Advances in Cancer Research Volume 86

Advances in

CANCER RESEARCH Volume 86

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Advances in

CANCER RESEARCH Volume 86

Edited by

George F. Vande Woude Van Andel Research Institute Grand Rapids, Michigan

George Klein Microbiology and Tumor Biology Center Karolinska Institute Stockholm, Sweden

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All Rights Reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the Publisher. The appearance of the code at the bottom of the first page of a chapter in this book indicates the Publisher’s consent that copies of the chapter may be made for personal or internal use of specific clients. This consent is given on the condition, however, that the copier pay the stated per copy fee through the Copyright Clearance Center, Inc. (222 Rosewood Drive, Danvers, Massachusetts 01923), for copying beyond that permitted by Sections 107 or 108 of the U.S. Copyright Law. This consent does not extend to other kinds of copying, such as copying for general distribution, for advertising or promotional purposes, for creating new collective works, or for resale. Copy fees for pre-2002 chapters are as shown on the title pages. If no fee code appears on the title page, the copy fee is the same as for current chapters. 0065-230X/2002 $35.00 Explicit permission from Academic Press is not required to reproduce a maximum of two figures or tables from an Academic Press chapter in another scientific or research publication provided that the material has not been credited to another source and that full credit to the Academic Press chapter is given.

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Contents

Contributors to Volume 86 vii

Coordinate Regulation of Translation by the PI 3-Kinase and mTOR Pathways Kathleen A. Martin and John Blenis I. II. III. IV. V.

Overview of Translational Regulatory Pathways 2 PI 3-K and mTOR Effectors which Regulate Translation 7 Regulation of Translational Effectors 10 Coordinated Translational Control 20 Conclusions 29 References 30

Histone Acetyltransferases and Deacetylases in the Control of Cell Proliferation and Differentiation Heike Lehrmann, Linda Louise Pritchard, and Annick Harel-Bellan I. II. III. IV. V. VI. VII. VIII. IX.

Introduction 42 Acetylation of Histones 43 Histone Acetyltransferases 44 Histone Deacetylases and Cell Cycle Regulation 48 Muscle Differentiation 51 Hematopoiesis 53 Huntington’s Disease 55 Histone Acetylation in Combination with Other Chromatin Modifications 56 Conclusion 57 References 58

Molecular Pathogenesis of Human Hepatocellular Carcinoma Michael A. Kern, Kai Breuhahn, and Peter Schirmacher I. Introduction 67 II. Morphology of Human Hepatocarcinogenesis 69

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Contents

Molecular Etiology 72 Host Carcinogenic Events 77 Functional Consequences 88 Therapeutic Implications 90 References 92

The Cell-Mediated Immune Response to Human Papillomavirus-Induced Cervical Cancer: Implications for Immunotherapy Gretchen L. Eiben, Markwin P. Velders, and W. Martin Kast I. II. III. IV. V.

Introduction 113 Human Papillomaviruses 114 Cellular Immunity to HPV 116 Immunotherapy against HPV-Induced Carcinomas 123 Conclusion 136 References 137

The T-Cell Response in Patients with Cancer Chiara Castelli and Markus J. Maeurer I. Definition of Immune Effector Functions 149 II. Defining the Bait for Antigen-Specific T Cells 152 III. The Role of the Coreceptor CD8 in Mediating Antitumor Restricted T-Cell Responses 158 IV. Tools to Measure ex Vivo T-Cell Avidity 160 V. Biomarkers or True Surrogate Markers? 163 VI. T-Cell Crossreactivity 172 VII. Questions of Specificity and Alternate T-Cell Effector Functions 173 References 176

The Life and Death of a B Cell ` and Peter H. Krammer Thierry Defrance, Montserrat Casamayor-Palleja, I. II. III. IV. V. VI.

Introduction 196 The Maintenance of B Cell Tolerance 197 The Regulation of B Cell Homeostasis 201 Control of the Specificity and Affinity of the Ab Response 206 Regulation of Survival in the Memory B Cell Compartment 212 Conclusive Remarks 216 References 218

Contributors

Numbers in parentheses indicate the pages on which the authors’ contributions begin.

John Blenis, Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115 (1) Kai Breuhahn, Institute of Pathology, University of Cologne, D-50931 Cologne, Germany (67) ` INSERM U404, “Immunity and VaccinaMontserrat Casamayor-Palleja, tion,” 69365, Lyon, Cedex 07, France (195) Chiara Castelli, Unit of Immunotherapy of Human Tumors, Istituto Nazionale per lo Studio e la Cura dei Tumori, 20133 Milano, Italy (149) Thierry Defrance, INSERM U404, “Immunity and Vaccination,” 69365, Lyon, Cedex 07, France (195) Gretchen L. Eiben, Cardinal Bernardin Cancer Center, Loyola University Chicago, Maywood, Illinois 60153 (113) Annick Harel-Bellan, CNRS UPR 9079, Institut Andr´e Lwoff, 94800 Villejuif, France (41) W. Martin Kast, Cardinal Bernardin Cancer Center, Loyola University Chicago, Maywood, Illinois 60153 (113) Michael A. Kern, Institute of Pathology, University of Cologne, D-50931 Cologne, Germany (67) Peter H. Krammer, Tumor Immunology Program, German Cancer Research Center, D-69120 Heidelberg, Germany (195) Heike Lehrmann, CNRS UPR 9079, Institut Andr´e Lwoff, 94800 Villejuif, France (41) Markus J. Maeurer, Department of Medical Microbiology, University of Mainz, 55101 Mainz, Germany (149) Kathleen A. Martin, Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115 (1)∗ Linda Louise Pritchard, CNRS UPR 9079, Institut Andr´e Lwoff, 94800 Villejuif, France (41) ∗ Present address: Departments of Surgery and of Pharmacology and Toxicology, Dartmouth Medical School, Lebanon, New Hampshire 03756

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Contributors

Peter Schirmacher, Institute of Pathology, University of Cologne, D-50931 Cologne, Germany (67) Markwin P. Velders, Cardinal Bernardin Cancer Center, Loyola University Chicago, Maywood, Illinois 60153 (113)

Coordinate Regulation of Translation by the PI 3-Kinase and mTOR Pathways Kathleen A. Martin and John Blenis Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115

I. Overview of Translational Regulatory Pathways A. The PI 3-Kinase Pathway B. The mTOR Pathway II. PI 3-K and mTOR Effectors which Regulate Translation A. 5’ TOP mRNA and S6K1 B. Capped mRNA and eIF-4E C. Other PI 3-K- and mTOR-Regulated Translation Initiation Factors III. Regulation of Translational Effectors A. 4E-BP1 Regulation B. Regulation of S6K1 C. S6K2 IV. Coordinated Translational Control A. Coordination of mRNA Splicing and Translation B. Coordinated Growth and Proliferation in Liver Regeneration C. PI 3-K, mTOR, Translation, and Cell Size D. Coordination of the PI 3-K and mTOR Pathways E. PI 3-K and mTOR Pathways in Cancer V. Conclusions References

Control of translation initiation is an important means by which cells tightly regulate the critical processes of growth and proliferation. Multiple effector proteins contribute to translation initiation of specially modified mRNAs that modulate these processes. Coordinated regulation of these translational effectors by multiple signaling pathways allows the integration of information regarding mitogenic signals, energy levels, and nutrient sufficiency. The mTOR protein, in particular, serves as a sensor of all of these signals and is thought to thus serve as a crucial checkpoint control protein. Signals from the mTOR pathway converge with mitogenic inputs from the phosphoinositide (PI) 3-kinase pathway on translational effector proteins to coordinately control cellular growth, size, and cell proliferation. The translational effectors regulated by the PI 3-kinase and mTOR pathways and their roles in regulation of cellular growth will be the primary focus of this review. C 2002, Elsevier Science (USA).

Advances in CANCER RESEARCH 0065-230X/02 $35.00

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Copyright 2002, Elsevier Science (USA). All rights reserved.

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I. OVERVIEW OF TRANSLATIONAL REGULATORY PATHWAYS A. The PI 3-Kinase Pathway Inputs from a wide range of extracellular signals converge on the phosphoinositide 3-kinase (PI 3-K) family enzymes, which, in turn, regulate a host of critical cellular processes, including proliferation, survival, motility, vesicle trafficking, transcription, and protein synthesis (Fig. 1) (Rameh and Cantley, 1999). The role of this pathway in cell survival, in large part through regulation of the effector kinase Akt/PKB, is widely appreciated (Downward, 1998). The importance of this pathway in maintaining the balance between proliferation, survival, and apoptosis is further underscored by the fact that several effectors are protooncogenes, and the functional antagonist of this pathway, PTEN, is a tumor suppressor gene frequently found to be mutated in human tumors (Cantley and Neel, 1999). Notably, the PI 3-K pathway coordinates the separable but related processes of cellular proliferation (an increase in cell number) and growth (an increase in cell mass) in large part through regulation of protein synthesis. In this review, we focus on the role of PI 3-K targets in regulation of protein synthesis and cell growth. Class I PI 3-Ks, including adapter (p85 family) and catalytic (p110 family) subunits, lie at the apex of an important growth factor stimulated signaling pathway that governs many aspects of cell behavior. While PI 3-kinase enzymes catalyze both lipid and protein phosphorylation, the lipid MITOGENS

PI 3-Kinase

phospholipid second messengers

PTEN

effector proteins

survival

proliferation

growth

motility

Fig. 1 The PI 3-kinase pathway regulates multiple cellular functions in response to mitogenic stimulation. Lipid second messengers generated by PI 3-K bind to and regulate multiple effector proteins, which in turn modulate numerous critical processes in mammalian cells.

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kinase-dependent functions are more thoroughly understood (Bondeva et al., 1998). PI 3-Ks phosphorylate phosphatidylinositols at the 3 -OH position to generate lipid second messengers that target a diverse array of downstream effector proteins, allowing coordinated modulation of multiple cellular processes. The major growth factor-induced PI 3-K-derived lipid messengers are phosphatidylinositol 3,4-bisphosphate (PI-3,4-P2) and phosphatidylinositol 3,4,5-trisphosphate (PI-3,4,5P3) (Rameh and Cantley, 1999). Binding of these lipids to effector proteins, especially those containing the plekstrin homology (PH) domain motif, may induce conformational changes and/or membrane targeting which alters their activities and access to other regulatory proteins or substrates. The PTEN lipid phosphatase dephosphorylates PI 3-K-generated phospholipid second messengers, and thus coordinately inhibits the activation of the numerous downstream effectors of PI 3-K (Cantley and Neel, 1999). The pharmacological inhibitors of PI 3-K, wortmannin and LY294002, have been important tools for dissecting the roles of this pathway and its effectors in vivo.

B. The mTOR Pathway The lipid-sensitive PI 3-K effectors that regulate translation will be discussed in detail in this review. Many of these mediators are also regulated by mTOR signaling, another pathway critical for translational control (Gingras et al., 2001b). The integration of signals from the PI 3-K and mTOR pathways is an important emerging theme in the coordinated regulation of cell growth and proliferation. A current model suggests that translational control is regulated by distinct, parallel signaling pathways that converge on common effectors: mTOR senses nutrient sufficiency, energy levels, and perhaps some mitogenic signals via phosphatidic acid (Fang et al., 2001) or PI 3-K/Akt (Fig. 2) (Nave, 1999; Sekulic et al., 2000), while growth factoror mitogen-induced translation is mediated largely by the PI 3-K pathway, with additional contributions from PKCs and MAPKs. Consistent with a role in a nutrient checkpoint, inhibition of the mTOR pathway can override PI 3-K and other growth factor-derived signals to multiple translational effectors. Thus, no discussion of PI 3-K signaling would be complete without an understanding of the inputs contributed by the mTOR pathway (Fig. 3). mTOR is the mammalian target of rapamycin (Sabers et al., 1995), also known as FRAP (FKBP and rapamycin-associated protein) (Brown et al., 1994), RAFT (rapamycin and FKBP12 target) (Sabatini et al., 1994), or RAPT (Chiu et al., 1994). This protein is a serine/threonine protein kinase that autophosphorylates and regulates exogenous substrates in translation pathways, including S6Ks and 4E-BPs (to be discussed in detail in following sections) (Brown et al., 1995; Brunn et al., 1997). mTOR signaling is

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NUTRIENTS (amino acids)

ENERGY (ATP)

MITOGENS (phosphatidic acid)

amino acid withdrawal

mTOR

TRANSLATION INITIATION

Fig. 2 Convergence of multiple upstream inputs on mTOR. Because mTOR integrates signals from multiple stimuli, and because its inhibition suppresses the activity of translation initiation effectors despite the presence of other mitogenic stimuli, mTOR can serve as an effective sensor in checkpoint control of protein synthesis.

inhibited by a complex formed between the lipophilic macrolide antibiotic rapamycin (also known as Sirolimus) and the ubiquitous cellular protein FKBP12 (FK506-binding protein 12 kDa) (Peterson et al., 2000). Rapamycin inhibits multiple important functions of mammalian cells, including protein synthesis, cell proliferation (Pyronnet and Sonenberg, 2001), growth factors PI3K

ERK

nutrients Rapamycin mTOR S6K

4E-BP

S6 eIF-4F structured-mRNA

40S 60S

5' TOP mRNA

Fig. 3 Coordinate regulation of translational effectors by growth factor and nutrient pathways. S6 kinases and 4E-BPs are regulated by multiple phosphorylations. Growth factor signals are transduced to these effectors by the PI 3-K, ERK, and mTOR pathways, and nutrient/energy sufficiency signals are mediated by the mTOR pathway. Both growth factor and nutrient/energy signals are required for full activation of these translational effector proteins.

