sevenless; PAK, p21-activated kinase.

to osmolarity changes, UV irradiation, y-irradiation, and cerarnide is mediated via a novel dual-specificity kinase termed SEKYMKK’I (Fig. 13) (380). The second activator of SAPKdJNKs operates independently from SEKl and defines a distinct signal transduction pathway. The substrates of SEKUMMK7 are likely to overlap with those of SEKl because overex-

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Oarnotic shock

+

v

?

MEKKl

3

i

SEKllMKK4 I-kB

NF-kB SAPK/JNKcr,p,y

t

Survival

I Survival

FIG.13. Two distinct signaling pathways relay different types of stress to SAPK activation. The liypothetieal point ofbifiircation, i.e., MEKK1, between the SAPK and NF-KBsignaling pathways, is indicated. For details see text. Although SAPK activation has been extensively implicated in the induction of apoptosis, recent genetic data indicate that SAPK activation could protect from premature T cell death during development and after activation.

pression of dominant inhibitory SEKl in COS cells can inhibit the SEKW MKK7-dependent activation of SAPWJNK in response to anisomycin, sorbitol, and UV irradiation (365). Interestingly, in transfection studies using COS cells, SAPWJNK responses to anisomycin are not affected by dominant-negative inhibitors of Racl or CDC42 (373-375). Moreover, UV irradiation and sorbitol-mediated osmolarity changes, but not anisomycin, trigger tyrosine phosphorylation of Pyk2 (also known as related adhesion focal tyrosine kinase or CAP-P), a tyrosine hnase that has been linked to SAPWJNK signaling in PC-12 cells (383). Thus, it appears that Racl, CDC42, and Pyk2 are involved in SEK1-independent SAPWJNK activation in response to sorbitol and UV irradiation but not anisomycin, adding to the multiplicity of stress kinase activating pathways. Besides activation of SAPWJNK signaling cascades in response to many types of cellular stress, SAPWJNK activity is induced in response to growth factors, heterotriineric G-proteins, phorbol esters, or costimulatory activation of T lymphocytes (284, 359-361, 384-386). Moreover, activation of SAPWJNKs leads to phosphorylation of c-Jun and activation of JudFos heterodimeric AP-1 transcriptional complexes, generally believed to be positive regulators of transcription (3Fj7, 369, 370, 372, 387). In T Iymphocytes, ligation of the TCR results in rapid activation of the Ras 4 Kaf + MEK -+ MAPK -+ Fos signaling cascade (Fig. 10) (358, 378,388). How-

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ever, activation of MAPK cascade is not sufficient for effective IL-2 production and proliferation, and T cells require a second costimulatory signal (389). Recently, it has been shown that coordinate stimulation of the TCR/ CD3 complex and the costimulatory receptor CD28 correlates with the activation of SAPKs/JNKs,phosphorylation of c-Jun, and AP1 activity (284). Activation via the TClUCD3 complex and CD28 costimulation can be mimicked using phorbol esters (PMA) and Ca2’- ionophores, respectively (390), and the simultaneous treatment with PMA and Ca2+-ionophore leads to SAPWJNK activation in T lymphocytes (378). These biochemical data imply that T cells utilize two distinct signaling cascades for antigen specific activation, a TCR-triggered Ras -+ Raf + MAPK + Fos cascade and aTClUCD28-induced SEKl -+ SAPWJNK+ c-Jun cascade. Recently, it has been demonstrated that the guanosine nucleotide exchange factor ~ 9 5 ” (316) ~ ’ and/or the TClUCD3-activated kinase Pyk2 (383, 391) might link TClUCD3 signaling to SAPK activation. Failure to activate SAPW JNKs in T cells might result in clonal anergy and induction of immunological tolerance (392-394). To determine the role of SEKl/MKK4 in SAPKs/JNKs activation in response to CD28 costimulation (284) and CD40 signaling (395), SEKl-’-RAG-’- chimeric mice have been generated (380). S E K P RAG2-’- chimeric mice exhibit a partial block in B cell maturation, whereas their peripheral B cells display normal responses to IL-4, IgM, and CD40 cross-linking. However, IL-2 production and proliferation are impaired in SEKl-’- T cells in response to suboptimal concentrations of CD28 costimulation and PMA/Ca2+ionophore activation, indicating that the stress signaling kinase SEKl is a downstream effector involved in TCW CD3 and/or CD28 auxiliary receptor signaling. The impairment of T cell growth and IL-2 production is not complete in response to CD28 costimulation and PMA/Ca2+ ionophore treatment, and strong activation via the TClUCD3 complex done leads to normal proliferation of SEKl-’- T cells. Whereas CD28 is absolutely crucial to generate vesicular stomatitis virus (VSV)-specific germinal centers, SEKl-’-RAG2-’- chimeras can mount a protective antiviral B cell response, exhibit normal IgG class switching, and make germinal centers in response to VSV. Interestingly, PMA/Ca2+ ionophore stimulation, which mimics TClUCD3 and CD28-mediated signal transduction, triggers SAPWJNK activation in peripheral T cells, but not in thymocytes, from S E K P mice (380). These data provided the first genetic evidence that SEKl is an important effector molecule that links CD28 signaling to IL-2 production and T cell proliferation. Most important, these results also show that signaling pathways for SAPK activation are developmentally regulated in T cells. Thymocytes use the SEKl/MKK4

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signaling pathway and peripheral T cells utilize SEKUMKK7 for SAPK activation in response to the same stimuli (380). 2. Stress Kinases: Universal or Restricted Inducers of Apoptosis? It has been proposed that SAPWJNK activation triggers apoptosis in response to many types of stress, including UV and y-irradiation, protein synthesis inhibitors (anisomycin),high osmolarity, toxins, ischemidreperfusion injury in heart attacks, heat shock, anticancer drugs (cisplatinum, adriamycin, or etoposide), ceramide, peroxide, or inflammatory cytokines such as TNF-a (363, 365, 367, 368, 396, 397). Moreover, nerve growth factor (NGF) deprivation in PC12 pheochromocytoma cells leads to sustained SAPUJNK activation and the induction of apoptosis (398). The overexpression of dominant-negative SEKlIMKK4 can block the induction of cell death by heat shock, irradiation, anticancer drugs (cisplatinum, adriamycin, and etoposide), peroxide, ceramide, or cytokine deprivation (364, 365, 397, 398). In addition, overexpression of inactive c-Jun or dominant-negative MEKKl was found to inhibit the induction of cell death by irradiation, ceramide, or heat shock in U937 and BAE cells (364) and to protect PC12 cells from apoptosis after NGF withdrawal (398). Overexpression of the novel SAPK-activating MAPKKK homolog apoptosis signal-regulating kinase-1 (ASK-1) can mediate apoptosis in mink lung epithelial cells (MvlLu cells), human 293 embryonal kidney cells, A673 rhabdomyosarcoma cells, KB epidermal carcinoma cells, and Jurkat T cells (399).ASK-1 is a familymemberofthe MAPKKKs Raf-1, Ksr-1, Tpl-2, Tak1, and MEKK1, and ASK-1 mediates SEKUMKK4 and SAPK activation in response to TNF-(r (Fig. 12). SAPK activation has also been linked to induction of ICE/CED-3-like protease and apoptosis in response to the DNA-damaging anticancer drugs etoposide (VP-16)or camptothecin (400). In addition to its importance in stress-induced cell death, SAPWJNK activation correlates with Fas-mediated apoptosis in human T lymphocytes (401, 402). The previous results might suggest that the ASK-UMEKKl +. SEKl +. SAPWJNK + c-Jun signaling cascade is a common pathway required for the induction of apoptosis. This possibility, however, is invalidated by experiments involving cell Iines or animals in which the SEKl gene has been inactivated. Apoptosis does occur in S E K P ES cells, SEK1-’thymocytes, and S E K P splenic T cells in response to anisomycin, serum depletion, UV and y-irradiation, sorbitol-mediated changes in osmolarity, heat shock, anticancer drugs (etoposide, adriamycin, and cisplatinum), CD3/CD28 ligation, and PMA/Ca2+ ionophore with similar kinetics and at similar doses as in SEKl+’+and SEKl+’- cells (380). In the absence of SEK1, anisomycin, heat shock, and PMA/Ca2+ ionophore treatment of

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thymocytes do not induce any detectable SAPWJNK activity, as would be expected. Similarly,in other models of apoptosis, cell death can be observed in the absence of SAPK activation; this applies to death induced via TNFRp55 (403, 404) or Fas (405). These results collectively invalidate the hypothesis that SEK1-mediated activation of SAPWJNK is required for the induction of cell death in response to all apoptosis inducers. Rather, this pathway must operate in a signal-specific (and perhaps cell typedependent) fashion. The question that remains to be examined is whether stress kinases may be involved in negative selection of thymocytes. It has been shown that overexpression of dominant-negative MEKl can inhibit the differentiation of CD4-CD8- double-negative (DN) thymocyte precursor cells to immature CD4+CD8+double-positive (DP) thymocytes (406), whereas the expression of activated Ras suffices to promote the differentiation of CD4-CD8- DN precursor cells to CD4+CD8+DP thymocytes in RAGBcomplementation assays (407). In contrast, it has been shown that positive thymocyte selection and maturation of immature DP thymocytes to mature CD4+ or CD8+ single-positive (SP) thymocytes is impaired in mice transgenic for dominant-negative Ras, Raf-1, and/or MEKl (408-411). However, negative selection via TCWCD3-mediated apoptosis is independent of Ras and MEKl(408-410), implying that signals for positive and negative selection are biochemically different (256). Because SEKl and SAPKs/ JNKs had been implicated as a common pathway required for the induction of apoptosis (363,365,367,368,396,397), the SAPWJNK signaling cascade was considered a prime candidate for the induction of cell death in thymocytes. What is the effect of a SEKl deficiency on thymocyte differentiation? SEK1-deficient RAG-’- chimeric mice have normal numbers and ratios of CD4’ and CD8+ T cells in lymph nodes and spleen (380). However, the thymi of S E K P chimeric mice were four to five times smaller than those of age-matched 129/J mice or SEK1’” chimeras. This reduction in thymus size was due to a significant decrease in the population of DP thymocytes and a relative (but not absolute) expansion of mature SP thymocytes. Moreover, the total and relative numbers of DN thymocytes were not increased in SEK1-I- mice, indicating that SEKl was not required for the progression of DN precursor cells to DP thymocytes. Surprisingly, SEK1-I- thymocytes and peripheral T cells were more susceptible to apoptosis in response to the physiological stimuli CD3/TCR and Fas (380). These data show that SEKl actually protects T cells from Fas- and C D 3 d TCRap-mediated cell death. Thus, the SEK-1 + SAPWJNK pathway appears dispensable for negative T cell selection.

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How is it possible that the S E K - 1 4 SAPUJNK cascade actually reduces thymocyte susceptibility to apoptosis induction? In the context of this question, it may be useful to recall that TNF-a, ionizing radation, or UV irradation do not only induce SAPK but also trigger the activation and nuclear translocation of NF-KB, a pleiotropic transcription factor that is important for lymphocyte responses to antigens and cytokines (412). Interestingly, the stress kinase activating MAPKKK MEKKl has been identified as a key regulator of NF-KBactivation through site-specificphosphorylation of the NF-KB inhibitor, I - K B ~which , binds and retains NF-KB in the cytosol (Fig. 13) (413). Thus, MEKKl is a critical component of both the SAPK and NF-KB stress response pathways. Moreover, NF-KB has been identified as a crucial survival factor and can protect human and mouse fibroblasts and T lymphocytes from apoptosis in response to ionizing radiation, the anticancer drug daunorubicin, and TNF-a, but not Fas, activation (414-416). Because TNF-a-mediated apoptosis is enhanced in many cell lines by drugs that inhibit protein synthesis, these results indicate that TNF-a mediates two signaling pathways, one that induces cell death and another that leads to the NF-KB-dependent transactivation of cytoprotective genes. It is conceivable that, in an analogous way, SEKl deficiency might entail the induction of antiapoptotic genes, which would account for the partial protection from anti-CD3- and anti-Fas-induced apoptosis observed in SEKl-’- thymocytes (380).

E CROSSTALKBETWEEN SIGNAL TRANSDUCTION MODULESA N D MOLECULES OF THE Bcl-2 COMPLEX

Bcl-2 is known to belong to a growing fiainily of apoptosis-regulatory gene products that may either be death antagonists (Bcl-2, Bcl-XL,Bcl-w, Bfl-1, Brag-1, Mcl-1, and A l ) or death agonists (Bax, Bak, Bcl-Xs, Bad, Bid, Bik, and Hrk) and that regulate the effector stage of apoptosis (4, 188, 189, 417). Knockout studies have revealed that Bcl-2-like apoptosisinhibitory proteins exert essential cytoprotective functions; their deficiency entails the ablation of determined cell types, for instance, that of lymphoid cells in Bcl-2-/- (418, 419) and that of postmitotic neurons in Bc1-X-l- mice (420). Transgene-mediated hyperexpression of Bcl-2 can protect lymphocytes against physiological and pathological insults in vivo (189), whereas overexpression of Bax favors lymphocyte apoptosis (126). The ratio of death antagonists (Bcl-2, Bcl-XIJ,Bcl-w, Bfl-1, Brag-1, Mcl1,arid A l ) to agonists (Bax, Bak, Bcl-Xs,Bad, Bid, Bik, and Hrk) determines whether a cell will respond to an apoptotic signal. This deatulife rheostat is mediated, at least in part, by cornpetetive dimerization between selective pairs of antagonists and agonists (417, 421).

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Activation of various growth factor receptors, antigen receptor stimulation in T and B lymphocytes, CD28-mediated costimulation in T cells (422, 423), or sensing of DNA damage via p53 (424-427) can modulate the expression levels of Bcl-2 family protooncogenes (189). However, the relative expression level of Bcl-2-related death agonists and antagonists is not the sole parameter to determine the resistance or susceptibility to apoptosis. Thus, in addition to changing the expression level of Bcl-2related proteins, signal transduction can influence the composition of the Bcl-2 complex and/or induce posttranslational modifications of death agonists and death antagonists of the Bcl-2 family. The homo- and heterodimers of Bcl-2 homologs mainly localize to the outer mitochondrial membrane, in particular to regions of inner-outer membrane contact sites, and face the cytosol, where they constitute targets of cytosolic effectors including signal transduction molecules. One of the main signal transduction molecules involved in growth factor receptor- and antigen receptor-mediated cell proliferation is the small GTP-binding molecule p21R"(428,429). P2lRaS is active in its GTP-bound form and GTP-Ras can directly bind to the serinekhreonine kinase Raf1. The growth-promoting activity of Ras is primarily due to Raf-1 + MEK1,2 + MAPK activation (354,430).Although expression of activated v-Ha-Ras and v-Ki-Ras can trigger apoptosis in fibroblasts and Jurkat T cells and although Ras-induced cell death can be prevented by overexpression of the death-protective protooncogene product Bcl-2 (431), it is generally assumed that the Ras protooncogene product and its downstream target Raf-1 mediate signals that protect cells from apoptosis (432, 433). p21R" (431), Raf-1 (434), and the Ras-related molecule R-ras p23 (435) coimmunoprecipitate with Bcl-2. Recent data imply that this interaction reflects a cross talk between the Ras/Raf-1 pathway and Bcl-2-like molecules (Fig. 14). Bcl-2 can target the Raf-1 kinase to the mitochondrial membrane (224) and mitochondria-associated, but not plasma membrane-bound, Raf1 can stimulate the phosphorylation of the death-promoting Bcl-2 family protein Bad (436), thereby favoring its distribution from the mitochondrial membrane to the cytosol. Growth factor (IL-3) receptor occupancy stimulates the phosphorylation of Bad on a serine residue, thereby favoring the sequestration of Bad in the cytoplasm through binding to the 14-3-3 molecule (which binds phosphorylated Bad but not nonphosphorylated Bad). (225). In contrast, growth factor withdrawal leads to dephosphorylation of Bad, thereby causing its release from 14-3-3 and subsequent translocation of Bad to the mitochondrial membrane where Bad interacts with Bcl-2 family members and ultimately triggers cell death (225). Another signal transduction pathway that alters the composition of the Bcl-2 complex influences the subcellular distribution of the Bcl-2 binding

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A

* *

Growth factor receptors occupied

+ +

Ras+

P13’K

Raf-1

PKB

PT-pore closed No apoptosis

B Growth factor withdrawal

p 1 i

1

Ras+

1

P13’K

Bcl-2 BCl-2

i

PT-pore open Apoptosis

FIG.14. Posttranslational niodificatioriof Bcl-2 and BAD through serine phosphorylation influences resistance or susceptibility to apoptosis. (A) Signaling via an essential growth factor receptor, e.g.,IL-3R, leads to phosphorylation of Bad and sequestration of phosphorylated Bad in the cytosol through binding to 14-3-3. Serine phosphorylation of Bad is probably regulated via the kinase Raf-1. The role of PKB in this phosphorylation events is purely hypothetical. In addition, growth Factor receptor occupancy (HGF-R and PDGF-R) may allow the death inhibitory molecule Bag-1 to activate the Raf-1 kinase. (B) Withdrawal of essential growth factors (IL-3) leads to inactivation of PKB and Raf-1 and subsequent dephosphorylation of Bad. Dephosphorylated Bad is released from 14-3-3binding and forms heterodimers with Bcl-UBcl-X,,,thereby triggering apoptosis. Moreover, growth factor (CFD and PDGF) withdrawal leads to recruitment of the death inhibitory molecule Bag1 to the plasma membrane and inactivation of Bcl-2 by phosphorylation. The direct effect of Bcl-2 molecules on the PT pores is speculative.

protein Bag-1, which cooperates with Bcl-2 to proIong cell survival (437). Bag-1 can interact with the cytoplasmic domains of the receptors for hepatocyte growth factor and platelet-derived growth factor. Upon growth factor withdrawal, Bag-1 binds to these receptors and hence Bag-1 is not

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available for binding to mitochondria1 Bcl-2 (438). In T cells, Bag-1 is upregulated in response to IL-2 stimulation (439). Bag-1 can also bind to and activate Raf-1 (431), suggesting the existence of complex interactions between members of the Bcl-2 family and kinases. Bcl-2 itself can also be the target of posttranslational modifications via serine phosphorylation (Fig. 14). Phosphorylation of Bcl-2 occurs within a 60-amino acid loop without defined structure (440). Although the functional significance of Bcl-2 phosphorylation is still disputed, recent data imply that Bcl-2 phosphorylation reverts its antiapoptotic effect, that is, phosphorylated Bcl-2 does not protect from apoptosis (441-445). The ability of chemotherapeutic agents that act on microtubules (taxol,vinblastine, and vincristine) to induce phosphorylation of Bcl-2 has been also implicated as a mechanism by which these anticancer drugs can neutralize the antiapoptosis function of Bcl-2 and promote death. The kinase responsible for Bcl-2 phosphorylation has not been identified yet and is probably not Raf-1 (445).Thus, the exact nature of the kinases acting on Bcl-2 (and other members of the Bcl-2 family?) remains elusive. Most studies suggest that Bcl-2-related proteins have to localize to mitochondria in order to regulate apoptosis (183,446,447). In addition to their probable pore-regulatory function (19, 147, 185, 186,448), Bcl-2 and BclXL can determine the subcellular localization of apoptosis regulators with which they interact. This applies to Raf-1, calcineurin (449), and CED-4. Homodimerized Bcl-2 and Bcl-XLcan bind to the mammalian homolog(s) of CED-4, a molecule that was originally identified to be involved in the cell death pathway in the nematode C. elegans (450-452). CED-4 may function as an adapter between the mitochondrial-bound Bcl-2/Bd-XL and some procaspases with large N-terminal prodomains (e.g., procaspase-1 and -8).As a possibility, CED-4 keeps caspases in an inactive state, provided that it is bound to BcI-YBcI-XL (Fig. 15). Disruption of the trimolecular complex between CED-4, Bc1-2/Bcl-XL, and the caspase would lead to the activation of the inactive zymogen. Heterodimerization of Bcl-2/Bcl-XL with the death promoters Bax, Bad, Bak, Bcl-Xs,or Bik has been speculated to disrupt binding of CED-4 to Bcl-YBcl-XL, thereby releasing CED-4, which might activate caspases and/or act on the nucleus to provoke chromatin condensation and other apoptosis-like changes (24, 450-452). Based on the findings detailed previously, the antiapoptotic effects of Bcl-2 and Bcl-XI,overexpression could be explained in terms of a ternary molecular complex involving, in addition to Bcl-WBcl-XL, the mammalian CED-4 analog arid procaspases (453). As an alternative possibility, the binding partners of Bcl-2 might influence its capacity to regulate mitochondrial membrane permeability. In this hypothetical scenario, Bcl-2 and its homologs would function as mere targets of signal transduction pathways

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Death

Mitochondrion Fic:. 15. Hypothetical model of Bcl-2. CED-4, and caspase interactions at the mitochontfrial nienibrane. Binding of CED-4 to Bcl-WBd-Xl,results in caspase inactivation.Activation of effector caspases, e . g , caspses 3 and 6, requires release of cytochroine c and/or AIF from the mitochondria. Caspases involved in the induction phase of apoptosis such as caspase-8 (FLICE/Mach-1)relay death receptor signaling (DISCS)to Bcl-2 family inembers to open the PT pore and release cytochroine c and AIF. Moreover. such activating caspases iniiy caiise the direct proteolytic activation of downstream easpases.

involving caspases, CED-4, and other molecules (kinases, and phosphatases, including calcineurin), which modulate the composition of the Bcl2 complex and hence determine its capacity to open and close pores in the mitochondria1 membrane (448).

