PROGRESS IN BRAIN RESEARCH VOLUME 56 BRAIN PHOSPHOPROTEINS Characterization and Function Proceedings of a Workshop at the State University of Utrecht, September 1981 edited by Willem Hendrik GISPEN Professor of Molecular Neurobiology, Rudolf Magnus Institute for Pharmacology and Institute of Molecular Biology. Padualaan 8, 3508 TB Vtrecht (The Netherlands) and Aryeh ROUTTENBERG Professor of Psychology and Neurobiology, Cresap Neuroscience Laboratory, 3021 Sheridan Road, Evanston. IL 60201 (V.S.A.) ELSEVIER BIOMEDICAL PRESS Amsterdam - New York 1982 PUBLISHED BY: ELSEVIER BIOMEDICAL PRESS MOLENWERF I. P.O. BOX I527 AMSTERDAM, THE NETHERLANDS SOLE DISTRIBUTORS FOR THE U.S.A. AND CANADA: ELSEVIER NORTH-HOLLAND INC. 52 VANDERBILT AVENUE NEW YORK, NY 10017, U.S.A. Ubrary of Congress Catploging in Publication Data Main entry under title: Brain phosphoproteins. ; (Progress in brain research v. 56) Bibliography: p. Includes indexes. 1. Brain chemistry--Congresses. 2. Phosphoproteins-- Congresses. I. Gispen, Willem Hendrik. 11. Routtenberg, Aryeh. 111. Series. CDNLM: 1. Brain chemistry-- Congresses. 2. Nerve tissue proteins--Congresses. - 1 WL 3. PhosphoProteins--ConRresses. Wl PR667J V. 56 300 ~81381 9813 ~376.~v7oi . 56 612l..8 2~ ~599.01~881 82-11501 ISBN 0-444-80412-9 (U.S ) 0 ELSEVIER BIOMEDICAL PRESS, 1982 ALL RIGHTS RESERVED. NO PART OF THIS PUBLICATION MAY BE REPRODUCED, STORED IN A RETRIEVAL SYSTEM, OR TRANSMITTED, IN ANY FORM OR BY ANY MEANS, ELECTRONIC, MECHANICAL, PHOTOCOPYING, RECORDING OR OTHERWISE, WITHOUT THE PRIOR PERMISSION OF THE COPYRIGHT OWNER. PRINTED IN BELGIUM Preface This volume contains the contributions of participants in a workshop on “Brain Phos- phoproteins : Characterization and Function”, which took place at the State University of Utrecht in September 198 1. The impetus for this meeting was based on our belief that the field had reached sufficient maturity to warrant a monograph devoted to this topic. Moreover, the rapid growth of information signalled to us increasing interest in brain phosphoproteins. The time was indeed auspicious, then, to inventory our field and discuss future directions. The chapters contained in this volume document the progress that has, in fact, been made in the past 3-5 years in the identification, characterization and in some cases purification of several different brain phosphoproteins. The discussions at the workshop were vigorous and prolonged, leading to a considerable interchange of ideas. The value of having the workshop directed at extended discussion will doubtless provide benefit to the participants’ research enterprise. We wish to acknowledge the encouragement and support provided by Dr. David de Wied. We also wish to thank the participants for their enthusiastic participation at the workshop and their cooperation in the final steps of preparing this volume. We like to thank Drs. Victor Wiegant and Henk Zwiers for taking care of the local organization and Miss Lia Claessens for her outstanding success in running the symposium desk. Finally, the meeting would not have been possible without the support of the Dutch Government, the University of Utrecht and the Dr. Saal van Swanenberg Foundation. We note with sadness the passing of Malcolm Weller, an early and important contributor to the brain phosphoprotein field. We feel that the contributions in this volume have grown out of his pioneering studies. The phosphorylation of brain proteins provides an interface between the neurophysiological signalling capacity of the brain and the metabolism of nerve cells. As such it represents a critical step in relating brain function to brain chemistry. The present volume, it is hoped, by summarizing much of the available evidence, will provide a basis for the accelerated develop- ment of the functional study of brain phosphoproteins. November 