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muscle hypertrophy (Rommel et al., 2001; Bodine et al., 2001), and cell growth/cell size (Fingar, et al., 2002). While mTOR and FKBP12 are ubiquitously expressed, the effects of rapamycin are more pronounced in certain cell types. Lymphocyte proliferation is highly sensitive to rapamycin, which likely accounts for the drug’s utility as an immunosuppressant that reduces organ transplant rejection in clinical trials (Podbielski and Schoenberg, 2001). mTOR contains a C-terminal domain with homology to the PI 3-K kinase domain. It does not appear to be a functional lipid kinase, but is a member of a growing family of proteins containing this homologous region known as PIKKs, or phosphoinositide kinase-related kinases (Hoekstra, 1997). Many members of the PIKK family are thought to serve checkpoint functions, including ATM, ATR, and DNA-PK, which act as sensors for DNA damage and repair (Hoekstra, 1997). A connection between DNA damage and mTORdependent signaling has been observed (Tee and Proud, 2000), suggesting that mTOR may also serve as a sensor of DNA damage. mTOR appears to function in a nutritional checkpoint that senses amino acid availability. Phosphorylation of critical translational effectors of mTOR, including S6K1 and 4E-BP1, is sensitive to the availability of leucine and other branched chain amino acids, and this effect is rapamycin-sensitive (Hara et al., 1998). Conversely, phosphorylation is inhibited by amino acid deprivation. Amino acid signaling and rapamycin may employ similar mTOR regulatory mechanisms, as a rapamycin-resistant mTOR mutant relieves the amino acid dependence of S6K1 (Iiboshi et al., 1999). The mechanism by which leucine and other amino acids signal to the mTOR pathway is not yet known but has been postulated to involve tRNA charging (Iiboshi et al., 1999), or leucine-stimulated increased mitochondrial metabolism via oxidative decarboxylation and activation of glutamate dehydrogenase (Xu et al., 2001; see Lynch, 2001, for review). While amino acid-sensitive mTOR effectors are also subject to PI 3-K regulation, a role for PI 3-K in amino acid signaling is unlikely, since PI 3-K and Akt activities are unaffected by amino acid stimulation or deprivation (Hara et al., 1998). As protein synthesis is the most energetically expensive function performed by the cell (Schmidt, 1999), integrated control of this process by a nutrientand energy-sensing checkpoint would be optimal. It has recently been suggested that mTOR may indeed serve as a sensor of energy levels (Dennis et al., 2001). Dennis et al. have shown that reduction of cellular ATP levels using the glycolytic inhibitor 2-deoxyglucose prevented insulin-induced activation of mTOR-regulated translational effectors. While all kinases require ATP, the apparent Km for ATP for mTOR was estimated to be greater than 1 mM, as opposed to 10–20 μM for most other known kinases (Dennis et al., 2001). This requirement for high, but physiological, concentrations of ATP may reflect the ability of mTOR to physically sense cellular energy

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levels, as well as explain the technical challenges that have faced researchers studying mTOR enzymatic activity in vitro. Nutrient regulation of the yeast mTOR homologs, TOR1 and TOR2, has been well documented (Rohde et al., 2001). The yeast TOR proteins have provided many important insights into the roles and regulation of this pathway in mammalian cells. While some mTOR functions may be mediated through phosphorylation of targets, yeast models have suggested that TOR 1/2 may have more sweeping effects on multiple downstream cellular targets through regulation of a phosphatase. In S. cerevisiae, TOR proteins stimulate the association of the phosphatases Sit4 (PP6 homolog) and Pph21/22 (PP2A homolog) with the regulatory protein Tap42. This association is inhibited by nutrient insufficiency or rapamycin, and different mutations in Tap42 can inhibit translation or confer rapamycin resistance, demonstrating the importance of this protein in TOR-mediated translational control (Di Como and Arndt, 1996; Jiang and Broach, 1999). α4, the murine homolog of Tap42, associates with the catalytic subunits of human phosphatases PP2A, PP6, or PP4 (Murata et al., 1997; Chen et al., 1998). α4 binding alters PP2A substrate specificity (Murata et al., 1997), and rapamycin inhibits the association of α4 and PP2A (Murata et al., 1997), suggesting that an analogous pathway may exist in mammals. Inhibition of S6K1 and 4E-BP1 by rapamycin may be mediated via phosphatase activity, and PP2A has been shown to associate with S6K1, but not with a rapamycin-resistant S6K1 mutant lacking the N- and C-terminal regulatory domains (Fig. 4) (Peterson et al., 1999). mTOR α4

Rapamycin or Amino acid withdrawal α4

PP2A-C PP2A-A

PP2A-C

altered substrate specificity

PP2A-B

S6K1

S6K1

(inactive)

(active)

Fig. 4 Hypothetical model for mTOR regulation of phosphatase activity and translational effectors in mammalian cells. Evidence from the TOR analogs in yeast, and from mammalian cell experiments, suggests a model by which mTOR may modulate the activity and/or substrate specificity of PP2A-family serine/threonine phosphatases by regulating binding of adapter subunits such as α4.

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Activation of a phosphatase, as opposed to inhibition of a kinase, is an attractive model to explain the rapid rapamycin-induced dephosphorylation of at least 12 different sites on 4E-BP1 and S6K1 with dissimilar motifs (Peterson et al., 1999). In this way, a nutrient-sensing checkpoint protein could coordinately inhibit multiple diverse translational effectors when the essential amino acid leucine is lacking, despite the continued presence of positive growth factor signals (i.e., from the active PI 3-K pathway). Recent work has suggested a novel mechanism by which serum mitogens may also signal through mTOR to translational effectors. Serum-induced phospholipase D (PLD) activity generates the second messenger phosphatidic acid (PA), which binds to the FRB domain of mTOR (the same domain that binds the FKBP12/rapamycin complex). This PA-mediated signaling stimulates phosphorylation and activation of S6K1 and 4E-BP1 in a rapamycinand wortmannin-sensitive manner. Interestingly, PA does not alter the kinase activity of mTOR. The authors suggest a model in which S6K1 and 4E-BP1 integrate nutrient signals through mTOR, as well as mitogenic signals via PI 3-K and PA/mTOR (Fang et al., 2001). The roles and regulation of translational effector targets of both the mTOR and PI 3-K pathways will be presented in detail below.

II. PI 3-K AND mTOR EFFECTORS WHICH REGULATE TRANSLATION Two downstream targets of PI 3-K and mTOR signaling, S6K1 and 4EBP1/eIF-4E, are major regulators of protein synthesis (Fig. 3). These effectors will be introduced here, followed by a discussion of the intermediates that transduce these signals to these targets.

A. 5’ TOP mRNA and S6K1 Translation initiation rates for different mRNAs can vary dramatically depending on the degree and type of secondary structure present in the 5 untranslated region of the message. One modification, the 5 terminal oligopyrimidine tract (5 TOP), a stretch of 4–14 pyrimidine bases at the extreme 5 end of the message, marks a particular subset of mRNAs for inefficient translation initiation under basal conditions (Meyuhas, 2000). Insulin or other growth factor stimulation rapidly induces translation of these messages in a rapamycin-sensitive manner (Jefferies et al., 1994). These 5 TOP mRNAs typically encode components of the protein synthetic machinery itself, including ribosomal proteins and translation elongation factors. Induction

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of 5 TOP mRNA translation upregulates ribosome biogenesis and overall translational capacity (Meyuhas, 2000). Translation initiation of 5 TOP mRNAs is thought to be mediated by the 40S ribosomal protein S6. Growth factor-stimulated phosphorylation of S6 at multiple C-terminal residues correlates with translation initiation of these 5 TOP mRNAs (Jefferies et al., 1997). S6 is phosphorylated by the 70-kDa S6 kinase 1 (p70 S6K1) (Kozma et al., 1990), a ubiquitously expressed mitogen- and amino acid-sensitive protein kinase. S6K1 activation and subsequent S6 phosphorylation is a conserved mitogenic response, as all mitogenic stimuli, including growth factors, protooncogene products, phorbol esters, and cytokines induce S6K1 activity (Dufner and Thomas, 1999). Enhanced ribosome biogenesis and translational capacity is a conserved response to growth signals. Recent data have questioned whether S6K1 regulates 5 TOP-mediated mRNA translation through S6 phosphorylation, suggesting that this process is instead dependent on PI 3-K and mTOR signaling, but not on S6K1 or the ribosomal S6 protein (Tang et al., 2001). Although many aspects of this study contradict the published work of others (Jefferies et al., 1997; Lee-Fruman et al., 1999), this and other studies suggest the likely possibility that other as yet unidentified targets for the S6 kinases exist that are important in mediating their physiological function (Shima et al., 1998; Montagne et al., 1999). Notably, mice lacking S6K1 exhibit a small-animal phenotype despite normal phosphorylation of S6 and 5 TOP translation (likely due to redundant function by S6K2) (Shima et al., 1998), thus other S6K1 targets may contribute to regulation of animal size. Whether or not ribosomal S6 phosphorylation modulates 5 TOP mRNA translation, it is likely an essential component of a proliferation checkpoint mechanism as revealed by conditional S6 deletion studies (Volarevic et al., 2000) (see Section IV.B).

B. Capped mRNA and eIF-4E A methyl cap structure (m7GpppN) is found at the 5 end of all mRNAs transcribed in the nucleus. Interaction of this cap with translation initiation factor eIF-4E (eukaryotic initiation factor-4E) is an important step in loading these messages on the ribosome (Sonenberg and Gingras, 1998). The capbinding subunit eIF-4E is present in rate limiting quantities relative to other components of the translational apparatus (Rau et al., 1996), and thus serves as a key regulatory translation factor. Notably, eIF-4E provides an additional degree of translational control toward mRNAs containing a high degree of secondary structure in the 5 UTR (Rousseau et al., 1996). These stable secondary structures include hairpin loops and upstream AUGs, modifications which mark mRNAs encoding proteins important for cell growth and cell

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Fig. 5 Regulation of eIF-4F formation by 4E-BP1. eIF-4E is sequestered by hypophosphorylated 4E-BP1. Phosphorylation of 4E-BP1 by growth factor- and nutrient-sensing signaling pathways allows release of eIF-4E, which subsequently associates with the scaffolding protein eIF-4G and helicase eIF-4A to form the eIF-4F complex.

cycle progression, including growth factors, receptors, cyclins, and signaling proteins (Sonenberg and Gingras, 1998). Efficient translation initiation of these highly structured mRNAs requires unwinding by a helicase, eIF-4A. eIF-4E links these messages to the helicase by binding both the mRNA cap and a scaffolding protein, eIF-4G, which also binds eIF-4A. This complex of eIF-4E, eIF-4G, and eIF-4A is referred to collectively as eIF-4F, and is important for recruiting the 40S ribosome to the message (Fig. 5) (Sonenberg and Gingras, 1998). Like 5 TOP mRNAs, eIF-4E-dependent growth regulatory messages are poorly translated in quiescent cells, and translation of these structured mRNAs is similarly upregulated following growth factor stimulation. In quiescent cells, eIF-4E is sequestered by the 4E-BP (eIF-4E-binding protein) family repressor proteins (Pause et al., 1994) (4E-BP1 is also known as PHAS-I; phosphorylated, heat- and acid-stable-Insulin responsive (Lin et al., 1994)). In their hypophosphorylated state, 4E-BPs bind eIF-4E in a manner mutually exclusive with eIF-4G, a scaffolding subunit of the eIF-4F initiation complex. This complex binds capped mRNAs (Haghighat, 1995; Marcotrigiano et al., 1997). 4EB-P1 binding to eIF-4E inhibits formation of eIF-4F complexes. This inhibition is relieved when 4E-BPs are phosphorylated in response to growth factors and dissociate from eIF-4E (Sonenberg and Gingras, 1998). In addition to regulation by 4E-BPs, phosphorylation of eIF-4E itself by the ERK effectors Mnk1/2 is thought to promote translation initiation (Waskiewicz et al., 1997; Scheper et al., 2001). Phosphorylation of eIF-4E alone, however, is not sufficient for eIF-4F assembly (Herbert et al., 2000). 4E-BP1, the best characterized 4E-BP, is regulated by signals from

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the PI-3K, ERK, and mTOR pathways, and these inputs will be discussed in detail below.

C. Other PI 3-K- and mTOR-Regulated Translation Initiation Factors Initiation factor eIF-4G also likely integrates PI 3-K and mTOR signals. It undergoes serum- or insulin-stimulated phosphorylation on at least three sites that are sensitive to wortmannin and rapamycin, but the upstream kinases are not yet known (Raught et al., 2000). The effects on eIF-4G function are also unknown, but it has been postulated that such phosphorylations may alter its structure, which may modulate its scaffold function during eIF-4F assembly. Another translational regulator responsive to both PI 3-K and mTOR signals is eIF-4B. This RNA binding protein may function as a link between ribosomal and messenger RNAs, and stimulates eIF-4A helicase activity (Rozen et al., 1990). eIF-4B is phosphorylated in response to growth factors in a rapamycin- and wortmannin-sensitive manner, and is a substrate for S6K1 in vitro (Morley and Traugh, 1993). The effect of phosphorylation is not known, but it is likely that regulation of eIF-4B may have the most profound effect on translation of mRNAs with highly structured 5 UTRs. A translational effector that appears to be regulated by the PI 3-K but not by the mTOR or MAPK pathways is eIF-2B. This guanine nucleotide exchange factor is a component of eIF2, which catalyzes binding of the initiator Met-tRNA to the ribosome, an important step in initiation (Price and Proud, 1994). The eIF-2Bε subunit is inhibited when phosphorylated by GSK3 (Welsh et al., 1998). Insulin relieves this suppression through an Aktinduced inhibition of GSK3 (Cross et al., 1995; Takata et al., 1999). Thus, the PI 3-K pathway regulates translation through multiple effectors of the initiation apparatus, including S6K1, 4E-BP1, eIF-4G, eIF-4B, and eIF-2B.

III. REGULATION OF TRANSLATIONAL EFFECTORS A. 4E-BP1 Regulation 4E-BPs are the major factors regulating eIF-4E activity, and thus, subsequent formation of the eIF-4F initiation complex. Hypophosphorylated 4E-BP1 binds eIF-4E with high affinity (Pause et al., 1994; Lin et al., 1994). The 4E-BP1–eIF-4E interaction is disrupted following sequential 4E-BP1

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Fig. 6 Regulation of 4E-BP1 phosphorylation. Sequential phosphorylation of 4E-BP1 is mediated by nutrient- and growth factor-sensing signaling pathways. Phosphorylation of the priming sites Thr37 and Thr46 precedes phosphorylation of Thr70 and Ser65, to allow release of eIF-4E.

phosphorylation at multiple sites. Thr37 and Thr46 are basally phosphorylated and are substrates for phosphorylation by mTOR in vitro (Fig. 6) (Gingras et al., 1999; Mothe-Satney et al., 2000a). 4E-BP1 undergoes ordered, sequential phosphorylation, with Thr37 and Thr46 serving as prerequisite priming sites for serum-induced phosphorylation at Thr70 and Ser65 (Gingras et al., 1999, 2001a; Mothe-Satney et al., 2000b). Studies with phosphospecific antibodies suggest that Ser65 may be the final site to be phosphorylated (Mothe-Satney et al., 2000a; Gingras et al., 2001a). This site is also likely critical for disruption of the eIF-4E interaction, as the eIF-4E binding site in 4E-BP1 is flanked by Thr46 and Ser65 (Haghighat, 1995), and phosphorylation of Ser65 prevents this association in vitro. Due to the multiple phosphorylations, 4E-BP1 from stimulated cells migrates as a triplet of bands in SDS-polyacrylamide gels. Only the most slowly migrating band reacts with phospho-Ser65, and this species fails to copurify with eIF-4E (Mothe-Satney et al., 2000a). Like S6K1, Ser65 and Thr70 are regulated by both PI 3-K and mTOR pathways, as they are sensitive to wortmannin and rapamycin (Gingras et al., 1998; Mothe-Satney et al., 2000a). S6K1 is not an in vivo 4E-BP1 kinase, but S6K1 and 4E-BP1 are thought to function in parallel pathways which bifurcate downstream of mTOR. S6K1 and 4E-BP1 may share a common rapamycin-sensitive activator, since overexpression of S6K1 can inhibit 4E-BP1 phosphorylation (von Manteuffel et al., 1997). One report states that Ser65 and Thr70 are also phosphorylated by mTOR in vitro, but only when