F PI3’ KINASEA N D Akt/PKB ACTIVATION THE KEY TO SURVIVAL? Recently, a novel signaling pathway, activation of the serinekhreonine

PKB (also known as Akt), has been shown to rescue cells from apoptosis (Fig. 16) (454).PKB is activated by the PI3’ kinase (455),which is involved in a variety of transmembrane receptor signaling cascades including signaling via antigen receptors in T and B cells, CD5, CD28 and CTLA4, receptors for IL-2, IL-4, insulin and insulin-growth factor, epidermal growth factor, platelet-derived growth factor, and basic fibroblast growth factor, or signaling via Gapy-coupled seven-membrane spanning receptors such as the major mitogenic factor present in fetal calf serum, LPA (454, 456, 457). Among the four distinct PI3’ Ks, three PI3’ Ks link tyrosine lanase receptor signaling to downstream effects and one is activated by induction of GPy subunits from G-protein-coupled receptors (458-460). PI3’ kinases are regulated via direct binding to transmembrane receptors or via Ras or

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FIG.16. The role of PI3’ kinase and protein kinase B (PKB or A h ) in growth factormediated survival. PKB might prevent apoptosis through inactivation of caspases. However, other downstream substrates of PKB might also have a critical role in the survival function of this kinase. GF-R, growth factor receptor; PtdIns-3,4-P2, phosphatidylinositol-3,4diphosphate. The scheme is adapated from Ref. (454).

R-Ras (461) and PI3’ kinases can induce a variety of downstream signaling events including p47phoXactivation required for cellular transformation, actin polymerization (462), and PBK/Akt activation (455). PI3’ kinase can phosphorylate the D3 position of phosphatidylinositol (PtdIns), phosphatidylinositol-4-phosphate (PtdIns-4-P), and phosphatidylinositol4,5-diphosphate (PtdIns-4,5-P2) to generate phosphatidylinositol-3phosphate (PtdIns-3-P), phosphatidylinositol-3,4-diphosphate(PtdIns-3,4P2), and phosphatidylinositol-3,4,5-triphosphate (PtdIns-3,4,5-P3), respectively (457). PtdIns-3,4-P2 can also be generated from PtdIns-3,4,5-P3 through the action of a 5’ phospholipid phosphatase such as the inositol polyphosphate 5-phosphatase SHIP, a negative regulator of B cell receptor and IgE recepter signal transduction (463-468). PtdIns-3,4,5-P3 and PtdIns-3,4-P2 interact with the plekstrin homology domain of PKB/Akt with high affinity (457, 469). High-affinity binding of PtdIns-3,4-P2 to PKB/Akt leads to the recruitment of PBWAkt to the plasma membrane, where PKB/Akt undergoes a conformational change and becomes phosphorylated on serine 473 and threonine 308 (454,457). Interestingly, the MAPK kinase p3WHOG-activated MAPKAP kinase 2 can phosphorylate serine 473 in PKB/Akt under conditions of cellular stress that do not activate P13’ kinase (470, 471), implying that the stress signaling kinase p38/HOG (258,378,472) can regulate PKB/Akt activity. The MAPK family kinase p38/HOG is activated by a plethora of different stimuli, such as irradiation, toxins, ischemia, heat shock, ceramide, or ligation of the TNF-

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Rp55 (Fig. 12) (258, 378, 379, 472). P38/HOG-regulated PKB/Akt activation provides another example of the multiple cross talks that exist between different signal transduction cascades. Overexpression of activated PI3’ kinase or membrane-targeted PKB/ Akt can protect Rat-1 fibroblasts and COS cells from UV-B light-mediated apoptosis (473). Insulin-like growth factor- 1 ( IGF-1)-mediated survival of neurons appears to depend on a functional PI3’K -+ PKB/Akt signaling pathway, and IGF-l-mediated neuronal survival can be blocked by overexpression of dominant-negative PKB/Akt (474). PKB/Akt signaling can also prevent NGF-induced apoptosis of pheochromocytoma PC12 cells (454). Moreover, Ras activation of PI3’ kinase suppresses c-Myc-induced apoptosis in fibroblasts.This protective effect depends on the activity of PKB/Akt (475). However, in the same experimental system Ras activation triggers apoptosis through induction of the Raf-1 kinase (475), implying that Raf1 activation does not necessarily lead to the suppression of cell death. Rather, Ras/Raf-1 can mediate antithetical intracellular signals for cell fate decisions. These data indicate that certain signals including the growth factor IGF1 can promote cellular survival via activation of the Ras + PI3’ kinase + PKB signaling cascade. The Ras-induced survival pathway is independent of MAPK activation and independent of ~ 7 0 ~and ~ GSK3, ~ ~ ” which ~ ’ ~are the only known downstream effectors of PKB/Akt activity (458). Thus, the identification of the downstream apoptosis regulatory targets of PBWAkt will be of the utmost importance. G. THETNF RECEPTORSUPERFAMILY The ever-growing TNF-R family includes the low-molecular-weight TNF-Rp55, the low-molecular-weight TNF-Rp75, LTPR (476), FadAPO1 (CD95) (343, 477), designated death receptor-3 (DR3 or wsl-1) (478, 479), designated death receptor-4 (DR4) (480), the cellular receptor for the cytopathic avian leukosis sarcoma virus termed CAR1 (481), ATAR (482), CD27 (483), 41.BB (484, 485), CD40, 0x40, nerve growth factor receptor (486), the Hodgkin’s lymphoma antigen CD30 (487),or the newly identified inhibitor of osteoclast differentiation osteoprotegerin (488).The TNF family of receptors is characterized by sequence homology at an extracellular cysteine-rich domain. In contrast, the cytoplasmic domains of these molecules lack homology, suggesting that the members of the family share similar ligand recognition properties but differ in signal transduction (485,489-491). The members of the TNF-R superfamily mediate a plethora of biological responses including induction of cellular proliferation, induction or suppression of programmed cell death, costimulation in T and B lymphocytes, osteoclast differentiation from bone marrow

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precursors, development of Peyer’s patches and lymph nodes, and inflammatory immune responses. Activation of mature T cells upregulates the expression of receptors that can receive death signals (Fas, CD30, and the low- and high-molecularweight TNF-Rp55 and TNF-Rp75) and simultaneouslycauses downregulation of apoptosis inhibitory genes such as bcl-2. Thus, antigen-driven activation and expansion generates T lymphocytes that are prone to apoptosis (257, 258, 492). This mechanism maintains leukocyte homeostasis in uiuo (Fig. 9). Activation-induced cell death of peripheral T cells is mainly mediated via the death receptor Fas, TNF-Rp55, and TNF-Rp75 (262266, 346, 477). Although these receptors have little if any impact on physiological thymocyte selection, another member of the TNF-R family, CD30, appears to determine negative selection in the thymus (256). The following sections will focus on the role of Fas, CD30, TNF-Rp55, and TNF-Rp75 in lymphocyte development and function and the general principles of signal transduction pathways that link these receptors to the central executioner or the survival factor NF-KB.

1 . TNF Receptors, Fas, and CD30: Impact on Central and Peripheral Tolerance The biological functions of TNF-a and TNF-P are mediated by three distinct surface receptors (TNF-RpSS, TNF-Rp75, and LTPR). TNF-a and TNF-P bind to either TNF-Rp55 or TNF-Rp75. These effects are pleiotropic, ranging from cell proliferation via costimulation to induction of programmed cell death and inflammatory immune responses (489-491, 493). The third distinct receptor that interacts with TNF, LTPR, preferentially interacts with TNF-/3 and controIs the development of Peyer’s patches and lymph nodes (494). Both TNF-Rp55 and TNF-Rp75 are expressed at low levels on the cell surface of resting peripheral T cells but upregulated upon antigen-specific activation, implying that these molecules may share a common mechanism for transcriptional induction and may function in a similar manner (491). Similarly, expression and function of other TNF receptor family members such as Fas, CD40, CD27, CD30, 41.BB, or OX-40, or their receptors are highly regulated and depend on the T cell lineage and stage of differentiation of thymocytes (489-491, 493). Within the thymus, both mature and immature T cells produce TNF-a constitutively (495, 496). TNF-a can augment IL-6-induced thymocyte proliferation or induce apoptosis of CD4CD8- precursor and mature single-positive thymocytes. Because murine, TNF-a but not human TNF-a, can induce thymocyte apoptosis (human TNF-a only binds to the inurine TNF-Rp55 but not the murine TNFRp75) (496), the apoptotic effect of TNF in thymocytes is most likely

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mediated by TNF-cdTNF-Rp75 interactions. However, t h p o c y t e development and negative selection are not altered in TNF-Rp55 (497), TNFRp75 (498), or LTPR (494) gene-deficient mice. In contrast to thymocyte selection, peripheral T cells from TNF-Rp55- and TNF-Rp75-deficient mice do exhibit a partial resistance to activation-induced cell death (264266). This phenomenon becomes particularly manifest when animals lacking both the TNF-R and the Fas receptor are analyzed (264,265), suggesting that various members of the TNF-R superfamily may be involved in the same pathway in a redundant fashion. Within the thymus, Fas is highly expressed in CD4+CD8+TCR""""'".''' cells, whereas Fas expression is downregulated in mature CD4' or CD8' thyniocytes (499, 500). Although Fas is probably not directly involved in negative selection of thyinoctes responding to peptide antigens (265, 501, 502) or superantigens (503),the peak of Fas expression correlates with the stage of thyniic maturation at which T cell selection occurs. Interestingly, CD4+CD8- or CD4-CD8- thyrnocytes expressing N K 1 . l and the TCRaP heterodimer are cytotoxicagainst CD4+CD8+thymocytes and this cytotoxic activity is mediated by Fas/Fas ligand interactions (504). Moreover, antiFas cross-linking can induce cell death of immature thymocytes in vitro (41,505) and in thymic organ cultures (506), implying that Fas might-at least to some extent-modulate death of thymocytes. However, most data suggest Fas is predominantly involved in peripheral T cell tolerance. A wealth of data point to a role of Fas in the maintenance of peripheral cell numbers and apoptosis of activated T ly~nphocytes(345, 346, 477, 507). Fas is rapidly upregulated upon stimulation of peripheral T cells. However, activated T lymphocytes are only susceptible to Fas-triggered apoptosis several days after the initial activation event (477, 508, 509). In addition, anti-CD4 induced apoptosis of peripheral T cells in vivo and Ca2+-independentT-cell cytotoxicity appears to depend on Fas expression (510, 511). In this context it is interesting to note that Fas does not only induce cell death but also can provide an IL-2 independent signal for proliferation of peripheral T cells and thymocytes (512). The 120-kDa glycoprotein receptor CD30 molecule is a member of the TNF-R superfamily and was originally identified as a diagnostic marker for Hodgkin's disease, the most frequent lymphoma in humans. CD30 is found on multinucleated Reed-Sternberg cells and Hodgkin's cells in Hodgkin's disease and on a variety of other tumor cells including embryonal carcinoma, melanoma, and some mesenchymal tumor cells (513-515). CD30 is also expressed on non-Hodgkin lymphomas and some virally transformed T and B cells. In addition, it has been suggested that CD30' T cells play a role in HIV pathogenesis, Epstein-Barr virus infections, measle virus infections, atopic disorders, or autoimmune diseases such as

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systemic lupus erythematosus and rheumatoid arthritis (516). In normal lymphoid tissues, CD30 is expressed on activated T and B cells and medullary thymocytes (517-519). Moreover, the CD30 ligand is expressed on activated T cells (520). Because some tumor cell lines undergo apoptosis after CD30 cross-linking, it has been proposed that the CD30 surface receptor triggers signal for cell death (521). By contrast, CD30 may also function as a receptor in activation and differentiation of T and B cells (518,522,523). Moreover, it has been proposed that CD30 is differentially expressed on Th2 T cells (516) and that CD30 induction through IL-4 might have a role in the differentiation and/or maintenance of Th2 cytokineproducing T lymphocytes (524, 525). Recent studies involving CD30 gene-deficient mice suggest a role for CD30 in central immune tolerance (256). In CD30-’- mice, thymocyte numbers are increased. Whereas positive selection and dexamethasone and y-irraditation-induced cell death of immature thymocytes are normal, CD30-’- thymocytes are resistent to anti-CD3-induced apoptosis in vitro and antigen-driven negative T cell selection is impaired in two different TCR transgenic mouse models in viva These results show that TCWCD3induced cell death can be mediated via the CD30 receptor in developing thymocytes. Despite impaired negative selection, peripheral T cells from CD30-’- mice are still tolerant to self-antigens, implying that multiple mechanisms control the induction and maintenance of immunological tolerance (256).However, these data provided the first evidence for a receptor that specifically interferes with negative thymocyte selection. The data for CD30-’- mice also confirmed that distinct signal transduction pathways exist for positive and negative selection of T cells. By contrast, CD30 can activate T and B cells (518,522,523) and may have a role in Th2 cytokine production (524, 525). It thus appears that CD30 signaling can induce contradictory signalingevents in terms of fate decisions for survival or apoptosis. 2. Death Domains and Death-Inducing Signaling Complexes Similar to most other transmembrane receptors, signaling of TNF-R family molecules requires clustering of the receptors after ligand binding (Fig. 17). Receptor clustering induces conformational changes, the recruitment of signal transduction molecules to the cytoplasmatic domain, and subsequent induction of cellular responses such as proliferation andlor apoptosis. The ensemble of signal transduction molecules that bind to the oligomerized death receptors and mediate cell death have been termed the “death-inducing signaling complex” (DISC) (65, 526, 527). One of the principles of DISC formation is the interaction between socalled death domains (DDs).The D D is a region ofhomology first identified

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FIG.17. Model of TNF-RpSS and Fas-mediated signal transduction for apoptosis (Fas and TNF-Rp5S) and NF-KB activation (TNF-Rp55). DD, death domain; DED, death effector domain: ICE, ICEKED homology domain. RAIDD also contains a DD domain and interacts with RIP via homotypic DD domain binding (not indicated in the figure). For more details see text.

in TNF-Rp55 and Fas in an approximately 80-amino acid-long cytoplasmic domain that is crucial for the induction of cell death (Fig. 17) (489, 528). Additional transmembrane receptors possess similar cytoplasmicDDs. This applies to DR3 (wsl-l),DR4, the chicken CAR1 protein (481), and possibly the p75 NGF-R (375,529,530). DDs are also found in cytoplasmic signaling molecules, namely, in the receptor interaction protein (RIP) (531), the Fas-associated protein with a death domain (FADD) (532, 533), the TNF-R associated protein with a death domain (TRADD) (534, 535), and the RIP-associated ICH1-homologous protein with a death domain (RAIDD) (536). Death domains are homotypic protein-interaction domains that permit binding of DD-containing intracellular molecules to the DD domains present in the transmembrane receptor. Thus, oligomerization of the TNF-

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Rp55 triggers the binding of the molecular adapters TRADD and FADD to the cytoplasmic tails of the TNF-Rp55 through homotypic D D interactions (532-535, 537). FADD contains a death effector domain (DED) that interacts with the DED domain of caspase-8 (46,65,526,538). Procaspase8, formerly called FLICE ( FADD-homologous ICEKED-3-like protease)/ Mach, contains a DED domain for binding to FADD and an additional region with high homology to ICE/CED-3 caspases (65, 538). During activation and assembly of the DISC, procaspase-8 gets proteolytically cleaved between the DED and the ICE homology domain and the activated caspase-8 can stimulate apoptosis induction. Similarly, the DR3 receptor triggers apoptosis via DD domain recruitment of TRADD and FADD and activation of caspase-8. The death-inducing signaling machinery associated with the DR4 receptor after binding to its ligand TRAIL has not been eluciated, but it does not appear to involve TRADD (65, 538). Similar to the TNF-Rp55, oligomerization of Fas through binding of its trimeric Fas-L induces the formation of a DISC. However, FADD binds directly to the D D of Fas and recruits caspase-8 (FLICE/Mach-1) via homotypic DED domain interactions (Fig. 17). Cleavage of FLICE at the assembled DISC then leads to the release of the active caspase component of caspase-8 and subsequent induction of Fas-mediated cell death (65, 538) in a pathway that may involve activation of other upstream caspases including caspase-1 (formely called ICE) (42, 45, 539). Caspase-1 may induce mitochondria1permeability transition in a fashion that is not antagonized by Bcl-2, at least in CEM-7 cells, (203), in accord with the fact that Bcl-2 is an inefficient inhibitor of Fas-induced apoptosis (540-542). Besides the recruitment of FADD and caspase-8, an additional molecule, RIP, interacts with the DD of Fas. RIP contains a DD at the NH2 terminal and a serinekhreonine kinase homology region at the COOH-terminal end (543). Moreover, a Fas-associated protein tyrosine phosphatase, FAP, has been identified that can directly bind to the COOH-terminal 15 amino acids of Fas and might protect cells from Fas-induced apoptosis (544). Although RIP can function as serinekhreonine kinase in vitro, the functional significance of this enzymatic activity is unclear. RIP interacts with the adaptor molecule RAIDD (536). RAIDD has an unusual bipartite architecture comprising a carboxy-terminal death domain that binds to the homologous domain in RIP and an amino-terminal domain homologous with the sequence of the prodomain of two procaspases, human caspase2 (ICH-1) and C. eleguns CED-3. Thus, homotypic interactions mediated by caspase prodomains can determine the specificity of binding of caspase zymogens to regulatory adaptor molecules (536). Selective activation of RAIDD by Fas might explain earlier reports that Fas and TNF-Rp55 can

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employ distinctive signal tranduction pathways to trigger programmed cell death (173, 545). Although clear evidence exists that CD30 plays a role in antigen-driven negative selection of thymocytes, the cytoplasmic domain of CD30 has no obvious homology to death domains present in Fas, TNF-Rp55, DR3, and DR4 receptors (see the following section). Nonetheless, CD30 activation leads to the recruitment of the TNF receptor-associated proteins TRAF1, TRAFB, and TRAFS and induction of NF-KB (546,547). In a Hodgkin's lymphoma cell line, ligation of CD30 is mitogenic, depending on activation of a protein tyrosine kinase and the MAPK signaling pathway (548). Thus, CD30, like many other receptors, can trigger both activating and Iethal signals.

