Protein Phosphorylation

Protein Phosphorylation Edited by F. Marks

0 VCH Verlagsgesellschaft mbH, D-69451 Weinheim (Federal Republic of Germany), 1996

Distribution: VCH, P. 0. Box 10 1161, D-69451 Weinheim (Federal Republic of Germany) Switzerland: VCH, P. 0. Box, CH-4020 Basel (Switzerland) United Kingdom and Ireland: VCH (UK) Ltd., 8 Wellington Court, Cambridge CB1 1HZ (England) USA and Canada: VCH, 220 East 23rd Street, New York, NY 10010-4606 (USA) Japan: VCH, Eikow Building, 10-9 Hongo 1-chome, Bunkyo-ku, Tokyo 113 (Japan) ISBN 3-527-29241-1

Protein Phosphorylation Edited by Friedrich Marks

-

-

-

Weinheim - New York Base1 Cambridge Tokyo

Prof. Dr. Friedrich Marks Deutsches Krebsforschungszentrum Forschungsschwerpunkt I1 : Tumorzellregulation Abteilung : Biochemie gewebsspezifischer Regulation Im Neuenheimer Feld 280 D-69120 Heidelberg This book was carefully produced. Nevertheless, authors, editor and publisher do not warrant the information contained therein to be free of erros. Readers are advised to keep in mind that statements, data, illustrations, procedural details or other items may inadvertently be inaccurate. Published jointly by VCH Verlagsgesellschaft mbH, Weinheim (Federal Republic of Germany) VCH Publishers, Inc., New York, NY (USA)

Editorial Director: Dr. Michael Bar Production Manager: Dip1.-Wirt.-Ing. (FH) Bernd Riedel

Library of Congress Card No. applied for.

* A catalogue record for this book is available from the British Library.

Deutsche Bibliothek Cataloguing-in-Publication Data :

Protein phosporylation I ed. by Friedrich Marks. - Weinheim ; New York ; Basel ; Cambridge ;Tokyo : VCH, 1996 ISBN 3-527-29241-1 NE: Marks, Friedrich [Hrsg.]

0VCH Verlagsgesellschaft mbH, D-69451 Weinheim (Federal Republic of Germany), 1996 Printed on acid-free and chlorine-free paper. All rights reserved (including those of translation into other languages). No part of this book may be reproduced in any form - by photoprinting, microfilm, or any other means - nor transmitted or translated into a machine language without written permission from the publishers. Registered names, trademarks, etc. used in this book, even when not specifically marked as such, are not to be considered unprotected by law. Composition: Mittenveger Werksatz GmbH, D-68723 Plankstadt Printing: Strauss Offsetdruck GmbH, D-69509 Morlenbach Bookbinding: Wilh. Osswald + Co., D-67433 NeustadtWinstr. Printed in the Federal Republic of Germany

Preface

This book deals with a biochemical reaction which despite its simplicity provides a major mechanism for the interaction of protein molecules in living cells. Such interactions are essential for a cell to receive and decipher messages from its environment, i.e. for its ability to communicate and thus to survive. In the living world, communication is organized in a hierarchal order at different levels, i.e. social systems, individuals, tissues, cells and - finally - molecules. While at the higher levels almost every physicochemical medium is used for the transmission of messages, at the cellular and especially at the molecular level signaling is brought about predominantly by chemical interactions, i.e. the reversible formation of covalent and non-covalent bonds between molecules. However, it is not so much the molecular mechanism of such interactions but the context in which they occur that lies at the heart of the cellular communication process. This is because the central aspect of communication is the meaning of a signal rather than its nature. It is most important to realize that meaning does not depend on the structure of a signal but on its interaction with the recipient. In other words: the medium is not the message, and communicative signals are symbols which have to be decoded by the recipients on the basis of suitable preinformation. This confers a high degree of arbitariness on any signal, i.e. one and the same event may be used for the transmission of completely different meanings. This arbitariness can be found at all levels of communication in biological systems. Thus, the autonomous nervous system does its numerous and diverse jobs mainly by employing only two signal molecules, i.e. acetylcholine and noradrenaline, and at the subcellular level the ambiguity of biological signaling is most impressively demonstrated by protein phosphorylation: here it is a very simple chemical signal that has a plethora of meanings depending on the context in which it occurs. It is becoming apparent now that protein phosphorylatioddephosphorylationprovides a major binary code for signal processing (i.e. decoding and interpretation) in cells. This mechanism, together with other chemical interactions, builds a tight communicative network between innumerable protein molecules. Such a network - which in many aspects resembles a neuronal network - shows an amazingly high degree of redundancy, cross-talk and feed-back control of the signaling pathways which is a prerequisite for its plasticity, i.e. its ability to adapt and to learn. A still widely mysterious aspect of complex signal processing networks is that they enable the emergence of a “reasonable” response out of apparently “chaotic” interactions. The perception of a sensory signal results in a diffuse excitation of millions of neurons scattered all over large parts of the brain, and the idea has been put forward that learning and memory operates in a manner which resembles holography rather than information storage on a disk. Evidence has been provided that proper and mean-

VI

Preface

ingful signal processing in the brain requires a “chaotic” neuronal baseline activity on which specific excitation patterns are superimposed upon signal perception. It has been proposed that this “chaotic” baseline activity provides the brain with a huge collection of potential response patterns. Signal processing would then be accomplished by the selection of a proper excitation pattern rather than by its de-novo formation. The same may be true for signal processing not only by other organs but even by single cells. It is becoming more and more evident that the perception of a signal in a cell, such as a hormone or a neurotransmitter, activates a large part of the signal processing network rather than a single signal-transducingpathway. As in the brain a diffuse excitation pattern is emerging rather than a precisely defined sequence of chemical reactions. It has still to be shown whether or not this excitation pattern is also based on a “chaotic” baseline activity. If so this might be a common principle of highly organized signal processors, be it a single cell, a brain or a society. This may also distinguish a biological information processor from a computer - at least one of the present generation - in a most fundamental way. The complexity of biological signal processing puts us rapidly at the limits of our capabilities to describe and to understand, a situation molecular biologists share with neurophysiologists. It is therefore mandatory to review the existing data and to try to put them into a framework and to provide a guide for students and other newcomers in this field. This is one of the aims of this book. It has been the editor’s responsibility to invite internationally renowned experts to provide substantial contributions to a selection of aspects of protein phosphorylation which he felt to be particularly suitable for an introduction into the field as a whole. I did not feel competent to dictate style, arrangement and content of the individual chapters. Therefore, the different contributions reflect the authors’ personal ways to handle the subject, ranging from rather broad and comprehensive overviews to more specialized in-depth treatments. Since current research on protein phosphorylation is focussed predominantly on the different classes of enzymes it seemed reasonable to base the book on a similar scheme. Due to the phenomenon of “signaling cross-talk”, i.e. tight interconnections between the signaling pathways of the cell, the reader has to put up with some overlap between the individual chapters. The selection of the different topics had to be done in an exemplary manner. Considering all classes of protein kinases and phosphatases in separate chapters would have been beyond the scope of this book and in conflict with the intention to provide an introduction into the field rather than to completely summarize the available data. For compensation, general reflections on the role of protein phosphorylation in cellular signaling are found in Chapters 1and 2 together with some information on subjects which are not treated in detail in the rest of the book. These include protein phosphorylation in prokaryotes and plants, receptor Ser/Thr kinases, the role of protein phosphorylation in the control of mRNA translation and DNA repair, structural aspects of protein kinase function, consequences of phosphorylation for protein structure etc. Again the selection of the topics may appear to be rather arbitrary because it is aiming at exemplarity rather than on completeness. Also in the other chapters emphasis is laid on more general subjects. This is immediately perceivable for chapter 10 and 11, dealing with signaling cross-talk and transcriptional control, respectively, but it also holds true for the articles focussing on individual enzymes.