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mTOR is incubated with an “activating” antibody, mTAb1 (Mothe-Satney et al., 2000a). Alternately, dissociation of eIF-4E from 4E-BP1 may involve 4E-BP1 phosphorylation by an mTOR-associated kinase (Heesom and Denton, 1999). Another model to explain 4E-BP1 inactivation is that these sites are phosphorylated by PI 3-K-regulated kinases, and dephosphorylated by PP2A-type phosphatases, which are activated following mTOR inhibition (Peterson et al., 1999). Akt appears to be an important regulator of 4E-BP1 in vivo, as expression of a constitutively active mutant induces 4E-BP1 phosphorylation, while a dominant negative is inhibitory (Gingras et al., 1998; Dufner et al., 1999; Takata et al., 1999). Akt may regulate a 4E-BP1 kinase, as Akt itself does not directly phosphorylate 4E-BP1 in vitro (Gingras et al., 1998). It has recently been shown that the mTOR-regulated nPKCδ (Parekh et al., 1999) can phosphorylate 4E-BP1 in a rapamycin-sensitive manner, although the target site has not yet been identified (Kumar et al., 2000a). Through an inhibitory association with mTOR, the c-abl tyrosine kinase is a negative regulator of 4E-BP1 function in response to DNA damage (Kumar et al., 2000b). Finally, 4E-BP1 phosphorylation may occur by a phorbol ester-stimulated, PI 3-K independent mechanism which may be mediated by the MEK/ERK cascade (Herbert et al., 2000, 2002). Basal MEK activity is also required for insulin-stimulated 4E-BP1 phosphorylation and eIF-4F assembly cascade (Herbert et al., 2000). ERK2 can phosphorylate 4E-BP1 in vitro (Fadden et al., 1997) and in vivo in vascular smooth muscle cells (Rao et al., 1999). Other 4E-BP family members, including 4E-BP2 and 4E-BP3 (Poulin et al., 1998) have been identified. These are related to 4E-BP1, with the greatest sequence conservation in the eIF-4E-binding motif (Gingras et al., 2001b). These proteins seem to serve a similar function and are likewise regulated by phosphorylation. 4E-BP1 and −2, but not 4E-BP3, contain a four-amino acid motif (Arg-Ala-Ile-Pro “RAIP”) in the N-terminus that is required for efficient phosphorylation (Tee and Proud, 2002). Early studies with 4E-BP2 suggest that slight differences in regulation may lead to different kinetics of eIF-4E dissociation (Gingras et al., 2001b; Grolleau, 1999). Thus, the function of these isoforms may allow for tissue-specific differences in growth factor stimulated translational responses (Grolleau, 1999).

B. Regulation of S6K1 The PI 3-K and mTOR pathways are major signaling pathways regulating S6K1 activity (Chung et al., 1992, 1994). The S6K1 (and 4E-BP1) requirement for inputs from both the PI 3-K and mTOR pathways, as well as by other mitogen activated signaling pathways, suggests a mechanism by which

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cells can integrate nutrient capacity and growth factor stimulated protein synthesis. S6K1 exists as two isoforms. The 70-kDa αII isoform is largely cytosolic in localization. An 85-kDa αI isoform is identical to p70 with the exception of an additional 23 amino acids at the N-terminus encoding a nuclear localization signal (Coffer and Woodgett, 1991; Reinhard et al., 1994). The function of nuclear S6K1 is unclear, but S6K1 can phosphorylate the transcription factor cremτ , suggesting a possible role in transcriptional control (de Groot et al., 1994). The two S6K1 isoforms appear to be regulated similarly in all systems examined, except for slightly delayed kinetics of p85 activation relative to p70 in response to pressure overload in cardiomyocytes (Laser et al., 1998). S6K1 kinase activity is regulated by at least nine growth factor-induced phosphorylation events. The first of these, targeting multiple sites in the C-terminus, is thought to relieve autoinhibition by intramolecular interactions (Cheatham et al., 1995; Weng et al., 1995). The kinase domain at the core of the molecule is flanked by N- and C-terminal regulatory domains (Fig. 7). A model of S6K1 activation based on structure/function mutagenesis studies suggests that acidic amino acids in the N-terminus interact with basic residues in the C-terminus to stabilize an autoinhibitory inactive conformation. In this inactive state, a pseudosubstrate region in the C-terminus with high homology to ribosomal S6 occludes the kinase

Fig. 7 Structure of S6 kinases. Shown is a schematic of the primary structures of the shorter isoforms of S6K1 (αII) and S6K2 (βII). Mitogen-stimulated phosphorylation sites are indicated by amino acid number. The activation loop (T229/228 TFCGT), linker region (S371/370 SPDD) and hydrophobic motif (T389/388 FLGFTY) sites are perfectly conserved between S6K1 and S6K2, but there is some divergence in the proline-directed C-terminal sites. S6K2 exhibits regions of divergence from S6K1 in the N- and C-terminal regulatory domains, including the presence of a C-terminal polyproline-rich domain and nuclear localization signal.

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domain (Cheatham et al., 1995; Weng et al., 1995). An early step in S6K1 activation is mitogen-induced phosphorylation of C-terminal regulatory sites (Ser404, Ser411, Ser418, Thr421, Ser424), which disrupt this interaction, perhaps mediated by ERK or p38 MAP kinases (Weng et al., 1998; A. Romanelli, unpublished results). Phospho-specific antibodies reveal that these sites are also sensitive to inhibition by rapamycin or wortmannin (Weng et al., 1998). Phosphorylation of sites in an internal regulatory domain and the kinase domain follow, including Ser371, Thr389, and Thr229. Ser371 phosphorylation is essential for S6K1 activity, and is mediated by an as yet undetermined mechanism but is insensitive to rapamycin and wortmannin (Moser et al., 1997; A. Romanelli, unpublished observations). Thr229, located in the catalytic activation loop, is essential for kinase activity and sensitive to wortmannin and rapamycin (Weng et al., 1998). This site is phosphorylated by phosphoinositide-dependent kinase 1 (PDK1), a constitutively active kinase whose subcellular localization and access to substrates is regulated by PI 3-K-derived phospholipids (Williams et al., 2000; Alessi et al., 1998; Pullen et al., 1998). The importance of Thr229 is highlighted by the fact that an inhibitor of PDK1 signaling, n-alpha-tosyl-lphenylalanyl chloromethyl ketone (TPCK), is a potent inhibitor of S6K1 activity (Grammer and Blenis, 1996; Ballif et al., 2001). Further, mutation of this site (T229A) abolishes S6K1 activity and subsequent Thr389 phosphorylation (Weng et al., 1998). Phosphorylation of Thr389 in the regulatory domain appears to be central to S6K1 activation, as it is exquisitely sensitive to inhibition by rapamycin, and thought to be the final and rate-limiting step in S6K1 activation (Weng et al., 1998; A. Romanelli, unpublished observations). While the importance of this amino acid in S6K1 function is universally accepted, the mechanism underlying Thr389 regulation is currently controversial. This site has been reported to be directly phosphorylated by mTOR in vitro (Burnett et al., 1998a). However, Thr389 is still phosphorylated in a mitogen-sensitive manner in a truncation mutant of S6K1 that is rapamycin resistant, indicating that mitogens can stimulate Thr389 phosphorylation independently of mTOR (S. Schalm, unpublished observations). The NIMA family kinases NEK6/7 have been shown to phosphorylate Thr389 in vivo and in vitro (Belham et al., 2001). Activity of these kinases, however, is only weakly stimulated by insulin and partially sensitive to wortmannin, while Thr389 phosphorylation is entirely wortmannin sensitive, suggesting that other mechanisms may contribute to Thr389 regulation. Regulation of the critical Thr389 site is also dependent on PDK1, as IGF-I fails to induce Thr389 phosphorylation in PDK1 null cells (Williams et al., 2000), and TPCK inhibits phosphorylation of both Thr229 and Thr389 (Ballif et al., 2001). It is also possible that S6K1 autophosphorylation

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contributes to the phosphorylation state of Thr389, as we have observed that optimal and prolonged Thr389 phosphorylation does not occur in a kinase inactive S6K1 (K100R) point mutant (A. Romanelli, unpublished observations). This mechanism is consistent with the requirement for PDK1 phosphorylation of the catalytic activation loop site (Thr229). Finally, the many mitogen-stimulated phosphorylation sites in S6K1, including Thr389, are rapidly dephosphorylated after rapamycin treatment. As discussed earlier, an mTOR-regulated phosphatase is a possible mechanism which could rapidly inhibit many sites regulated by diverse upstream kinases (Peterson et al., 1999). A highly conserved sequence in the N-terminus of S6K1 and S6K2 (amino acids 5 to 9 of S6K1) has recently been revealed to be a critical TOR signaling (TOS) motif that is essential for mTOR-dependent signaling. The TOS domain mediates mTOR regulation of two distinct regions of S6K1: this motif is required for phosphorylation of Thr389, as well as for release of a negative regulatory mechanism mediated by the S6K1 C-terminus. The importance of the TOS motif in mTOR signaling is underscored by its identification in other mTOR-regulated proteins. This motif is conserved throughout evolution in the C-termini of 4E-BPs. Mutation of the TOS motif in 4E-BP1 similarly blocks its mitogen-dependent phosphorylation (Schalm and Blenis, 2002). It has been long known that conventional PKCs (cPKCs) can also contribute to S6K1 activation (Blenis and Erikson, 1986), but the precise molecular mechanism is still not well documented. PDGF receptor mutants deficient in PLCγ binding and subsequent cPKC activation are impaired in signaling to S6K1, while mutants lacking the PI 3-K binding site suggest that the PI 3-K pathway is the major contributor to S6K1 activation (Chung et al., 1994). Separable PI 3-K and cPKC inputs were also documented in B cells (Monfar et al., 1995). Similarly, S6K1 activation by B cell antigen receptor cross-linking is dependent on both PI 3-K and cPKC-dependent pathways and involves the tyrosine kinase Syk (Li et al., 1999).

1. SUBCELLULAR LOCALIZATION AND REGULATION OF S6Ks S6K1 is activated by multiple PI 3-K effectors, including Cdc42, Rac (Chou and Blenis, 1996), PDK1 (Alessi et al., 1998; Pullen et al., 1998), and atypical PKCζ (Romanelli et al., 1999) and PKCλ (Fig. 8) (Akimoto et al., 1998). While PDK1 directly phosphorylates S6K1 (Alessi et al., 1998; Pullen et al., 1998), the mechanism of activation by these other effectors is an ongoing subject of investigation. The low-molecular-weight G proteins Cdc42 and Rac activate S6K1 independent of the p38 or JNK pathways (Chou and Blenis, 1996). These G proteins, however, can bind to S6K1 in vivo in a

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PI3Kp110

Cdc42/Rac

PKCζ

mTOR

PI3K p85

PDK1

Akt

PP2A?

S6K1

Fig. 8 Regulation of S6K1 by PI 3-K and mTOR. The PI 3-K effectors Cdc42, Rac, PKCζ , PDK1, and Akt have all been implicated in regulation of S6K1. Only PDK1 is known to directly phosphorylate S6K1 (at Thr229). Evidence suggests that mTOR may directly phosphorylate S6K1 at Thr389, and/or may regulate all S6K1 phosphorylation sites through regulation of a PP2A-type phosphatase. Multiple PI 3-K effectors exist in a complex with S6K1, and the PI 3-K p85 adapter subunit may mediate association of mTOR and S6K1.

rapamycin- and wortmannin-insensitive manner. This association is required for Cdc42 activation of S6K1. Isoprenylation of the G protein is also required (Chou and Blenis, 1996). These data suggest that interaction of these G proteins may activate S6K1 by inducing a conformational change in the kinase, and/or by targeting the kinase to a membrane, bringing it in proximity with other membrane-localized activators including PDK1, aPKCs, and Akt. We have also reported that S6K1 coimmunoprecipitates with PDK1 or PKCζ , and that PDK1 and PKCζ can associate with each other (Romanelli et al., 1999). Constitutively active mutants of Akt support a membrane targeting model, as only those which localize to the plasma membrane are capable of activating S6K1 (Dufner et al., 1999). These combined data suggest that S6K1 may participate in a complex of PI 3-K-regulated effector proteins which facilitates signaling in this pathway. The existence of such scaffolding mechanisms has been suggested for other pathways, including MAPK pathways (Kholodenko et al., 2000; Garrington and Johnson, 1999; Burack and Shaw, 2000). It is not known whether a common central scaffolding molecule exists in this PI 3-K model, but other evidence suggests that these interactions may take place at the plasma membrane and/or cytoskeleton. Finally, coprecipitation of S6K1, mTOR, and the p85 subunit of PI 3-K suggest that a ternary (or larger) complex integrates S6K1 activation by the PI 3-K and mTOR pathways (Gonzalez-Garcia et al., 2002) (see Section IV.D for further detail).

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The S6K1 activators Cdc42 and Rac are also important regulators of the cytoskeleton that mediate many structural changes contributing to cell motility (Erickson and Cerione, 2001). Several studies suggest that S6K1 might also play a role in motility, as it has been shown to colocalize with stress fibers, and that thrombin-induced elongation and organization of stress fibers is rapamycin sensitive in fibroblasts (Crouch, 1997). Furthermore, S6K1 colocalizes with actin arc structures at the leading edge of migrating cells (Berven and Crouch, 2000). Interestingly, several factors known to regulate and associate with S6K1, the atypical PKCs ζ or λ (Romanelli et al., 1999; Akimoto et al., 1998) and Cdc42 (Chou and Blenis, 1996), have recently been reported to associate with each other in a GTP-dependent manner (Coghlan et al., 2000). This study demonstrated that activated Cdc42 induced stress fiber loss, and that this process required active aPKCs. A role for S6K1 in this process was not addressed, but these data further document the clustering of similarly regulated signaling proteins at cytoskeletal structures. Two-hybrid and biochemical analyses identified the F-actin binding protein neurabin as a binding partner for S6K1 in neural cells (Burnett et al., 1998b). A PDZ domain in this neural-specific cytoskeletal-associated protein binds to the extreme C-terminus of S6K1 in a serum- and rapamycinindependent manner. The mRNAs for neurabin and S6K1 colocalize in various brain structures, including the hippocampus and cerebellum, and it has been suggested that these proteins colocalize at nerve terminals. Coexpression of S6K1 and neurabin in nonneural tissues leads to a modest induction of S6K1 activity (Burnett et al., 1998b), supporting the model that “scaffold”-mediated targeting of S6K1 to cellular locales enriched in regulatory molecules may facilitate S6K1 activation. Subcellular localization is important for regulation of the mTOR pathway, as well as for PI 3-K effectors, and perhaps especially so for common effectors of both pathways such as S6Ks. The ubiquitously expressed protein gephyrin serves to cluster glycine receptors at postsynaptic nerve terminals, and has been identified as a binding partner for mTOR (Sabatini et al., 1999). mTOR interaction with gephyrin mediates its subcellular localization and is essential for its ability to regulate S6K1 and 4E-BP1. Furthermore, an intriguing study has suggested that nuclear shuttling of mTOR may be necessary for mitogen-stimulated activation of translational effectors, as inhibition of nuclear export using leptomycin B inhibited phosphorylation of both S6K1 and 4E-BP1 (Kim and Chen, 2000). Such nuclear/cytoplasmic shuttling of mTOR suggests a possible regulatory mechanism for primarily nuclear isoforms such as p85-S6K1 or the S6K2 proteins. It is likely that future studies will further clarify the mechanisms by which components of signaling pathways are brought together to function efficiently and specifically.