198 I Utrecht, Willem Hendrik Gispen Evanston, Aryeh Routtenberg vi Participants Aloyo, V.J. Div. of Molecular Neurobiology, Institute of Molecular Biology and Rudolf Magnus Institute for Pharmacology. State University of Utrecht, Padualaan 8,3508 TB Utrecht (The Netherlands) BL, P.R. Institute of Molecular Biology and Rudolf Magnus Institute for Pharmacology, State University of Utrecht, Padualaan 8, 3508 TB Utrecht (The Netherlands) Baudry, M. Psychobiology Department, University of California Irvine, Ca 92717 (U.S.A.) Bowling, Allen Dept. of Neurology, Yale Medical School, 333 Cedar Street, New Haven, CN 06510 (U.S.A.) Browning, M. Dept. of Pharmacology, Yale University, School of Medicine, Cedar Street 333, New Haven, CN 06510 (U.S.A.) Carlin, Richard Dept. of Cell Biology, The Rockefeller University, New York, NY (U.S.A.) K. Cheung, Wai Yiu Dept. of Biochemistry, St. Jude Children's Research Hospital and the University of Tennessee Center for the Health Sciences, Memphis, TN 38101 (U.S.A.) Cohen, Rochelle S. Dept. of Anatomy, University of Illinois at the Medical Center, Chicago, IL 60612 (U.S.A.) DeLorenzo, Robert J. Dept. of Neurology, Yale Medical School, 333 Cedar Street, New Haven. CN 06510 (U.S.A.) Edwards. P.M. Div. of Molecular Neurobiology, Institute of Molecular Biology, State University of Utrecht, Padualaan 8, 3508 TB Utrecht (The Netherlands) Ehrlich, Yigal H. Dept. of Psychiatry, Medical Alumni Building, University of Vermont, College of Medicine, Burlington, VE 05405 (U.S.A.) Farber, Debra B. Jules Stein Eye Institute, UCLA School of Medicine, Los Angeles, CA 90024 (U.S.A.) Frankena. H. Div. of Molecular Neurobiology , Institute of Molecular Biology, State University of Utrecht, Padualaan 8, 3508 TB Utrecht (The Netherlands) Garfield. Mark K. The Neuroscience Unit, Dept. of Psychiatry and Pharmacology, University of Vermont, College of Medicine, Burlington, VE 05405 (U.S.A.) Gispen, W.H. Division of Molecular Neurobiology, Institute of Molecular Biology and Rudolf Magnus Institute for Pharmacology, State University of Utrecht, Padualaan 8, 3508 TB Utrecht (The Netherlands) Goldering, James Dept. of Neurology, Yale Medical School, 333 Cedar Street, New Haven, CN 06510 (U.S.A.) Gonzalez, Basilio Dept. of Neurology, Yale Medical School, 333 Cedar Street, New Haven, CN 06510 (U.S.A.) Grab, Dennis J. Dept. of Biochemistry, Ilrad, Nairobi (Kenya) Graber, Stephen G. The Neuroscience Research Unit, Dept. of Psychiatry and Biochemistry, University of Vermont, College of Medicine, Burlington, VE 05405 (U.S.A.) Guy, Paul Dept. of Biochemistry, University of Dundee, Medical Sciences Institute, Dundee, DDI S. 4HN (Scotland) Hardie, D. Grahame Dept. of Biochemistry, University of Dundee, Medical Sciences Institute, Dundee DDI 4HN (Scotland) Harrison, Janie J. Cancer Center and Dept. of Molecular Biology, Northwestern University Medical School, 303 East Chicago Avenue, Chicago, IL 6061 I (U.S.A.) Hawkes. Richard Friedrich Miescher-Institut. P.O. Box 273 CH-4002, Basel (Switzerland) Heron, Davis Dcpt. of Isotope Research, The Weizmann Institute of Science, Rehovot 76100 (Israel) , Hershkowitz, Moshe Dcpt. of Isotope Research, The Weizmann Institute of Science, Rehovot 76100 (Israel) Jacobson, Ronald Dcpt. of Neurology, Yale Medical School, 333 Cedar Street New Haven, CN 06510 ( U.S.A .) Jolles, J. Division of Molecular Neurobiology, Rudolf Magnus Institute for Pharmacology, and Institute of Molecular Biology, State University of Utrecht, Padualaan 8, 3508 TB Utrecht (The Netherlands) Jungmann, Richard A. Cancer Center and Dept. of Molecular Biology, Northwestern University Medical School, 303 East Chicago Avenue, Chicago, IL 6061 1 (U.S.A.) ... Vlll Kaibuchi, Kozo Dept. of Biochemistry, Kobe University, School of Medicine, Kobe 650 (Japan) Kikkawa, Ushio Dept. of Biochemistry. Kobe