Preface

VII

Such general aspects include, for instance, elucidation of protein kinase structure (chapter 2), multienzyme families (chapter 3), brain function (chapter 5), cell cycle control (chapter 6), cancer (chapters 1, 3, 7, 8, 9, and 12), developmental aspects (chapter 9), molecular genetic analysis of signaling pathways (chapters 4, 7, and ll), and signal extinction (chapter 12). We are just at the beginning of acknowledging the immense complexity of the molecular “brain of the cell”. It is highly questionable whether we will be able to describe and understand this subject in every detail in due time. Thus, this book offers a snapshot of one out of many different aspects of cellular communication. Many of the questions raised today will certainly have been answered in the near future (some of them already when the book will finally be published!), whereas other problems not yet recognized will become apparent. I nevertheless hope that for a reasonable period of time this book will serve as a useful guideline for students and scientists who are looking for an introduction to one of the most rapidly developing fields in biomedical science. Heidelberg, March 1996

Friedrich Marks

Contents

v

Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . List of contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

XV

List of Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

XIX

1

Thebrainofthecell Friedrich Marks

...................................

Signals and symbols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Proteins as communicative molecules . . . . . . . . . . . . . . . . . . . . . . . . The discovery of protein phosphorylation . . . . . . . . . . . . . . . . . . . . . Protein phosphorylation in prokaryotes . . . . . . . . . . . . . . . . . . . . . . Protein phosphorylation in eukaryotes . . . . . . . . . . . . . . . . . . . . . . . Eukaryotic protein kinases: common features and diversities . . . . . . . . Control of protein kinase activity . . . . . . . . . . . . . . . . . . . . . . . . . . . The problem of substrate specificity . . . . . . . . . . . . . . . . . . . . . . . . . Regulatory signals for protein kinases and examples of signaling crosstalk . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5.5 Protein tyrosine phosphorylation and the integrity of multicellular organisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.6 Signal extinction by protein dephosphorylation . . . . . . . . . . . . . . . . . . 1.7 Cancer: a cellular ‘psychosis’ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.8 Advancing beyond the metaphor: proteins as non-trivial machines . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1.1 1.2 1.3 1.4 1.5 1.5.1 1.5.2 1.5.3 1.5.4

2

2.1 2.2 2.2.1 2.2.2 2.2.3 2.3 2.3.1 2.3.2 2.4 2.4.1 2.4.2

CAMP-dependent protein kinase: structure. function and control . . . . . . Dirk Bosserneyer, Volker Kinzel and Jennifer Reed Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Biochemistry of cAPK . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Principles of purification of cAPK . . . . . . . . . . . . . . . . . . . . . . . . . . The catalytic subunit (C-subunit) . . . . . . . . . . . . . . . . . . . . . . . . . . . Control of cAPK . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cellular aspects of cAPK function and control . . . . . . . . . . . . . . . . . . In vivo control of cAPK . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cellular location of cAPK subunits . . . . . . . . . . . . . . . . . . . . . . . . . Structural aspects of cAPK function . . . . . . . . . . . . . . . . . . . . . . . . . Dynamics of substrate-induced fit in solution . . . . . . . . . . . . . . . . . . . Crystal structure of cAPK C-subunit . . . . . . . . . . . . . . . . . . . . . . . .

1 1 2 7 8 12 12 15 17

19 23 26 27 28 31 37 37 38 38 39 42 49 49 51 53 53 54

X

Contents

2.4.3 Aspects of future research on cAPK . . . . . . . . . . . . . . . . . . . . . . . . . 2.5 A quick look at the cGMP-dependent protein kinase: a close relative ofcAPK . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.6 Structural consequences of protein phosphorylation in general . . . . . . . 2.6.1 Immediate physical consequences . . . . . . . . . . . . . . . . . . . . . . . . . . indirect evidence . . . . . . . . . . . . . . . . . . . . 2.6.2 Conformational change . direct evidence . . . . . . . . . . . . . . . . . . . . . 2.6.3 Conformational change . 2.6.4 Structural effects in peptides. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 3.1 3.2 3.2.1 3.2.2 3.2.3 3.3 3.3.1 3.3.2 3.3.3 3.3.4 3.3.5 3.3.6 3.4 3.4.1 3.4.2 3.4.3

4

4.1 4.2 4.2.1 4.2.2 4.2.3 4.2.4 4.2.5 4.2.6 4.2.7 4.2.8 4.3 4.3.1

Protein kinase C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Friedrich Marks and Michael Gschwendt Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The protein kinase C isoenzyme family . . . . . . . . . . . . . . . . . . . . . . . The PKC subfamilies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . PKC isoenzyme structures: common features and differences . . . . . . . . Regulation of PKC activity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cellular functions of protein kinase C . . . . . . . . . . . . . . . . . . . . . . . . Activators and inhibitors as tools in PKC research . . . . . . . . . . . . . . . Phorbol ester effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Are effects of phorbol esters and DAG reliable indicators of PKC action? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Effects of altered PKC expression on cellular functions . . . . . . . . . . . . PKC substrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . How PKC may acquire substrate specificity . . . . . . . . . . . . . . . . . . . . Protein kinase C in disease. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Involvement of PKC expression in benign and malignant hyperproliferative diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Oncogenic and anti-oncogenic effects of protein kinase C expression . . . Protein kinase C and skin tumor promotion . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Casein kinases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Walter Pyerin. Karin Ackerrnann and Peter Lorenz The different classes of casein kinases . . . . . . . . . . . . . . . . . . . . . . . Protein kinase CK2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . History . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Biochemical features . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Molecular structures. interaction of subunits and regulation mechanisms . . CK2 genes and their chromosomal locations . . . . . . . . . . . . . . . . . . . Transcribed CK2 messages and transcription control . . . . . . . . . . . . . . Cell physiological roles of CK2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . CK2 in mitogenic signal transmission . . . . . . . . . . . . . . . . . . . . . . . . CK2 and the cell cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Protein kinase CK1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Biochemical features and molecular structures of CK1 . . . . . . . . . . . . .