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C. S6K2 For many years it was thought that S6K1 was the only in vivo S6 kinase. However, several groups recently identified a homolog closely related to S6K1, now called S6K2 (also called p70β, SRK) (Shima et al., 1998; Gout et al., 1998; Lee-Fruman et al., 1999; Koh et al., 1999). In addition to isolation based on database searches for novel proteins with homology to S6K1 (Shima et al., 1998; Gout et al., 1998; Lee-Fruman et al., 1999; Koh et al., 1999), S6K2 was identified when normal S6 phosphorylation and 5 TOP mRNA translation was discovered in cells derived from mice lacking both p70 and p85 isoforms of S6K1 (Shima et al., 1998). As the mRNA for S6K2 was found to be upregulated in these mice, it is likely that S6K2 may supply the S6 phosphorylation function in vivo in the absence of S6K1. Using a rapamycin-resistant S6K2 mutant, we find that S6K2 is indeed an in vivo S6 kinase, as S6 phosphorylation persists in rapamycin-treated cells stably overexpressing this mutant, when S6K1 and other mTOR-effectors are maximally inhibited (K. Martin, unpublished observations). Whether additional in vivo S6 kinases might also exist remains to be determined. Mice lacking S6K1 through homozygous deletion demonstrate a smallanimal phenotype despite normal S6 phosphorylation and 5 TOP mRNA translation (Shima et al., 1998), suggesting that S6 regulation is not the critical determinant of animal size, but that S6K1 may mediate other important nonredundant functions that contribute to size regulation. Because, unlike S6K1, both S6K2 isoforms encode a common C-terminal nuclear localization signal (Koh et al., 1999), it is likely that S6K2 mediates unique nuclear functions. Several structural features and differential regulation further suggest that S6K2 mediates distinct functions. While S6K2 is highly homologous to S6K1 overall, there are regions of divergence in both the amino- and carboxyl-terminal regulatory regions (Fig. 7). In addition to the unique Cterminal NLS, S6K2 contains a C-terminal polyproline-rich region absent in S6K1. The role of the polyproline domain is not yet known, but deletion of this domain reportedly does not affect wortmannin or rapamycin sensitivity (Gout et al., 1998). Chimeras swapping the region of the polyproline domain between S6K1 and S6K2 failed to reveal an obvious function for this domain (S. Schalm, unpublished observations). S6K2 is activated by the same stimuli that activate S6K1, and is potently inhibited by wortmannin and rapamycin (Shima et al., 1998; Gout et al., 1998; Lee-Fruman et al., 1999; Koh et al., 1999), suggesting that it also functions as an effector of the PI 3-K and mTOR pathways. Regulation of S6K2 is largely similar to S6K1, with some intriguing differences, likely arising due to sequence variations and/or differential subcellular localization. Despite the primarily nuclear expression of S6K2, it is regulated

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by the PI 3-K effectors Cdc42, Rac, PDK1, PKCζ , (Martin et al., 2001a), and Akt (Koh et al., 1999). One difference in S6K regulation was that atypical PKCζ was a more potent activator of S6K2. Interestingly, point mutation which destroys the S6K2 nuclear localization signal modestly potentiates its activation by PI 3-K effectors (Martin et al., 2001a). This regulation by cytosolic effectors suggests that S6K2 may exit the nucleus during the course of activation. Like S6K1, S6K2 exists as two alternately spliced isoforms, which differ by an additional N-terminal 13 amino acids present in S6K2β1 but lacking in S6K2β2 (Gout et al., 1998). Although the C-terminal nuclear localization signal is common to both S6K2 isoforms (Koh et al., 1999), it has been suggested that the unique basic sequence at the N-terminus of S6K2β1 contributes to its nuclear localization as well, as overexpressed S6K2β1 is found primarily in the nucleus, while overexpressed S6K2β2 can be found in both the nucleus and the cytosol (Minami et al., 2001). It has been suggested that S6K2 is less sensitive to rapamycin or wortmannin than is S6K1 (Gout et al., 1998; Minami et al., 2001). It is likely, however, that this reflects a difference in inactivation, as opposed to initial activation. Minami et al. observed that S6K2 overexpressed in cells continually cultured in 10% serum was less sensitive to inhibition by addition of rapamycin or wortmannin to the medium. We and others, however, have found that activation of S6K2 in serum-starved cells by the addition of insulin or serum is nearly completely inhibited by rapamycin or wortmannin (Lee-Fruman et al., 1999; Koh et al., 1999). The most notable functional difference between S6K1 and S6K2 is the role of the C-terminal autoinhibitory domain. Deletion of this regulatory region, which lies just C-terminal to the kinase domain, has a modest inhibitory effect on S6K1, yet has a potent stimulatory effect on S6K2 that is dramatically enhanced by coexpression of PI 3-K effectors, such as Cdc42 or PDK1 (Martin et al., 2001b). These data suggest that this domain participates in potent repression of S6K2 kinase activity. Furthermore, this repression of S6K2 may be relieved by inputs from the MEK/ERK1/2 pathway, as fulllength S6K2 is highly sensitive to inhibition by the MEK inhibitor U0126, while S6K1 is far less sensitive to MEK inhibition (Martin et al., 2001b). The S6K2 U0126 sensitivity was evident for EGF, a potent ERK agonist, but not for insulin, a poor ERK agonist in the HEK293 cells used. Activation of S6K2 by G protein-coupled receptor agonists in cardiomyocytes was also found to be highly MEK dependent (Wang et al., 2001). The MEK pathway has been implicated in regulation of the C-terminal phosphorylation sites in both S6K1 and S6K2 (Lenormand et al., 1996; Scott and Lawrence, 1997; Mukhopadhyay et al., 1992; Herbert et al., 2000; A. Romanelli, unpublished results), but may play a more critical role in the initial steps of S6K2

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activation. It is also possible that the presence of the polyproline-rich domain in proximity to the C-terminal regulatory phosphorylation sites may confer differential S6K2 regulation. While S6K1 contains a motif at its C-terminus which may allow its regulation by PDZ domain proteins such as neurabin (Burnett et al., 1998b), S6K2 lacks such a motif. The absence of a C-terminal PDZ-binding sequence may account for some differences in S6K2 regulation by this domain (Martin et al., 2001b). However, both S6K1 and S6K2 are regulated by isoprenylated Cdc42 and Rac (Chou and Blenis, 1996; Martin et al., 2001a), suggesting that membrane association may be important in regulation of both kinases.

IV. COORDINATED TRANSLATIONAL CONTROL A. Coordination of mRNA Splicing and Translation Recent work on regulation of mRNA splicing by the low-molecular-weight G protein Cdc42 suggests that the PI 3-K and mTOR pathways may coordinate the related processes of mRNA splicing and translation, in part, through S6K1 (Wilson et al., 2000). Cdc42 has been shown to be an effector of the PI 3-K pathway, and an activator of both S6K1 and S6K2 (Chou and Blenis, 1996; Martin et al., 2001a). Activated Cdc42 stimulates binding of the capbinding complex (CBC) to nuclear capped mRNAs (Wilson et al., 1999) and stimulates pre-mRNA splicing independent of its PAK, ACK, or WASP effector functions (Wilson et al., 2000). Notably, splicing stimulated by an activated Cdc42 mutant is sensitive to rapamycin, but not to wortmannin. Signaling from Cdc42 to S6K1 has been proposed to mediate this effect, as S6K1 was found to phosphorylate the CBC subunit CBP80 in vitro at growth factor-stimulated rapamycin-sensitive sites. It is likely that PI 3-K lies upstream in this pathway, but that the downstream constitutively activated Cdc42 mutant employed is resistant to the effects of wortmannin, as overexpression of p70 S6K1 and constitutively active PI 3-K enhances splicing in the presence of endogenous Cdc42. The S6K1 phosphorylation sites flank a nuclear localization signal, but ablation or acidic substitution of the sites does not alter nuclear localization of CBC. S6K1 phosphorylates CBP80 in vitro, but it has not yet been determined whether the cytosolic p70 or nuclear p85 S6K1, or even the nuclear S6K2 isoforms, may mediate this event in vivo. One model suggests that the nuclear CBC escorts the capped mRNA complex from the nucleus to the cytosol, where it binds to the eIF-4E capbinding translation initiation factor (Wilson et al., 2000; Visa et al., 1996). Thus, Cdc42/S6K inputs may coordinate mRNA splicing with PI 3-K- and mTOR-regulated translation initiation. Cdc42 has been implicated in cell

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transformation (Lin et al., 1997). Interestingly, an activated Cdc42 double mutant deficient in splicing regulation is also deficient in transformation, suggesting that this new S6K-mediated function of Cdc42 may contribute to the transformed phenotype (Wilson et al., 2000).

B. Coordinated Growth and Proliferation in Liver Regeneration p85 alpha PI 3-K knockout mice reveal that these enzymes play an essential role in the liver (Fruman et al., 2000). Liver regeneration following partial hepatectomy provides a model system for assessing the intertwined processes of cell growth and proliferation. Partial hepatectomy increases circulating levels of hepatocyte growth factors, and markedly induces the activities of PI 3-K, Akt, and S6K1 (Michalopoulos and DeFrances, 1997; Hong et al., 2000; Jiang et al., 2001), and expression of a novel liver-specific PI 3-K, termed PI 3-KIIγ (Ono et al., 1998). Mice with diminished levels (CRE/lox conditional knockout) of ribosomal S6 protein in liver are impaired in recovery from partial hepatectomy (Volarevic et al., 2000). Hepatocytes from these livers exhibit abnormal ribosome profiles and progress through early G1 phase, as indicated by normal induction of cyclin D. The cells do not progress to S phase, as cyclins E and A mRNA and protein are lacking. The authors propose that this arrest may be due to activation of a checkpoint which senses abnormal ribosome biogenesis. This study raises interesting questions regarding the coordination of protein synthesis and proliferation. Interpretation of these studies is complicated, however, by the long half-life of the S6 protein. Because S6 protein was present at even 5 days after CRE induction, it should be noted that the data were observed in the presence of reduced levels, but not complete lack, of S6. A complete knockout of S6 protein, if viable, may have a more immediate and potent effect on liver regeneration following both starvation and hepatectomy. Another interesting finding from the partial hepatectomy model is that 4E-BP1 is not required for rapamycin-sensitive liver regeneration. Partial hepatectomy induces S6K1 activation and 4E-BP1 phosphorylation and subsequent reduction in eIF-4E binding and repression (Jiang et al., 2001). Normal animals treated with rapamycin suffer impaired liver regeneration (Francavilla et al., 1992; Jiang et al., 2001). It was found that under these conditions, S6K1 phosphorylation and activity is markedly inhibited, while 4E-BP1 phosphorylation persists, and eIF-4E cap-binding activity consequently increases in hepatocytes from rapamycin-treated rats after partial hepatectomy (Jiang et al., 2001). These data suggest not only an mTORindependent phosphorylation of 4E-BP1, but also that S6K1 may be an essential target in rapamycin-sensitive regeneration in vivo. The role of the

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rapamycin-sensitive related kinase S6K2 in hepatocyte models has not yet been addressed, but this kinase may be another candidate for regulation of protein synthesis and proliferation in regenerating liver. These findings differ from reports of rapamycin-sensitive 4E-BP1 phosphorylation in vitro, and suggest that the in vivo environment following hepatectomy is not mirrored in tissue culture experiments. The Erk pathway has been implicated in 4E-BP phosphorylation in other cell types (Fadden et al., 1997; Rao et al., 1999), but Erk1/2, p38, or Jnk activation was not detected following hepatectomy (Jiang et al., 2001). Other rapamycin-insensitive effector kinases in the PI 3-K or PKC pathways known to regulate 4E-BPs in other systems may be responsible for the rapamycin-insensitive phosphorylation following hepatectomy. In light of these studies suggesting the importance of S6K and S6 in liver regeneration, it will be of interest to determine the effect of partial hepatectomy in S6K1 knockout mice. Embryonic fibroblasts from these mice are reported to have normal S6 phosphorylation, perhaps due to compensation by S6K2 (Shima et al., 1998). Whether S6 phosphorylation is entirely normal in adult hepatocytes in vivo, however, has not yet been established. This may shed light on the relative roles of S6K1 versus S6K2 in the dynamic liver model. Comparison of individual and combined knockouts of S6K1 and S6K2 would be the best tool to address this question.

C. PI 3-K, mTOR, Translation, and Cell Size The critical role of PI 3-K in cell growth and proliferation is underscored by the conservation of this pathway from C. elegans to humans. This conservation has allowed for elegant genetic studies in multiple organisms, which have revealed the importance of the PI 3-K pathway and specific effectors in control of cell size. Mutations of the Drosophila insulin receptor (INR) (Chen et al., 1996), IRS homolog (chico) (Bohni et al., 1999), PI 3-K (dp110) (Leevers et al., 1996), Akt (dAkt) (Verdu et al., 1999), TOR/FRAP (dTOR) (Zhang et al., 2000; Oldham et al., 2000), or S6K1 (dS6K) (Montagne et al., 1999) give rise to a small cell phenotype. Similarly, overexpression of a d4EBP mutant having high affinity for deIF4E reduces Drosophila cell size (Miron et al., 2001). In the dS6K mutant flies, cell number is preserved, but each individual cell is smaller in size than wild-type cells, producing normally proportioned animals with a 50% reduction in body size (Montagne et al., 1999). These data suggest that dS6K regulates cell size and, subsequently, organ and body size, but does not influence patterning, cell-fate, or spatial decisions. S6-dependent control of ribosome biogenesis and, translational capacity is an attractive hypothesis to explain these phenotypes, but it is more likely that other targets of S6K1 are involved in cell size control: Homozygous deletion of S6K1 in mice results in a small animal phenotype