3. NF-KB Activation by TNF: Activation of u Survival Signal Activation via TNF does not always induce apoptosis, and many cell types are fairly resistant to TNF-mediated cell death. Most TNF-resistant cells, however, are hlled by TNF in the presence of protein synthesis inhibitors. This indicates that TNF induces two pathways, one that mediates apoptosis and a second that induces transcription of cytoprotective genes. One crucial molecule that is activated by almost all TNF-R superfamily receptors (with the exceptions of Fas and DR4) is the transcription factor NF-KB. NF-KB is important for lymphocyte responses to antigens and cytokine-inducible gene expression (412). NF-KB activation can protect fibroblasts and T lymphocytes from apoptosis in response to ionizing radiation, the anticancer drug daunorubicin, and TNF-(I!treatment (414-416). Activation of NF-KB appears to be mediated by TNF-R-associated factors (TRAFs) (Fig. 17) (549). Six distinct TRAF molecules have been identified to date. TRAFs bind via their COOH-terminal TRAF domains to the TRAF domain of TRADD or directly to the cytoplasmatic region of the receptor (for example, in the case of TNF-Rp75) (550-554). Thus, activation of the TNF-Rp55 leads to recruitment of TRADD via the DD domains and TRADD recruits TRAFB via homotypic TRAF domain interactions. By contrast, TNF-Rp75 oligomerization leads to the recruitment of TRAFl and TRAFB to the cytoplasmatic region of the receptor and TRAFl and TRAFB forin heterooligomers through interactions of their TRAF domains. Similarly, CD40 activation leads to the recruitment of TRAF2, which then forms oligomers with TRAF3 (555). In addition to the TRAF domain, some TRAF molecules, e.g., TRAFB and TRAFS, contain a NH2terminal ring-finger domain that is required for NF-KB induction. Importantly, a doininant-inhibitory mutation of TRAF2 leads to TNF-mediated apoptosis instead of activation, i.e., in the absence of a TRAF2-mediated signal, TNF-treated cells trigger a default signaling

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pathway that culminates in cell death (552). Moreover, it has been shown that TRAF2 mediates TNF-induced activation of SAPK (403, 404). The bifurcation between TRAF2-induced SAPK and NF-KB activation is probably the serinekhreonine kinase MEKK1, a MAPKKK that directly activates SEKl and NF-KB through site-specific phosphorylation of the NF-KB inhibitor, I - K B (Fig. ~ 13) (413). Recently, it has been shown that a variety of other molecules bind to the signaling complex that relays TNF-R stimulation to cell death and NFKB. For example, a novel TRAF-interacting protein, I-TRAF, has been identified that binds to the conserved TRAF-C domain of TRAF1, TRAFB, and TRAF3. Overexpression of I-TRAF inhibits TRAF2-mediated NF-KB activation signaled by CD40 and both TNF receptors. Thus, I-TRAF appears as a natural regulator of TRAF function that may act by maintaining TRAFs in a latent state (549). Moreover, four mammalian molecules, termed cellular inhibitors of apoptosis (c-IAPs), have been identified that interact with TRAFl and TRAF2 (552, 556), suggesting the existence of multiple endogenous inhibitors acting on specific apoptosis induction pathways. Although c-1APs have homology to viral inhibitors of apoptosis such as the baculovirus p35 protein, the physiological role of c-IAPs in mammalian cells is still elusive. 4. TNF-Receptors, Sphingomyelinases, and Ceramide

TNF-Rp55 signal transduction for NF-KB activation does not only involve TRAFZ but also sphingomyelin breakdown into ceramide by the acidic sphingomyelinase (557, 558). The second messenger ceramide is produced through either the induction of sphingomyelin (SM) hydrolysis via cytosolic acidic or membrane-bound neutral sphingomyelinases (SMase) or de novo biosynthesis (559, 560). Ceramide transduces signals mediating differentiation, growth, growth arrest, apoptosis, cytokine biosynthesis and secretion, and a variety of other cellular functions (559). Rapid sphingomyelin hydrolysis to ceramide correlates with irradiation, Fas, and TNF-R-induced apoptosis in various cellular systems (561-563). In particular, it appears crucial for irradiation-induced apoptosis because lung epithelial cells, but not thymocytes, from mice deficient for acidic sphingomyelinase and some human cell lines from patients lacking this enzyme fail to undergo apoptosis in response to y-irradiation (564). Ceramide is a second messenger that induces a membrane-bound ceramide activated serinekhreonine protein kinase (CAPK) (565) and a cytoplasmic ceramide-activated protein phosphatase (566). Ceramid can also lead to the induction SAPK (357, 494) and p38/HOG (472). Ceramidemediated SAPK activation has been functionally linked to the induction of apoptosis (363, 364, 397, 567). Other downstream targets for ceramide

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action include Cox, IL-6 and IL-2 gene expression, PKCS, Vav, the retinoblastoma protein, c-Myc, c-Fos, and a variety of other transcriptional regulators (568).Although a number of ceramide-activated effector molecules have been described, it will be important to identify additional cellular targets that link the activation of SMases to the regulation of nuclear events. Cross talk between ceramide-induced signal transduction cascades and other signaling pathways adds to the inherent difficulty in distinguishing the specific effects of complex and multifaceted signal transduction pathways (559, 569). The human TNF-Rp55 initiates at least two independent ceramide signaling cascades via activation of acidic or neutral sphingomyelinases (570). The acidic sphingomyelinase (A-SMase) pathway involves a phosphatidylcholine-specific phospholipase C , an endosomal A-SMase, and controls expression of multiple TNF-responsive genes through induction of transcription factors including NF-KB (557,558,571).Acidic SMase-triggered NF-KB activation is probably mediated by rapid degradation of I - K B ~ through a serine-like protease. Activation of A-SMase by TNF-Rp55 stimulation was mapped to the region of the death domain (572) and may require a crmA-inhibitable caspase (78). The neutral sphingomyelinase (N-SMase) pathway comprises a membrane-bound N-SMase, CAPKs, and phospholipase A2 and appears critical for the inflammatory responses induced by TNF. An ll-amino acid region (aa 309-319) of the human TNF-Rp55 is necessary and sufficient for activation of N-SMase and a molecule termed FAN has been cloned that couples the TNF-Rp55 to the N-SMase signaling pathway (571). The N-SMase activation domain is distinct from the death domain and incapable of induction of A-SMase, NF-KB, and cytotoxicity suggesting that N-SMase and A-SMase control nonoverlapping pathways of TNF receptor signal transduction (573). Interestingly, CAPKs can phosphorylate and activate Raf-1 (574) and mediate MAPK activation (575), implying that N-SMases initiate the proinflammatory actions of TNF-a via a CAPK + Raf-1 -+ MAPK signaling cascade. In C. elegans and Drosophila melanogaster, a potential CAPK has been genetically identified as the kinase suppressor of Ras (KSR) (576). In synthesis, it appears that ceramide derived from neutral SMase activation mediates proinflammatory responses through the activation of CAPK (KSR), Raf-1, and MAPK, whereas ceramide generated through acidic SMase activation appears to be primarily involved in NF-KB and SAPK activation and correlates with the induction of apoptosis (567).It is tempting to speculate that neutral, membrane-associated sphingomyelinases may be involved in cell proliferation and proinflammatory responses, whereas the induction of acidic sphingomyelinases may mediate cell death. In contrast

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to this hypothesis, however, irradiation of bovine aortic endothelial cells induces rapid sphingomyelin hydrolysis and apoptosis through a neutral sphingomylinase pathway (561). Moreover, CD28 costimulation, which results in T cell proliferation and prolonged survival, leads to the activation of an acidic sphingomylinase (568). The mechanism by which activation of shingomyelinasesand the generation of ceramide activate the central executioner is unknown. According to one report (217),ceramide can induce mitochondrial permeability transition, but this finding has not been confirmed by another group (577), who reports that ceramide enhances the mitochondrial generation of reactive oxygen species. In a cell-free system that includes mitochondria and nuclei, ceramide itself is an inefficient inducer of nuclear apoptosis. However, cytosols from cells treated with ceramide contain an activity that provokes mitochondrial permeability transition in vitro (203). Thus, ceramide may either have a direct effect on mitochondria or provoke the generation of second messengers that activate the mitochondrial executioner. V. Conclusions

DeatMlife decisions are crucial for the homeostasis of both the thymic and the peripheral compartments of T cells. During the past few years, major progress has been achieved in understanding how signal transduction pathways may influence this decision. Whereas certain signal transduction pathways are mainly involved in conferring apoptosis resistance (e.g., the P13’ kinase +. PKB signaling cascade and activation of NFKB),others may have a major role in mediating death signals. One such pathway involves activation of stress-activated protein kinases (SAPWJNK)and/or p38HOG kinase via a plethora of different stimuli, such as irradiation, toxins, ischemidreperfusion, heat shock, ceramide, or surface death receptors such as the TNF-Rp55 and Fas may trigger apoptosis. However, recent genetic evidence indicates that SAPK signaling prevents premature T cell death during activation and development. Another predominantly deathinducing pathway involves raises in cytosolic Ca2+levels, which together with additional but ill-defined factors can trigger apoptosis. Recent data indicate that the stoichiometry of Bcl-2-related death agonists and antagonists is not the sole parameter to determine apoptosis regulation of these proteins. Posttranslational modifications of Bcl-2-related proteins, such as BAD and Bcl-2, have a major impact on apoptosis regulation and might be a critical crossroad that links surface receptor signals to the regulation of apoptosis. Multiple signaling pathways may feed into the posttranslational modification of these molecules, in particular signaling via the serine hnase Raf-1. A series of receptors belonging to the

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TNF-R superfamily, namely, TNF-Rp55, Fas, DR3, and DR4, assemble a death-inducing signaling complex upon ligation. Oligomerization of these receptors leads to the recruitment of death-inducing adaptor molecules through so-called death domains and eventually triggers certain caspases. It would be an oversimplification to asssuine that these receptors mediate only apoptotic responses. Thus, the TNF-Rp55 triggers conflicting signals that either induce apoptosis (caspase activation and ceramide) or trigger activation of the apoptosis-inhibitory transcription factor NF-KB. Altogether, it becomes increasingly clear that the initiation of apoptosis mostly does not involve just a simple linear sequence of biochemical events. Rather, multiple parallel and sometimes antagonistic pathways are activated. Cross talk between different signal transduction pathways is frequent and contributes to augment the complexity of the system. Recent progress suggests that after the heterogeneous, signal transduction-dependent initiation phase, apoptosis employs one (or a few) common pathways, in line with the fact that the biochemical and ultrastructural features of apoptosis are similar in all cell types, independent from the initial apoptosis trigger. The common pathway can be subdivided into two phases: the effector phase, during which the central executioner is activated, and the degradation phase, beyond regulation, during which cellular catabolism gives rise to the apoptotic phenotype. The central executioner involves major changes in mitochondrial membrane permeability, including the opening of so-called PT pores. Recent evidence suggests that a number of signaling molecules facilitate such changes in mitochondrial membrane permeability: Ca2+,reactive oxygen species, nitric oxide, perhaps ceramide, and some caspases that are activated in particular pathways of apoptosis (e.g., caspase-1). In addition, mitochondrial membrane permeability is regulated by the members of the Bcl-2 family and/or Bcl-2 associated proteins, and thus constitutes a target of multiple pathways that affect the level of expression of such Bcl-2-related proteins, influence their subcellular distribution, or introduce posttranslation modification affecting their apoptosis regulatory potential. At this latter level, lunases and phosphatases may act on Bcl-2 and Bcl-2-binding proteins. The involvement of mitochondria in the central executioner is attractive for several reasons. First, it opens the possibility of detecting apoptosis at a relatively early stage by monitoring mitochondrial membrane integrity. Second, the mitochondrion can function as a sensor for changing (stressful) metabolic conditions as well as for certain second messengers. Third, the molecules involved in the mitochondrial executioner cannot be mutated because they are necessary for normal cell function, thus avoiding the selection of tumor cells that would be completely resistant to apoptosis induction. Fourth, the mitochondrial membrane changes may have some

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self-amplifying properties, which may allow them to act in a switch-like fashion. Finally, activation of the mitochondrial executioner has multiple lethal consequences. Once the mitochondrial membrane permeability has been perturbed and vital mitochondrial functions are disrupted, the cell is irrversibly committed to death. At this stage, mitochondria release apoptogenic proteins that are normally well secluded. Such proteins trigger the activation of downstream caspases and endonucleases and thus induce the typical pattern of morphological and biochemical changes that accompanies the late stage of apoptosis. Inhibition of caspases and endonucleases does not prevent cytolysis and rather determines a necrotic type of cell death. Thus, the degradation phase is probably not a useful target for pharmacological interventions. Any attempt to modulate apoptosis must aim at interfering with apoptosis-triggering signal transduction pathways and/or the activation of the central executioner.

ACKNOWLEDGMENTS We thank Dr. Maurice Geuskens (Free University of Bruxelles, Belgium) for electron microscopic data and Drs. Catherine Brenner, Didier Decaudin, Tamara Hirsch, Philippe Marchetti, Isabel Marzo, Patrice X. Petit, Santos Susin, Naoufal Zamzami, Young-Yun Kung, Klaus Fischer, Ivona Kozieradzki, and Takehiko Sasaki for sharing unpublished data and helpful discussion. This work has been partially supported by Agence Nationale pour la Recherche contre le Sida, Association pour la Recherche contre le Cancer, Centre National de la Recherche Scientifique, Fondation de France, Fondation pour la Recherche MBdicale, Institut National de la SantB et de la Recherche MBdicale, Ligue Franqaise contre le Cancer, North Atlantic Treaty Organization, and the French Ministry of Science (to GK), and grants by the Medical Research Council and Amgen (to JMP).

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This chapter was accepted for publication on July 2, 1997.

ADVANCES I N I M M U N O L O G Y . V O L 68

Prenylation of Ras GTPase Superfamily Proteins and Their Function in lmmunobiology ROBERT B. LOBEU Merck Research labomtories, hporhnent of Cancer Research, Merck and Company, Inc., West Point, Pennsybanio 19486

I. Introduction

The Ras superfamily of GTPases comprises a diverse group of proteins that play critical regulatory roles in a variety of cellular processes involved in immune system function (see Fig. 1).Although members of this family play diverse roles in cells, they carry out their functions via similar biochemical mechanisms. First, these proteins cycle between GDP and GTP-bound states and rely on accessory proteins to regulate this GDP/GTP cycle. Second, many members of the Ras superfamily can regulate multiple signaling pathways through interactions with different downstream effector molecules. Third, these proteins all function at membrane surfaces and are localized to membranes via C-terminal lipid moieties that are added to the protein posttranslationally in a process commonly referred to as prenylation. Lipidation of the Ras superfamily of proteins involves a family of prenyltransferases, which attach isoprenoid-derived lipids consisting of 15 carbon units (farnesyl) or 20 carbons (geranylgeranyl) to C-terminal cysteine residues. In contrast to membrane insertion via transmembrane domains, membrane association via prenylation can be a transient and regulatable phenomenon. This transient association is essential to the function of some of the prenylated GTPases, in particular the Rab GTPases, which catalyze the intracellular flow of membrane compartments and that must cycle on and off membrane sites to function. This review will give an overview into the biochemistry and function of the Ras superfamily members, discuss the enzymology and functional consequences of protein prenylation, detail specific roles of the three major subfamilies of the Ras superfamily in immune system function, and discuss inhibitors of protein prenylation and their effects on the function of the Ras superfamily of proteins. II. The Ras Superfamily Members

The Ras superfamily can be subdivided into three major subfamilies, the Ras proteins, the Rho/Rac proteins, and the Rab proteins (Boguski and 145

Copynght 0 1998 by Acdrmir Press All nghts of reprmiuction in Any form reservc=d W65-277fiN8 $25 00

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FIG.1. Some ofthe functions of Ras superfamily proteins in immune cells. Ras superfamily members are indicated by the solid circles.

McCormick, 1993).The Ras proteins play a key role in signal transduction processes that regulate cell proliferation, activation, and differentiation. The Ras subfamily includes the mammalian ras alleles Harvey (H), N-ras, and Kirsten (K). The K-ras gene codes for two alternatively spliced variants, K4A and K4B, which are distinguished by the presence of a highly charged C-terminal region in K-Ras4B known as the polybasic domain (Barbacid, 1987). The expression of the different rus alleles vanes in different tissues (Leon et al., 1987), and there is some evidence to suggest that these different forms of Ras have somewhat different biochemical activity because mutant forms of these proteins differ in their ability to transform cells (Maher et al., 1995). The Ras proteins are closely related to the Rap proteins (RaplA, -1B, -2A, and -2B) and the R-Ras, RalA, RalB, and TC21 proteins. The Rho/Rac subfamily of proteins play many regulatory roles, including regulation of the actin cytoskeleton (Ridley, 1995),and regulation of the c-Jun kinasehtress activated protein kinase ( JNWSAPK) pathway.

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The Rho/Rac family includes RhoA, RhoB, RhoC, RhoD, RhoE, RhoG, Racl, Rac2, CDC42Hs, and TC10, which are -50% identical with each other and share -30% identity with other Ras-like GTPases (Nobes and Hall, 1994). The Rab subfamily of proteins consists of -30 members, which regulate the trafficking of intracellular membrane compartments (Pfeffer, 1994);this includes a role in regulating endocytosis and exocytosis, two types of membrane transport that are particularly important in immune cell function. 111. The GTPase Cycle

The members of the Ras superfamily of GTPases function as on-off switches that cycle between GTP-bound and GDP-bound states. They are in the “resting state” or “off” position when bound to GDP and activate their respective cellular processes when in the GTP-bound state. Turning these molecular switches on and off requires accessory proteins that are specific for the different members of the family (reviewed in Boguski and McCormick, 1993). The activation step involves guanine nucleotide exchange factors (GEFs),which facilitate dissociation of the bound GDP. Dissociation of the bound GDP enables the GTP to bind due to the high concentration of GTP in the cell relative to GDP (Bourne et al., 1991). The signaling pathway leading from transmembrane receptors such as the EGF or platelet-derived growth factor (PDGF) growth factor receptors to the activation of Ras is fairly well understood and proceeds through the activation of the guanine nucleotide exchange factor SOS. The initiating event is the binding of ligand to the receptor, which induces tyrosine autophosphorylation of residues in the receptor’s intracellular domain. The phosphorylated tyrosines of the receptor serve as docking sites for adapter proteins, such as Grb2 or Shc, which bind to the phosphotyrosines via their SH2 domains (Burgering and Bos, 1995).These adapter proteins also contain SH3 domains that mediate a binding interaction with polyproline stretches found on SOS (Quilliam et ul., 1995).Thus, receptor activation recruits the Grb2-SOS complex to the membrane, leading to guanine nucleotide exchange and activation of membrane-bound Ras. GTP binding is thought to induce a change in conformation that exposes the so-called effector domain, allowing the Ras protein to interact with downstream signaling effectors. A fairly detailed molecular description of the activation of Ras and its interaction with downstream effectors has been provided from crystallographic studies of Ras and Ras-related proteins (for a review, see Wittinghofer and Nassar, 1996). The members of the Ras family of proteins remain activated until their bound GTP is hydrolyzed. The Ras family members have weak intrinsic

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GTPase activity and require an interaction with another auxiliary protein, called a GTPase activating protein (GAP), to hydrolyze the bound GTP. In the case of Ras, Ras-GAP can accelerate the Ras GTPase reaction by almost five orders of magnitude (Gideon et al., 1992). Rho family members are activated by Rho-GAP domains, which are found in a variety of large, multifunctional proteins (Lamarche and Hall, 1994). The crystal structure of the Ras-GAP domain of p12OGAP, and the Rho-GAP domain from p50rhoGAP, have been solved recently (Barrett et al., 1997; Scheffzek et al., 1996). The deactivated GDP-bound form of the protein remains dormant until the proper activation stimulus is received and the protein can repeat the GTPase cycle. In the case of the Rho and Rab proteins, a third type of auxiliary protein, called a guanine nucleo'ide dissociation inhibitor (GDI),is involved in the GTPase cycle. GDI binds the GDP-bound form of the protein and, as the name implies, inhibits the dissociation of GDP. More important, the interaction of GDI with the Rho or Rab proteins prevents their binding to cellular membranes and, additionally, GDI can extract the Rho or Rab proteins from membranes (Wu et al., 1996). The ability of GDI to extract Rab proteins from membranes is critical to Rab protein cycling. GDI functions in retrieving the Rab protein from an acceptor membrane after a membrane vesicle fusion event has occurred and in delivering the Rab protein back to the donor membrane for another round of transport (Pfeffer, 1994; Soldati et al., 1994; Ullrich et al., 1994). IV. Downstream Signaling Effectors: Ras and the Rho/Rac Connection