66 67 68 68 68 69 71 74 81 81 82 82 83 85 90 90 91 93 94 95 100 101 102 104 104 109 117 117 118 118 118 119 124 127 130 132 135 141 141

Contents

4.3.2 Substrates. cell physiological roles. subcellular location. and regulation ofCK1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

XI

142 143

5

Ca’+/calrnodulin-dependentprotein b a s e and neuronal function . . . . . 149 Mark Mayford

5.1 5.2 5.2.1 5.2.2 5.2.3 5.2.4 5.2.5 5.3 5.4 5.5 5.5.1 5.5.2 5.6 5.6.1 5.6.2 5.7 5.7.1 5.7.2 5.8

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Biochemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Physical structure and distribution . . . . . . . . . . . . . . . . . . . . . . . . . . Functional domains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Regulation of Ca’+-dependent activity . . . . . . . . . . . . . . . . . . . . . . . Cooperative effects in the holoenzyme . . . . . . . . . . . . . . . . . . . . . . . CaM kinase as a frequency detector and a memory molecule . . . . . . . . Regulation of CaM kinase in vivo . . . . . . . . . . . . . . . . . . . . . . . . . . CaM kinase substrate proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . Presynaptic mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Neurotransmitter release and presynaptic facilitation . . . . . . . . . . . . . . Serotonin and aggression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Postsynaptic regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Long-term potentiation, learning, and memory . . . . . . . . . . . . . . . . . Modification of glutamate receptors . . . . . . . . . . . . . . . . . . . . . . . . . The control of gene expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . Regulation of transcription via CREB . . . . . . . . . . . . . . . . . . . . . . . Regulation of transcription via C/EBP-P . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

149 150 150 151 153 156 157 159 161 162 162 165 166 166 168 170 170 172 173 174

Cyclin-dependentprotein kinases and the regulation of the eukaryotic cell cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ingrid HofSmann

179

6.1 6.2 6.2.1 6.2.2 6.3 6.3.1 6.3.2 6.3.3 6.3.4 6.3.5 6.4 6.5 6.6 6.7

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179 Phosphorylation sites on cdc2 kinase . . . . . . . . . . . . . . . . . . . . . . . . 180 Phosphorylation events inhibiting cdc2 kinase activity . . . . . . . . . . . . . 182 Phosphorylation on Thrl61 is required for activation of cdc2 kinase . . . . 183 Regulation of cdc2 phosphorylation . . . . . . . . . . . . . . . . . . . . . . . . . 183 Cyclin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 184 The cdk-activating kinase (CAK) . . . . . . . . . . . . . . . . . . . . . . . . . . 185 The protein kinases wee1 and mikl . . . . . . . . . . . . . . . . . . . . . . . . . 186 The Thrl4 kinase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 188 The cdc25 protein phosphatase . . . . . . . . . . . . . . . . . . . . . . . . . . . . 188 Regulation of other cyclin-dependent kinases . . . . . . . . . . . . . . . . . . . . 191 Other regulators of cdks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 194 Substrates of cyclin-dependent kinases . . . . . . . . . . . . . . . . . . . . . . . 196 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197

XI1 7

Contents

Raf protein serinelthreodne kinases . . . . . . . . . . . . . . . . . . . . . . . .

203

Ulrike Naumann. Angelika Horneyer, Egbert Flory and Ulf R . Rapp

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Raf: its role in disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . O v e ~ i e w. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Raf in retroviruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Gene structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C-raf genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-Raf . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B-Raf . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Raf genes in invertebrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Raf genes in plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chromosome mapping of Raf family proto-oncogenes . . . . . . . . . . . . . C.raf.1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-raf . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B-raf . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Tissue distribution of Raf . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C.raf.1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-raf . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B-raf . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Protein structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Raf-1 : role and function in signal transduction . . . . . . . . . . . . . . . . . . Raf-1 and the cytoplasmic kinase cascade . . . . . . . . . . . . . . . . . . . . . Regulation of Raf function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Downstream of Raf . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Raf in the regulation of cellular processes . . . . . . . . . . . . . . . . . . . . . Proliferation and transformation . . . . . . . . . . . . . . . . . . . . . . . . . . . Cell differentiation and development . . . . . . . . . . . . . . . . . . . . . . . . Proliferation versus apoptosis versus differentiation the role of Raf in cell fate determination . . . . . . . . . . . . . . . . . . . . . . Future perspectives. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

203 203 203 205 207 207 209 210 210 211 211 211 211 212 212 212 213 213 213 216 216 216 220 222 222 223

8

Non-receptor protein tyrosine kinases . . . . . . . . . . . . . . . . . . . . . . . Geraldine M . Twamley and Sara A . Courtneidge

237

8.1 8.2 8.2.1 8.2.2 8.2.3 8.2.4 8.2.5 8.2.6 8.3 8.4

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Src familiy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Evolution and history . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Subclassification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Src structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Src family regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Substrates. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The members of the Src family . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Csk family . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The JAK family . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

237 238 238 239 241 243 244 245 253 253

7.1 7.2 7.2.1 7.2.2 7.3 7.3.1 7.3.2 7.3.3 7.3.4 7.3.5 7.4 7.4.1 7.4.2 7.4.3 7.5 7.5.1 7.5.2 7.5.3 7.6 7.7 7.7.1 7.7.2 7.7.3 7.8 7.8.1 7.8.2 7.8.3 7.9

226 227 228

Contents

XI11

The SYK family . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Btk family . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The FAK family . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Abl family . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Fps family . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

255 255 256 257 257 258 258

9

Receptor protein tyrosine kinases . . . . . . . . . . . . . . . . . . . . . . . . . . Deborah L . Cadena and Gordon N . Gill

265

9.1 9.2

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 265 Specific functions of receptor protein tyrosine kinases are provided by structural features . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 265 Ligand binding domains have evolved by combining various structural motifs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 266 The tyrosine kinase domain is required to mediate biological responses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267 Subdomains of the intracellular domain regulate diverse biological functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 268 Separate membrane-bound ligand-binding subunits and soluble protein tyrosine kinases also mediate intracellular signaling . . . . . . . . . 270 Receptor protein tyrosine kinases couple to signal transduction complexes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 270 Receptor protein tyrosine kinases dimerize in response to ligand . . . . . . 271 Intracellular signaling is mediated through interactions with tyrosine 271 phosphorylated proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Integrated responses to receptor protein tyrosine kinases . . . . . . . . . . . 276 Receptor protein tyrosine kinases function in development . . . . . . . . . . 277 Inappropriate expression of receptor protein tyrosine kinase activity leads to diseases including cancer . . . . . . . . . . . . . . . . . . . . . . . . . . 279 Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 280 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 281

8.5 8.6 8.7

8.8 8.9 8.10

9.2.1 9.2.2 9.2.3 9.2.4 9.3 9.3.1 9.3.2 9.4 9.4.1 9.4.2 9.5

10

Hierarchal phosphorylation of proteins . . . . . . . . . . . . . . . . . . . . . . . Carol J . Fiol and Peter J . Roach

285

10.1 10.2 10.3 10.4 10.5 10.6 10.7

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Phosphorylation of glycogen synthase . . . . . . . . . . . . . . . . . . . . . . . . Ordered versus hierarchal phosphorylation of proteins . . . . . . . . . . . . . Other examples of hierarchal phosphorylation . . . . . . . . . . . . . . . . . . Molecular mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Structural elements in phosphoserine/phosphothreoninerecognition . . . . Hierarchal phosphorylation and the integration of cellular information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

285 287 288 289 291 293

10.8

294 294 295

XIV

11

Contents

Phosphorylation of transcription factors . . . . . . . . . . . . . . . . . . . . . . Mathias Treier and Dirk Bohmann

11.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.1.1 Eukaryotic transcription factors . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.1.2 General transcription factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.1.3 Promoter-selective transcription factors. . . . . . . . . . . . . . . . . . . . . . . 11.1.4 Biological role of transcription factors . . . . . . . . . . . . . . . . . . . . . . . 11.2 The CREB familiy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.2.1 CAMP-inducible transcription regulation by CREB . . . . . . . . . . . . . . . 11.2.2 Integration of signals by CREB transcription factors . . . . . . . . . . . . . . 11.2.3 Antagonists of CREB: turning off the CAMPresponse . . . . . . . . . . . . 11.3 Jun . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.4 Serum response factor and ternary complex factors . . . . . . . . . . . . . . . 11.4.1 Serum response factor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.4.2 Ternary complex factors; Elk-1 and SAP-1 . . . . . . . . . . . . . . . . . . . . 11.5 STATs, JAKs and cytokine signaling . . . . . . . . . . . . . . . . . . . . . . . . 11.6 Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