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despite the presence of normal S6 phosphorylation and 5 TOP mRNA regulation, presumably mediated by the intact function of the homolog S6K2 (Shima et al., 1998). These mice have pancreatic β-cells of reduced size, rendering them hypoinsulinemic (Pende et al., 2000). Notably, the dS6K mutant phenotype is far more severe in flies, where it includes female sterility, developmental delay, and early death (Montagne et al., 1999). The S6K1-/- mice are viable, fertile, and only 20% smaller than wild type (Shima et al., 1998). This may reflect the role played by S6K2 in mammalian cells (Shima et al., 1998), while there is thought to be only one S6K species in Drosophila. The PI 3-K pathway has also been implicated in cell size regulation in a cardiac hypertrophy model, where the variable of cell proliferation is eliminated due to the terminally differentiated status of cardiomyocytes (Shioi et al., 2000). Cardiac-specific expression of activated or dominant negative PI 3-K resulted in transgenic mice with larger or smaller hearts, respectively, with corresponding regulation of Akt and S6K1. Similarly, overexpression of active PTEN inhibited, and catalytically inactive PTEN promoted, cardiomyocyte hypertrophy (Schwartzbauer and Robbins, 2001). Genetic studies demonstrate that upstream PI 3-K components may play more diverse roles in regulation of both growth and proliferation. While chico and dS6K mutant flies are viable, deletions of INR, PI 3-K, or dAkt result in embryonic lethality (Chen et al., 1996; Leevers et al., 1996; Verdu et al., 1999). Mutations in INR, chico, PI 3-K, and dAkt affect cell number as well as cell size (Leevers, 1999). dPTEN has been shown to act as a tumor suppressor in flies, reducing cell number and cell size, by antagonizing the PI 3-K pathway (Goberdhan et al., 1999). Other evidence, however, points to a role for S6K1 in proliferation as well as translational control and cell size. A rapamycin-resistant S6K1 mutant rescued rapamycin-sensitive inhibition of E2F transcriptional responses in lymphocytes (Brennan et al., 1999). Others suggest that S6K1 is not essential for proliferation after observation of rapamycin-sensitive S6K1 activity in cells whose proliferation is rapamycin resistant (Hosoi et al., 1998; Louro et al., 1999; Slavik et al., 2001). We propose that there may be multiple downstream targets of the mTOR pathway that may confer rapamycin resistance to proliferation. A lesion in the pathway downstream of S6K1 does not necessarily exclude a role for S6K1 in proliferation. While genetic data from Drosophila and in vivo mouse models have begun to identify signaling molecules that function to regulate cell growth and cell size, the biochemical signaling pathways that cooperate to control cell growth in mammalian cells are less well understood. Treatment of asynchronously cycling cultured mammalian cells with rapamycin or LY294002 reduces cell size as well as cell cycle progression and proliferation, implicating a role for mTOR and PI3-K in control of mammalian cell size (Fingar et al., 2002). It is the inhibition of mTOR that mediates

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rapamycin’s effect on cell size, as expression of a rapamycinresistant mutant of mTOR rescues the rapamycin-induced small cell size phenotype. Similarly, treatment of differentiated C2C12 myotubes or isolated rat skeletal muscle with rapamycin blocks muscle hypertrophy (Bodine et al., 2001; Rommel et al., 2001). Consistent with a role for mTOR and PI3-K in control of cell size, overexpression of either S6K1 or eIF4E increases cell size, while coexpression of both proteins additively increases cell size, demonstrating that the mTOR to S6K1 and mTOR to 4EBP/eIF4E pathways function in parallel downstream of mTOR to coordinately regulate cell size (Fingar et al., 2002). Similarly, overexpression of S6K1 or Akt1 in differentiated C2C12 myotubes induces muscle cell hypertrophy (Rommel et al., 2001). Consistent with the reduction in cell size observed upon overexpression of a d4EBP mutant with high affinity for deIF4E in Drosophila (Miron et al., 2001), overexpression of a phosphorylation site mutant of 4EBP1 (Thr37/46Ala) in cultured mammalian cells also reduces cell size (Fingar et al., 2002). Further evidence supporting a role for mTOR-mediated signaling in control of mammalian cell size is that an S6K1 phosphorylation site acidic substitution mutant (E389D3E) that exhibits partial rapamycin resistance, or overexpression of eIF4E, partially rescues the reduced cell size phenotype induced by rapamycin (Fingar et al., 2002). Lastly, while cell cycle progression and cell growth are normally tightly coordinated during cellular proliferation (cells tend to remain fairly constant in size through multiple cell division cycles), the two processes can be experimentally dissociated in mammalian cells (Fingar et al., 2002), confirming what Lee Hartwell and colleagues first observed in budding yeast 25 years ago (Johnston et al., 1977). When cell cycle progression is blocked upon expression of cell cycle inhibitory proteins (such as the cdk inhibitors p16 or p21, or dominant-negative cdk2), cells continue to grow to increased size, indicating that cell cycle progression and cell growth are separable and distinct processes. Importantly, this growth to increased cell size is blocked by rapamycin or LY294002. The mechanisms that allow tight coordination of cell cycle progression and cell growth are poorly understood (see Section IV.D). Data from numerous experimental systems all point to an important role for signaling molecules that regulate protein translation as important regulators of cell growth and cell size.

D. Coordination of the PI 3-K and mTOR Pathways Two recent genetic studies of mutations in the Drosophila TOR (dTOR) protein elegantly assessed the relationship between the PI 3-K and TOR pathways (Oldham et al., 2000; Zhang et al., 2000). Both studies conclude that the dTOR pathway serves a nutrient checkpoint function that converges

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on growth factor-regulated translational effectors of the PI 3-K pathway. Disruption of dTOR function prevents flies from developing past the larval stage (Zhang et al., 2000). The cellular phenotypes perfectly mimic the effects of amino acid starvation (Oldham et al., 2000). As with mutations in the PI 3-K pathway, including dS6K (Montagne et al., 1999), loss of dTOR function results in a cell autonomous cell size defect, resulting in reductions in organ and body size (Zhang et al., 2000; Oldham et al., 2000). In dTOR mutants, proliferation rates are also reduced, characterized by an increased number of cells in G1 phase, and fewer in S and G2 (Zhang et al., 2000). Cyclin E overexpression rescued the G1 arrest, and cyclin E protein levels were greatly reduced in dTOR mutant flies, suggesting that this cyclin is a critical downstream target of TOR regulation (Zhang et al., 2000). The dTOR mutants provide important observations on the role and regulation of dS6K. Oldham et al. observed diminished S6 phosphorylation, but an upregulation of dS6K protein levels in dTOR mutants or following amino acid deprivation, suggesting dTOR-mediated feedback regulation of dS6K. Interestingly, mutations in the PI 3-K pathway do not affect dS6K levels (Oldham et al., 2000). The role of dS6K as an essential downstream target of dTOR was emphasized by Zhang et al. (2000), who demonstrated that overexpression of dS6K could rescue development of flies with diminished dTOR function, allowing them to develop to adulthood. The dS6Krescued flies were fertile and developed normally, but were slightly reduced in size, suggesting that while dS6K is a major in vivo effector of the TOR pathway, other dTOR-mediated functions may be necessary. Consistent with this hypothesis, constitutively activated dS6K could not rescue the most severe dTOR mutants to adulthood, but did allow progression to the pupal stage (Zhang et al., 2000). Oldham et al. also report a failure of dS6K to rescue severe dTOR mutants. Thus, a low level of dTOR activity is necessary to supply functions of dTOR effectors in addition to dS6K. One such function may be eIF-4E activation, as eIF-4E mutants exhibit a growth arrest phenotype (Zhang et al., 2000). However, eIF-4E overexpression alone was not sufficient to rescue the dTOR mutant phenotype (Zhang et al., 2000). The common effectors of both PI 3-K and mTOR have lead some to hypothesize that these proteins may function in a linear signaling pathway. The dTOR studies in Drosophila, along with other evidence, suggest that this is not the case. dTOR mutations complement mutations in PTEN, suggesting that dTOR affects downstream components in the PI 3-K pathway (Oldham et al., 2000; Zhang et al., 2000). Mutations in dTOR are more severe than those in PI 3-K, however, as dTOR mutants arrest at an earlier larval stage than those of the Drosophila PI 3-K subunits Dp110 or Dp60 (Weinkove et al., 1999; Zhang et al., 2000). In contrast to dTOR mutants, PI 3-K

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mutants fail to upregulate dS6K, and exhibit dissimilar larval phenotypes (Oldham et al., 2000). Thus, the Drosophila genetic data are inconsistent with a role for dTOR as a downstream effector of PI 3-K. Studies in mammalian cells also support the model that PI 3-K and mTOR regulate independent but parallel pathways that converge on common effectors. While the autokinase activity of mTOR is indeed wortmannin sensitive, this inhibition requires a concentration 100-fold greater than the dose that effectively inhibits PI 3-K activity (Brunn et al., 1996). Other studies have suggested that the PI 3-K effector Akt phosphorylates mTOR in a putative negative regulatory domain (Ser2448) (Nave et al., 1999; Sekulic et al., 2000). Mutation of this site, however, does not inhibit mTOR activation of S6K1 (Nave, 1999; Sekulic et al., 2000), and, notably, this site is not conserved in dTOR, despite high sequence conservation between dTOR and mTOR in other regions, including the kinase and HEAT domains (Zhang et al., 2000). In addition, growth factor activation of the PI 3-K pathway and Akt induces little to no change in mTOR kinase activity (Scott et al., 1998; Sekulic et al., 2000). Notably, S6K1 is activated by constitutively active Akt mutants only when these mutants are targeted to the plasma membrane (Dufner et al., 1999), and dominant negative Akt inhibits S6K1 (Takehashi, et al., 2002). This Akt regulation of S6K1 may be explained by its inhibition of TSC2 (Manning, unpublished observations, see Section IV.E.). S6K1 itself suggests separable mTOR and PI 3-K pathways, as an S6K1 truncation mutant is rapamycin resistant, yet retains sensitivity to wortmannin (Cheatham et al., 1995; Weng et al., 1995). Genetic studies, however, suggest that the PI3K and mTOR pathways may also function linearly, as rapamycin analogs inhibit malignancies induced by lesions in PI3K/PTEN signaling (Podsypanina et al., 2001; Neshat et al., 2001; Aoki et al., 2001) (see Section IV.E). The mTOR gene has been mutated, but not deleted, in mice. This study reveals mTOR is essential for embryonic development (Hentges et al., 2001). Mutation of mTOR or treatment of embryos with rapamycin results in a “flat top” embryonic lethal phenotype, characterized by lack of the telencephalon. Interestingly, cells from the mutant mouse exhibited a defect in proliferation, but not in cell size. It should be noted, however, that S6K1 and 4E-BP1 phosphorylation and activities were reduced, but not absent, and may have been sufficient to maintain cell size regulation. This study suggests, however, that mTOR is an important mediator of mitogenic signaling in the developing mouse. The issue of the relationship between PI 3-K and mTOR remains controversial, as experimental evidence is difficult to interpret for several reasons. Much of the controversy revolves around the ability of the PI 3-K pathway, and specifically, Akt, to regulate mTOR kinase activity. Measurement

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of mTOR kinase activity is technically challenging and may account for disparate results from different groups. A larger issue, however, is the misleading assumption that mTOR kinase activity always mediates mTOR function. For example, PA activation of mTOR substrates is dependent upon PA binding to mTOR, but stimulation or inhibition of PLD/PA signaling does not alter mTOR kinase activity (Fang et al., 2001). There is evidence for direct phosphorylation of S6K1 and 4E-BP1 by mTOR in vitro, but there is also compelling evidence that mTOR-mediated phosphatase regulation may be more important in regulating translational effectors. Equally plausible is involvement of both mechanisms. Finally, whether or not PI 3-K can signal to mTOR is difficult to address by pharmacologic means, because rapamycin completely overrides all other signals that regulate mTOR effectors. Much of the data regarding PI 3-K and mTOR can be explained by two possible models. One model suggests that PI 3-K and mTOR function in distinct and parallel signaling pathways, converging upon common downstream effectors. Inputs from both pathways are required, as inhibition of either pathway is sufficient to override incoming signals from the other. This model is consistent with a checkpoint function for mTOR. Another model would suggest that in addition to functioning independently in parallel, PI 3-K-derived signals may also contribute to activation of mTOR, creating a linear pathway from PI 3-K to mTOR. Because of the technical concerns described, it is difficult to discriminate between these views. Finally, a physical basis by which parallel PI 3-K and mTOR inputs converge upon activation of S6K1 has been proposed in a recent study, which suggests that the p85 adapter subunit of PI 3-K nucleates a complex between S6K1 and mTOR (Gonzalez-Garcia et al., 2002). A p85 truncation mutant (p65PI3K) which lacks the C-terminal SH2 domain was shown to retain the ability to activate the p110 PI 3-K catalytic subunit and Akt. However, this mutant failed to promote serum-stimulated activation of S6K1. While p85PI3K forms a complex with S6K1 and mTOR, p65PI3K does so inefficiently. Interestingly, a rapamycin-resistant S6K1 truncation mutant can be activated by either p65PI3K or p85PI3K, suggesting that the crucial function of the PI 3-K regulatory subunit is recruitment of mTOR. This study suggests a model by which PI 3-K is necessary for (1) phosphorylation of S6K1 at Thr229 and Thr389 by PI 3-K-regulated kinases, and (2) recruitment of mTOR to an S6K1-containing complex, which confers protection from mTOR-regulated phosphatases (Peterson et al., 1999). This model suggests a physical basis by which the PI 3-K and mTOR pathways integrate growth factor- and nutrient-derived signals at the level of the translational effector S6K1. It will be interesting to determine whether this complex formation is mediated by the TOS domain of S6K1, whether this regulation applies to activation of primarily nuclear S6 kinases (p85 S6K1 and S6K2 isoforms), and whether

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PI 3-K and mTOR signals similarly converge in a physical complex on 4E-BP factors.

E. PI 3-K and mTOR Pathways in Cancer The importance of these signaling pathways in regulating cell growth and proliferation is underscored by the presence of mutations in multiple components of these pathways in human cancers. Gain of function mutations in PI 3-Kp110, Akt, mTOR, and eIF-4E, and loss of PTEN function lesions have been detected in multiple human cancers (Vogt, 2001). Germline mutation of PTEN has been implicated as the lesion in Cowden’s disease, a human genetic disorder characterized by multiple hamartomas and susceptibility to multiple benign and malignant tumors (Marsh et al., 1999). Elevated S6K activity has been observed in uterine tumors from mice heterozygous for PTEN, and treatment with the rapamycin ester CCI 779, an analog designed for intravenous delivery, reduced tumor size and proliferation (Podsypanina et al., 2001). The growth and proliferation of tumor cell lines lacking PTEN were found to be more sensitive to CCI 779 than PTEN+/+ cells (Neshat et al., 2001). These PTEN null cells also exhibited increased S6K1 activity and 4E-BP1 phosphorylation. Interestingly, oncogenic transformation of chick embryo fibroblasts by PI 3-K or Akt gain of function could be inhibited by rapamycin (Aoki et al., 2001). This study found that mTOR was essential for PI 3-K-induced oncogenesis, but not for transformation induced by oncogenes that function in other signaling pathways. These studies suggest that rapamycin may be an effective anticancer agent, particularly for tumors arising due to lesions in the PI 3-K pathway. The tuberous sclerosis complex (TSC) genes have been recently identified as tumor suppressors that negatively regulate both cell growth and proliferation. These genes are responsible for a human inherited disorder characterized by development of benign hamartomatous tumors in multiple organs, and predisposition to malignant tumor formation (Jones et al., 1997). Loss of function of TSC1 or 2 results in benign tumors characterized by large cells. Genetic analyses in Drosophila suggest that the TSC1/2 complex lies on a parallel pathway that inhibits insulin signaling downstream of Akt (Gao and Pan, 2001). Interestingly, genetic epistasis experiments place dS6K downstream of TSC1/2 and demonstrate that dS6K and TSC1/2 serve antagonistic functions (Fig. 9) (Potter et al., 2001; Tapon et al., 2001). This also appears to be true in mammalian cells, as S6K1 activity is upregulated in mouse cells lacking TSC1 (Kwiatkowski et al., 2002) and in human cells lacking TSC2 (Goncharova et al., 2002). Akt phosphorylation and inhibition of the TSC2 gene product tuberin suggests a mechanism by which Akt contributes to S6K1 activation (Manning, unpublished observations), and

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Fig. 9 The tuberous sclerosis complex (TSC) opposes insulin signaling. The TSC1/2 genes have been identified as negative regulators of S6K function and cell growth. The PI 3-K effectors Akt, PDK1, and PKCζ contribute to S6K activation. Akt relieves TSC inhibition by phosphorylation and inhibition of the TSC2 gene product tuberin. It is not yet known whether tuberin inhibits S6K1 directly or via mTOR.

may explain S6K1 inhibition by dominant negative Akt (Takehashi et al., 2002). These examples from the cancer literature further highlight the interdependence of the PI 3-K and mTOR pathways, and the contribution of their effectors to growth and proliferation.