University, School of Medicine, Kobe 650 (Japan) Kleine. Leonard P. Animal and Cell Physiocology Group, Biological Sciences M54, National Research Council of Canada, Ottawa, Ont. KIA OR6 (Canada) Lee, Seung-Ki Cancer Center and Dept. of Molecular Biology, Northwestern University Medical School, 303 East Chicago Avenue, Chicago, 1L 6061 1 (U.S.A.) Lenox, Robert M. The Neuroscience Research Unit, Dept. of Psychiatry, University of Vermont, College of Medicine, Burlington, VE 05405 (U.S.A.) Lopes da Silva, F.H. Dept. of Animal Physiology, University of Amsterdam, Kruislaan 320, 1098 SM Am- sterdam (The Netherlands) Lynch, G. Psychobiology Department, University of California, Irvine, CA 927 17 (U.S.A.) Mahler, Henry R. Dept. of Chemistry and the Molecular, Cellular and Developmental Biology Program, Indiana University, Bloomington. IN 47405 (U.S.A.) Matsubara, Tsukasa Dept. of Biochemistry, Kobe University School of Medicine, Kobe 650 (Japan) Matus, Andrew Friedrich Miescher-Institut, P.O. Box 273, CH-4002, Basel (Switzerland) Miles, Michael F. Cancer Center and Dept. of Molecular Biology, Northwestern University Medical School, 303 East Chicago Avenue, Chicago, 1L 6061 1 (U.S.A.) Milkowski, Deborah Cancer Center and Dept. of Molecular Biology, Northwestern Univ. Medical School, 303 East Chicago Avenue, Chicago, IL 6061 1 (U.S.A.) Minakuchi, Ryoji Dept. of Biochemistry, Kobe University School of Medicine Kobe 650 (Japan) Naito, Shigetaka Mental Health Research Institute and the Department of Psychiatry and Pharmacology, The University of Michigan, Ann Arbor, MI 48109 (U.S.A.) Ng, Meelian Friedrich Miescher-Institut, P.O. Box 273, CH-4002, Basel (Switzerland) Niday, E. Friedrich Miescher-Institut, P.O. Box 273, CH-4002 Basel (Switzerland) Nishizuka, Yasutomi Dept. of Biochemistry, Kobe University School of Medicine, Kobe 650 (Japan) Oestreicher, A.B. Division of Molecular Neurobiology , Rudolf Magnus Institute for Pharmacology and Instituteof Molecular Biology, State university of Utrecht, Padualaan 8,3508T B Utrecht (The Netherlands) Petrali, Elena H. Dept. of Physiology, College of Medicine, University of Saskatchewan, Saskatoon, S7N OW0 (Canada) Ratner, Nancy Dept. of Chemistry and the Molecular, Cellular and Development Biology Program, Indiana University. Bloomington, IN 47405 (U.S.A.) Roberts, Sidney Dept. Biol. Chemistry, School of Medicine and Brain Research Institute, University of California Center for the Health Sciences Los Angeles, CA 90024 (U.S.A.) Rodnight, Richard Dept. of Biochemistry, Institute of Psychiatry De Crespigny Park, London SE5 8AF (England) Routtenberg, Aryeh, Cresap Neuroscience Laboratory, 202 1 Sheridan Road, Northwestern University, Evan- ston, IL 60201 (U.S.A.) Samuel, David Dept. of Isotope Research, The Weizmann Institute of Science, Rehovot, 76100 (Israel) Sano, Kimihiko Dept. of Biochemistry, Kobe University, School of Medicine Kobe 650 (Japan) Schlichter, Doris J. Dept. of Biochemistry, University of Tennessee Knoxville, TN 37916 (U.S.A.) Schotman. P. Div. of Molecular Neurobiology, Institute of Molecular Biology, State University of Utrecht, Padualaan 8, 3508 TB Utrecht (The Netherlands) Schrama, L.H. Div. of Molecular Neurobiology , Institute of Molecular Biology, State University of Utrecht, Padualaan 8, 3508 TB Utrecht (The Netherlands) Schweppe, John S. Cancer Center and Dept. of Molecular Biology, Northwestern University Medical School, 303 East Chicago Avenue, Chicago IL 6061 I (U.S.A.) Shinitzky, Meir Dept. Of Membrane Research, The Weizmann Institute of Science, Rehovot 76100 (Israel) Siekevitz, Philip Dept. of Cell Biology, The Rockefeller University, New York, NY (U.S.A.) Sorensen, Roger G. Dept. of Chemistry, Texas Christian University, Fort Worth, TX 76129 (U.S.A.) Sulakhe, Prakash V. Dept. of Physiology, College of Medicine, University of