Protein phosphatases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . Van HooJ J . Goris and W Merlevede

297 297 298 299 300 302 303 303 306 307 308 313 314 315 317 321 323 329

12.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.2 The protein serinelthreonine phosphatases . . . . . . . . . . . . . . . . . . . . 12.2.1 General classification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.2.2 Protein phosphatase type 1. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.2.3 Protein phosphatase type 2A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.2.4 Protein phosphatase type 2B . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.2.5 Protein phosphatase type 2C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.2.6 Novel members of the protein serinekhreonine phosphatase families . . . 12.3 The protein tyrosine phosphatases . . . . . . . . . . . . . . . . . . . . . . . . . . 12.3.1 Receptor-like PTPases. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.3.2 Cytosolic PTF'ases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.4 The dual-specificity protein phosphatases . . . . . . . . . . . . . . . . . . . . . 12.4.1 Protein tyrosine phosphatase displaying Ser/Thr phosphatase activity . . . 12.4.2 PP2A, a Ser/Thr phosphatase displaying Protein tyrosine phosphatase activity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.4.3 Interaction of protein phosphatases with viral tumor antigens . . . . . . . . 12.5 Alkaline and acid phosphatases . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.6 Protein histidine phosphatases . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.7 Historical events versus new perspectives . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

351 355 357 357 358 358

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

367

329 330 330 331 334 340 341 342 343 343 346 349 349

List of Contributors

Dr. Karin Ackermann Deutsches Krebsforschungszentrum Forschungsschwerpunkt I1 Tumorzellregulation Projektgruppe Biochemische Zellphysiologie Im Neuenheimer Feld 280 D-69120 Heidelberg Germany Phone: +49 (6221) 4232 17 Fax: +49 (6221) 423261 e-mail: K.Ackermann @ dkfz-Heidelberg.de

Dr. Dirk Bohmann Differentiation Programme European Molecular Biology Laboratory Postfach 1022 09 69012 Heidelberg Germany Phone: +49 (62 21) 38 74 16 Fax: +49 (6221) 387306 e-mail : [email protected]

Dr. Dirk Bossemeyer Deutsches Krebsforschungszentrm Forschungsschwerpunkt I1 Tumorzellregulation Abteilung Pathochemie Im Neuenheimer Feld 280 D-69120 Heidelberg Germany Phone : +49 (62 21) 42 32 57 Fax: +49 (6221) 423259 e-mail: [email protected]

Dr. Deborah L. Cadena Department of Medicine Division of Endocrinology and Metabolism University of California, San Diego La Jolla, California 92093-0650 USA Phone: +1 (619) 5342100 Fax: +1 (619) 5348193 e-mail: [email protected] Dr. Sara A. Courtneidge SUGENInc. Research 515 Galveston Drive Redwood City, CA 94063 USA Phone: +1 (501) 3 0676 12 Dr. Carol J. Fiol Indiana University, School of Medicine Department of Biochemistry and Molecular Biology Van Nuys Medical Science Building 410 635 Barnhill Drive Indianapolis, Indiana 46202-5122 USA Phone: +1 (3 17) 2 74 66 47 Fax: +1 (317) 2744686 e-mail: [email protected] Dr. Egbert Flory Insitut f i r Meduin, Strahlenkunde und Zellforschung der Universitat Versbacher StraBe D-97078 Wurzburg Germany Phone: +49 (931) 201 5141 Fax: +49 (931) 2013835 e-mail: [email protected]

XVI

List of Contributors

Dr. Gordon N. Gill Department of Medicine Division of Endocrinology and Metabolism University of California, San Diego La Jolla, California 92093-0650 USA Phone: +1 (619) 5342100 Fax: +1 (619) 5348193 e-mail: [email protected]

Dr. Angelika Hoffmeyer Institut fiir Medizin, Strahlenkunde und Zellforschung der Universitat Versbacher StraBe D-97078 Wurzburg Germany Phone: +49 (9 31) 2 01 51 41 Fax: +49 (931) 2013835 e-mail: [email protected]

Dr. J. Goris Katholieke Universiteit Leuven Faculteit der Geneeskunde Afdeling Biochemie Herestraat 6 B-6000 Leuven Belgium Phone: +32 (16) 34 57 00 Fax: +32 (16) 345995 e-mail: Biochem@ MED .KULeuven.ac. be

Prof. Dr. Volker Kinzel Deutsches Krebsforschungszentrum Forschungsschwerpunkt I1 Tumorzellregulation Abteilung Pathochemie Im Neuenheimer Feld 280 D-69120 Heidelberg Germany Phone: +49 (62 21) 42 32 53 Fax: +49 (6221) 423259

Dr. Michael Gschwendt Deutsches Krebsforschungszentrum Forschungsschwerpunkt I1 Tumorzellregulation Abteilung : Biochemie gewebsspezifischer Regulation Im Neuenheimer Feld 280 D-69120 Heidelberg Germany Phone : +49 (62 21) 42 45 05 Fax: +49 (6221) 424406 e-mail: m. [email protected]

Dr. Ingrid Hoffmann Deutsches Krebsforschungszentrm Forschungsschwerpunkt Angewandte Tumorvirologie Im Neuenheimer Feld 280 D-69120 Heidelberg Germany Phone : +49 (62 21) 42 48 00 Fax : 49 (62 21) 42 49 02 e-mail: ihoffmann@dkfzheidelberg. de

+

Dr. Peter Lorenz Cold Spring Harbor Laboratory PO. Box 100 Cold Spring Harbor, New York 11724 USA Phone: +1 (5 16) 3 67 84 78 Fax: +1 (516) 3678876 e-mail: [email protected]

Prof. Dr. Friedrich Marks Deutsches Krebsforschungszentrm Forschungsschwerpunkt I1 Tumorzellregulation Abteilung : Biochemie gewebsspezifischer Regulation Im Neuenheimer Feld 280 D-69120 Heidelberg Germany Phone: +49 (6221) 424531 Fax: +49 (6221) 424406 e-mail: [email protected]

List of Contributors

Dr. Mark Mayford Center for Neurobiology and Behavior College of Physicians and Surgeons of Columbia University and Howard Hughes Medical Institute 722 West 168th Street New York, N. Y. 10032 USA

Prof. Dr. Wilfried Merlevede Katholieke Universiteit Leuven Faculteit der Geneeskunde Afdeling Biochemie Herestraat 6 B-6000 Leuven Belgium Phone : +32 (16) 34 57 00 Fax: +32 (16) 345995 e-mail: Biochem@MED. KVLeuven. ac.be.