V. CONCLUSIONS The PI 3-K pathway mediates many essential cellular functions, including survival, proliferation, and cell growth/cell size. We have described PI 3-K effectors which transduce signals to regulate translation initiation, ribosome biogenesis, and translational capacity. These include the S6 kinases, 4E-BPs and eIF-4E, eIF-4G, and eIF-4B. These PI 3-K effectors also integrate signals from other growth factor-stimulated pathways, including conventional PKCs and MAPKs. Importantly, these effectors integrate the incoming growth factor signals with mitogenic inputs from the mTOR pathway, a crucial nutrient- and energy-sensing checkpoint pathway, ensuring adequate amino acid resources to meet the demand for new protein synthesis. Common subcellular localization of effectors and regulatory molecules may facilitate such integrated signal transduction. The influence of growth factorand nutrient-sensitive pathways on growth and proliferation is a continuing subject of investigation that will likely reveal important new insights into normal physiology and pathological conditions including hypertrophy, diabetes, and cancer.

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ACKNOWLEDGMENTS The authors thank Diane Fingar, Angela Romanelli, Stefanie Schalm, Celeste Richardson, and Andrew Tee for their contributions to this manuscript.

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Weng, Q., Andrabi, K., Kozlowski, M. T., Grove, J. R., and Avruch, J. (1995). Multiple independent inputs are required for activation of the p70 S6 kinase. Mol. Cell. Biol. 15, 2333–2340. Weng, Q. P., Kozlowski, M., Belham, C., Zhang, A., Comb, M. J., and Avruch, J. (1998). Regulation of the p70 S6 kinase by phosphorylation in vivo. Analysis using site-specific anti-phosphopeptide antibodies. J. Biol. Chem. 273, 16,621–16,629. Williams, M. R., Arthur, J. S., Balendran, A., van der Kaay, J., Poli, V., Cohen, P., and Alessi, D. R. (2000). The role of 3-phosphoinositide-dependent protein kinase 1 in activating AGC kinases defined in embryonic stem cells. Curr. Biol. 10, 439–448. Wilson, K., Fortes, P., Singh, U. S., Ohno, M., Mattaj, I. W., and Cerione, R. A. (1999). The nuclear cap-binding complex is a novel target of growth factor receptor-coupled signal transduction. J. Biol. Chem. 274, 4166–4173. Wilson, K., Wu, W. J., and Cerione, R. A. (2000). Cdc42 stimulates RNA splicing via the S6 kinase and a novel S6 kinase target, the nuclear cap-binding complex. J. Biol. Chem. 275, 37,307–37,310. Xu, G., Kwon, G., Cruz, W. S., Marshall, C. A., and McDaniel, M. L. (2001). Metabolic regulation by leucine of translation initiation through the mTOR-signaling pathway by pancreatic beta-cells. Diabetes 50, 353–360. Zhang, H., Stallock, J. P., Ng, J. C., Reinhard, C., and Neufeld, T. P. (2000). Regulation of cellular growth by the Drosophila target of rapamycin dTOR. Genes Dev. 14, 2712–2724.

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Histone Acetyltransferases and Deacetylases in the Control of Cell Proliferation and Differentiation Heike Lehrmann, Linda Louise Pritchard, and Annick Harel-Bellan CNRS UPR 9079, Institut Andr´e Lwoff, 94800 Villejuif, France

I. Introduction II. Acetylation of Histones III. Histone Acetyltransferases A. GNAT-Family (Gcn5-Related N-acetyltransferases) B. MYST-Family C. CBP/p300 Family IV. Histone Deacetylases and Cell Cycle Regulation A. Mad /Max and HDACs B. Rb and HDACs C. HDACs and Cancer V. Muscle Differentiation A. Interaction of MyoD with CBP/p300 and PCAF B. MyoD and HDAC1 C. MEF2 and Deacetylases VI. Hematopoiesis A. GATA-1 B. EKLF C. CBP/p300 and Hematopoietic Disorders VII. Huntington’s Disease VIII. Histone Acetylation in Combination with Other Chromatin Modifications IX. Conclusion References

Histone acetylation and deacetylation are chromatin-modifying processes that have fundamental importance for transcriptional regulation. Transcriptionally active chromatin regions show a high degree of histone acetylation, whereas deacetylation events are generally linked to transcriptional silencing. Many of the acetylating and deacetylating enzymes were originally identified as transcriptional coactivators or repressors. Their histone-modifying enzymatic activity was discovered more recently, opening up a whole new area of research. Histone acetyltransferases such as CREB-binding protein

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Copyright 2002, Elsevier Science (USA). All rights reserved.

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(CBP) and PCAF are involved in processes as diverse as promoting cell cycle progression and regulating differentiation. A controlled balance between histone acetylation and deacetylation seems to be essential for normal cell growth. Both histone acetyltransferases and deacetylases are involved in the development of diseases, including neurodegenerative disorders and cancer. Treatments that target these enzymes are already under clinical investigation. C 2002, Elsevier Science (USA).

I. INTRODUCTION Packaging of DNA is based on interactions between the negatively charged DNA and positively charged histones. The fundamental unit of this packaging system is the nucleosome, consisting of 146 base pairs of DNA helix wrapped around one histone octamer. Although this packaging system was originally believed to serve mainly scaffolding functions (e.g., condensation of DNA), this view has changed dramatically in recent years, and a complex interplay between histone modifications, chromatin structure, and transcriptional regulation has emerged. Two main chromatin-modifying principles have been identified: ATP-dependent helicases and histone-modifying enzymes. The former can induce changes in nucleosomal structure by ATPdependent processes. Based on homology to yeast and Drosophila ATPases, these chromatin remodeling complexes can be divided into three subclasses (SWI/SNF, ISWI, Mi-2) (Kingston and Narlikar, 1999). Chromatin-remodeling complexes can interact with and recruit the second class of chromatinmodifying complexes, leading to covalent modifications of histones. Of special interest are enzymes that modify the N-terminal histone tails that protrude from the histone octamer, and serve functions in histone–DNA and/or histone–histone interactions. An intensively studied modification is acetylation of the epsilon-amino group of lysine residues in histone tails. Acetylation of histones is effected by histone acetyltransferases (HATs) and counteracted by histone deacetylases (HDACs). Acetylation and deacetylation of histone tails are rapidly changing events that mediate a link between cellular signaling pathways, on the one hand, and activation or repression of transcription due to changes in the chromatin structure, on the other hand. Additional modifications of histone tails include methylation, phosphorylation, ubiquitination, and ADP-ribosylation. Although these modifications are less well studied, recent evidence suggests a complex regulation system involving different types of histone modifications. The importance of acetylation and deacetylation events in cell cycle regulation and differentiation is the subject of this review.

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II. ACETYLATION OF HISTONES Histone acetylation is generally associated with transcriptionally active genomic regions (Allfrey, 1977). The most relevant acetylation sites map to the tail regions of histones H3 and H4, although H2A and H2B are also acetylated in vivo. Modified lysine residues of these regions include lysines 9, 14, 18, and 23 of H3 and lysines 5, 8, 12, and 16 of H4 (Strahl and Allis, 2000). Acetylation of lysines neutralizes the charge of the lysine amino group. This is thought to weaken the interaction between histone tails and DNA or adjacent histones. Weakening of these interactions can lead to a more open chromatin structure and can facilitate the access of transcription factors to their cognate DNA binding site. In addition, several transcription factors have themselves been shown to be substrates for HATs. In some cases, acetylation of transcription factors is suspected to increase their DNA binding activity through an uncharacterized mechanism and to result in more stable protein–DNA complexes. Alternatively, acetylated transcription factors can recruit additional proteins or protein complexes that are essential for the initiation of promoter activation. HATs target either single lysine residues or combinations of them. The specificity of HATs for one or the other lysine residue is determined not only by the enzyme itself but also by interacting proteins. Recombinant yeast Gcn5 preferentially acetylates lysine 14 of H3 and lysine 8 of H4 (Kuo et al., 1996). Gcn5 in cells, however, is part of the multiprotein complexes SAGA and ADA. In this context, Gcn5 not only efficiently acetylates histones within assembled nucleosomes but also targets histone H2B as well as most lysine residues of the H3 tail (Grant et al., 1999). Certain patterns of lysine acetylation in histone tails have already been linked to physiological functions. Acetylation of lysines 5 and 12 of histone H4 is characteristic for newly synthesized histones and seems to be necessary for nuclear transport and deposition of histones on replicated DNA (Sobel et al., 1995). Acetylation of lysine 16 of H4 is linked to dosage compensation in Drosophila (Bone et al., 1994; Turner et al., 1992), whereas acetylation of H4 lysine 12 is found in heterochromatic regions of the genome (Turner et al., 1992). The involvement of histone acetylation in transcriptional activation has been demonstrated in yeast: mutation of acetylatable lysine residues in the H4 tail resulted in loss of gene expression from the GAL1 and PHO5 promoters (Durrin et al., 1991). Characterization of the enzymes involved in the acetylation process has led to intensified research in the field, so that a large body of information on histone acetylation is now available. Many of these enzymes were first identified as transcriptional coactivators, again emphasizing the importance

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of acetylation for transcriptional regulation. Nuclear HATs are classed in several families; three of them will be discussed here.

III. HISTONE ACETYLTRANSFERASES A. GNAT-Family (Gcn5-Related N-acetyltransferases) Gcn5, the founding member of this family, was the first HAT to be identified (Brownell et al., 1996). Using an in-gel HAT assay, Brownell and colleagues isolated a protein from Tetrahymena macronuclear extracts that exhibited histone acetyltransferase activity. Characterization of this protein revealed homology to a known transcriptional regulator, the yeast Gcn5 protein. Mutation of yeast Gcn5 demonstrated a direct correlation between its HAT activity and transcriptional control (Wang et al., 1998). Mammalian cells express two homologues of yGcn5, hGcn5 and PCAF. Both yGcn5 and hPCAF have been shown to be members of multisubunit complexes. Gcn5 can be found in the yeast ADA and SAGA complexes. The SAGA complex shows a subunit composition similar to the mammalian PCAF complex (Schiltz and Nakatani, 2000). In addition to proteins of the Ada and Spt families, both complexes contain TBP associated factors (TAFs). Deletion of specific TAFs renders the whole complex inactive for nucleosomal acetylation, emphasizing the importance of regulated protein–protein interactions for proper HAT activity (Grant et al., 1998). Homozygous deletion of Gcn5 in mice leads to embryonic lethality, whereas PCAF null mice show no aberrant phenotype. The GNAT family contains additional members that are less well characterized than Gcn5 and PCAF. Elp3, for example, has been identified as part of the yeast elongator complex and associates with RNA polymerase II transcription complexes (Wittschieben et al., 1999). Characteristic for the GNAT family is the presence of four conserved protein domains. Of special interest is the acetyl-coenzyme A binding site, which is conserved between distinct HAT families, and the presence of a C-terminal bromodomain (Fig. 1). No involvement of GNAT family members in diseases has been reported.

B. MYST-Family This family is named for its founding members MOZ, Ybf2/Sas3, Sas2, and Tip60. Sas2 and Sas3 (Sas for something about silencing) are yeast HATs that have been identified as members of multiprotein complexes, similar to HATs of the GNAT family. Sas2 is part of the SAS-I complex that also

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Fig. 1 Schematic presentation of human HATs with conserved protein motifs and binding sites for associated proteins. HAT: histone acetyltransferase enzymatic domain; Bromo: bromodomain; Zn: zinc finger region; Poly-Q: polyglutamine region.

contains Sas4 and Sas5 proteins. Recent findings demonstrate an association of Sas2 with chromatin assembly factors. Sas2 associates with Cac1, a component of the yeast chromatin assembly factor-1 complex (CAF-I) and with the nucleosome assembly factor Asf1. Association with the chromatin assembly factors targets SAS-I to newly replicated DNA. Deletions encompassing either components of the SAS-I complex or Cac1 or Asf1 all have similar effects on derepression of silenced telomeric heterochromatin. Requirement of lysine 16 of histone H4 for Sas2 function suggests an involvement of histone acetylation in the biologic activity of Sas2, although HAT activity of Sas2 remains to be demonstrated (Meijsing and Ehrenhofer-Murray, 2001; Osada et al., 2001). Sas3 is part of the yeast H3 specific NuA3 complex (John et al., 2000). While deletion of Sas3 shows almost no detectable phenotype in yeast, combined deletion of Sas3 and Gcn5 (both of which target H3 for acetylation) results in extensive loss of H3 acetylation and G2/M-phase arrest. These findings lead to the conclusion that acetylation of histone H3 is essential for yeast viability and that this need is governed by several complexes with at least partially overlapping activities (Howe et al., 2001). In contrast, H4 in yeast is only acetylated by the NuA4 complex, which contains Esa1p (a MYST family member, Esa for essential Sas family acetyltransferase) as the catalytic HAT (Allard et al., 1999). Deletion of Esa1p is lethal, demonstrating also the requirement of H4 acetylation for yeast viability (Clarke et al., 1999; Smith et al., 1998). Esa1p is homologous to the Drosophila MOF (males absent on the first) protein. MOF is required

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for dosage compensation in Drosophila. Members of the MSL complex (see below) target MOF to the male X chromosome, where acetylation of H4 lysine 16 leads to increased transcription. Deletion of members of the MSL complex leads to male-specific lethality (MSL) (Akhtar and Becker, 2000; Smith et al., 2000). Tip60 (Tat-interacting protein) represents the catalytic subunit of the human TIP60 complex (Ikura et al., 2000). Tip60 has been identified as an HIV-Tat interacting protein; and Tat inhibits Tip60 HAT activity (Creaven et al., 1999; Kamine et al., 1996). The predicted involvement of Tip60 in DNA repair could have implications for cancer development in the event of loss or mutation of members of the Tip60 complex.