A common feature of the Ras superfamily of GTPases, exemplified by both the Ras and Rho/Rac proteins, is the ability to activate multiple downstream effector pathways. Proteins that interact with the Ras effector domain include the Raf serinekhreonine kinase, phosphoinositide 3'kinase, MEK kinase (a kinase in the JNUSAPK kinase cascade), Ras GAP, Ral-GEF, and two proteins of unknown function (Rin, for Ras interacting; and Rsb; for Ras binding, Marshall, 1995; Wittinghofer and Nassar, 1996). Although the physiological importance of many of these effector interactions to the function of Ras remain unclear, the activation of the mitogenactivated protein kinase (MAPK) pathway via the Ras-Raf interaction is clearly important in transducing growth proliferation signals. The Ras-Raf interaction serves to localize Raf to the plasma membrane (Moodie et al., 1993; Van Aelst et al., 1993; Vojtek et al., 1993), where Raf itself becomes activated (Stokoe et al., 1994).One mechanism of Raf activation is through phosphorylation by a ceramide-activated protein (CAP) kinase (Yao et al., 1995). Recently, CAP kinase was shown to be the KSR (kinase suppressor

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of Ras) protein (Y. Zhang et al., 1997), which had been identified through genetic studies in Caenorhabditis elegans and Drosophila mlanogaster as being a modulator of the Ras-Raf pathway (Kornfeld et al., 1995; Sundaram and Han, 1995; Therrien et al., 1995).Once activated, Raf phosphorylates MEK (Dent et al., 1992; Howe et al., 1992; Kyriakis et al., 1992),a tyrosine/ threonine kinase that in turn phosphorylates MAP kinases such as Erk2. Phosphorylated Erk2 can then translocate to the nucleus, where it can phosphorylate transcription factors such as Elk-1. Elk-1 in turn can activate genes involved in cell growth, such as cfos. The ras genes, particularly K-ras and N-ras, are frequently mutated in a variety of human cancers; for example, N-ras in the case of acute myelogenous leukemia, and K-ras in carcinomas of the pancreas, lung, and colon (Bos, 1990).These mutations inactivate the GTPase activity of Ras, leaving the Ras switch stuck in the “on” position. The inability to turn off Ras leads to the transformed phenotype of the cancerous cells because they no longer require growth factor-induced transmembrane signals to initiate the signaling pathways leading to cell proliferation. In addition to stimulating uncontrolled proliferation via activation of the MAPK pathway, oncogenic versions of Ras also have a profound effect on cellular morphology. Ras-transformed cells growing in monolayer cell cultures are not contact inhibited as are normal cells, and they have a refractile appearance in the light microscope. The effect of Ras on cell morphology is most likely mediated through the Rho/Rac family. The involvement of Rho and Rac proteins in Ras-mediated cell transformation is illustrated by the ability of dominant-negative inhibitors of these proteins to block Ras transformation (Khosravi-Far et al., 1995; Prendergast et al., 1995; Qiu et al., 1995a,b). Although the exact molecular link between Ras and the Rho/Rac pathway is unclear, mutations in the Ras effector domain reveal that the Ras-Rho/ Rac connection is independent of the Ras-Raf interaction (White et al., 1995).One such Ras mutant is defective in its interaction with Raf and is unable to stimulate the MAP kinase pathway but still causes the change in cellular morphology typical of activated Ras (Joneson et al., 1996b; Khosravi-Far et al., 1996). Several mechanisms have been proposed to account for the effect of Ras on cell morphology. One possible mechanism is through stimulation of a Rho-GAP activity; the Ras effector domain can interact with Ras-GAP, which in turn has been shown to bind the p190 Rho-GAP protein (Foster and Hu, 1994). Alternatively, the Ras effector, phosphoinositide 3’ kinase (PIS-K), might be involved in linking Ras to the Rho/Rac pathway (Rodriguez-Viciana et al., 1997). This is indicated by the correlation between the ability of Ras effector domain mutants to affect the actin cytoskeleton and to bind to PI3-K, and by the finding that inhibition of PI3-K blocks Ras induction of membrane ruffling.

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V. Rho/Rac Effectors

Like Ras, the Rho family of proteins have multiple cellular effectors. These proteins were first noted for their effects on the cell cytoskeleton. Microinjection studies in fibroblasts have shown that Rho induces actin stress fiber formation, whereas Rac and Cdc42 induce membrane ruffling and the formation of filopodia and lamellipodia (Nobes and Hall, 1995). In addition to their regulation of the cytoskeleton, Rho family members regulate several different protein kinases. For example, CDC42, as well as Racl, regulates the JNWSAPK kinase cascade via an interaction with the P6EiPAK kinase (Coso et al., 1995; Minden et al., 1995). Racl and Cdc42 bind and activate the 70-kDa S6 kinase (Chou and Blenis, 1996),which has been shown to play an important role in cell cycle progression in many cell types including lymphocytes. Other downstream signaling targets of the Rho/Rac family include the pl2OAcKtyrosine kinase (Manser et al., 1993) and the p160HoCK serinekhreonine kinase (Ishizaki et al., 1996). Rho family members also function through signaling systems that involve lipid metabolism. For example, stress fiber formation induced by growth factors involves leukotriene generation via the metabolism of arachidonic acid. An activated (GTPase-defective) Rac mutant induces stress fiber formation and leukotriene generation in a growth factor-independent manner, and leukotriene synthesis inhibitors abrogate Rac-induced stress fiber formation (Peppelenbosch et al., 1995). Additionally, Cdc42Hs binds to the p85 subunit of PI3-K and regulates its activity (Zheng et al., 1994), whereas RhoA has been implicated in phospholipase D activation (see below). The list of Rho/Rac effectors is likely to grow because Burbelo et al. (1995) have identified a motif found in the GTPase binding sites of p120ACK and P65PAKthat is present in more than 25 proteins from a variety of eukaryotic species. Similar to what has been found for Ras, mutations in the effector domain of Rac and Cdc42 have been defined that prevent their ability to interact with P6SPAK and activate the JNK kinase pathway but do not affect their ability to regulate the cytoskeleton (Lamarche et al., 1996; Joneson et al., 1996a). VI. Prenylation of the Rar Superfamily Members

Since 1980 it had been known that Ras localized to the plasma membrane of cells and that this localization required posttranslational modification of the protein (Lowy and Willumsen, 1993). In 1984, it was shown that a cysteine residue in a CaaX motif found at the C terminus of Ras played a role in its membrane localization because a Cys to Ser substitution

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PHEKYLATION OF R a GTP'sr PROTEINS

abolished membrane binding (Willumsen et al., 1984). Furthermore, this Cys to Ser mutation abolished the transforming ability of a GTPasedefective H-Ras mutant, suggesting that membrane localization was critical to the function of Ras. A number of observations made in the mid-1980s led to the definition of the nature of the Ras C-terminal posttranslational modification. It had been shown that an inhibitor of HMG-CoA reductase, the rate-limiting enzyme in the cholesterol biosynthetic pathway (Fig. Z), blocked entry of cells into the S phase of the cell cycle (Schmidt et al., 1982). Furthermore, metabolites of exogenously added [3H]mevalonate, an intermediate in the cholesterol synthetic pathway, were incorporated into proteins (Schmidt et al., 1984).In 1986, genetic studies in yeast showed that posttranslational modification of yeast Ras and the yeast a-mating Factor, which also contained a CaaX at its C terminus, was controlled by the same genes (Powers et al., 1986).In 1988, the precise chemical structure of yeast a-mating factor was elucidated and shown to contain a C-terminal cysteine that was farnesylated via a thioether linkage and methylesterified

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-c-

s-9s

S - geranylgeranyl -C-OMe

s-99 s-99 , -C-X-C-OMe

s-99 C -OH

Frc;. 2. Biosynthetic pathway of prenylated proteins. The pathway is shown initiating from an early intermediate in the cholesterol biosynthetic pathway (HMG-CoA), leading to the prenylation enzyme snbstrdtes farnesyl diphosphate (FPP) and geranylgeranyldiphosphate (GGPP).The pathway branches through the three different prenyltransferase enzyrues and the subsequent processing enzymes. Sites of inhibition by existing pharinacologic agents, including HMG-CoA reductase inhibitors, FTIs, GCTIs, the CaaX protease inhibitor BPI, and the methyltrailsferase inhibitor AFC, are indicated. Abbreviations used: S, serine: M, rnethionine; Q, glutamine; L, leucine: X, any amino acid; gg, geranylgeranyl: -OMe, methylated carboy terminus.

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ROBERT B. LOBELL

on its carboxylate group (Anderegg et al., 1988). From this precedent, similar modifications on the Ras C-terminal CaaX were demonstrated (Casey et al., 1989; Hancock et al., 1989; Schafer et al., 1989). VII. Prenylation and Processing of CaaX Substrates

Protein prenylation involves the covalent addition of two types of isoprenoids, farnesyl pyrophosphate or geranylgeranyl pyrophosphate, to cysteine residues at or near the C terminus. The farnesyl isoprenoid, a 15-carbon lipid, is an intermediate of the cholesterol biosynthetic pathway and is derived from the basic 5-carbon isoprenoid unit, isopentyl pyrophosphate (Fig. 2). Geranylgeranyl pyrophosphate contains an additional isoprenoid unit and is derived directly from farnesyl pyrophosphate. Three different enzymes, or prenyltransferases, have been identified that carry out these modifications (Zhang and Casey, 1996). Farnesyltransferase ( FTase) and geranylgeranyltransferase type-I (GGTase-I) are sometimes referred to as the CaaX prenyltransferases, because they catalyze the addition of farnesyl diphosphate (FPP) and geranylgeranyl diphosphate (GGPP), respectively, to the cysteine residue in the sequence CaaX found at the C terminus of prenyltransferase substrates. A variety of proteins are substrates for the CaaX prenyltransferase enzymes (Table I), many of which are members of the Ras superfamily of small GTP-binding proteins. The third prenyltransferase enzyme, known as Rab geranylgeranyltransferase or geranylgeranyltransferase type I1 (GGTase-I1), adds geranylgeranyl groups to each cysteine in the XXCC, CCXX, and XCXC motifs at the C terminus of Rab proteins. VItI. CaaX Prenyltransferases

FTase and GGTase-I are both heterodimeric proteins that share a common 48-kDa a! subunit (Reiss et al., 1990; Seabra et al., 1991). The p subunits of FTase and GGTase-I are 46 and 43 kDa, respectively, and are approximately 30% identical at the amino acid level (Zhang et al., 1994). The genes for the yeast and mammalian prenyltransferases have been cloned and expressed in heterologous systems (Chen et al., 1991a,b; Fujiyama et al., 1987; Kohl et al., 1991; Omer et al., 1993; Powers et al., 1986). The a and /3 subunits of mammalian FTase are 30 and 37% identical to the corresponding yeast enzyme, encoded by the RAM2 and RAMl genes, respectively (Chen et a!., 1991a,b; Kohl et al., 1991; Omer et al., 1993). Mutations in either RAMl or RAM2 abolish the activity of FTase in yeast. FTase and GGTase-I both recognize the cysteine in CaaX motifs as the site for prenylation. In general, whether a protein is prenylated by FTase

PRENYLATION OF Ras CTPase PROTEINS

153

or GGTase-I is defined by the X residue in the C a d motif; proteins with = serine, methionine, or glutamine are FTase substrates, whereas X = leucine for GGTase-I substrates (Casey et d.,1991; Moores et al., 1991; Yokoyama et al., 1991). The specificity of prenylation of CaaX substrates by FTase or GGTaseI is not always absolute because some proteins, such as K-Ras4B, can be both farnesylated by FTase and geranylgeranylated by GGTase-I in vitro (James et al., 1995; Moores et al., 1991). However, the catalytic efficiency (ke&,,,) for farnesylation of K-Ras4B by FTase is -140-fold greater than that for the geranylgeranylation of the protein by GGTase-I (F. L. Zhang et al., 1997). The preference for farnesylation of K-Ras4B is also reflected in vim, in which the protein is normally found in the farnesylated state (Casey et al., 1989). Other Ras isoforms, including K-Ras4A and N-Ras but not H-Ras, are also prenylated by both enzymes in uitro, with farnesylation being the preferred reaction (F. L. Zhang et al., 1997). The RhoB protein is another exception to the prenyltransferase specificity “rules” because this protein is both farnesylated and geranylgeranylated in vivo (Adamsonet al., 1992). RhoB is not an FTase substrate in vitro but rather is both farnesylated and geranylgeranylated by GGTase-I, with farnesylation being the preferred reaction (Armstrong et aZ., 1995). Some proteins, such as the heterotrimeric Gia subunit, contain an apparent CaaX motif but are not prenylated (Mumby et al., 1990). In the case of Gia, sequences upstream of its CGLF CaaX box apparently inhibit prenylation because the CGLF sequence confers prenylation when transferred to rus sequences (Cox et al., 1993). These data illustrate potential inaccuracies in predicting the nature of the prenyl group attached to a putative CaaX substrates based solely on prediction from the sequence of the CaaX. Characterization of the prenyl group on a CaaX substrate can be suggested by analysis of FTase and GGTase-I prenylation reactions on the substrate in vitro but should ultimately rely on characterization of the protein from cells or tissues. One commonly used method for characterization of the prenyl group involves labeling cells with [3H]mevalonate,which will incorporate into both farnesylated and geranylgeranylated proteins. After isolation of the labeled protein of interest, its labeled isoprenoid can be released via chemical means and identified by chromatographic separation and coelution with known standards (Casey et al., 1989). The potential for cross-prenylation of CaaX substrates in vivo, i.e., the farnesylation of GGTase-I substrates by FTase and vice versa, is suggested by the ability of proteins such as K-Ras4B to be prenylated by both FTase and GGTase-I. Studies in yeast illustrate the potential for cross-prenylation. It was shown that overexpression of the GGTase-I p subunit partially suppressed the growth defect of cells lacking FTase-P (Trueblood et al.,

X

TABLE I A CATALOG OF PRENYLATED PROTEINS Protein Ras proteins H-Ras N-Ras K-Ras4A K-Ras4B Ras-related proteins RaplA RaplB Rap2A Rap2B R-ras RalA RalB TC21 Rheb Rho proteins RhoA RhoB RhoC RhoD RhoE RhoG Cdc42Hs Racl Rac2 TClO

C terminus

Prenylation

CVLS CWM CIIM CVIM

F F F F

CLLL CQLL CNIQ CVIL CVLL CCIL CCLL CVIF CSVM

GG GG F GG GG GG GG GG F

CLVL CCKVL CPIL CCLAT CTVM CILL CVLL CLLL CSLL CLIT

GG FIGG GG F F GG GG GG GG GG

Protein Heterotrimeric G proteins yl (bovine, transducin) Y2 (ui2 a i3 Nuclear lamins Lamin A Lamin B cGMP phosphodiesterase (asub.)

C terminus

Prenylation

CVIS CAIL CGLF CGLF

F GG Not prenylated Not prenylated

CSIM CAIM

F F

CCIQ

F

Phosphorylase kinase (rabbit) CAMQ PXF (CHO cell) CLIM Interferon-inducible GTP binding proteins GBPl CTIS GBP2 CNIL Yeast YDJl CASQ CQTS Human HDJ2 Hepatitis delta virus large antigen CRPQ Protein tyrosine phosphatase, PRL-1 CCIQ Inositol triphosphate 5' phosphatase CWQ 2',5'-oligo(A) synthetase CTIL

F F F GG F F F F F GG

Rab proteins (selected members) Rabla cc cc Rab2 Rab3a CAC Rab3b csc Rab4a CGC Rab5 CCSN Rab5b CCSN Rab6 csc Rab’i dog csc Rab8 CVLL Rab9 dog CC RablO dog cc Rabll CCQNI

cli-GG di-GG di-GG di-GG di-GG di-GG di-GG di-GG di-GG GG di-GG di-GG di-GG

Note. The C-terminal amino acids (in standard amino acid code) and the prenylation state of each protein [farnesyl ( F ) or geranylgermyl (GG)]are indicated

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ROBERT B. LOBELL

1993). Similarly, overexpressionof two essential GGTase-I substrates, Rho1 and Cdc42, allows for growth of GGTase-I P-deficient cells: presumably, overexpression allows the cells to survive due to at least some level of farnesylation of these proteins (Ohya et al., 1993; Trueblood et al., 1993). Cross-prenylation has important implications in the context of pharmacological inhibition of prenyltransferases; alternative protein prenylation might rescue protein function in cells treated with a specific prenylation inhibitor (see Section XX). Structural information on prenyltransferases will further our understanding of the determinants of substrate specificity of FTase and GGTase-I and, in this regard, the structure of Rat FTase was recently solved by X-ray crystallography (Park et al., 1997). Although the crystal structure was determined in the absence of either substrate, the location of the active site of the enzyme was surmised based on the location of a bound zinc atom. It has been shown that the cysteine thiol of a CaaX peptide substrate coordinates to the zinc metal in a ternary complex consisting of enzyme, peptide, and FPP, indicating a direct role of the zinc in catalysis (Huang et al., 1997). The structure showed that the zinc atom of FTase is in close proximity to a hydrophobic pocket found in the P subunit of FTase. This hydrophobic pocket is likely the binding site for FPP because it is of sufficient length to accommodate FPP but not the larger GGPP molecule, consistent with the observation that FPP binds 15-fold tighter than GGPP to FTase (Yokoyama et al., 1997). In addition, Park et al. (1997) proposed a model for the interaction of the CaaX motif with active site residues of FTase. The model was based on the location of the nine C-terminal amino acids of the P subunit, which for some reason inserted into the active-site region of the adjacent aJP dimer in the crystal structure. Although this model provides a useful starting point for further studies, it is not supported by recent site-directed mutational data, which showed that mutation of three residues, Ser159, Tyr362, and Tyr366, changed the substrate specificity of yeast FTase to that of GGTase-I (DelVillar et al., 1997). The crystal structure model did not implicate these residues as being directly involved in CaaX substrate binding, although it cannot be ruled out that mutation of these residues changes substrate specificity through indirect effects on neighboring residues. Evidence from circular dichroism analysis indicates that FTase undergoes conformational changes upon binding CVIM peptide, FPP analogs, or tetrapeptide inhibitors of the enzyme (Wallace et al., 1996). Thus, the structure of the apoenzyme might not accurately reflect the structure of the active site with ligands bound. Further structural information, particularly data derived from enzyme-substrate or enzyme-inhibitor complexes, will enable a more precise

PRENYLATION OF Ras GTPase PROTEINS

157

definition of the molecular interactions involved in substrate recognition by FTase and GGTase-I. IX. CaaX Protease and Carboxymethyltransferase

Proteins modified by FTase and GGTase-I undergo additional Cterminal processing steps (Fig. 2). The C-terminal aaX is cleaved from the protein by a microsomal protease and the resulting C-terminal prenylated cysteine is carbolo/methylated. A protein activity that binds both farnesylated and geranylgeranylated proteins that contain an intact aaX C terminus has been postulated to play a role in these additional processing steps by localizing prenylated proteins to the membrane surfaces where the CaaX protease and methyltransferase activities reside (Thissen and Casey, 1993). Two Saccharomyces cerevisiae genes, Rcel and Afcl, are required for the C-terminal proteolysis of prenylated proteins (Boyartchuk et al., 1997). The AFCl protein is a zinc-dependent metalloprotease that is required for proteolysis of the yeast mating pheromone, a-factor, but is not essential for processing of yeast Ras. RCEl is essential for processing of both afactor and Ras. The mammalian protease activity responsible for processing of prenylated proteins has only been partially characterized. Proteolytic activity capable of releasing the Val-Ile-Met tripeptide from the tetrapeptide substrate, N-acetyl-S-farnesyl-L-Cys-Val-Ile-Met, is localized to membranes, is not affected by standard protease inhibitors, and displays properties consistent with a serine or cysteine protease (Ma et al., 1993). This proteolytic activity requires detergent for its solubilizationfrom membranes and chromatographs as a single peak of activity over gel filtration and anion exchange chromatography (Chen et al., 1996). Tetrapeptide inhibitors of the CaaX protease, such as RPI (Fig. 3), have been developed and have been reported to block Ras processing and function in cells (Chen et al., 1996). The mammalian enzyme(s) responsible for C-terminal methylation of prenylated proteins is also incompletely characterized. In yeast, the methyltransferase for CaaX proteins has been identified as Stel4 (Hrycyna and Clarke, 1990; Marr et al., 1990).Prenylcysteine-directed mammalian methyltransferase enzyme(s) is associated with microsomal membranes and utilizes S-adenosylmethionine as the methyl donor (Stephenson and Clarke, 1992). Methylation reactions are potentially reversible and, in this regard, a methylesterase has been described that is selective for prenylated cysteines containing methylesters (Tan and Rando, 1992). The ability to add or remove methyl groups from the C terminus of prenylated proteins suggests that these events could regulate the function of prenylated proteins, and there is some evidence to suggest that this does occur (see Section XIV).