Saskatchewan, Saskatoon S7N OW0 (Canada) Takai. Yoshimi Dept. of Biochemistry, Kobe University School of Medicine, Kobe 650 (Japan) Thomas, G. Friedrich Miescher Institut, P.O. Box 273, CH 4002 Basel (Switzerland) Tielen, A.M. Institute for Medical Physics, MFI-TNO, Da Costakade 45, 2861 PN Utrecht (The Netherlands) Ueda, Tetsufumi Mental Health Research Institue and the Department of Psychiatry and Pharmacology, The University of Michigan, Ann Arbor, MI 48109 (U.S.A.) ix Whittemore, Scott R. The Neuroscience Research Unit, Dept. of Psychiatry and Physiology-Biophysics, Uni- versity of Vermont, College of Medicine, Burlington, VE 05405 (U.S.A.) Wiegant, V.M. Rudolf Magnus Institute for Pharmacology, State University of Utrecht, Vondellaan 6, 3521 GD Utrecht (The Netherlands) Yu, Binzu Dept. of Biochemistry, University, School of Medicine, 650 (Japan) Kobe Kobe Zwiers, Div. of Molecular Neurobiology. Institute of Molecular Biology and Rudolf Magnus H. Institute for Pharmacology State University of Utrecht, Padualaan 8, 3508 TB Utrecht (The Netherlands) Aspects of Protein Phosphorylation in the Nervous System with Particular Reference to Synaptic Transmission RICHARD RODNIGHT Department of Biochemistry, Institute of Psychiatry, De Ctespigny Park, London SES 8AF U.K. INTRODUCTION Few fields of biochemistry have seen such explosive progress in recent years as the one we are concerned with in this book. Some 15 years ago the phosphorylation of hydroxyamino acids in proteins was thought to be a relatively uncommon biochemical event, involving only a few acceptor proteins and catalysed by one or two protein kinase enzymes. Now we recognize several families of protein kinases dependent on a variety of co-factors, as well as kinases which on present evidence do not require an activating factor. Even more impressive is the very wide range of polypeptides now known to accept phosphate from ATP as a result of kinase action, a range that includes at least 25 enzymes and several structural proteins (Weller, 1979; Krebs and Beavo, 1979; Cohen, 1980). In the nervous system the great importance of this ubiquitous process of cellular control is demonstrated by numerous observations showing that structures derived from mammalian synapses are probably the richest and most diversified known source of protein phosphorylating systems (defined as complexes of protein kinase, acceptor protein and protein phosphatase). For recent reviews on various aspects of protein phosphorylation in neural tissue see Williams and Rodnight (I 977), Greengard (I 978), Kometiani et al. (1978), Routtenberg (1979), Rodnight (1979, 1980 a,b, 1981), Dunkley (1981) and Rodnight et al. (1982). In parallel with the discovery of the great diversity of phosphoproteins in mammalian cells, and particularly in cells of neural origin, has come a revolution in techniques for detecting and characterizing the polypeptide chains of proteins on a micro scale. I refer to the development of powerful two-dimensional electrophoretic methods for the analysis of cellular proteins. At a recent EMBO workshop the possibility was seriously discussed of eventually making by this technique a complete catalogue of all animal cell proteins (Clark, 1981). The formidable nature of such a task is indicated by the fact that two-dimensional studies have already shown that membrance structures from E. coli possess in excess of 600 distinct polypeptides. In mammalian tissues the number is believed to be of the order of 10 OOO in given cell type, of which many are presumably located in the plasma membrane. Such knowledge is of great significance for students of the nervous system, where the limiting cell membrane is a key structure for functional processes. As to how many neuronal membrane polypeptides are normally phosphorylated, clearly we have little idea at present, but the number must greatly exceed the 20 or so major phosphate acceptors described in the literature; a conservative guess would suggest several hundred. Moreover, many of these minor phosphoproteins may be characteristic of specific neuronal cell types and therefore only appear as minor in heteroge- neous membrane preparations made from whole tissue. 