C. CBP/p300 Family CBP (CREB-binding protein) and p300 are considered to be functional homologues. CBP was originally characterized as a transcriptional coactivator. Both proteins contain N- and C-terminal regions that are able to activate transcription when artificially tethered to promoters, even though these regions are devoid of HAT activity. These regions contain zinc finger domains that are characterized interaction sites for a variety of proteins. Viral proteins, transcription factors, and other HATs have been shown to bind to CBP/p300 through these regions (Fig. 1). Some of the interactions seem to be mutually exclusive (e.g., interaction of p300 with either E1A or PCAF). Due to limiting cellular CBP/p300 levels, competition for CBP/p300 is thought to be a means for regulating promoter activity and to account for the changes observed during viral infections or differentiation processes (Kamei et al., 1996). CBP and p300 are also characterized HATs, with the catalytic domain residing in the central region of the protein (Bannister and Kouzarides, 1996; Ogryzko et al., 1996). Therefore, CBP/p300 exhibit their transcriptional coactivator function through multiple facets: through offering a surface for the binding of various proteins, CBP/p300 contribute to the formation of a multiprotein activation complex and ensure the integration of different signaling pathways. In addition, the enzymatic activity conferred by the HAT domain of these proteins can influence promoter activity directly by acetylating histones in the corresponding promoter region. Besides opening of the chromatin structure due to reduced interaction between DNA and acetylated histone tails, acetylation of histones can also influence the histone composition of nucleosomes. p300 has been shown to interact with NAP-1, a histone chaperone involved in nucleosome remodeling (Ito et al., 2000). p300 acetylates the H2A/H2B dimer, which facilitates the transfer of H2A/H2B from the nucleosome to NAP-1. It is not clear whether acetylation reduces the interaction between histones in the octamer, or if it increases the affinity of H2A/H2B for NAP-1. Nucleosomes depleted

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of H2A/H2B histones may be responsible for the altered chromatin structure of actively transcribed regions and facilitate the access for the transcription machinery (Baer and Rhodes, 1983). p300 and CBP are implicated in a variety of cellular processes. A wide range of promoters can be activated by overexpression of either of these proteins. Pathways as different as cell cycle progression, apoptosis, and differentiation seem to require CBP and/or p300. Both proteins are also targets for viral transforming proteins, which highlights their importance in the regulation of cellular processes. E1A, for example, has been shown to interact with p300 (Eckner et al., 1994). The binding site of E1A on CBP/p300 overlaps the interaction site for PCAF (Yang et al., 1996). This interaction is essential, since deletion of the N-terminal p300-binding region of E1A eliminates the transforming potential of E1A (Harlow et al., 1986). CBP/p300, and to a lesser extent PCAF, also acetylate nonhistone substrates. Among them are many transcription factors (e.g., E2F, MyoD, HNF-4) (MartinezBalbas et al., 2000; Polesskaya et al., 2000; Sartorelli et al., 1999; Soutoglou et al., 2000), but also structural proteins such as alpha-tubulin (MacRae, 1997) and the cytoplasmic-nuclear shuttle protein importin alpha (Bannister et al., 2000).

1. INTERACTION WITH p53 Of special interest here is the interaction of CBP and p300 with p53. p53 is a classical tumor suppressor. Its function as “guardian of the genome” ensures proper transmission of the genomic information from cell division to cell division. Aberrations such as DNA damage due to UV irradiation or deregulated gene expression after viral infection lead to activation of p53 and concomitant cell cycle arrest or apoptosis. Activation of p53 is regulated at the posttranslational level by multiple mechanisms, including phosphorylation and association with the ubiquitin ligase MDM2. Gu and Roeder (1997) recently demonstrated that this regulation cascade also includes acetylation of p53 by CBP/p300 and PCAF. In vivo, acetylation of p53 increases upon DNA damage and activates the transcriptional activity of p53. As for many other acetylated transcription factors, initial studies suggested that an increase in DNA binding due to acetylation of p53 might be the molecular explanation for the observed increase in transcriptional activity. However, it is not clear whether these data, which are based on electrophoretic mobility shift assays in vitro, reflect the in vivo situation. Acetylation of p53 might rather influence the recruitment of specific factors to a given promoter region or alter the subnuclear localization of p53 (i.e., association with PML and localization to nuclear bodies). In contrast, p300 also participates in the degradation of p53. p53 degradation involves a ternary complex of p300, MDM2, and p53. Interestingly, TAFII250, another HAT, shows

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ubiquitin-conjugating activity (Wassarman and Sauer, 2001). The potential for competition between acetylation and ubiquitination on certain lysine residues is especially intriguing. Furthermore, the participation of p300 in both activation and degradation of p53 puts it in a critical position for cell cycle regulation and reflects its dual role in promoting cell cycle progression and acting as a tumor suppressor gene.

2. CBP/p300 AND CELL CYCLE REGULATION A direct involvement of CBP/p300 in the regulation of S-phase entry has been demonstrated by Ait-Si-Ali et al. (1998). Serum stimulation of arrested cells leads to phosphorylation of CBP/p300 and an increase in its intrinsic HAT activity; E1A binding to CBP seems to mimic the effect of phosphorylation although under different conditions, maybe depending on the dose of E1A, it can have inhibitory effects. Both phosphorylation and HAT activity of CBP/p300 peak at the G1/S boundary. A candidate enzyme for phosphorylating CBP is the cell cycle regulated cyclinE/cdk2 complex. Cyclin E can interact directly with p300 (Perkins et al., 1997). Additionally, p300 regulates the expression of cyclin D1, and its HAT activity is required for this effect (Albanese et al., 1999). p300 also coactivates the transcription factor E2F, which is critically involved in the regulation of S-phase specific genes (Martinez-Balbas et al., 2000; Trouche et al., 1996). Inhibition of CBP/p300 by antibodies results in reduced numbers of cells in S phase, strongly suggesting the necessity of CBP/p300 in cell cycle progression (Ait-Si-Ali et al., 2000). An involvement of CBP and p300 in the regulation of proliferation was also predicted from knockout mice, based on the observation that CBP or p300 null cells show reduced proliferation (Yao et al., 1998).

IV. HISTONE DEACETYLASES AND CELL CYCLE REGULATION Histone deacetylases can be grouped into three classes based on their homology to yeast enzymes. The first class contains HDACs 1–3 and 8 and shows homology to the yeast enzyme RPD3. HDACs 4–7, 9, and 10 are grouped in the second class with similarity to the yeast HDA1 enzyme, while the third class of HDACs displays NAD-dependent deacetylation and homology to the yeast enzyme Sir2. Extensive studies in yeast have demonstrated an involvement of histone deacetylases in transcriptional control. The transcriptional repressor Tup1 recruits HDA1 to the promoter regions of ENA1 and STE6 genes. Recruitment of HDA1 leads to a specific deacetylation of H3 and H2B that is limited to the promoter’s vicinity (Wu et al., 2001).

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The transcription factor UME6, on the other hand, represses target promoters by recruitment of RPD3, which deacetylates all four core histones of nucleosomes in the entire promoter region. The specificity of mammalian HDACs is less well documented. However, association with many transcriptional repressors has been reported (Cress and Seto, 2000).

A. Mad/Max and HDACs The Mad/Max and Myc/Max complexes are thought to be involved in cell cycle control. In particular, Mad/Max is induced upon cell terminal differentiation (Ayer and Eisenman, 1993). Mad inhibits cell cycle progression through association with Max and binding to E-box containing promoters (McArthur et al., 1998). Promoters that are repressed by Mad/Max include E2F and cdc25 promoters. The repressive function of Mad/Max requires the association with a repressor complex that contains mSin3, N-CoR, and HDAC (Alland et al., 1997; Ayer et al., 1995; Hassig et al., 1997; Heinzel et al., 1997; Laherty et al., 1997; Nagy et al., 1997; Schreiber-Agus et al., 1995).

B. Rb and HDACs An intensively studied interaction is the association of HDAC with the retinoblastoma protein (RB). RB is frequently mutated in human tumors and plays a key role in regulating cell cycle progression from G1 to S phase. The main mechanism of this regulatory function depends on the association of RB with the transcription factor E2F. E2F regulates the expression of many genes involved in progression from G1 to S phase. Association of E2F with RB inhibits transcriptional activation by E2F via several mechanisms. First of all, RB interacts with the transcriptional activation domain of E2F and masks its activity. Active repression of promoters additionally requires an association with HDAC. This interaction depends on the pocket region of RB and leads to deacetylation of histones in the promoter region of repressed genes (Ferreira et al., 2001). Recent findings suggest a concomitant association of RB with the DNA methyltransferase DNMT1 (Robertson et al., 2000) as well as with the histone methyltransferase SUV39 and methylation of histones (Nielsen et al., 2001). However, methylation is currently thought to be an irreversible mechanism, and methylation of promoters regulated during the cell cycle would require periodic methylation and demethylation. If this occurs, it would extend the properties of histone methylation from an epigenetic marker to a flexible label comparable with acetylation and

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phosphorylation. Characterization of an enzyme involved in the demethylation process has not yet been reported. In addition to regulation of G1/S progression, RB is also involved in regulating additional stages of the cell cycle. M-phase entry of cells requires the expression of cyclin A and cdc2. RB controls the expression of these genes through association with Brg1 or Brm, components of the histone remodeling complex SWI/SNF. Progressive phosphorylation of RB during the G1 phase of the cell cycle disrupts its association with Brg1 and leads to expression of cyclinA and cdc2 and, as a result, to progression into M phase (Zhang et al., 2000). Although these examples show an involvement of HDAC proteins in the control of cell cycle progression, the cellular regulation system seems to be complex. Administration of HDAC inhibitors like TSA leads to cell cycle arrest in G1 and G2. TSA treatment induces histone hyperacetylation and activates expression of the cdk inhibitor p21/WAF/Cip1 (Nakano et al., 1997; Sowa et al., 1997). In addition, Rb accumulates in its hypophosphorylated form. Although cyclin D1 levels increase following TSA treatment, no associated kinase activity is detectable. This is potentially due to eleveated levels of the cyclin-dependent inhibitor p27. However, since even p21- and p27-deficient cells respond to TSA treatment by growth arrest, additional factors must be regulated by TSA, and the molecular mechanism of growth arrest triggered by TSA remains elusive (Wharton et al., 2000).

C. HDACs and Cancer A direct involvement of HDACs in human cancers is well documented in the case of promyelocytic leukemia (PML)– and PLZF–RAR fusion proteins (Melnick and Licht, 1999; Minucci and Pelicci, 1999). In certain types of acute promyelocytic leukemia, genomic translocations result in fusion of PML or PLZF to the retinoic acid receptor (RAR), thus preventing hematopoietic differentiation. Both fusion proteins are known to associate with transcriptional repressor complexes (NcoR/SMRT) that contain HDAC activity. Wild-type RAR is also found to associate with these repressor complexes. However, whereas physiological doses of retinoic acid (RA) release the transcriptional repression mediated by wtRAR, the PML– and PLZF–RAR repressive complexes are resistant to these doses of RA. Increased doses of RA release the repressor complex from PML–RAR fusion proteins, leading to progression in the differentiation process, whereas PLZF-RAR is resistant. Both PML– and PLZF–RAR fusion proteins form oligomers that result in increased concentrations of repressor complex at the promoter region of target genes, explaining the need for higher doses of RA to overcome the augmented repressor activity (Lin and Evans, 2000; Minucci et al., 2000);

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however, for PLZF–RAR fusion proteins repressor complexes also associate with the PLZF part of the protein that does not respond to RA administration, and release from transcriptional repression can only be achieved by additional administration of HDAC inhibitors (Melnick and Licht, 1999). Treatment of PLZF–RAR patients with a combination of HDAC inhibitors and pharmacological doses of RA has been proven efficient in inducing myeloid differentiation (Warrell et al., 1998) but often results in the emergence of resistant clones. For a detailed description of HDAC inhibitors in clinical trials readers are referred to Wang et al. (2001).

V. MUSCLE DIFFERENTIATION A. Interaction of MyoD with CBP/p300 and PCAF Muscle differentiation depends on two families of transcription factors: myogenic basic helix-loop-helix proteins (bHLH) and myocyte enhancer factors-2 (MEF2s). Overexpression of myogenic bHLHs (i.e., MyoD, Myf-5, Myogenin, MRF-4) is sufficient to induce the muscle-specific differentiation program. Expression of MyoD and Myf-5 results in chromatin remodeling at muscle-specific promoters (Gerber et al., 1997). MyoD has been shown to interact with p300; and p300 coactivates MyoD-dependent transcription of muscle-specific promoters (Eckner et al., 1996; Sartorelli et al., 1997; Yuan et al., 1996). The N-terminal KIX domain and the C-terminal CH3 region of p300 are characterized binding sites for MyoD (Riou et al., 2000; Yuan et al., 1996). The CH3 region of p300 is involved in the interaction with several cellular and also viral proteins that regulate processes as diverse as proliferation (E2F-1), differentiation (GATA-1), apoptosis (p53), and transformation (E1A) (Goodman and Smolik, 2000). Competition for this binding region is likely to be involved in cellular decision processes. MyoD and PCAF seem to be able to bind to the CH3 region of p300 simultaneously, whereas the adenoviral protein E1A, which also interacts with the CH3 region of p300, inhibits binding of MyoD and PCAF to p300. Injection of antibodies against PCAF and p300 demonstrated that both HATs are necessary for the muscle differentiation process (Eckner et al., 1996; Puri et al., 1997). Although initial studies with deletion mutants of p300 and PCAF suggested that only the enzymatic HAT activity of PCAF was required for muscle differentiation (Puri et al., 1997), this model has been revised recently. Administration of p300-specific HAT inhibitors demonstrated the necessity of the p300 HAT domain for the muscle differentiation process. The requirement for PCAF vs. p300 HAT activity might be regulated in a timely ordered manner. p300, for

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example, shows specificity for induction of late muscle genes, concomitant with an increase in its intrinsic HAT activity (Polesskaya et al., 2001a). Interestingly, MyoD itself can serve as a substrate for p300 and PCAF (Polesskaya et al., 2000; Sartorelli et al., 1999). The importance of MyoD acetylation in the differentiation process is not completely understood. Sartorelli et al. have proposed a structural change in the conformation of MyoD and increased DNA binding of MyoD following acetylation by PCAF (Sartorelli et al., 1999). Polesskaya et al., however, demonstrated an increased affinity of acetylated MyoD for CBP (Polesskaya et al., 2001b). This interaction requires the bromodomain of CBP/p300 and is likely to involve a recognition process similar to that observed for acetylated histone tails and the bromodomains of Gcn5 and TAFII250. Tighter binding of CBP/p300 to muscle-specific promoters might lead to increased histone acetylation in the promoter region and enhanced transcription of muscle-specific genes.

B. MyoD and HDAC1 In undifferentiated myoblasts, MyoD is found in association with HDAC1, which represses the transcriptional activation potential of MyoD. Upon differentiation, HDAC1 dissociates from MyoD; and MyoD associates with PCAF (Mal et al., 2001). The interactions of HDAC1 and PCAF with MyoD might therefore be mutually exclusive. The mechanism responsible for release of HDAC1 from MyoD is not completely understood. Puri et al. (2001) have suggested a link to the Rb pathway: during the differentiation process, Rb becomes progressively hypophosphorylated and associates with HDAC1. A mutant HDAC1 protein, deficient for Rb-binding, was unable to relieve repression of transcription from muscle-specific promoters. They postulate that redistribution of HDAC1 into Rb-containing complexes during the muscle differentiation process could be one of the means that regulates MyoD/HDAC repressor complexes (Fig. 2).