Dr. Ulrike Naumann Institut fiir Medizin, Strahlenkunde und Zellforschung der Universitat Versbacher StraBe 5 D-97078 Wurzburg Phone: +49 (931) 201 5141 Fax: +49 (931) 2013835 e-mail: [email protected]

Prof. Dr. Walter Pyerin Deutsches Krebsforschungszentrm Forschungsschwerpunkt I1 Tumorzellregulation Projektgruppe Biochemische Zellphysiologie Im Neuenheimer Feld 280 D-69120 Heidelberg Germany Phone : +49 (62 21) 42 32 54 Fax: +49 (6221) 423261 e-mail : W.Pyerin@DKFZHeidelberg .de

XVII

Prof. Dr. Ulf R. Rapp Institut fiir Medizin, Strahlenkunde und Zellforschung der Universitat Versbacher Stral3e 5 D-97078 Wiirzburg Germany Phone: +49 (9 31) 2 01 5141 Fax: +49 (9 31) 2 01 38 35 e-mail : [email protected] .de

Dr. Jennifer Reed Deutsches Krebsforschungszentrm Forschungsschwerpunkt I1 Tumorzellregulation Abteilung Pathochemie Im Neuenheimer Feld 280 D-69120 Heidelberg Germany Phone: +49 (62 21) 42 32 56 Fax: +49 (6221) 423259 e-mail: j [email protected]

Dr. Peter J. Roach Indiana University, School of Medicine Department of Biochemistry and Molecular Biology Van Nuys Medical Science Building 410 635 Barnhill Drive Indianapolis, Indiana 46202-5122 USA Phone: +1 (3 17) 2 74 1583 Fax: +1 (317) 2744686 e-mail : Peter [email protected]. edu.

Dr. Mathias Treier HHMI at the University of California, San Diego CMM. RM 34 9500 Gilman Drive La Jolla, CA 92093-0648 USA Phone: +1 (619) 5340758 Fax: +1 (619) 5348180 e-mail : [email protected]

XVIII

List of Contributors

Dr. Geraldine M. Twamley Tralee Regional Technical College Co. Kerry Ireland Phone: +353 (66) 246661 12

Dr. C. van Hoof Katholieke Universiteit Leuven Faculteit der Geneeskunde Afdeling Biochemie Herestraat 6 B-6000 Leuven Belgium Phone : +32 (16) 34 57 00 Fax: +32 (16) 345995 e-mail : Biochem@MED . KULeuven.ac.be

List of Abbreviations

AA AKAP AMPA AMP-PNP ANP AP-1 ARIA ATF

arachidonic acid A-kinase anchoring protein a-amino-3-hydroxy-5-methyl-isoxazol propionic acid adenylyl imidophosphate, non-hydrolysable ATP analog atrial natriuretic peptide activator protein 1 acetylcholine receptor-inducing activity activating transcription factor

BDNF bHLH bZip

brain-derived neurotrophic factor basic region helix-loop-helixdomain basic region leucine zipper domain

Ca, Ca2, Cp, Cp2, Cy C-subunit c*, CB CAK CaMK CaMRE cAMP CAP cAPK CBP CD cdc cdi cGMP cdk CEBP-0 cGPK CK1 (CKI) CK2 (CKII) CNTF CRE CREB CREM CSF-1

cAPK catalytic subunit isoforms catalytic subunit of cAPK isoelectric variants of catalytic subunit isoform Ca cdk-activating kinase calciudcalmodulin-activated kinase CaM kinase response element cyclic adenosine3!5'-monophosphate catabolite gene activator protein cyclic AMP-dependent protein kinase (= PKA) CREB-binding protein circular dichroism cell division cycle cyclin-dependent kinase inhibitor cyclic guanosine-3!5'-monophosphate cyclin-dependent kinase CCAAT enhancer binding protein$ cyclic GMP-dependent protein kinase (=PKG) casein kinase 1 casein kinase 2 ciliary neurotrophic factor cAMP response element cAMP response element binding protein cAMP response element modulator colony stimulating factor 1

XX

List of Abbreviations

CTF

carboxy-terminal domain of the largest subunit of RNA polymerase I1 CAAT-box transcription factor

DAG DARPP-32 DNA-PK dsI

diacyl glycerol dopamine- and CAMP-regulated phosphoprotein DNA-dependent protein kinase double-stranded RNA-activated inhibitor

eEF EF hand E2F EGF eIF EPO ERK

ribosomal elongation factor helix-loop-helixCa2+binding motif adenovirus E2 gene factor epidermal growth factor ribosomal initiation factor erythropoetin extracellularly (signal) regulated kinase (= MAPK)

F,/GSK3 FAK FGF FGFR FMTC

activating factor/glycogen synthase kinase 3 focal adhesion kinase fibroblast growth factor fibroblast growth factor receptor familial medullary thyroid carcinoma

GABA GAF GAP GAS Gas 2 GEF GHF-1 GM-CSF Grb GRE GSK-3

y-amino butyric acid y-interferon activated factor GTPase activating protein y-interferon activation site growth arrest-specific protein 2 Golgi-enriched fraction growth hormone transcription factor (=pit 1) granulocyte macrophage colony stimulating factor growth factor receptor binding protein glucocorticoid response element glycogen synthase kinase 3

HB-EGF HETE HGF HODE HRI HTH

heparin binding EGF hydroxy eicosatetraenic acid hepatocyte growth factor hydroxy octadecaenoic acid heme-regulated inhibitor helix-turn-helix domain

1-1 1-2 ICER IFN

inhibitor 1 inhibitor 2 inducible CAMPearly repressor interferon

CTD

List of Abbreviations

IGF IxB IL IL-2 IL-4 IL-6 IL-10 IP3 IRS-1 ISGF ISPK ISRE 3JHN

insulin-like growth factor inhibitor of NFxB interleukin interleukin-2 interleukin-4 interleukin-6 interleukin-10 inositol trisphosphate insulin receptor substrate-1 interferon-stimulated growth factor insulin-stimulated protein kinase interferon (a@)-stimulated growth element vicinal coupling constant between protons bonded to amide nitrogen and the a-carbons of an amino acid

JAK JNK Jun-ppase

Janus kinase Jun amino-terminal kinase (=SAPKs) Jun phosphatase

KCIP

Kinase C inhibitor protein

LCR LIF LT LTD LTP LTR

long control region leukemia inhibitory factor polyoma large T antigen long term depression long-term potentiation long terminal repeat

MAP-2 MAPK MAPKK MARCKS MBP MCSF-1 MDR MEK MEKK MEN MPF mT

microtubule-associated protein 2 mitogen-activated protein kinase (= ERK) MAPK kinase (= MEK) myristoylated alanine-rich-c-kinase substrate myelin basic protein macrophage colony stimulating growth factor multidrug resistance MAP ERK kinase (= MAPKK) MEK kinase multiple endocrine neoplasia maturation promoting factor polyoma middle T antigen

N-CAM NDF NGF NFxB NK

neural cell adhesion molecule neu differentiation factor nerve growth factor nuclear factor of the x enhancer B natural killer (cell)

XXII

List of Abbreviations

NLS NMDA NMR ~H-NMR 31P-NMR NT-3 NT-4

nuclear localization signal N-methyl-D-aspartate nuclear magnetic resonance proton NMR phosphorus NMR neurotrophin-3 neurotrophin-4