2 The reason why synaptic structures have evolved such a complex array of protein phos- phorylating systems is presumably related to a great complexity in the molecular events concerned with neuronal communication. In fact, as has been pointed out elsewhere (Green- gard, 1978; Neary et al., 198 1) the post-translational modification of membrane-located proteins in synapses represents an attractive molecular mechanism for the modulation of both short- and long-term aspects of synaptic transmission. This general statement seems particu- larly true of protein phosphorylation, since this is a readily reversible process that occurs rapidly with a high degree of specificity, and can reasonably be assumed to lead to conforma- tional change and therefore to modulation of membrane functions with the potential for signal amplification ; moreover through the control of protein dephosphorylation reversibility can conceivably be delayed and thus lead to long-lasting modifications in membrane properties. In the present Chapter I shall give a broad overview of certain aspects of the subject, based mainly on recent work from my own laboratory, but also filling in on certain facets that are not covered in other chapters. SUBCELLULAR STUDIES It is logical to start with these aspects since only at the subcellularlevel is the full potential of the tissue’s phosphorylating systems displayed. The general approach of incubating subcel- lular fractions of brain tissue with [Y-~~PIATanPd observing the transfer of phosphate to protein as a result of endogenous protein kinase activity is well known and widely used. However, strictly speaking this approach does not directly measure protein kinase activity : tissue fractions invariably contain protein phosphatase activity, and the observed incorporation is therefore compounded of the net transfer of phosphate to vacant sites and the turnover of previously phosphorylated sites. Since dephosphorylation is rate-limiting in turnover (Weller and Rodnight, 1971) the speed and magnitude of 32Pi ncorporated into a polypeptide depends upon the extent to which it has been dephosphorylatedd uring tissue fractionation, as well as on the activity state of its associated phosphatase. A further complication is the very rapid breakdown of ATP in the reaction mixture catalysed by ATPase activity which may result in the hydrolysis of the major part of the donor within seconds of initiating the reaction (Wiegant et al., 1978). Clearly these considerations limit the extent to which data from this approach can be interpreted in terms of the kinetics of protein kinase action. In theory the most meaningful results should be obtained from reaction conditions using very short (e.g. < 1 sec) incubation times and a high ratio of ATP to tissue protein. In practice most workers compromise and use low ATP concentrations (<20 pM) in order to maintain an acceptable specific radioactivity and incubation times of 15-60sec. Apart from these methodological considerations concerning the labelling reaction, there are several cogent reasons for questioning the physiological significance of endogenous protein phosphorylation in membrane fragments that require discussion. (1) All synaptic membrane preparations are contaminated to some extent with non-synaptic structures The question of the purity of membrane preparations putatively derived from synapses is a vexed one, but there is nevertheless compelling evidence to suggest that with few exceptions the main phosphate acceptors in typical preparations are located in fragments of synaptic plasma membranes. Some of this evidence is mentioned later and for a more detailed discussion see Sorensen et al. (1981) and Rodnight et al. (1982). 