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ROBEHT 8 . LOBELL

FPP

P

7--

0 P-0-P-0 I

0

1

0

PPP-Competitive m ' s

L-704,272

Manurnycin 0

L bi substrate FTi

BMS-186511 Fic;. 3. Representative inhibitors of prenyltransferases and other enzymes in the biosynthesis of prenylated proteins. Shown at the top are the substrates for the farnesylation of k-Ras (FPP) and the k-Ras CaaX (CVIM). See text for details and references.

X. Rab GGTase-ll

Rab proteins are digeranylgeranylated by GGTase-11, a heterodimeric enzyme containing a 50-kDa a subunit and a 38-kDa 6 subunit that share approximately 30% identity in amino acid sequence with the cdfi subunits

CVlM

CaaX Competitive FTl‘s

I

L-731,734

I L-739,749 (kCH3) L-744,832 (R=CHz(CH&

FTI-276 (R=H) sFTI-277 (R=CH3)

I

SOzCH,

sHwc

HzN

% “O H

FTI-265

NHz

n

/

8956 (R=H) 81086 (R=CHa)

L-745,631

0

SCH44342

160

ROBERT B. LOBELL

GGTase-I lnhlbitor

GGTI-286: R=OCHB

I

GGTI-287: R=O-

HMG-CoA Reduction Inhibitor

Lovastatin

L

CaaX Proteare Inhibitor

Carboxymbthyitransferaselnhibltor

t?+n L-AFC(1)

"y 0

AFC

FIG.3-Continued

of FTase and GGTase-I (Armstrong et al., 1993).GGTase-I1 adds geranylgeranyl groups to each cysteine residue at the C terminus of Rab proteins, which end in CCXX, XXCC, or CXC sequences (Farnsworth et al., 1994).

PRENYLATION OF

Ray

GTPase PROTEINS

161

Rab proteins are not proteolyzed at their C termini, and only Rab proteins

with the CXC motif are carboxymethylated (Smeland et al., 1994). Prenylation of Rab proteins by GGTase-I1 requires a third protein called Rab escort protein or Rep. Rep functions by presenting the unprenylated Rab protein to the catalytic GGTase-I1 d p heterodimer (Andres et al., 1993; Seabra et al., 1992). Mutational analysis of Rab proteins has shown that in addition to the C-terminal cysteines, internal Rab protein sequences are involved in the prenylation reaction (Wilson and Maltese, 1993). The effect of these mutations may be due to effects on the Rab-Rep interaction. Rep binds both unprenylated and prenylated Rab proteins; due to this property, the GGTase-I1 reaction in uitro is limited by the concentration of Rep. MonogeranylgeranylatedRab protein remains tightly bound to Rep, even in the presence of detergents, ensuring that the second geranylgeranyl group can be added by GGTase-11. Although digeranylgeranylated Rab exhibits a somewhat greater propensity to dissociate from Rep in the presence of detergents or phospholipids (Shen and Seabra, 1996) in vim the prenylated Rab likely remains bound to Rep until it is delivered to the correct intracellular membrane compartment (Alexandrovet al., 1994). In uiuo, delivery of the Rab protein to the correct membrane compartment presumably facilitates the dissociation of the prenylated Rab protein from Rep, although it is not known what directs the Rab protein to its correct membrane compartment. Another aspect of the Rab-Rep interaction that is not well understood concerns the low affinity of Rep for Rab-GTP. Because GTP is found at much higher concentrations than GDP in cells, newly synthesized, unprenylated Rab might bind GTP and then would be less able to bind Rep. One possibility is that a chaperone protein might bind newly synthesized Rab in a conformation that prevents GTP binding and allows the Rab to bind Rep (Desnoyers et al., 1996). Two Rep proteins, Repl and Rep2, have been identified. A defect in Repl function is responsible for choroideremia, a human retinal degenerative disease (Andres et al., 1993). Repl and Rep2 are 75% identical and are generally redundant in activity except for two known examples. Rab27, a protein found in high levels in the retina, has a somewhat higher affinity for Repl compared to Rep2, which might explain why the effects of choroideremia are limited to the retina (Seabra et al., 1995). Additionally, the prenylation rate of Rab3a is lower when Rep2 is the escort in the reaction (Cremers et al., 1994). Rep proteins are 30%identical in amino acid sequence to the Rab-GDI protein (Wu et al., 1996). X-ray crystallography of Rab-GDI has revealed that two of the most highly conserved regions between Rep and GDI are found on one face of GDI (Schalk et al., 1996). Mutational analysis of GDI suggests that this protein surface is involved in the interaction of

162

ROBERT B. LOBELI,

GDI, and presumably Rep, with Rab proteins (Wu et al., 1996). Although there are similarities to the interaction of Rep and GDI with Rab proteins, there must be significant differences because only Rep can bind unprenylated Rab and thus only Rep can facilitate the geranylgeranylation reaction (Pfeffer et nl., 1995). In addition, GDI but not Rep apparently interacts with the effector domain on the Rab protein. A single mutation in the RablB effector domain abolishes the GDI-Rab binding interaction but does not affect the geranylgeranylation reaction, indicating that the mutant protein retains the ability to interact with Rep (Wilson et al., 1996). In cells, this RablB effector domain mutant was targeted to the correct intracellular compartment but was unable to cycle between membrane and cytosolic compartments (Wilson et al., 1996). This result is consistent with a model in which Rep functions in the delivery of prenylated Rab proteins to donor membranes, whereas GDI functions specifically in the recycling of Rab back to donor membranes after the vesicle fusion process has occurred. XI. Role of Prenylation in Membrane Binding and in Prokin-Prokin Interactions

Ras proteins containing Cys to Ser mutations in the CaaX are not prenylated and are not membrane bound (Willumsen et nl., 1984). Although farnesylation of Ras is clearly a critical component to its membrane localization, the proteolytic cleavage of the a& and the methylation of the farnesyl cysteine at the mature C terminus are also important. An in vitro system utilizing rabbit reticulolysates, reconstituted with or without microsomal membranes containing CaaX protease and methyltransferase activity, can produce Ras in various states of posttranslational processing. Utilizing this system, it was found that farnesylation of K-Ras4B in the absence of proteolysis and methylation results in only 20% of the K-Ras4B protein being associated with membrane fractions (Hancock et al., 1991a). Forty percent of farnesylated and proteolyzed K-Ras4B associated with membranes, whereas addition of the carboxymethylation activity led to 80% of the fully processed protein associated with the membrane. Similarly, a KRas4B CaaX mutant that can be farnesylated but not further processed is approximately 50% membrane associated in cells compared to a >90% association of the wild-type protein (Kato et ul., 1992). The importance of the carboxymethylation event to membrane binding is further illustrated by in vitro analysis of the binding of prenylated peptides to liposomes. Farnesylated peptides bind poorly to liposomes unless the farnesyl cysteine is methylated, whereas geranylgeranylated peptides bind reasonably well in the absence of methylation (Silviusand L’Heureux, 1994).The difference between farnesylated and geranylgeranylatedpeptides in their requirement

PRENYLATION OF Ras GTPasv PROTEINS

1ti3

for carboxymethylation for binding to membranes reflects the greater lipophilicity of the geranylgeranyl group. In addition to C-terminal farnesylation, proteolysis, and methylation, other mechanisms, including the addition of other lipid moieties in the case of H-Ras or the presence of multiple-charged amino acid residues in the case of K-Ras4B, contribute to the binding of Ras and other prenylated proteins to cellular membranes. A specific palmitoyltransferase covalently modifies H-Ras and N-Ras on cysteine residues in their C-terminal region with the 16-carbon lipid, palmitate (Liu et al., 1996). The palmitoylation reaction apparently requires that the proteins are first farnesylated and further processed because nonfarnesylated, bacterial-expressed H-Ras is not a substrate for the palmitoyltransferase (Liu et al., 1996). C h a n p g the two cysteines in H-Ras that are normally palmitoylated to serine residues prevents palmitoylation, results in a 10-foldreduction in membrane binding compared to the palmitoylated protein (Hancock et al., 1990), and significantly impairs its signaling ability (Dudler and Gelb, 1996).The C-terminal polybasic domain of K-Ras4B, which contains a stretch of six lysine residues adjacent to the CaaX, was shown to contribute significantlyto its membrane binding in cells (Hancock et al., 1990).Changing these lysines to glutamine impairs the membrane binding of K-Ras4.B; as the number of lysines in the polybasic region is progressively decreased, the affinity of the protein for membranes is also progressively decreased. Other prenylated proteins in the Ras superfamily also contain polylysine stretches adjacent to their Cadi. These studies indicate that all the C-terminal processing events, including prenylation, proteolysis, carboxymethylation, palmitoylation, as well as the polybasic domain of K-Ras4B, all contribute to the membrane binding of Ras proteins. However, mutations that abolish palmitoylation, proteolysis, or the polybasic domain only slightly impair the transforming ability of Ras proteins, whereas Cys to Ser mutations in the CaaX completely abolish the transforming ability (Hancock et al., 1990; Kato et al., 1992). These mutational studies show that the only processing event that is absolutely critical to the transforming ability of Ras is the farnesylation step. However, these transformation studies should be interpreted with caution because they involve overexpression of the Ras protein. A mutant version of Ras that is partially impaired in its membrane binding might reach the threshold level of signaling at the plasma membrane that is required for transformation of the cell only when the protein is overexpressed. Although the C-terminal lipidation of Ras is critical for its function, this requirement can be abrogated by artificially targeting the protein to membranes via introduction of a lipid functionality at the N terminus of the protein. This has been accomplished by introducing the v-Src N-

164

ROBERT B. LOBELL

myristoylation sequence at the N terminus of Ras (Buss et at., 1989). Furthermore, it has been suggested that the sole function of Ras is to tether the signaling molecules downstream of Ras to the plasma membrane. This is supported by the finding that a Raf kinase construct containing a CaaX motif is transforming to cells even in the presence of a dominantnegative Ras protein that can normally inhibit transmembrane signaling to the MAPK pathway (Leevers et al., 1994; Stokoe et al., 1994). Although the processed, prenylated C terminus of Ras mediates its membrane association, it is unclear what directs Ras to the plasma membrane rather than to other intracellular membrane compartments. It would seem that other signals are required for targeting Ras to the proper membrane compartment because farnesylated proteins other than Ras can be localized to other membrane structures such as the nucleus in the case of the lamins (Brown et al., 1992; Chen et al., 1991b; Reiss et aE., 1991) and the cytoplasmic surface of peroxisomes in the case of a protein of unknown function known as PXF (James et al., 199413). Although prenylation of Ras is important in localization of the protein to membrane surfaces, prenylation also plays a role in protein-protein interactions. For example, yeast Ras2 regulates the enzyme adenylyl cyclase, and farnesylation is required for this interaction. The interaction of unprocessed Rase with solubilized adenylyl cyclase is approximately 100fold less than when Ras2 is farnesylated (Kuroda et al., 1993). The interaction of H-Ras and K-Ras with the guanine nucleotide exchange protein, SOS, is influenced by prenylation (Porfiri et al., 1994; McGeady et al., 1997). SOS fails to catalyze nucleotide exchange of unprocessed H-Ras and K-Ras, and addition of the 10-carbon geranyl group fails to reconstitute the interaction. The exchange reaction occurs with Ras modified with farnesyl, analogs of farnesyl such as tetrahydrofarnesyl, and geranylgeranyl groups, and proceeds to a greater extent when it is fully processed (McGeady et al., 1997). Although these results indicate that SOS interacts, at least in part, with the prenyl group of Ras, they do not exclude the possibility that the prenyl group induces a structural change in Ras that enables it to interact with SOS. The contribution of prenylation and carboxymethylation to membrane binding is further illustrated from studies of other prenylated proteins, notably the heterotrimeric G-proteins. These GTPases are localized to the plasma membrane via myristoylation and palmitoylation of the G, subunit and prenylation, either farnesylation or geranylgeranylation, of the Gy subunit (see Table I and Higgins and Casey, 1996). Transducin, a Gprotein found in the retina, contains a farnesylated G, that is found in both methylated and unmethylated forms. Both farnesylation and methylation

PRENYLATION OF Ras GTPase PROTEINS

165

of G, contribute to the membrane binding of this protein (Parish and Rando, 1994). Prenylation also plays an important role in many aspects of proteinprotein interactions involving heterotrimeric G-proteins, including intrasubunit interactions, interactions between the G-protein and the transmembrane receptor, and perhaps interaction of the G-protein subunits and downstream signaling effectors. Heterotrimeric G-proteins dissociate into G, and Gpysubunits when ligand binds to the seven-transmembrane receptor to which the G-protein was originally bound. The G, dimer is extremely stable, and its initial assembly appears to be influenced by prenylation. The assembly of the Gpycomplex is thought to occur prior to prenylation and proteolytic processing of G,; this is suggested by the finding that proteolysis of the a& of prenylated G, impedes complex formation with G, (Higgins and Casey, 1994). The high-affinity interaction of Gp, with G, requires the prenylation of G, (Higgins and Casey, 1994) and the myristoylation of G, (Linder et al., 1991). Farnesylated peptides correspondmg to the C terminus of G, can inhibit the interaction of Gpywith G, and the degree of inhibition increases as the hydrophobicity of the prenyl group is increased by either methylation of the farnesylated peptide or geranylgeranylation of the peptide (Matsuda et al., 1994). The prenylation and methylation state of the G, subunit can also influence the interaction of the heterotrimeric G-protein with the seven-transmembrane receptor. In the case of transducin, both farnesylation and methylation are required for high-affinity binding to the receptor rhodopsin (Fukada et al., 1994), and as with the G,, and G, interaction, a farnesylated peptide corresponding to the C terminus of G, can disrupt the rhodopsin-transducin complex (Kisselevet al., 1994). Prenylation and methylation of G, is also critical for the interaction of G, with downstream effectors, as has been demonstrated in the regulation of a phospholipase CP, (Parish et al., 1995), although it is not clear if this effect is due to enhanced membrane binding of the fully processed Go, or to a specific G,,-phospholipase CP, interaction. In the case of Rab proteins, prenylation plays a role in the interaction of Rab with Rep and GDI. In addition to promoting the association of Rab with membranes (Overmeyer and Maltese, 1992), prenylation of Rab is required for binding to GDI (Musha et al., 1992).Although both monoand digeranylgeranylated Rab proteins can associate with GDI, the length of the prenyl group affects the ability of Rab to bind GDI because a Rab5 mutant with a farnesylation site in place of the geranylgeranylation sites binds weakly to GDI (Ullrich et al., 1993).Another system in which prenylation has been shown to affect protein-protein interactions is in the biosynthesis of hepatitis delta virus. Prenylation of the large antigen of hepatitis

166

ROBERT B. LOBELL

delta virus is required for its assembly with the hepatitis B surface antigen in the formation of hepatitis D virus particles (Hwang and Lai, 1993). XII. Role of Ras GTPase Family Members in Immunobiology: The Ras Pathway

Many of the components of the Ras signaling pathway, which were originally defined from work involving growth factor signaling in fibroblasts, have now been demonstrated in cells of the immune system. In T lymphocytes, the Ras pathway has been shown to be important in the immediate activation events triggered via the T cell antigen receptor (TCR), which leads ultimately to IL-2 secretion and upregulation of IL-2 receptors (IL2R). The Ras pathway is also involved in the proliferative events in T cells triggered by binding of IL-2 to its receptor (IL-2R) (Pastor et al., 1995). The Ras pathway is activated in other antigen receptor signaling systems related to the TCR, including the B cell antigen receptor (Cambier et al., 1994), and in mast cell activation via the high affinity receptor for IgE, FceRI (Fukamachi et al., 1993; Turner et al., 1995). The Ras pathway is involved in other aspects of lymphocyte biology, including the regulation of B cell function by the CD40 receptor (Gulbins et al., 1996a) and T cell activation via engagement of the GD3 disialoganglioside (Ortaldo et at., 1996). As is the case in growth factor receptor signaling in fibroblasts, signaling from lymphocyte antigen receptors and the IL-2R involves activation of Ras via the SOS guanine nucleotide exchange factor through a series of protein-protein interactions that initiates with tyrosine phosphorylation events (Quilliam et al., 1995). In the case of growth factor receptors, intrinsic tyrosine kinase domains in the receptor autophosphorylate receptor tyrosine residues which serve as adapter sites for the GrbYSOS complex, which in turn activates Ras. The TCR and the IL-2R lack intrinsic kinase activity but initiate the Ras pathway through receptor-associated kinases of the Src family, including ZAP-70, p56Ick,and ~ 5 9 s in " the case of the TCR, and through activation of ~ 5 6 'in ' ~the case of the IL-2R (Weiss and Littman, 1994).These kinases phosphorylate multiple substrates, including proteins that couple to the GrbYSOS complex. The phosphorylated proteins that link SOS/Grb2 to the TCR and IL-2R appear to be different. In the case of the TCR, a 36-kDa protein serves as the phosphoprotein adapter to Grb2 (Buday et al., 1994; Reif et al., 1994; Sieh et al., 1994), whereas for IL-2R, the Shc protein is phosphorylated by p56Ick,coupling the activated kinase to the GrbYSOS complex(Ravichandran and Burakoff, 1994). FcsRI activation of Ras in mast cells has also been shown to involve the GrbYSOS pathway (Turner et al., 1995); it is not clear which phospho-

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protein adapter couples the Src kinases associated with this receptor to GrbWSOS. The events downstream of Ras are fairly well understood in the case of signaling via the TCR (Pastor et d., 1995). Ras activation in response to TCR activation leads ultimately to upregulation of a transcription factor that activates the IL-2 promoter, known as nuclear factor of activated T cells (NFAT). NFAT is a complex of AP-1, itself a complex of the Fos/ Jun factors, and NF-ATp, a member of the c-re1 family of transcription factors, (€320, 1994). The pathway leading to activation of NFAT by Ras most likely involves activation of the MAPK pathway. Activation of Erk2 via Raf and MEK has been demonstrated in T lymphocytes (Izquierdo et al., 1993, 1994; Franklin et d., 1994). It is likely that NFAT activation by Ras ultimately involves activation of AP-1 through the induction of the cfos gene; this could occur via activation of the Elk-1 transcription factor by the Erk2 map kinase (Marais et al., 1993).As in the T cell, IgE receptor activation in the mast cell leads to activation of NFAT via the Ras-RafMek-map kinase-Elk-1 cascade (Turner and Cantrell, 1997). Additionally, another member of the Ras superfamily, Rac-1, has been implicated in NFAT activation in mast cells (Turner and Cantrell, 1997). In granulocytes such as the neutrophil, Ras is activated in response to proinflainmatory mediators. For example, Ras and its downstream effectors, Raf and MAPK, are activated in human neutrophils in response to the chemoattractants FMLP and C5a (Buhl et al., 1994; Worthen et al., 1994). The FMLP and CSa receptors are seven-transmembrane spanning G-protein coupled receptors, and the activation of the Ras pathway in neutrophils via these receptors is sensitive to pertussis toxin. The linkage between the G-protein coupled receptors and the Ras pathway in the neutrophil has not been firmly established but appears to involve the Src-family kinase Lyn. FMLP stimulates Lyn, which in turn binds and phosphorylates Shc; this could lead to Ras activation via phosphorylated Shc binding to GrbWSOS (Ptasznik et al., 1995). There is some data to suggest that the expression of proteins in the Ras pathway can be modulated in the neutrophil in viva in response to inflammatory stimuli. Neutrophils from bum patients contain elevated levels of Ras and Ras-GAP but reduced levels of Rapl, a Ras superfamily member that regulates the NADPH oxidase (Brom et al., 1993). Neutrophils from burn patients exhibit impaired chemotactic and phagocytic function, although it is not clear what role, if any, the elevation of Ras protein levels has in this impaired function. Although Ras activation plays a growth stimulatory role in T cells, there is evidence that in some settings, it can actually transduce growth inhibitory and apoptotic signals. For example, in a recent study it was shown that Ras negatively regulates calcium-dependent immediate early gene induction in