OA OSM

okadaic acid onconstatin M

PA PAK PCR PC-PLC PDGF PEP PGP PH PI-3-K pit 1 PI 3-K PI (43 )P2 PK PKA PKI PKI(5-24) PKC PKG PKR PLA2 PLCy Pleckstrin PP1 PP2A PP2B PP2C PPlG PPlM PPlN PPF PRb PR55 PR65 PR72 PR130

phosphatidic acid p21-(Rac)activated kinase polymerase chain reaction

phosphatidylcholine-specificphospholipase c platelet-derived growth factor phosphoenol pyruvate plasma membrane phosphoglycoprotein pleckstrin homology phosphatidylinositol-3-kinase pituitary transcription factor 1 (=GHF-1) phosphatidylinositol3-kinase

phosphatidylinositol4,5-bisphosphate

protein kinase cydic AMP-dependent protein kinase (= cAPK) heat- and acid-stable protein kinase inhibitor for cAPK inhibitory 20-amino acid peptide derived from PKI protein kinase C cyclic GMP-dependent protein kinase (= cGPK) RNA-dependent protein kinase phospholipase A2 phospholipase C-gamma platelet and leukocyte c-kinase substrate protein phosphatase type 1 protein phosphatase type 2A protein phosphatase type 2B protein phosphatase type 2C PP1 associated with glycogen-binding subunit (G subunit) PP1 associated with myosin-binding subunit (M subunit) PP1 associated with the nuclear inhibitor polypeptide paired pulse facilitation retinoblastoma protein phosphatase regulatory subunit of 55 kDa phosphatase regulatory subunit of 65 kDa phosphatase regulatory subunit of 72 kDa phosphatase regulatory subunit of 130kDa

List ofAbbreviations

XXIII

PRE PRTase PTP PTS IJTPA PTPase PTS

progesterone response element receptor-like PTPase protein-tyrosine phosphatase phosphotransferase system phosphotyrosyl phosphatase activator protein-tyrosine phosphatase phosphotransferase system

RACK RI , RII subunits RI2C2, RIIzCz RIa, RIIa, RIB, RIIP Rb RBD RSK RSV

receptor for activated C-kinase regulatory subunits of cAPK type I and type I1 tetrameric holoenzymes type I and type I1 cAPK regulatory subunit isoforms retinoblastoma gene product (=retinoblastoma protein) Ras binding domain ribosomal S6 protein kinase Rous sarcoma virus

SAP SAPK SF SH2 SH3 SIE SIF sos, sos SPl SRE SRF st STAT Stat91 SV40

SRF-associated protein stress-activated protein kinase (=JNKs) scatter factor Src homology domain 2 Src homology domain 3 Sis-inducible element Sis-induciblefactor son of sevenless (GDP-GTP exchange factor) specificity factor 1 serum response element serum response factor polyoma small T antigen signal transducer and activator of transcription signal transducer and activator of transcription, 91 kDa simian virus 40

TCF TCR TGFa TGFf3 TNFa TPA TRE TSE

ternary complex factor T cell receptor transforming growth factor a transforming growth factor f3 tumor necrosis factor a 12-0-tetradecanoyl-phorbol-13-acetate TPA-response element tissue-specificextinguisher

UBF

upstream binding factor

VEGF

vascular endothelial cell growth factor

XLA

X-linked agammaglobulinemia

Protein Phosphorflation Edited by Friedrich Marks copyright 0 VCH Vdagsgesfllahaft mbH,IYL)h

1 The brain of the cell Friedrich Marks

1.1 Signals and symbols The ability to communicate is one of the characteristic properties of cells and may actually be considered the fundamental condition of life. Communication takes place through the exchange of signals between transmitters and receivers. Biological signals are symbols, i. e. they have a distinct meaning. To respond adequately the receiver has to both recognize and decipher a signal. For this purpose prior information is required, which may either have been acquired or is genetically fixed. Signal transduction requires a physical medium. However, the significance of a signal is by no means encoded in its structure (the medium is not the message). Rather there exists an arbitrary connection between form and meaning resulting in an enormous flexibility of communicative systems. This principle prevailing in human language [l] holds equally true for cellular communication. Depending on the target tissue a hormonal signal such as adrenaline, for instance, has quite different meanings, because differentiation takes place exclusively in the receptor cells. The same holds true for intracellular signal processing, where a simple and apparently monotonous signaling reaction such as protein phosphorylation plays a central role resulting in countless functional consequences which depend on the particular target proteins. It is finally the receiver who coordinates the signal and its specific meaning. Thus, cellular communication has both a syntactic and a semantic aspect. To provide a symbol, e. g. a word or a picture, with a distinct meaning we need our brain. During the processing of sensory input signals rather diffuse patterns of excitation are observed in the brain which involve innumerable interconnections between millions of cells and do not allow a precise cellular localization of individual events [2]. Nevertheless, the result is generally a rather exact allocation of meaning which in turn is the precondition for a proper response. Signal processing is not restricted to neuronal networks but is a general property of every single cell where molecular networks do the job. In the following I shall use the metaphor of ‘the brain in the cell’ in order to emphasize the close relationship between signal processing in neuronal and molecular networks. Occasionally it has been stated that the genome resembles a ‘brain’ on the subcellular level. This is certainly not true: the genome is nothing but a memory store for primary protein structures. The brain, however, is much more, namely a device which uses memory for the interpretation of symbols aiming at proper responses to environmental influences. Thus, the cellular brain would at least include both the genome and the network of molecular interactions required for signal processing. Only by an interplay between these two entities can the meaning of a symbol be deciphered.

2

1

The brain of the cell

A term such as ‘meaning’ has, of course, a certain teleological after-taste which may be inacceptable for some scientists. However, if we restrict ourselves solely to describing structures and molecular interactions in physicochemical terms we will certainly fail to cope with the complexity of living systems, a situation similar to that with which a behavioristic hard-liner is confronted in psychology. It must be emphasized, however, that this does not imply anything like a ‘ghost in the molecule’, but that we are talking about the phenomenon of communicative interactions between biomolecules and cell structures resulting in the emergence of complexity and of properties which can neither be explained on the basis of molecular structures alone nor be reduced to structural parameters without losing exactly what proves to be their biological significance [3]. Each biomolecule and each molecular transformation gets its meaning only out of its ‘semantic milieu’, i. e. from the living organism, just as a word does from the framework of language. Although we are just at the beginning of understanding these relationships, some basic principles have become apparent already that indicate a high degree of similarity between intercellular (for instance, neuronal or endocrine) and intracellular (i. e. molecular) signal processing. On both levels we see a complex network pattern which operates in a non-linear manner due to a high degree of feedback interactions as well as multiplicity and redundancy of the processing units. Moreover, the processing units - cells or protein molecules - undergo permanent changes of their ‘internal state’ and, thus, of their receptive and responsive capabilities. This provides the systems with plasticity, i. e. it enables them to adapt and to learn. Both molecular and cellular signal-processing systems decipher the meaning of signals by adjusting them to the information they have previously acquired, be it the memory of the brain, or the genome and other molecular memory stores in a single cell. The nervous and endocrine systems establish long-distance communication along nerve fibers and blood vessels. On the subcellular level such connections result from interactions between molecules and substructures (Fig. 1.1).

1.2 Proteins as communicative molecules Intracellular signal processing depends on specific interactions between proteins. Such interactions include a direct non-covalent contact (as, for example, between receptors and G-proteins) as well as communication by diffusible signals (such as second messengers) or covalent changes (such as protein phosphorylation). Actually, signal transduction is a characteristic property not only of signaling proteins proper, but of all types of proteins. This property is based on an extraordinary structural flexibility and chemical reactivity which allows an enormous variability of the ‘internal state’. As input signals, such regulatory factors induce conformational changes which in turn result in specific alterations of protein function, i. e. the output signal. Thus, any enzyme, for instance, may be looked upon not only as a catalyst but also as a signal-transducing entity, i.e. the network of metabolic reactions is entirely superimposed by a signalprocessing network. Among the events involved in the communication between proteins, protein phosphorylation occupies a central position in that it appears to be the most variable and

1.2 Proteins as communicativemolecules

Neurons

6

Neurotransmitter

Q .’.....