C. MEF2 and Deacetylases The class II HDACs 4 and 5 interact directly with MEF2 (Miska et al., 1999), and overexpression of HDACs 4 and 5 inhibits muscle differentiation (Lu et al., 2000a). Activation of MEF-2 dependent promoters requires dissociation of the MEF-2/HDAC II complex. Phosphorylation of class II HDACs by CaMK triggers the release from MEF-2 (Lu et al., 2000b). In addition, phosphorylation of HDACs 4 and 5 induces cytoplasmic localization of these proteins, via interaction with proteins of the 14-3-3 family (Grozinger and Schreiber, 2000; McKinsey et al., 2000; Wang et al., 2000).

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Fig. 2 Activation of MyoD regulated promoters during muscle differentiation. In proliferating myoblast, MyoD and MEF-2 are associated with HDAC proteins. Induction of differentiation leads to release of HDAC proteins and the formation of transcriptional activator complexes that contain CBP and PCAF. HD: HDAC; P: phosphorylation; Ac: acetylation.

VI. HEMATOPOIESIS A. GATA-1 GATA-1 is a zinc finger-containing transcription factor that is required for the erythroid and megakaryocytic differentiation processes (Blobel, 2000). GATA-1 interacts with CBP in vivo and in vitro; and GATA-1-dependent transcription is augmented by overexpression of CBP/p300 (Blobel et al., 1998; Boyes et al., 1998). The GATA-1 binding site on CBP has been mapped to the CH3 region of CBP (Blobel et al., 1998). Overexpression of the adenovirus protein E1A, which also interacts with the CH3 region of CBP, blocks the erythroid differentiation process and inhibits GATA-1-dependent transcription. CBP and p300 acetylate GATA-1 in vitro (Boyes et al., 1998; Hung et al., 1999). The importance of this acetylation event in vivo, however, is controversial. Whereas Boyes et al. showed increased DNA binding by acetylated GATA-1, the second study failed to detect this effect.

B. EKLF ¨ The erythroid Kruppel-like factor EKLF is essential for the regulation of beta-globin expression (Miller and Bieker, 1993). In overexpression experiments, EKLF can interact with CBP, p300, and PCAF. However, only CBP and p300 seem to acetylate EKLF in vitro, and only CBP and p300 enhance transcriptional activation by EKLF in vivo (Zhang and Bieker, 1998).

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Acetylation of EKLF does not affect its DNA-binding affinity. Mutation of one of the target lysine residues of EKLF resulted in reduced transactivation potential and nonresponsiveness to coactivation by CBP or p300. Zhang et al. (2001) demonstrated that acetylation of EKLF results in higher affinity for the SWI/SNF complex. The transcription factor EKLF therefore seems to integrate different chromatin modifying complexes at specific target promoters. Transcriptional activation in this case is most likely a result of histone acetylation combined with remodeling of the nucleosomal structure of the promoter region.

C. CBP/p300 and Hematopoietic Disorders 1. HEMATOPOIETIC DISORDERS IN CBP/p300 KNOCKOUT MICE Although CBP and p300 show many overlapping functions, divergence has been observed for certain differentiation processes. Common features of CBP and p300 knockout mice are growth retardation and developmental defects such as lack of neural tube closure and embryonic lethality (Tanaka et al., 1997; Yao et al., 1998). p300 homozygous animals show characteristic defects in heart development, whereas heterozygous CBP knockout mice display a high incidence of hematological malignancies. In addition, some of the defects observed in CBP knockout mice are reminiscent of alterations seen in the Rubinstein–Taybi syndrome (RTS). This human disorder is characterized by cranial and digital malformations, mental retardation, and hematopoietic abnormalities. Patients show mutation of one CBP allele (Petrij et al., 1995) and are predisposed to certain types of cancer, specifically, childhood tumors of neural crest origin. Tumor development is usually accompanied by loss of the second CBP allele (Miller and Rubinstein, 1995).

2. GENOMIC TRANSLOCATIONS AND LEUKEMIAS Genomic translocations of CBP and p300 are also associated with certain subtypes of leukemia. As discussed below, fusion of CBP to MOZ results in the development of acute myeloid leukemia (AML). Another genomic translocation fuses CBP to the mixed lineage leukemia gene MLL/ALL-1, a homologue of the Drosophila Trithorax protein. MLL is found in several translocations in childhood leukemias and in chemotherapy-induced secondary leukemias (Dimartino and Cleary, 1999). All of the MLL fusion proteins contain the N-terminal region of MLL and the C-terminal part of the translocation partner, for example, CBP (Sobulo et al., 1997). In many cases,

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MLL retains its DNA binding domain, transcriptional repression domain, and the SET domain, a region conserved in methyltransferases. Fusion to CBP adds the C-terminal part of this protein, including the bromodomain and HAT region. Although the molecular mechanism that leads to tumorigenesis is not understood, transcriptional deregulation due to aberrant chromatin remodeling is a likely hypothesis. Mutations of p300 have also been found in certain tumors (Gayther et al., 2000; Muraoka et al., 1996). Although the incidence of CBP/p300 mutations in tumors is low, their occurrence suggests a possible role for both proteins in tumor suppression.

3. MYST-FAMILY MEMBERS AND LEUKEMIAS As mentioned above, another member of the MYST family is MOZ (monocytic leukemia zinc finger protein), which is a human homologue of the yeast Sas3 protein (Reifsnyder et al., 1996). Although an actual HAT activity has not been demonstrated for MOZ, such a catalytic activity is strongly suggested due to homology to other MYST family members. MOZ is essential for proper differentiation of hematopoietic cells. Genomic translocations of MOZ and fusion of the gene to that encoding other HATs (CBP, p300, TIF2) is observed in some leukemias (Borrow et al., 1996; Carapeti et al., 1998; Kitabayashi et al., 2001). All of these fusion proteins contain the N-terminal region of MOZ, including its potential HAT domain, linked to the C-terminal regions of CBP, p300, or TIF2, containing a second HAT domain (Fig. 1). In addition, TIF2 has been shown to associate with CBP/p300, potentially generating a protein complex with three functional HAT domains. The increased number of HAT domains and/or the lost regulatory regions can lead to aberrant promoter activation and development of leukemias. Interestingly, like MOZ, many of the other MYST family proteins contain zinc finger domains and chromodomains.

VII. HUNTINGTON’S DISEASE Huntington’s disease is a neurodegenerative disorder that is characterized by movement disorders and progressive dementia (Gusella et al., 1996). The only identified mutation implicated in this disease is the expansion of a polyglutamine stretch in the N-terminal region of huntingtin (Htt). Expression of polyglutamine-containing N-terminal fragments of Htt is sufficient to trigger the disease phenotype in mice (Mangiarini et al., 1996). Histologically, the disease is accompanied by the appearance of intranuclear and cytoplasmic inclusions that contain Htt and ubiquitin (Davies et al., 1997; DiFiglia et al., 1997). Recent reports demonstrate that the polyglutamine form of Htt

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associates with a glutamine repeat sequence of CBP and sequesters it in nuclear and cytoplasmic inclusions both in transgenic mice and in the brains of Huntington patients (Nucifora et al., 2001; Steffan et al., 2000). In addition, it has been observed that mutant Htt reduces the transcriptional coactivating function of CBP (Nucifora et al., 2001; Steffan et al., 2000). Overexpression of CBP, on the other hand, rescues some of the disease-related cellular phenotypes (McCampbell et al., 2000; Nucifora et al., 2001). Furthermore, Steffan et al. have shown that mutant Htt interacts directly with the HAT domains of CBP and PCAF and represses the HAT activities of CBP, p300, and PCAF in vitro (Steffan et al., 2001). Expression of the mutated Htt protein in cell culture leads to a reduction in the acetylation levels of H3 and H4, and this effect is reversed by the administration of histone deacetylase inhibitors (Steffan et al., 2001). It should be mentioned that Htt also associates with transcriptional repressor complexes such as mSin3A and N-CoR that can recruit histone deacetylase activity (Boutell et al., 1999; Steffan et al., 2000). Although the molecular mechanism leading to the development of Huntington’s disease is not completely understood, the use of HDAC inhibitors shows encouraging effects. This is especially important since, to date, no effective treatment exists for this lethal disease.

VIII. HISTONE ACETYLATION IN COMBINATION WITH OTHER CHROMATIN MODIFICATIONS Although it is not the subject of this review, it should be kept in mind that other histone modifications exert profound influences on transcriptional regulation. Interplay between different chromatin-modifying systems has been observed in several diseases. The combination of DNA methylation and histone deacetylation in the silencing of promoters is especially intriguing. The genetic loci of MLH1, TIMP3, and CDKN2A (p16) are hypermethylated in the colorectal carcinoma cell line RKO. CDKN2B is hypermethylated in the leukemic cell line KG1a. A combined treatment by demethylating agents and histone deacetylase inhibitors has been proven most efficient for activation of transcription from these loci (Cameron et al., 1999). Similar experience was reported from studies of the fragile X-syndrome. The genomic locus for this disease (FMR-1) is mutated and inappropriately silenced. Loss of FMR-1 expression is manifested by mental retardation. Recent studies have revealed that silencing of FMR-1 is linked to cytosine methylation of the DNA and deacetylation of histones H3 and H4 in the 5 region of the gene. Although treatment with the HDAC inhibitor TSA restores acetylation of histone H4, this does not result in activation of gene expression. Administration of methyltransferase inhibitors, however,

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resulted in both absence of methylation and acetylation of the genomic region, with activation of gene expression (Coffee et al., 1999).

IX. CONCLUSION Although histones were long considered as simple packaging units for DNA, it is now clear that modifications of histones and nucleosome architecture actively participate in transcriptional regulation and thus in many pathways involved in cell fate control. Recent studies demonstrate close interactions between different modification systems. Sequential recruitment of SWI/SNF and HAT complexes has been demonstrated for the HO locus in yeast (Cosma et al., 1999). Cell cycle-dependent recruitment of chromatin remodeling complexes has been reported in several systems. Whereas Gcn5 alone can activate certain promoters during interphase, transcriptional activation of promoters during mitosis also requires the presence of SWI/SNF complexes (Krebs et al., 1999). Multiple modifications on histone tails suggest the existence of a histone “code” (Strahl and Allis, 2000) that is deciphered by specific protein modules (e.g., chromo- and bromodomains). Additionally, Thomson and colleagues have postulated a dynamic model for histone modifications, with specific modifications actively changing as transcription proceeds (Thomson et al., 2001). Acetylation of histones can occur on a small number of nucleosomes in the promoter region of a gene or extend over several hundreds of base pairs of DNA, and the interrelationship between the acetylation and methylation status on specific histones is currently under active investigation. The association of distinct site-specific histone H3 methylation patterns with heterochromatic vs. euchromatic compartments, and the fact that boundary elements indeed prevent the spread of silencing into neighboring regions of the chromosome have now been directly demonstrated in a fission yeast model (Noma et al., 2001). Deletion of these boundary elements can lead to silencing of normally active genes in adjacent regions, and it might be anticipated that, in some human diseases, chromosomal translocations could result in inappropriate silencing of normally active genes (or vice versa) through disruption of boundary elements. Downregulation of cell cycle regulators and tumor suppressor genes and loss of differentiation-specific gene expression are frequently associated with tumor development. Analysis of the molecular defects that lead to inappropriate promoter activation or silencing will be necessary in order to design specific tools for treatments. In this respect it will be important to identify the enzymes and complexes that are crucially involved in histone modification and promoter regulation. The recent success in unraveling HAT and HDAC

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complexes and the growing information on histone and DNA methyltransferases are encouraging. Use of HDAC inhibitors and demethylating agents in the treatment of cancer has already proven successful. Since both treatments may cause severe side effects, the design of disease- and cell type-specific drugs is likely to prove necessary. The development of specific inhibitors for certain HATs or HDACs is in progress. However, the documented involvement of these enzymes in multiple complexes makes it necessary to target the inhibitors specifically to one or the other complex or promoter region. Misfunctioning enzymes (i.e., fusion proteins that arise from genomic translocations) could be targeted for specific degradation or inactivation through complexing with dominant negative proteins.

ACKNOWLEDGMENTS Work from the authors is supported by the European Community, 5th Framework Programme (QLG1-CT-1999-00866 and QLGA-CT-2000-51259) and by La Ligue Nationale Contre le Cancer.

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Molecular Pathogenesis of Human Hepatocellular Carcinoma Michael A. Kern, Kai Breuhahn, and Peter Schirmacher∗ Institute of Pathology, University of Cologne, D-50931 Cologne, Germany

I. Introduction II. Morphology of Human Hepatocarcinogenesis III. Molecular Etiology A. General (Nonspecific) Mechanisms B. Hepatitis B Virus C. Hepatitis C Virus D. Other Causes IV. Host Carcinogenic Events A. Genomic Alterations B. Alterations in Specific Tumor-Relevant Host Genes C. Oncogenic Molecular Cross-Talk V. Functional Consequences A. Cell Growth B. Neoangiogenesis C. Invasion and Metastasis VI. Therapeutic Implications References

I. INTRODUCTION Hepatocellular carcinoma (HCC) is one of the most frequent human cancers. Depending on the type of statistic, it is the fifth to seventh most frequent malignancy worldwide (Bosch, 1997). It is most predominant in subsaharan Africa and far eastern Asia, but its presence is not limited to developing countries; it is also high in some industrialized countries, such as Japan. In Europe there is a strong north-to-south gradient, with relatively high incidence in Spain, Italy, and Greece. The incidence appears to be rising, even in countries with low HCC-frequencies. In more than 80% of the cases, a ∗ Address correspondence to Peter Schirmacher, M.D., Institute of Pathology, University of Cologne, Joseph-Stelzmann-Str. 9, D-50931 Cologne, Germany; Tel: +49-221-478-5257; Fax: +49-221-478-6360; E-mail: [email protected]

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Table I Etiology of Human HCC

High frequency Moderate frequency Low frequency

High risk

Low risk

Chronic HBV infection Chronic HCV infection (stage 4) Haemochromatosis (stage 3) [Aflatoxin B1] Glycogen-storage disease (type1) Hereditary tyrosinemia

Chronic alcohol abuse “Healed” HBV infection

Wilson’s disease α1–AT-deficiency Chemicals (vinyl chloride, Thorotrast)

well-defined etiology causes chronic liver disease and leads to the development of HCC (Table I). In the western world, where infections with hepatitis viruses are usually obtained during adulthood, 70–80% of the HCCs are accompanied by complete cirrhosis in the nontumorous liver. In areas with endemic hepatitis B virus (HBV) and hepatitis C virus (HCV) infection, the time point of infection is frequently around birth in a state of immunological tolerance and therefore the cirrhosis rate in these HCC patients is frequently below 50%. Overall, the geographic distribution of HCCs matches well the prevalence of chronic hepatitis virus infections (Szmuness, 1978). Clinical diagnosis of HCC is difficult, especially in early stages (