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lymphocytes (Chen et al., 1996). Furthermore, FAS-induced apoptosis in lymphocytes involves Ras activation through a pathway involving ceramide generation via a sphingomyelin signaling pathway (Gulbins et al., 1995). This was indicated by an increase in Ras-GTP levels upon FAS stimulation of Jurkat cells and by the inhibition of FAS-induced apoptosis by a dominant-negative Ras mutant. FAS-induced events downstream of Ras involve the generation of superoxide anions (Gulbins et al., 1996b), which have also recently been implicated in signaling events downstream of Ras induced by mitogenic stimulation of NIH-3T3 fibroblasts (Irani et al., 1997). Ras activation is also involved in apoptosis induced by the cytokine, tumor necrosis factor (TNF) (Trent et al., 1996). Like FAS, TNF activates a sphingomyelin pathway leading to ceramide production, which has been shown to cause phosphorylation and activation of Raf via a CAP kinase (Yao et al., 1995). It remains to be seen whether FAS-induced apoptosis, which involves ceramide generation, also results in CAP kinase and Raf activation. It is apparent that further studies are needed to sort out the tangle of signaling pathways in lymphocytes and other cells that involve Ras, which can result in a variety of responses, including activation, growth, or cell death. XIII. The Rho/Rac Pathway and leukocyte Function

Most cells of the immune system are motile and migrate in response to specific chemotactic stimuli. Leukocyte migration involves integrindependent adhesioddeadhesion events and changes in the cell cytokeleton, including actin polymerization and membrane ruffling (Stossel, 1993). There is direct evidence from studies in immune cells that integrindependent adhesion events and changes in the cytoskeleton involve Rho proteins. The involvement of Rho proteins in chemoattractant-induced effects on integrin-dependent adhesion was illustrated in a recent paper in which a lymphoid cell line, transfected with the FMLP or IL-8 chemoattractant receptors, showed agonist-stimulated activation of nucleotide exchange on RhoA within seconds (Laudanna et al., 1996). Furthermore, in this paper it was demonstrated that Clostridium botulinurn toxin C3 ADP ribosyltransferase, an enzyme that inhibits Rho function through ADP ribosylation, blocked agonist-induced lymphocyte a 4 p l integrin-mediated adhesion to vascular cell adhesion molecule-1 and also blocked neutrophil p2 integrin-mediated adhesion to fibrinogen. The involvement of Rho proteins in cytoskeletal organization in leukocytes is further supported by the finding that the C3 ADP ribosyltransferase inhibits actin microfilament formation and chemoattractant-induced motility in neutrophils (Stasia et al., 1991). The C3 ADP ribosyltransferase also inhibits events that involve

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leukocyte cell-cell interactions, including the CD1ldCD18-dependent homotypic aggregation of B cells (Tominaga et al., 1993) and the cytolytic function of cytotoxic T cells (Lang et al., 1992). Additionally, CDC42Hs is required for the polarization of T cells toward antigen presenting cells (Stowers et al., 1995). Further evidence for the involvement of Rho family members in leukocyte cytoskeletal organization and cell motility comes from studies of the Wiskott-Aldrich syndrome (WAS).WAS is a hematopoietic disorder characterized by thrombocytopenia, recurrent infections, and eczema (Ammann and Hong, 1989).The cellular abnormalities in WAS patients include cytoskeletal defects in T cells and platelets (Molina et al., 1992) and defective neutrophil chemotaxis (Ochs et al., 1990). The genetic defect in WAS has been mapped by positional cloning (Derry et al., 1994). The involvement of the WAS protein (WASP) in regulation of actin polymerization was demonstrated by the recent finding that WASP binds to CDC42Hs (Symons ef al., 1996). Overexpression of WASP produced intracellular clusters of the protein that were highly enriched in polymerized actin; formation of these clusters was inhibited by coexpression of dominantnegative CDC42Hs-Nl7. Thus, mutation of WASP, a downstream effector of CDC42Hs, can have profound effects on immune cell functions that involve regulation of the cytoskeleton. XN. Regulation of the Neutrophil NADPH Oxidose by Roc and Rap

Activation of neutrophils by proinflammatory mediators, including the chemoattractant peptides FMLP and C5a, leads to a number of cellular responses including the generation of toxic and microbiocidal oxygen metabolites such as superoxide anion and hydrogen peroxide. This event, termed the respiratory burst, is due to activation of the multisubunit NADPH oxidase complex (Chanock et al., 1994). The oxidase consists of a heterodimeric flavocytochrome b that consists of 22- and 91-kDa transmembrane protein components (Parkos et al., 1987). The oxidase is also composed of two cytoplasmic proteins, p47P""' and p67P"", that bind tightly to the transmembrane oxidase components upon cell activation (Clark et al., 1990) . The inability to generate the respiratory burst seriously compromises the host defense system, as evidenced by individuals with chronic granulomatous disease, a hereditary condition caused by a mutation in one of the NADPH oxidase components (Dinauer, 1993). The NADPH oxidase is regulated by two geranylgeranylated GTPbinding proteins, RaplA and Rac. The involvement of these GTP-binding proteins was first suggested by the requirement for guanine nucleotides in the activation of the oxidase in a cell-free system (Gabig et al., 1987),

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and the involvement of RaplA in the NADPH oxidase system was first suggested by its association with purified neutrophil flavocytochrome b (Quinn et al., 1989).This interaction is functionally important in NADPH oxidase function because cytosol immunodepleted of RaplA is unable to reconstitute oxidase activity unless recombinant RaplA is added back (Eklund et al., 1991). Furthermore, dominant inhibitory mutants of RaplA inhibit the NADPH oxidase when expressed in differentiated HL-60 cells and EBV-transformed B cells (Gabig et al., 1995; Maly et al., 1994). Rac was first shown to play a role in regulation of the NADPH oxidase by experiments in the cell-free system (Abo d. al., 1991; Knaus et al., 1991).In unstimulated cells, Rac is present in the cytosol and is complexed with RhoGDI (Aboet al., 1994). Upon immunologic activation, Rac dissociates from RhoGDI and translocates to the plasma membrane (Abo et al., 1994; Quinn et al., 1993). Rac translocates independently of the p47PhoX and p67ptiox proteins and interacts with both the p2Wp91 flavocytochrome subunits and with p67P’’0xvia its effector domain (Diekmann et al., 1994; Heyworth et al., 1994). The role of the Rac geranylgeranyl group in activation of the NADPH oxidase has been examined. Unprenylated Racl was found to activate the oxidase in the cell-free system, but only when it was preloaded with GTPyS (Heyworth et al., 1993).This suggested that prenylation of Rac is required only in the activation of Rac itself, presumably through an interaction of Rac with a guanine nucleotide exchange protein, but that prenylation is not absolutely required for the activation of the oxidase by Rac. However, recent evidence suggests that prenylation of Rac is an important determinant in the activation of the oxidase. It was shown that prenylated Racl and Race are significantly more effective in activating the oxidase in vitro than the nonprenylated forms (Kreck et nl., 1996). Racl is a more effective activator than Race; this is likely due to the presence of a polybasic domain near the CaaX of Rac, as is found in K-Ras4B. The polybasic domain of Racl is an important determinant of membrane binding because elimination of only one of the charged residues markedly reduces its activation of the oxidase. The polybasic domain contributes to the membrane binding of Rac via electrostatic interactions. This is indicated by the finding that the activation of the oxidase by Racl but not by Rac2 is sensitive to salt concentration, and that addition of acidic phospholipids to reconstituted oxidase subunits enhances the activation by Racl but not by Rac2. The exact role that Rac plays in the regulation of the NADPH oxidase remains unclear, although it is reasonable to propose that its function may be to anchor the soluble p47Phohat the plasma membrane in proper orientation to the transmembrane oxidase components (Kreck et al., 1996).

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XV. Regulation of Phospholipase D by RhoA

There may be another level of regulation of the NADPH oxidase by a Rho protein. Phosphatidic acid, a product of the hydrolysis of phospholipids by phospholipase D (PLD),enhances oxidase activity (Agwu et al., 1991). PLD activity in human neutrophils is activated by GTPyS and this effect was suggested to be mediated by a Rho protein because Rho-GDI can inhibit the activation of PLD (Bowman et nl., 1993). Depletion of Rho from membranes with Rho-GDI, followed by add back of recombinant Rho to the membrane, showed that RhoA but not Cdc42Hs could reconstitute PLD activity in rat liver membranes and in human neutrophil membranes(Kwak et al., 1995; Malcolm et nl., 1994). Evidence for Rho protein involvement in receptor-mediated PLD activation in intact cells has been obtained through the inactivation of Rho proteins with either the Clostridiurn botulinurn C3 ADP ribosyltransferase or the Clostridiurn dificile toxin B, a Rho glucosylation enzyme (Malcolm et al., 1996; Schmidt et al., 1996). RhoA activation of PLD has also been implicated in IgE receptor-mediated mast cell activation because the C. dificile toxin B abolishes antigeninduced PLD activation and granule enzyme release (Ojio et al., 1996). However, the involvement of RhoA in the activation of PLD has been questioned by a recent study involving HL-60 cells; this study reported that depletion of RhoA from membranes with Rho-GDI had no effect on PLD activity and attributed the activation of PLD by GTPyS to the GTPbinding protein, Arf (ADP ribosylatioii factor) (Martin et al., 1996). XVI. Role of C-Terminal Methylation of Prenylated Proteins in NADPH Oxidase Regulation and Other Leukocyte Functions

It has been suggested that the C-terminal inethylation of Rac and Rap proteins is regulated and plays a role in the translocation of these proteins to the plasma membrane upon FMLP-induced activation of the NADPH oxidase in neutrophils. The amount of carboxymethylation of Ras-related proteins in neutrophils, including Rac and Rap, increases in response to FMLP or nonhydrolyzable GTP analogs, both in intact cells and in cell lysates (Philips et al., 1993). Furthermore, N-acetyl-S-trans,trans-farnesylr,-cysteine (AFC) (see Fig. 3), an inhibitor of the carboxyinethyltransferase, effectively inhibits FMLP-induced superoxide generation, whereas N acetyl-geranylcysteine, a poor inhibitor of the methylase, does not inhibit superoxide generation. In neutrophils, prenylcysteine-directed carboxymethyltrarisferase activity is localized to the plasma membrane (Pillinger et nl., 1994). This methylase activity is dependent on phosphatidic acid, a lipid that increases in concentration upon neutrophil activation. These data suggest that upon activation of neutrophils with FMLP, Rac is released

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from its interaction with the GDI protein and then translocates to the plasma membrane where it becomes carboxymethylated and participates in the activation of the NADPH oxidase. Carboxymethylation of prenylated proteins has also been suggested to play a role in other aspects of leukocyte activation. The heterotrimeric Gprotein subunit Gy2 is carboxymethylated in response to FMLP, and AFC inhibits the reaction (Philips et al., 1995). In addition to its inhibition of FMLP-mediated superoxide generation in neutrophils, AFC inhibits FMLP-mediated homotypic aggregation but enhances both the FMLPinduced upregulation of CDllbICD18 and the granule enzyme release in these cells (Philips et al., 1995). Furthermore, agonist-mediated activation of human platelets (Huzoor-Akbar et al., 1993) and the chemotaxis of mouse peritoneal macrophages toward lipopolysaccharide (LPS)-activated serum are also inhibited by AFC (Volker et al., 1991). Although AFC inhibits a variety of leukocyte activation-dependent responses, the role of the carboxymethyltransferase in these processes has been questioned (Ma et al., 1994). These authors show that several AFC analogs that are not inhibitors of the methyltransferase can nonetheless inhibit agonist-induced platelet aggregation. Furthermore, they found that the KM of farnesylcysteine for the platelet methyltransferase in vitro is -28 PM, whereas AFC inhibits platelet aggregation in the range of 1-10 PM (Huzoor-Akbar et al., 1993), suggesting that the methyltransferase is not the target of AFC in platelets. Additional studies to delineate the mechanism of inhibition of leukocyte activation by AFC are clearly necessary. XVII. Role of Rab Proteins in Membrane Transport in Leukocytes

Membrane transport functions that play particularly important roles in the biology of the immune system include endocytosis mediated via immunoglobulin Fc receptors and complement receptors. These types of receptor-mediated endocytosis are important in the clearance of foreign antigens by phagocytic cells and in the presentation of antigens via the MHC class I1 pathway by antigen presenting cells. At least four distinct Rab proteins, Rab4, Rab5, Rab7, and Rab9, play a role during various stages of endocytosis (Bottger et al., 1996; Rybin et al., 1996; Soldati et al., 1995). However, studies on the role of these proteins in the endocytic process in cells of the immune system are lacking. Another important immune system function that involves membrane transport is exocytosis, also known as degranulation or regulated secretion. For example, many inflammatory events including allergic reactions are triggered by antigen binding to high-affinity IgE receptors on mast cells, resulting in the rapid release of proinflammatory mediators from preformed

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secretory granules. Other granulocytes, including basophils, neutrophils, and eosinophils, degranulate in response to inflammatory stimuli. There is significant data from studies in leukocytes demonstrating that Rab proteins play an important role in exocytosis. The involvement of Rab proteins in exocytosis was first suggested by the ability of nonhydrolyzable GTP analogs to trigger mast cell degranulation when delivered through a patch pipette (Fernandez et al., 1984). GTP analogs have also been shown to trigger exocytosis in other granulocytes, including neutrophils (Nusse and Lindau, 1988) and eosinophils (Nusse et al., 1990). Several studies have implicated the Rab3A protein in the GTP-dependent exocytotic process. Mast cell degranulation is triggered by the injection of peptides corresponding to the effector domain of Rab3A (Oberhauser et al., 1992). Rab3A appears to play a role in regulated exocytosis in other cells because application of the Rab3A effector domain peptide to permeabilized pancreatic acini, chromafin cells, and insulinsecreting cells also triggers secretion (Nuoffer and Balch, 1994). Further pharmacological evidence for the involvement of Rab3A in exocytosis comes from the finding that prenylcysteine analogs stimulate exocytosis in permeabilized HIT-T15 cells (Regazzi et al., 1995). These authors suggest that Rab3A might normally inhibit exocytosis, and that the Rab3A effector peptide or the prenylcysteine analogs stimulate exocytosis by disrupting an inhibitory interaction between the prenylated Rab3A protein and a Rab effector protein. That Rab3A might act as a negative regulator of exocytosis is supported by the finding that microinjection of Rab3A antisense oligonucleotides enhanced exocytosis in adrenal chromaffin cells (Johannes et al., 1994). However, another study found the opposite result for a different isoform of Rab; in anterior pituitary cells microinjection of Rab3B antisense oligonucleotides inhibited regulated exocytosis but did not affect constitutive secretion or endocytosis (Liedo et al., 1993). The involvement of the Rab3 protein in exocytosis is equivocal. Although rat peritoneal mast cells express the Rab3B and Rab3D isoforms (Oberhauser et al., 1994), guinea pig eosinophils do not express any known isoforms of Rab3, even though they degranulate in response to nonhydrolyzable GTP analogs (Lacy et al., 1995). It has been suggested that other Rab proteins could play a role in exocytosis. In resting human neutrophils, Rab5A is localized to both membranes and cytosol, and upon challenge with PMA there is increased membrane association of the protein and a concomitant decrease in the cytosolic pool (Vita et al., 1996). The time course for the increased membrane association parallels the time course of exocytosis, suggesting that Rab5A might play a role in the secretory process. Alternatively, regulation of exocytosis by GTPases might involve proteins other than, or in addition to, Rab proteins because some members

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of the heterotrimeric G-protein family have been implicated in this process (Aridor et al., 1993). Although there is significant evidence to suggest a role of Rab proteins in exocytosis, further studies are required to understand their exact involvement in this process. XVIII. Regulation of Vesicular Transport by Rho Proteins

Although the Rab family of GTP-binding proteins is well known for its function in vesicular transport, there is an increasing appreciation for the involvement of Rho family members in these transport processes. For example, activated mutants of RhoA and Racl impair the formation of clathrin-coated vesicles in cells and in a reconstituted cell-free system (Lamaze et al., 1996). Cdc42Hs may also be involved in membrane transport because it is localized to the Golgi apparatus and its intracellular distribution is affected by brefeldin A, an agent that has profound effects on vesicular transport (Erickson et al., 1996).Additionally, a newly discovered Rho family member, RhoD, was shown to regulate cell morphology and endosome dynamics in a variety of mammalian cell types, including the macrophage cell line J774 (Murphy et al., 1996). Overexpression of wild-type RhoD or a GTPase-defective RhoD caused striking changes in cell morphology, including the formation of extended membrane processes that protruded from the body of the cell that were enriched in F-actin. This was accompanied by the disappearance of actin stress fibers and the disassembly of focal adhesion complexes in the cell body. Furthermore, wild-type RhoD and the RhoD mutant were localized to the plasma membrane and endosomes, and the RhoD mutant dramatically reduced the motility of endosomes in the cell. These studies indicate that RhoD regulates the movement of endosomes via a process that may depend on actin stress fibers. XIX. Other Prenylated Proteins

Several other prenylated proteins that might play roles in leukocyte function have been identified. Two interferon-? ( IFN-.)I)inducible GTPbinding proteins of unknown function have been identified in human fibroblasts (Cheng et al., 1991). One of these proteins, huGBP1, contains a C-terminal CTIS sequence predictive of modification by farnesyltransferase, whereas the other, huGBP2, contains a CNIL sequence at its C terminus that predicts modification by GGTase-I. HuGBPl and its murine homolog are induced by IFN-y and LPS in human monocytes, the human promyelocytic HL-60 cell line, and in murine macrophages, and their prenylation is sensitive to farnesyltransferase inhibitors (Nantais et al.,

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1996).Another potentially important prenylated protein is the yeast YDJl protein and its human homolog, hDJ2. The yeast YDJl protein is a farnesylated protein that functions as a molecular chaperone and is involved in cell cycle regulation (Yaglom et al., 1996).Other prenylated proteins that could be involved in immune cell function are two protein tyrosine phosphatases referred to as PTPcm (Cates et al., 1996).These phosphatases can transform human epithelial cells when overexpressed, and thus may normally play a role in regulating cell growth. XX. Prenyltransferase Inhibitors

The importance of the Ras pathway in cellular transformation and cancer, and the discovery that Ras requires farnesylation for its function, sparked the development of FTase inhibitors as potential chemotherapeutic agents. HMG-CoA reductase inhibitors such as lovastatin that inhibit prenylation of both farnesylated and geranylgeranylated proteins through their inhibition of isoprenoid synthesis, existed even before the discovery of protein prenylation and have been valuable tools for understanding the biological roles of prenylation. However, HMG-CoA reductase inhibitors have been considered unsuitable as clinically useful inhibitors of Ras function because they inhibit the biosynthesis of downstream metabolites in the mevalonate pathway, including cholesterol, dolicliol, and ubiquinone, as well as the prenylation of both farnesylated and geraiiylgeranylatedproteins. The findings that tlie CaaX motif itself is the minimal essential element for substrate recognition and catalysis by FTase and GGTase-I and that substitution of tlie second aliphatic amino acid within the CaaX with an aromatic amino acid converts the CaaX peptide substrate into a competitive inhibitor (Brown et al., 1992; Goldstein et al., 1991; Reiss et al., 1990; Schaber et al., 1990) serve as a starting point for the development of potent, cell active inhibitors of FTase. A number of CaaX peptidomimetic compounds that display excellent selectivity for FTase inhibition compared to GGTase-I inhibition have been reported, including L-731,734,BZA-SB, and BS81 (see Fig. 3) (Garcia et al., 1993;James et al., 1993; Kohl et al., 1993).These farnesyltransferase inhibitors ( FTIs) are modified CaaX peptides that lack peptide bonds and are therefore resistant to hydrolysis by proteases. Additionally, many of the first peptidomimetics were made as prodrugs, containing an esterified C-terminal carboxylate group that eliminates the charged nature of the molecule, making it permeable to cell membranes. They are prodrugs because significant activity against FTase requires generation of the free carboxylate through the action of cellular esterases. Nonpeptide mimetics that lack the C-terminal carboxylate and/or tlie sulfhydryl moeity of the