3 (2) Cell disruption may lead to association of kinases and phosphatases with unphvsiological protein substrates Matus et al. (1980) have drawn attention to this problem with evidence that certain cytoplasmic proteins tend to stick to the postsynaptic densities after cell disruption. It has also been investigated in another tissue (Brunner et al., 1978). However, artifactual associations of kinases with unphysiological substrates during preparative procedures are likely to be reversed by exposure of the membrane fragments to solutions of high ionic strength, and as will be mentioned later, the majority of protein phosphorylating systems are not affected by such treatment. (3) Protein dephosphorylation and proteolysis may occur during preparation It must be assumed that extensive protein dephosphorylation of membrane proteins occurs during subcellular fractionation and it is unfortunate that no satisfactory inhibitor of this process is available that does not also seriously interfere with fractionation procedures. Proteolysis during preparation is a serious risk and has hardly been considered, except by Burke and DeLorenzo (198 1) with respect to cytosolic phosphorylating systems. (4) Redistribution of regulatory factors may occur during preparation The possibility of a redistribution of regulatory subunits of cyclic AMP-dependent protein kinases occumng during preparation needs to be considered seriously, although evidence from the erythrocyte membrane suggests that the regulatory subunit of the membrane kinase is more tightly bound than the catalytic subunit (Rubin et al., 1972). (5) ATP has access to the external membrane surface during incubation Current evidence from experiments with intact synaptosomes supports the conclusion that virtually all of the cyclic AMP (CAMP)-dependent phosphorylating activity is located intra- terminally, indicating no support for the existence of ecto-kinases dependent on cyclic AMP (Weller, 1977; Sorensen et al., 1981 ;R odnight et al., 1982; see also below). However, most of the activity appears to be non-occluded in synaptosomal Ca2+-calmodulin-dependent preparations and must therefore be located either in the postsynaptic densities (for which there is considerable evidence) or on the external surface of the terminal membrane. The fact that ATP is released from synaptic terminals (Potter and White, 1980), either as a purinergic transmitter, or along with other transmitters, indicates that the concept of ecto-kinase activity may have functional implications. There are several reports of ecto-kinase acitivity occurring in other tissues (e.g. Kang et al., 1978). (6) Vesicle formation from membrane fragments may limit access of ATP to sites of enzyme action Electron microscopy of typical preparations suggests that vesiculation of the membrane fragments does indeed occur (Jones and Matus, 1974; Rodnight, 1981). Probably this results in some restriction of the access of ATP to all sites since it is well known that detergents (e.g. Triton X-100) increase the activity of many phosphorylating systems, particularly those dependent on CAMP. However, detergents do not uncover activity towards substrates not seen under normal conditions, although not all the CAMP-dependent systems are equally affected. For example in our experience, 0.5 % Triton X-100 trebles the CAMP-stimulated activity towards an acceptor identified an autophosphorylatedr egulatory subunit of a CAMP-depen- as dent protein kinase (mol. wt. 52 OOO- see Table 11) while only having a minimal effect on the activity of other CAMP-dependent systems. Moreover the Ca2+-activated systems are either