3

Molecules

2 n d Messenger Phosphorylation

6 t

Figure 1.1 Common principles of chemical signal transduction between cells and between mol-

ecules. Cells - as for example neurons - communicate either via diffusible signal molecules (such as neurotransmitters,hormones, cytokines, etc.) or by means of direct contact via adhesion molecules. The same principles of communication hold true for the protein molecules involved in intracellular signal processing. Like cells, they may communicate either via diffusible signal molecules (second messengers) and chemical reactions (in particular protein phosphorylation) or by direct contact.

versatile mechanism for changing the ‘internal state’ of a protein in a reversible manner. Moreover, protein phosphorylation provides the major binary code for the processing of intercellular and environmental signals. Such exogenous signals are discriminated by receptor proteins, which directly or indirectly modulate the intracellular machinery of protein phosphorylation (Fig. 1.2). Thus, protein phosphorylation is of vital importance for intercellular communication in that it is required for the processing and proper interpretation of communicative signals. It is, therefore, all but surprising that a disease of intercellular communication such as cancer is to a great extent the result of defects in the network of protein phosphorylation. As a signal-transducing molecule a protein comprises at least two parts, a receiver module for discrimination of the input signal (regulatory domain) and a transmitter module for the emission of an output signal (functional domain). As far as receptor proteins are concerned the output signal is generally transduced in the form of an allosteric conformational change which is ‘recognized’by other signal-transducing proteins or protein domains located downstream in the signaling cascade. Many intercellular signal molecules such as proteins, peptides, amino acids, amines, and nucleotided nucleosides cannot penetrate the lipid barrier of the plasma membrane but interact with receptors at the cell surface (Fig. 1.2). Other signal molecules such as thyroid hormones, steroids and retinoids are able to enter the cell, finding their receptors in the cytoplasm or in the nucleus [4]. The effector molecules which are directly controlled by activated receptors include enzymes such as guanylate cyclases, protein kinases and GTPases (G-proteins), but

4

1

The brain of the cell

(A) SIGNALS

Ion channels

Enzymes, cytoskeleton, ion channels etc.

Responsive genetic elements

+

Gene products

CELLULAR EFFECTS

Figure 1.2 Standard pathways of outside-inside signaling in cells. Depending on their chemical structures signal molecules interact with receptor proteins localized either at the cell surface or in the cytoplasm. The signal-activated receptors are able to contact effector molecules such as (from the left to the right) tyrosine-specific protein kinases (Tyr-PK), G-proteins, ion channels, and gene-regulatory DNA sequences. In certain cases receptor and effector may be localized on one and the same protein molecule. Effector molecules modulate the input signal in amplitude and frequency and translate it into the cell’s signaling language. The latter makes use of direct interactions between proteins, a variety of second messengers and covalent protein modifications, in particular phosphorylation. It must be emphasized that the left diagram (A) and its schematic sketch (B, upper sketch) represent an extremely simplified picture. The variability of cellular signal cascades is actually much greater. Moreover, there are no linear pathways but a complex pattern of feedback interactions, and cross-talking (B, lower sketch). Signal reception thus results in diffuse excitation patterns rather than in precisely defined sequences of chemical interactions.

1.2 Proteins as communicative molecules

5

also ion channels and regulatory genomic sequences. Effector molecules modulate the signals and translate them into an intracellular ‘language’ of signal molecules (second messengers) and molecular interactions. The variability of second messengers can not yet be estimated. Well-known representatives such as the cyclic nucleotides (CAMP, cGMP), diacylclycerol (DAG), the inositol phosphates, and Ca2+ions, certainly only represent the tip of an iceberg. Second messengers control the function of other downstream effector proteins, in particular protein kinases, but also ion channels, components of the cytoskeleton, etc. For a large number of receptors the immediate downstream-effectors are Gproteins. G-proteins are guanine nucleotide-binding proteins with an intrinsic GTPase activity [5-71. Their main function is signal transduction and modulation. For this purpose G-proteins make contact with other effector proteins, in particular with ion channels and enzymes which catalyze second messenger formation, such as adenylate cyclase and phospholipases. Activation of a G-protein by an input-signal such as an activated receptor molecule results in an exchange of bound GDP by GTP. Receptorcoupled G-proteins are heterotrimeric molecules which upon activation dissociate into two subunits, a and p/y. Both subunits have been shown to influence different pathways of signal transduction [S]. The active state of a G-protein is only short-lived since the bound GTP is rapidly hydrolyzed by the intrinsic GTPase activity. Both the activating GDP/GTP exchange reaction and the inactivating GTP hydrolysis are under the control of accessory proteins which are components of other signaling pathways [9]. There is a striking analogy between this system and the presynaptic modulation of neurons. The enormous variability of such regulatory GTPases and their interactions with other signal-processing elements of the cell can not yet be assessed’. Beside being receptor-controlled effector molecules, GTPases also control mRNA translation and microtubuli association. Moreover, the Ras superfamily of so-called small (or monomeric) G-proteins [lo] represents a large group of regulatory GTPases with key functions in the control of cellular vesicle transport, organization of the cytoskeleton, and transduction of mitogenic signals. Together with the associated activator, inhibitor, and effector molecules, G-proteins form a signal-processingnetwork of their own, which transforms, integrates and modulates input signals in respect to their amplitude and frequency. Since the interactions between different G-proteins provide biochemical ‘AND’ and ‘BUTNOT’ logic gates, the G-protein network has been looked upon as a molecular microcomputer [ll]. Moreover, due to their intrinsic GTPase activity, GTP-activated G-proteins are exponential timers which become inactivated at characteristicrates. This may help to transform the digital mode of signal processing at the molecular level into an analog behavior of the cell. The receptors connected to G-proteins form a large family consisting of several hundred members [12]. They interact with numerous hormones and neurotransmitters, but also with environmental signals such as light (rhodopsin [13]), odorants [14], and taste stimulants. G-protein-coupledreceptors exhibit a common structural ‘serpentinemotif’, i. e. seven transmembrane domains. Like most other signal-transducing pro-

’ The most recent progress in this exciting field has been reviewed by Chant and Stowers [171](Added in proof).