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cysteine residue in the CaaX have also been developed (Bishop et al., 1995; Hunt et al., 1996; Vogt et al., 1995;Williams et al., 1996).In addition to CaaX competitive inhibitors, compounds competitive with FPP such as manumycin, as well as bisubstrate analogs competitive with both CaaX and FPP, have been developed (Fig. 3) (Haraet al., 1993;Pate1 et al., 1995). The CaaX competitive FTIs can block the famesylation of Ras and other FTase substrates in cells (Garcia et al., 1993; James et al., 1993; Kohl et al., 1993) and, in general, are more potent in cells than FPP competitive compounds (Hara et al., 1993).The efficacy of these compounds in inhibiting farnesylation in cells is illustrated by their effects on the Ras signaling pathway. FTIs inhibit many aspects of the transformed phenotype that are induced through the introduction of oncogenic H-ras into rodent fibroblasts, including anchorage-independent cell growth, rapid growth in monolayer culture, and alterations in cell morphology (James et al., 1993; Kohl et al., 1993; Prendergast et al., 1994). FTIs inhibit the formation and growth of rodent and human xenograft tumors in nude mice (Hara et al., 1993; Kohl et al., 1994; Sun et al., 1995). Additionally, the FTI L-744,832 is efficacious in a transgenic mouse model of mammary cancer (Kohl et al., 1995). In this model, oncogenic H-ras is expressed under control of the MMTV promoter, which induces mammary and salivary carcinomas (Sinn et al., 1987). Daily treatment of tumorbearing mice with L-744,832 induced a rapid regression of the tumors, and continual treatment prevented the reappearance of new tumors (Kohl et al., 1995). Cultured cells growing under anchorage-independent conditions undergo apoptosis in response to FTI treatment, suggesting that the rapid tumor regression induced by FTI treatment in the H-ras oncomouse model might also be due to apoptosis (Lebowitz et al., 1997). No detectable toxicity has been reported in animal studies involving FTI treatment. The lack of toxicity was unanticipated, given the ubiquitous importance of Ras in cell proliferation. One explanation for the lack of global toxicity in the face of dramatic effects on tumor growth is that many of the published studies used tumors that are driven by activated H-Ras, which is a relatively poor substrate for FTase and is thus easily inhibited. For example, the &, of FTase for H-Ras, which has a CaaX where X = ser, is much higher than the K,,, of FTase for K-Ras, where X = met (James et al., 1995). Additionally, 10-fold higher concentrations of the BZA-5B FTI are required to block farnesylation of the nuclear lamins compared to those for H-Ras (Dalton et al., 1995). Another explanation for the lack of toxicity in normal tissues is that transduction of growth proliferative signals in normal cells may rely on other forms of Ras, such as K-Ras and N-Ras, or Ras-related proteins such as R-RasmC21. This is suggested by studies that showed that FTIs did not inhibit the EGF-

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stimulated activation of MAPK in nontransformed cells but did inhibit the MAPK activation induced by oncogenic H-ras (James et al., 1994a). Additionally, cross-prenylation of some farnesylated proteins by GGTaseI in the presence of an FTI blockade might explain the lack of toxicity. R-RasZflC21, which is capable of triggering malignant transformation (Graham et al., 1994), as well as K-Ras4B are prenylated by both FTase and GGTase-I in vitro (James et al., 1995; Carboni et al., 1995) and might remain functional and transduce growth signals in normal tissues treated with an FTI. The finding that K-Ras4B, which is the predominant form of mutated Ras associated with cancer (Barbacid, 1987), is prenylated by GGTase-I as well as FTase in vitro (James et al., 1995) has raised important questions concerning the development of FTIs as chemotherapeutic agents. Although K-Ras4B is found as a farnesylated protein in vivo (Casey et al., 1989),it remains prenylated in FTI-treated cells (James et al., 1996), and preliminary data suggest that this is due to cross-prenylation by GGTase-I (Lerner et al., 1997; Pai et al., 1996; Rowel1 et al., 1997). Furthermore, K-Ras4B containing an altered CaaX sequence (CVIL) that is presumed to be exclusively a GGTase-I substrate is transforming to cells (Hancock et al., 1991b; Kato et al., 1992), suggesting that geranylgeranylated K-Ras4B in FTItreated cells would be functional. Sebti, Hamilton, and coworkers have further explored the issue of K-Ras4B cross-prenylation through their development of prenylation inhibitors that are more specific for GGTase-I compared to FTase. These compounds were derived from FTI peptidomimetics by replacing the methionine residue of an FTI peptidomimetic with leucine in the X position of the CaaX (Fig.3) (Lerner et al., 1995). Although one of these compounds was reported to block K-Ras processing in NIH-3T3 cells and to inhibit MAP kinase activation, recent data suggest that a combination treatment with both an FTI and a GGTase-I inhibitor is required to effectively inhibit K-Ras4B prenylation in human tumor cell lines (Lerner et al., 1997). Although FTIs alone may not inhibit K-Ras4B prenylation, FTIs can inhibit the anchorage-independent growth of a variety of cell lines derived from human tumors including those containing K-Ras4B mutations (SeppLorenzino et al., 1995; Nagasu et al., 1995). The sensitivity of these tumor lines to growth inhibition by the FTI varied greatly and was independent of the ras mutational status of the cell. This suggests that human tumor cell proliferation can be regulated by farnesylated proteins in addition to Ras. One such protein may be RhoB, a member of the Ras superfamily of GTPases that can be both farnesylated and geranylgeranylated in vivo (Adamson et al., 1992). FTI treatment of cells disrupts the intracellular localization of this protein, and cells transformed with an FTI-resistant

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form of RhoB containing an N-myristylation site require 10-fold higher concentrations of FTI to be growth inhibited (Lebowitz et al., 1995). Growth inhibition by FTIs might involve multiple mechanisms because the processing of at least 18 cellular proteins is affected by FTI treatment (James et al., 199413). In addition to the potential of FTIs for cancer treatment, GGTase-I inhibitors might also have potential as chemotherapeutics. HMG-CoA reductase inhibitors and the GGTase-I inhibitor, GGTI-287 (Fig. 3),inhibit the proliferation of cultured cells through a mechanism that involves growth arrest in the GI phase of the cell cycle (Vogt et al., 1996). Progression of a cell from GI into S phase involves the ubiquitin-dependent degradation of the p27 cyclin-dependent kinase inhibitor (Pagan0 et al., 1995), and the HMG-CoA reductase inhibitor pravastatin prevents the elimination of p27 through a mechanism that appears to involve geranylgeranylated Rho proteins (Hirai et d., 1997). The involvement of geranylgeranylated Rho proteins in p27 elimination is suggested by the finding that in pravastatintreated cells, addition of liposomes containing the GGTase-I substrate GGPP but not the FTase substrate FPP results in a decrease in p27 protein levels and progression of the cells through GI into S. Furthermore, the Rho inactivator, C3 ADP ribosyltransferase,prevents the ability of GGPP to stimulate progression into S phase in pravastatin-treated cells. Additionally, both lovastatin and a GGTase-I inhibitor block the PDGF-induced tyrosine phosphoylation of the PDGF receptor (McGuire et al., 1996). A Rho protein could be involved in this aspect of signaling because the PDGF type B receptor has been found to associate with Rho (Zubiaur et al., 1995). The ability of GGTase-I inhibitors to induce G, arrest, to inhibit multiple aspects of signal transduction, and to block the prenylation of KRas4B in conjunction with FTI treatment suggests that these agents might also be suitable as chemotherapeutics. In this regard, preliminary studies indicate that GGTase-I inhibition can block the growth of several human tumor lines in nude mice (Sun et al., 1997). MI. Effects of Prenylation Inhibitors on Leukocyte Function

Although FTase inhibitors show little toxicity in animal models, the potential effect of these inhibitors on the function of the immune system has not been adequately addressed. The HMG-CoA reductase inhibitor lovastatin inhibits both proximal and distal signaling events in the human Jurkat T cell line and in normal human peripheral blood mononuclear cells activated through the TCR (Goldman et al., 1996). In Jurkat cells, lovastatin inhibited both the processing of Ras and the activation of MAPK. Additionally, TCR signaling events that are presumably independent of

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Ras were inhibited, including mobilization of intracellular calcium, inositol phosphate production, and tyrosine phosphorylation, suggesting the involvement of a prenylated protein other than Ras in these aspects of TCR-mediated signaling. The effect of lovastatin on these Ras-independent signaling events was specific to the T cell receptor because calcium signaling and inositol metabolism triggered by transfected type-1 muscarinic receptors were unaffected in these cells. The potential for prenylation inhibitors to affect diseases involving lymphocyte proliferation and/or differentiation is suggested by several other studies involving HMG-CoA reductase inhibitors. For example, lovastatin showed some efficacy in inhibiting chronic allograft rejection in an animal model (O’Donnell et al., 199.5). IIMG-CoA reductase inhibitors and zaragozic acid, an FTI isolated from natural products, can inhibit signaling, specifically inositol lipid metabolism, in human keratinocytes induced by inflammatory mediators such as PAF and bradykinin (Alaei et al., 1996). This suggests that prenylation inhibitors could ameliorate the symptoms of inflammatory skin diseases. Another cell of the immune system that is responsive to HMG-CoA reductase inhibition is the human macrophage; it has been shown that lovastatin inhibits the expression of the type I lipoprotein scavenger receptor gene in these cells (Umetani et al., 1996). The inhibition of lipoprotein scavenger receptor expression in niacropliages is likely not related to the efficacy of this cholesterol-lowering agent in cardiovascular disease management because the inhibition of gene expression occurred at concentrations (5-15 ~ L ) Mfar higher than the peak plasma concentration commonly achieved in patients treated with this agent. XXII. Conclusion

Members of the Ras superfainily of GTP-binding proteins regulate a wide variety of cellular processes, and many of members of this family have been shown to play an iinportant role in tlie function of irninune system cells. I expect that appreciation of the importance of these proteins in immunobiology will only continue to grow as studies of these proteins and tlie discovery of new family members progress. The C-terminal processing of the Ras superfainily of proteins, which depends on the action of prenyltransferases and other processing enzymes including the CaaX protease, the prenylcysteine-directed methyltransferase, and in some cases palmitoyltransferase, is critical to the function of these proteins. The development of specific inhibitors of these C-terminal processing enzymes, in particular, inhibitors of farnesyltransferase, has proceeded rapidly in recent years and has helped to illustrate the importance of protein prenylation in various cell functions. Studies of these prenylation inhibitors in irninune

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system function should be expanded. These studies would be valuable not only from a clinical standpoint but also to aid in our understanding of the importance of Ras superfamily members in the proper functioning of the immune system. ACKNOWLEDGMENTS I thank Dr. Jay Gibbs and Dr. Charles Omer of the Merck Research Labs for their advice and suggestions on the manuscript.

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A I W A U C h 5 IN IMMUNOLOCY VOL hH

Generation and TAP-Mediated Transport of Peptides for Major Histocompatibility Complex Class I Molecules FRANK MOMBURG AND GUNTER J. HhMERLING Deparhnent of Molecular Immunology, German Cancer Research Center (DKFZj,

69120 Heidelherg, Germany

1. Iniroduction

During evolution of the adaptive immune system as a defense against environmental pathogens two major groups of microbes had to be dealt with that differ with regard to their intracellular location: microbes replicating in the cytosol, e.g., viruses and some bacteria, and those propagating in vesicular compartments, e.g., some bacteria, or their toxic products that enter endosomal/Iysosomal compartments via the endocytic pathway. Therefore, different pathways needed to be developed for the presentation of antigens located in distinct subcellular compartments. This is achieved by the two classes of major histocompatibility complex (MHC) molecules that are specialized peptide receptors and serve to display antigenic peptides at the cell surface for recognition by T lymphocytes. Peptide antigens generated in the vesicular compartments of the endocytic pathway are usually loaded onto MHC class I1 molecules. These are targeted to endosomaVlysosoina1 loading compartments with the help of the invariant chain, which carries an endosoinal sorting signal. After binding of peptides the resulting MHC class 11-peptide complexes proceed to the cell surface for screening by CD4+ T cells. In contrast, pathogens dwelling in the cytosol will be subject to degradation by the major cytosolic proteolytic machinery, the proteasome, which is an evolutionary ancient protease that is found in eu- and archaebacteria. The immune system makes use of the peptidic degradation products generated in the cytosol by translocating them into the lumen of the endoplasmic reticulum (ER) where they assemble with newly synthesized MHC class I molecules and are transported to the cell surface for recognition by CD8+ T lymphocytes. This process allows T cells to continuously sample the cell surfaces for the presence of peptides derived from a potentially harmful intruder. Translocation of peptides into the ER is achieved by the TAP transporter (transporter associated with antigen processing), which belongs to a large family of membrane translocators containing an ATP-binding cassette (ABC). TAP appears to be a specialized ABC transporter that serves exclusively the immune system as indicated by the observation that 191

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TAP deficiency in human patients or in knockout mice seems to solely affect the immune system, in which it plays a pivotal role in the class I presentation pathway. II. TAP as the Principal Peptide Supplier for MHC Class I Molecules

A. EVIDENCE FOR PEPTIDE TRANSLOCATION BY TAP Approximately a decade ago, strongly reduced levels of cell surface class I molecules had been detected in various cell lines that had undergone radiation or chemical mutagenesis or that had been treated with class I antibodies and complement (Kavathas et al., 1980; DeMars et al., 1984, 1985; Ljunggren and Karre, 1985; Karre et al., 1986; Salter et al., 1985). By culturing the mutant cells in high concentration of class I-binding peptide, class I surface expression could be reconstituted (Townsend et al., 1989; Cerundolo et al., 1990). Unstable complexes of class I h e a y chains and &-microglobulin (P2m)were largely retained in the ER of the mutant cells (Salter and Cresswell, 1986; Ljunggren et al., 1989). Incubation at ambient temperature, which has a stabilizing effect on the dimers, was found to induce their transport to the cell surface (Ljunggren et al., 1990; Schumacher et al., 1990). Furthermore, the mutants were unable to present intracellular antigens to cytotoxic T cells, whereas exogenously added peptides or peptides introduced into the ER by a signal sequence were efficiently presented (Townsend et al., 1989; Ohkn et al., 1990a; Cerundulo et al., 1990; Hosken and Bevan, 1990; Anderson et al., 1991). Addition of peptide ligands to detergent extracts of mutant cells induced class I molecules to assemble with Pem (Townsend et al., 1990; Schumacher et al., 1990; Elliott et al., 1991). From these results it was concluded that the supply of peptides into the ER was grossly disturbed in the mutant cells. With the cloning of cDNAs coding for molecules with homology to ABC transporters, TAPl and TAP2 (see Section V), the defects in assembly and antigen presentation by class I molecules could be linked to the function of a peptide transporter in the ER membrane. The defective phenotypes of different mutants were found to be reversible by transfection of TAPl (Spies and DeMars, 1991; Spies et al., 1992), TAP2 (Powis et al., 1991a; Attaya et al., 1992; Kelly et al., 1992), or both TAPl and TAP2 cDNAs (Arnold et al., 1992; Momburg et al., 1992). The underlying defects in TAP genes have been determined as transcriptional inactivation of TAPl in human LCL721.134 B lymphoblastoid cells (Spies et al., 1990);deletion of TAPl and TAP2 in LCL721.174 (Spies et al., 1990) and its derivative, the BxT hybrid .174xCEM.T2 (T2), a frameshift mutation in TAP2 leading to a functionally defective molecule of extended length in the human B

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lymphoblastoid line BM36.1 (Kelly et a1 , 1992); and a premature stop codon early in the TAP2 sequence present in the mouse T lymphoma cell line RMA-S (Yang et al., 1992a). These transfection studies also indicated that both TAPl and TAP2 molecules need to be functional for the bulk of class I peptide loading and presentation to occur. Independent evidence for the existence of genes that influence the repertoire of class I-bound peptides came from studies with intra-MHC recombinant inbred rat strains. The antigenicity of the rat class I molecule, RTl.A', both as alloantigen and as restriction element, was modified by a locus in the MHC class I1 region termed cini (for class I modifier) existing in two allelic phenotypes, cirrio and cim" (Livingstone et al., 1989, 1991). Furthermore, the transit of RT1.Ad molecules to the cell surface was retarded in the presence of cim" but not &ma,leading to the worhng hypothesis that the cirri6 product supplied RT1.Adwith ill-suited peptides (Powis et al., 1991b).cini was demonstrated to be the TAP2 product, which has an extensive allelic polymorphism in the rat (Powis et al., 1992a; see Section VI). Transfection of TAP2l (cim")cDNA into c i d host cells restored the cim" antigenic phenotype of RT1.Ad molecules and caused a significant shift in the high-performance liquid chromatography (HPLC) spectrum of peptides extractable from RT1.Adtoward more hydrophilic peptide species (Powis et al., 1992a).This finding was fully consistent with TAP molecules acting as peptide transporters, although direct proof was still laclung.

B. TAP-DEPENDENT versus -INDEPENDENT ANTIGENPRESENTATION For avaiiety of peptides presented by classical or medial class I molecules it was shown that their presentation is abrogated in TAP deficiency mutants. Among these epitopes were Kd, Kk, Dd, and HLA-A1 and -A2-restricted peptides derived from the influenza virus nucleoprotein or matrix protein, a K"-restricted epitope from vesicular stomatitis virus (VSV) nucleocapsid protein, the minor histocompatibility antigen HA-2 presented by HLAA2, the N-formylated mitochondrial-derived peptide MTF" presented by the mouse class Ib molecule M P 3 (HMT')),and unknown allostimulatory peptides presented by Kh or Qal" (Powis et al., 1991a; Anderson et al., 1991; Aosai et a ! , 1991; Hermel et al., 1991; Kelly et d., 1992; Attaya et al., 1992; Spies et al., 1992; Momburg et al., 1992; Eisenlohr et a1 , 1992; Zhou et al., 1993a, 1994; Bacik et al., 1994).Also, Qa-2, another mouse class Ib molecule, required functional peptide transporters for stable assembly of its soluble and the glycosylphosphatidylinositol-anchoredisoforms (Tabaczewski and Stroynowski, 1994). In mice harboring a disrupted TAPl gene, class I cell surface levels were severely reduced, lymphoblasts were unable to present endogenous

194

FRANK MOMBUHC: AND CUNTER J. HAMMEHLINC

antigen, and the number of peripheral CD8+ T cell was reduced to E 144) results in prevention of EAE upon immunization with the native PLP 139-151 due to the induction of T cells that are cross-reactive with the native hgand and produce Th2-like cytokines (IL-4 and IL-10) and ThO-like cytokines (IFN-y and IL-10) (Kuchroo et al., 1994). Of note, the adoptive transfer of T cell lines that are generated upon exposure to the altered peptide ligand confers protection from EAE (Nicholson et al., 1995).

IMMUNOLOGICAL TREATMENT OF AUTOIMMUNE DISEASES

387

These results suggest that immune deviation is one important mechanism by which altered peptide ligands of the TCR inhibit autoimmune diseases. A major effect on the niodulation of cytokine responses is supported by a study of h4BP 87-99-induced EAE; of three altered peptide ligands, only the MBP 87-99 (91 K > A) substitution results in protection and reversal of EAE. This is accompanied by suppression of IFN-y and TNF-a responses in cells isolated from draining lymph nodes upon in uitro challenge with the native MBP 87-99 peptide (Karin et al., 1994). A MBP 87-99 (96 P > A) peptide also exerts dramatic therapeutic effects on established EAE induced by adoptive transferral of MBP 87-99-specific T cells. The therapeutic benefit is not only clinical but also histopathological, with regression of inflammatory infiltrates and disappearance of heterogeneous T cell infiltrates; only T cells with characteristics similar to those used to induce E.4E in the first place remain present in the lesions of the CNS (Brocke ot al., 1996). In this study, neutralization of IL-4 reverses the tolerant state induced by the MBP 87-99 (96 P > A) peptide. The altered peptide ligation thus seems to provide selective signals that result in an efflux of T cells recruited in the initial lesions; this wash out may be the result of intralesional downregulation of TNF-a and concomitant rise of IL-4. TCR antagonism or MHC competition, on the other hand, are unlikely to account for the therapeutic effects because MBP 87-99 (96 P > A) is a partial agonist for the encephalitogenic T cells and fails to inhibit their in vitro proliferation. The most likely explanation for the therapeutic effect of the altered peptide ligand approach remains immune deviation (Fig. 6). Specific inimunotherapy based on altered peptide ligation, however promising, faces the obvious problem that in some experimental models, especially in human autoiminiine diseases, the autoreactive pathogenic T cells and their antigen specificities are still unknown; this clearly hampers the rational design of appropriately modified peptide ligands. In the case of multiple sclerosis, however, this promising approach may find its way into clinical trials because there is already some information available 011 the fine specificity of myelin-specific autoreactive T cells. Another theoretical obstacle is that the therapeutic effectiveness may fade in the case of substantial determinant spreading, which leads by definition to diversity ofthe T cell repertoire. In this respect, encouragng data show that targeting immunoclominant T cell clones in EAE lesions may transactingly suppress encephalitogenic T cells with a diverse repertoire (Brocke et al., 1996).

K. ACTIVATION OF REGULATORY T CELLSBY T CELLAND TCR PEF~TIDE VACCINATION T cell vaccination has been proposed in pioneer studies in EAE, adjuvant arthritis, and EAT (Ben-Nun et al., 1981; Holoshitz et al., 1983; Maron

388

J. R. KALDEN et al.

et al., 1983), with the attenuation protocols varying from irradiation to pressure or chemical cross-linking. The disease resistance resulting from T cell vaccination is transferrable to naive recipients by regulatory T cells of both CD4+ and CD8' phenotype (Lider et al., 1988). Anticlonotypic T cells are the major regulators that recognize clonotypes of the TCR. Protection by these cells is mediated through cytotoxic effects but may include other suppressive mechanisms. Regulatory T cells recognizing cellular markers other than the TCR clonotype may also be important, in concert with the antiidiotypic response. It is conceivable that T cell vaccination acts through the same regulatory pathways preexisting and naturally operating in the periphery. Thus, clonotypic networks may be preshaped in the physiological situation to accommodate and regulate a limited number of autoreactive T cells within a certain tolerable limit. This hypothesis is supported by recent data showing that inactivation of TCR peptide-specific CD4 regulatory T cells induces chronic EAE in an otherwise self-limiting model induced by MBPAc 1-9 (Kumar et al., 1996). Thus, a failure to generate regulatory T cells that participate in the reestablishment of peripheral tolerance after an acute flare may cause or favor chronic autoimmune conditions. The aim of T cell vaccination is therefore the reestablishment of a physiologic balance in the clonotypic network. Once preclinically tested, the effects of T cell vaccination remain limited to protection against the development of disease rather than reversal of the established signs, with the possible exception of adjuvant arthritis (Lider et al., 1987). Another problem is illustrated by vaccination with attenuated AChR-specific T cell lines in EAMG; this treatment induces AChR-specific suppressor cells in the spleen, but it also enhances AChR antibody responses (Kahn et al., 1990). Thus, T cell vaccination can turn out a seemingly double-edged sword. A novel approach in TCR vaccination is represented by the injection of DNA encoding the Vb 8.2 region of a TCR critical to the pathogenesis of EAE (Waisman et al., 1996). This approach ameliorates MBPAc 1-20induced EAE, inducing the surge of peptide-specific regulatory T cells characterized by a T h l > Th2-like shift of their cytokine production. Of note, the efficacy of this approach extends to EAE induced by the entire MBP molecule, although the latter certainly shapes a more diverse T cell repertoire than the 1-20 peptide. The TCR peptide vaccination also protects DBN1 mice from developing CIA, as shown by the use of a recombinant TCR domain derived from the Val1.l-JA17 gene, a gene used by a T cell hybridoma specific for an immunodominant epitope of collagen I1 (Rosloniec et al., 1995). Similarly, TCR peptide vaccination with VplO peptides blocks the development of

IMMUNOLOGICAL TREATMENT OF AUTOIMMUNE DISEASES

389

CIA in BUB/BnJ mice (Haqqi et al., 1996). This approach elicits the formation of antibodies that react with self-TCR; these are likely to account for the downregulation of both T cell and B cell pathogenic responses observed in the study. Antibodies against certain V gene segments of the TCR effectively protect against autoimmune diseases characterized by limited V gene usage. The administration of anti-VP 8.1,2 or anti-VP 5.1,2 mAbs protects DBA mice from developing CIA (Chiocchia et al., 1991);likewise, a combination of anti-Vp 8.2 and anti-Vp 13 protects completely against MBP-induced EAE (Zaller et al., 1990). However, this type of treatment becomes much less effective in models characterized by diverse T cell repertoire, for example, in the case of the relapsing form of EAE induced by PLP 139-151 (Whitham et al., 1996). The attempt to treat autoimmune diseases by activation of regulatory cells is very challenging. However, the therapeutic effects of T cell vaccination, as well as those of TCR peptide vaccination, are mostly confined to prevention of disease models. It remains to be demonstrated how this type of therapy influences established phases of chronic disorders.

L.

ANTIGEN-SPECIFIC TOLERANCE

EAE is the most extensively used model for the study of orally induced tolerance. Initial studies in Lewis rats, with oral administration of high dose of MBP (in four feedings a total of 20 mg in 1 week) before immunization, show delayed onset, reduction of incidence, and clinicalhistopathological improvement of the disease; these effects are accompanied by a decrease of T cell proliferation and a slight reduction of antiMBP antibodies in response to MBP (Bitar and Whitacre, 1988). Notably, high doses (10-20 mg) are necessary to cause the effect, whereas 2 mg remains ineffective; heterologous MBP is also necessary (human or pig), whereas rat MBP is ineffective. Similar therapeutic benefits can also be achieved with a low-dose regimen of oral MBP (9 proteins of human, 211-213, 22.5, 227 species differences, 211-213, 219, 225-228 structure and fiinction, 231-2

427

Permeability transition pore. 74, 80-88. 110, 123 Peyer’s patches, macrophages in, 273 p59’”’,93, 95, 96, 97-98 p53, 7, 69-70, 91 Phagocytes, phospliatidylserine receptors on, 64 Phagocytosis, 167, 282 Phosphatidylserine, 64,70. 76. 80 Phospliolipase C-71, 9-3-95 Phosphorylation, :32. 35, 86. 93, 109-110 7, 10, 17, 31. 35 Pli~~ohemaglutinin, Pituitary cells, 173 PKB, see Protein kinase B PKC, sce Protein kinase C Plasma cells, 17; see also Lymphoid cells Plasma ineinbrane, 54-55 64, 65, 164 Platelet-derived growth factor, 8 Platelets, 14, 169, 172 Ynetcnzncystis carinii, 282, 3f;l Polychontiritis. 352 Polysolnes, 4, 21, 33, 35 Potassiiim ions, in apoptosis. 65, 95, 175 Prenylation, of Has GTPase proteins, 145-189 Prenykransferase, 152- 157, 175-178 Procoagulant enzymes, 64 Prostaglandins, 13, 345 I’roteases. 54, 61-64, 199-200; see &J Caspases; Proteasomes Proteasomes, 61-62, 199-205 Protein l)inding, 22-23 Protein kiirase B. 111-113 Protein kinase C, 11, 12. 19, 32, 9.5 Proteins AIF. 85, 87, 88 in apoptosis, 54, 74, 76, 85, 124 Anf-1, 33-34 A U U U A HNA-binding, 30-35 cataluse-specific, HNA-binding, 25 cytokine and protooncogene inHNA-binding, 35 digestion of, by caspases, 60 heat shock, 197, 205 interactions with mHNA, 22-23, 29-30 myelin basic, 389-391 non-AUUUA RNA-binding, 35-36 of permeability transition pore. 80 prenylation of Has GTPase C-terminal methylation of, 157-1.58, 171- 172

428 heterotrimeric G, 164-165 Rab, 148, 158-162, 165-166, 172-174 Rac, 147, 169-170 Rap, 169-170 Ras, 146, 148-149, 163-164, 166-168 Rep, 161-162 Rho, 147, 171, 174, 177-178 Rho/Rac, 148-150, 168-169 Protein synthesis, 15, 16, 19, 36 Protein tyrosine kinases, 93, 97-98 Protein tyrosine phosphatase, 93, 98-101 Proteolysis, 163 Protonophores, 80 Protooncogenes, 5-8, 19, 22, 27-29, 36; see also specific protooncogenes Psoriasis, 238, 357, 361 FTK,see Protein tyrosine kinases Pyknosis, 54

Rab proteins, 148, 158-162, 165-166, 172- 174 Rac proteins, 169-170 Radiotherapy, 53 Rapamycin, 9, 12, 333 Rap proteins, 169-170 Ras-GAP, 94 Ras proteins activation hy GTPase cycle, 147-149 in apoptosis, 168 cancer, and mutation of, 177 C-terminal events and, 163-164 function in immune cells, 146, 164 growth inhibition and, 167 prenylation of, 150- 152 Rho-Rac pathway, link to, 148-149 signaling pathway of, 146, 166-168 Ras superfamily, 145-157 Rauscher virus, 194-195 Reactive oxygen species, in apoptosis. 64-65, 70-71, 77, 123 Redox status, apoptosis and, 54, 64-65, 70-71, 88 Reiter’s syndrome, 238-239 Rep proteins, 161-162 Respiratory burst, 169 Respiratory chain inhibitors, 80 Retina, 161, 164

INDEX

Rhabdomyosarcoma cells, 105 Rheumatoid arthritis animal studies on, 339, 340, 369-371 anti-CD4 therapy, 349-353 anti-CD5 therapy, 356-358 anti-CD7 therapy and, 358-359 anti-CD52 therapy and, 362-364 anti-116 therapy and, 344-345 anti-TNF-a therapy, 338-341 apoptosis induction therapy, 337 CD30 and, 115-116 CD51C trials in, 356-358 cytokines and, 345-347 erosive versus non-erosive, 327 gene therapy, 337 and ICAM-1,369-371 interferon-y therapy, 346 interleukin-1 blockade and, 339, 341, 344 interleukin-2 targeting therapy, 359-360 juvenile, and oral collagen, 335 monoclonal antibody therapy, 336, 347-353 prognostic indicator for, 326-327 role of peptide transporter in, 238-239 susceptibility genotype, 315-332 Rho proteins, 147, 171, 174, 177-178 Rhomac proteins, 148-150, 160-161, 168-169 Ribonucleases, 20, 31 Ribonucleotide reductase, 24-25, 35, 36 Ribosomes, 20, 24

Schizosaccharomyces pomnbe, 56 Selectins, 285 Sendai virus epitopes, 194, 196 Septic shock, 12 Serine proteases, 61, 62-64 Serine-threonine kinases, 6, 93, 101, 121 Serine threonineRKB, 111-113 Serine-threonine protein kinases, 120 Serine-threonine tyrosine kinases, 6, 93, 120-121, 122 Sialoadhesin, 275-276 Signal transduction pathways antigen receptor-mediated, 93-95 in apoptosis and apoptosis-resistance, 122 Bcl-2 and modules of, 107-111

INDEX

Kas role in, 146-147, 166-168 resulting in permeability transition, 83 surface receptor to death effector, 89 Skeletal muscle, macrophages in, 284 Skin, macrophages in, 284 Somatostatin, 95 SOS, see Guanine nucleotide exchange factor Sphingomyelin, 120-122, 168 Spleen, 272, 275-276 Splenocytes, 56, 239, 262-266, 273 Splenomegaly, 51 Staphylococcus atireus, 351 Staurosporine, 69, 70, 71, 75 Stress fiber formation, 150 Stress kinases, 101-107 Substance P; 11-12 Superantigeris, 97 Superoxide production, 25, 85 Systemic lupus erytheinatosus animal studies of, 337, 377-378 CD30 and, 115-116 CD51C studies on, 357 CD4 monoclonal antibody and, 352 and tumor necrosis factor, 338

T T. acidophihtm, 200 TAP, see Peptide transporter Tapasin, 207-208 T cell hybridomas, 78-80, 97 T cell-mediated killing, 27 T cell receprors antagonists, and autoimmune disease, 386-,387 and CD4, 381-385 CD3 and, 16, 34, 93-96, 100-101, 266 and CD2H costirnulation, 12 costimulalion, interference with, 381-385 Ras pathway and, 166-168 and rheumatoid arthritis susceptibility, 322-324 self-recognizing, 89-91 and thymocyte deletion, 68-69 transgenic 11-Y, 97, 99 T cells activation 8-9, 16-17 antigen-receptor signaling in, 111 apoptosis in, 51-144

429

as apoptosis inducers, 53 Bcl-2, and mitochondria1 function of, 75 CD8+and, 191, 193-194 CD 45 and. 98-101 CD4 monoclonal antibody and, 348 cytoskeletal defects in, 169 differentiation of, 17, 89-91 elimination of, and rheumatoid arthritis, 353-358 Fc receptors in, 278-279 FV receptors and, 258-266 GM-CSF production and, 8, 258 HLA class 11 expression in, 16-17 homeostasis of, 91-93 interleukin-lWNK effect on human, 10 and interleukins, 13, 14 major histocompatibility complex molecules and, 89-91 memory, 91-92, 285, 356 mHNA regulation and function of, 1-50 peptides from intruders and, 191, 230 pokeweed mitogen effect on, 5 protooncogene levels, 5 Kas pathway in, 166-168 self-reactive, 93, 321 specific antigen recognition, 89, 93 transferrin receptors in, 16 tumor eradication and, 257-258 tumor necrosis factor and, 14 vaccination with, 387-389 TCWCD3 receptor complex, 93-97, 100-101, 266 Tetracycline, as mRNA promoter, 3 Thalidomide, 13 Themnoplasma acidophilum, 200 Thymocytes antigen-specific activation of, 95-96 apoptosis in, 78-80, 90-91 Bcl-2, and mitochondria1 function of, 75 CD8' and, 97 CD45 and, 99-100 chromatin condensation in rat, 56 differentiation and selection of, 17-18, 89-91, 106-107 etoposide effect on, 62 gfucocorticoid-treated, 52, 56, 57, 61-62, 68-69 immature, and thymic stromal cells, 95 major histocompatibility complex and, 89-91,97

430

INDEX

mitochondria1transmembrane potential, 91 positive and negative selection, 89-91 with self-recognizing T cell receptors, 89-91 sphingomyelinase and, 120 T cell receptors and, 68-69, 95-97 thymic stromal cells and, 95 Thymus, 17, 115, 239; see also Thymocytes Thyroid disease, 346, 371 Tissue destruction, autoimmune, 368, 378 T lymphoma cells, murine, 7, 193, 195 Tolerance, antigen-specific, 389-395 Toxins, 53, 122, 359-361 Transferrin receptors, 15-16, 24, 297 Transforming growth factor ligation of receptor, and apoptosis, 53 transforming growth factor a, 17 transforming growth factor b, 6, 24-25, 36, 377, 378 Tumor necrosis factor and cytokine stimulation, 12 and encephalomyelitis,373-374 sphingomyelinases, ceramide and, 120-122 and TCWCD3-triggered thymocyte apoptosis, 91-92 Tumor necrosis factor-a; see also Tumor necrosis factor; Tumor necrosis factor receptor Actinomycin D and mRNA of, 19 in activated murine T cell clones, 14 apoptotic effect in thymocytes, 114 AU-A protein and, 34 in collagen 11 arthritis, 377 in Crohn’s disease therapy, 342 effect on interleukin-1 mRNA, 11 ligation of, and apoptosis, 53, 72 mRNA regulation in, and T cell activation, 8-9 NF-KB activation and, 119-120 produced by T cells with Fv receptor, 258 and rheumatoid arthritis, 338-342 in systemic lupus, 338 Tumor necrosis factor receptor, and apoptosis in thymocytes, 114-116 Tumors; see also Carcinoma; Lymphoma and adoptive immunity, 257-269 CD30 and, 115 FTase inhibitors and, 177-179 Ioss of TAP expression in human, 236-238 lymphocyte-derived,51 macrophages, binding to, 284

Tyrosine kinases, 6, 17; see also Protein tyrosine kinases Tyrosine phosphorylation, 17, 93

Ubiquitin system, 204 Ultraviolet light, 27, 102, 103, 105 U-rich sequence-binding proteins, 34 Uveitis, experimental autoimmune, 371

v Vaccination, T cell receptor peptide, 387-389 Vasculitis, 352, 364 Vesicular transport, 174 Vinblastine, 110 Vincristine, 110, 224 Viruses, see also specijic virus as apoptosis inducers, 53 caspase inhibitors in, 60 and cytotoxic T cells, 53 peptide transport inhibition by, 234-235 and T cell death, 93 VP-16, 62, 105, 224

Wiskott-Aldrich syndrome, 169

Y Yeast a-mating factor, 151 endoplasmic reticulum protein in, 198 genes for prenykransferases of, 152 methylation in, 157 prenylated proteins in, 175 proteasomes of, 200-201 Ras2 and adenylyl cyclase, 164

z Zinc, 55, 56, 156, 157 Zinc finger proteins, 32

CONTENTS OF RECENT VOLUMES

Volume 64

Volume 65

Proteasomes and Antigen Processing NF-ILG and NF-KB in Cytokine Gene KElJl TANAKA, NORUYUKI T A N ~ H A S I I I , Hegulation CHIZUKO T5UKUMI, KIN-YAY0KO.I.A. A N D SHIZUO AKIKAA N D TADAM~TSU KIStIIMOTO NAOKI SIIIMRAHA Transporter Associated with Antigen Processing TIMELLIOTT

Recent Advmces in Understanding V(D)J Recombination MAKTI bt GELI .E HT

NF-KB as a Frequent Target for Inirnuno~uppressiveand Anti-Inflammatory Molecules PATKICK A. BAEUENLE A N D VIJAY R.

The Role of Ets Transcription Factors in the Developnrent and Function of the Mammalian Immune System A L E X A ~ D Ec. H BASSllK A N D JEFFHEY M. LEIDEh

BAICIIW4L

Mouse Mammary Tumor Virus: Immunological Interplays between Vinis and IIost A. LUTIIEH AND H A MACIIASANJIV OKBEA

Mechanism of Class I Assembly with PMicroglotlin and Loading with Peptide A N D DAVID R. LEE TEDH HANSEN Ilow Do Ljmphocytes Know Where to Go?: Current Concepts and Enigmas of Lymphocyte Homing MAHKOSALMI AND SIRPA JALKANEN

IgA Deficiency PETEHD. BUKKOWS A N D MAXD. COOPER

Plasma Cell Dyscrasias NOHlIii KO NISIIIMOTO, SACIIIKO SUEMATSU, A N D TADAMITSU KISIIIMOTO

Role of Cellular Immunity in Protection against HIV Infection SAKAII ROWLAND-JONES, RUSUNGTAN, A N D ANDREWMCMICHAEL

Anti-Tumor Necrosis Factor-a MAHCFELDUANN, MI(:IIAELJ. ELLIOTT, JAMES V . WOODY, AND RAVINUEK N. MAINI

High Endothelial Venules: Lymphocyte Traffic Control and Controlled Traffic KKAALA N D REINAE. MEBIUS GEOKC

INDEX

INDEX

431

432

CONTENTS OF RECENT VOLUMES

Volume 66 The Role of CD45 in Signal Transduction LOUISB. JUSTEMENT

The Intrinsic Coagulatioflinin-Forming Cascade: Assembly in Plasma and Cell Surfaces in Inflammation ALLENP. KAPLAN,KUSUMAM JOSEPH, Y O J ~ SHIBAYAMA, SESHA REDDIGARI, BERHANE CHEBHEHIWET, AND MICHAEL SILVERBEHC

HLA Class I1 Peptide Binding Specificity and Autoimmunity JUEHGEN HAMMER, TIZIANA STURNIOLO, CDW Cells in Human Immunodeficiency AND FRANCESCO SINIGAGLIA Virus Type I Pathogenesis: Cytolytic and Noncytolytic Inhibition of Viral Replication Role of Cytokines in Sepsis OTTO0. YANGAND BRUCED. WALKER C. ERIKHACK, LUCIEN A. AAHDEN, AND LAMBERTUS G. THIJS INDEX Role of Macrophage Migration Inhibitory Factor in the Regulation of the Immune Response CHRISTINE N. METZ AND RICHARD BUCALA

Volume 67 CUMULATIVE INDEX

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