6

1

The brain of the cell PROTEIN KINASE ATP

\

ADP

P

Protein

Protein-@

PROTEIN PHOSPHATASE

Figure 1.3 ATP-dependent protein phosphorylation and dephosphorylation as catalyzed by protein kinases and phosphoprotein phosphatases. In most cases, ATP enters the reaction as Mp-salt. @, phosphate.

teins these receptors are subject to a sophisticated feedback control of their activity which resembles an analogous situation in sensory nerve systems (see Fig. 1.9). Besides G-protein interactions, the reversible phosphorylation of proteins has evolved into the most efficient and versatile signal of intermolecular communication, being found in the simplest prokaryotes and the most sophisticated brain neurons alike. In fact in eukaryotes there is almost no cellular protein, which does not at least potentially provide a target for phosphorylatioddephosphorylation thus undergoing functional modulation. This holds true in particular for most components of the cellular signal processing machinery. Together with G-protein interactions, protein phosphorylation actually seems to be the major chemical code on which the function of the ‘brain in the cell’ is based. Phosphorylation brings about an abundant modification of the protein structure’. The phosphoryl group is covalently bound to amino acid residues such as serine, threonine and tyrosine (ester bond), histidine and lysine (amide bond), cysteine (thioester bond) or glutamic and aspartic acid (mixed anhydride bond). Phosphorylation used for signaling purposes has to be distinguished from the formation of phosphorylated proteins as short-lived transition states in enzymatic catalysis. An example is provided by phosphoglucomutase, which is phosphorylated transiently at a Ser residue when catalyzing the isomerization of glucose-1-P to glucose-6-P (cited in [15]). On the other hand, there is some overlapping between both types of protein phosphorylation, as shown, for instance, by two-component signaling in bacteria (see Section 1.4). Signaling protein phosphorylation is catalyzed by specific enzymes, the protein kinases, and canceled by corresponding phosphoprotein phosphatases (Fig. 1.3). Protein kinases appear to be the most variable and comprehensive enzyme family known. Several hundreds of such enzymes have been found already and the discovery rate is still in its exponential phase. Thus, the assumption that a eukaryotic cell may express at A detailed discussion of the consequences of protein phosphorylation for protein structure is found in Chapter 2.

1.3 The discovery of protein phosphorylation

7

least ‘1001’ different protein kinases [16] does not seem to be an overestimation. The same may be true for the corresponding phosphatases [17], although they have not yet been investigated as thoroughly as the kinases (see Chapter 12). It has been estimated that up to 5 % of the human genome may code for protein kinases and phosphatases [181. This would correspond to several thousand different enzymes!

1.3 The discovery of protein phosphorylation The first phosphoproteins to be discovered as early as in the 19th century were the milk proteins of the casein family and the egg yolk protein phosvitin, which may contain between 1-10 % of phosphorus, mainly as seryl phosphate. (In fact, phosvitin is the most highly phosphorylated protein known.) Whether or not the multiple phosphorylation of these proteins serves any regulatory purposes is questionable. It is generally believed that phosphorylation favors their role as nutrients, i. e. as rich sources of amino acids, phosphorus and ions [19]. Thus, phosphorylation alters the physiochemical properties of casein in such a way that it is kept in dispersion forming micellar structures which are able to bind large amounts of Ca2+ and other ions. Casein phosphorylation is catalyzed by a casein kinase found in the Golgi fraction of mammary gland cells [20]. This enzyme is not identical with the so-called casein kinases types I and 11, which fulfil important regulatory functions in many different cell types and which were so named after the traditional use of casein as a suitable protein kinase substrate [21] (see also Chapter 4). Actually, a liver casein kinase was the first protein kinase to be discovered [21]. For almost a century casein, phosvitin, and some related milk and egg yolk proteins were the major representatives of the phosphoprotein family. Consequently, protein phosphorylation was mainly recognized as a gross metabolic reaction. The first example of a regulatory role of protein phosphorylation was provided by the control of glycogen phosphorylase activity. As shown by Fischer and Krebs [22], and Sutherland and Wosilait [23] in 1955, the inactive b-form of this enzyme is converted into the active aform by reversible phosphorylation.The enzyme phosphorylase kinase which catalyzes this activation was the first regulatory protein kinase to be studied extensively [24,25]. It was found that the activity of this enzyme was itself controlled not only by reversible phosphorylation, but also by Ca” ions. In the course of these studies cyclic AMP, as an intracellular mediator of glycogenolytic and lipolytic hormones (adrenaline, glucagon, etc.), was discovered [26] and the second messenger concept of hormone action formulated [27]. Efforts to arrive at an understanding of cAMP action led to the discovery of a CAMP-stimulatedphosphorylating enzyme responsible for the activation of phosphorylase kinase [28]. The term ‘protein kinase’ was introduced to emphasize the broader substrate specificity of this enzyme as compared with phosphorylase kinase. This, and the widespread distribution of CAMP-stimulatedprotein kinase activity in mammalian tissues, led Kuo and Greengard [29] to propose that, in eukaryotic cells, all effects of the second messenger cAMP are mediated by protein phosphorylation. Apart from a few exceptions, for example the cyclic nucleotide-controlled ion channels

8

1

The brain of the cell

in sensory cells [30], this assumption has been fully confirmed. When it became apparent that even hormones which do not induce CAMPformation as well as other intracellular effectors such as cGMP, heme, and Ca” ions, could stimulate protein phosphorylation, Greengard extended this hypothesis by postulating that protein phosphorylation is not restricted to CAMP-dependent processes but plays a ubiquitous role in biological regulation [31]. In the early 1980s, when the discovery of protein kinases began to grow exponentially,protein phosphorylationbecame recognized as ‘the major general mechanism by which intracellular events in mammalian tissues are controlled by external physiological stimuli’ [32] and the existence of an integrated network of regulatory pathways in cells, mediated by reversible phosphorylation, became apparent. In the meantime this concept has been supported by a steadily increasing body of evidence. Landmarks in the elucidation of protein phosphorylation as a major signaling reaction are described in detail in the individual chapters of this book.

1.4 Protein phosphorylation in prokaryotes While in eukaryotes protein phosphorylation was detected already in the mid-l950s, it took almost 20 more years to acknowledge its role fully as a signaling reaction also in prokaryotes. Today, the existence of a wide variety of phosphoproteins, protein kinases, and phosphatases has been established for more than 50 different prokaryotic species including eu-, archae- and cyanobacteria [33, 341. In fact, bacteria now provide one of the best-studied and clearest examples of a signal-processing function of protein phosphorylation. This reaction appears to be a general and versatile mechanism by which prokaryotes transform environmental signals into behaviour patterns and metabolic adaptation. Thus, protein phosphorylation obviously provides an advantageous means of signal transduction which had been developed early in evolution. It may be speculated that this ‘invention’was facilitated by the fundamental role that phosphoric acid residues play in transport processes, energy conservation, and nucleic acid structure, i. e. by the early availability of enzymes of the phosphotransferase and phosphatase types. In bacteria, protein phosphorylation has been found to be targeted to Ser, Thr, Tyr, His, Arg, Lys, Asp, Glu, and Cys residues

WI.

Autophosphorylation of His residues and the phosphate transfer from histidine to Asp residues provides one of the major pathways of prokaryotic protein phosphorylation. This is a rather primeval mechanism in that it is more related to the formation of short-lived phosphoprotein intermediates in certain enzymes than to the catalytic function of eukaryotic protein kinases. Therefore, the autophosphorylation of bacterial His-kinases is considered to resemble the formation of a high-energy transition state in the phosphate transfer from a donor (ATP or PEP) to an acceptor protein [35]. This view is confirmed by the energetics of protein phosphorylation at different amino acid residues. In proteins only the hydrolysis of phospho-His residues provides sufficient free energy to elicit subsequent substrate phosphorylation, whereas the other phospho-amino acid residues are considerably more stable [36].This is in particular true for phospho-Asp, which as a free amino acid is an energy-rich molecule (free energy of hydrolysis -10 to -13 kcal mol-’) whereas in proteins it becomes extremely

1.4 Protein phosphorylation in prokaryotes

9

Plasma membrane

PEP