Cellular Regulation by Protein Phosphorylation

Edited by

Ludwig M.G. Heilmeyer, Jr. Universität Bochum Medizinische Fakultät Institut für Physiologische Chemie Universitätsstr. 150 W-4630 Bochum, FRG

Springer-Verlag

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¥_ I Published in cooperation with NATO Scientific Affairs Division

CONTENTS

I. BACKGROUND AND GENERAL INTRODUCTION

Signal Integration in Phosphorylase Kinase L. M. G. Heilmeyer, Jr .

Physical Approaches to Conformation and Assembly of Biological Macromolecules

Μ. H. J . Koch

A Vibrational Raman Spectroscopic Study of Myosin and Myosin-Vanadate Interactions

J . J . C. Teixeira-Dias, Ε. Μ. V. Pires, P. J . A. Ribeiro-Claro, L. A. E. Batista de Carvalho, M. Aureliano and A. M. Amado

Phosphorylation and the Frequency Encoding of Signal-induced Calcium Oscillations

A. Goldbeter and G. Dupont

II . METHODOLOGY

Determination of Phosphorylated Amino Acids in Protein Sequences

Η. E. Meyer, E. Hoffmann-Posorske, H. Korte, T. R. Covey and A. Donella-Deana

Back-Phosphorylation - a Sensitive Technique to Study Protein Phosphorylation in the Intact Heart

P. Karczewski

Thermal Transitions in Cardiac Troponin and Its Subunits L. A. Morozova, V. L. Shnyrov, Ν. B. Gusev and E. A. Permyakov

Dynamic Phosphorylation of a Small Chloroplast Protein Exhibiting So Far Undescribed Labelling Properties

J . Soil, A. Steidl and I. Schröder

VIII

Identification of Phosphorylation Sites in the Nicotinic 79 Acetylcholine Receptor by Edman Degradation and Mass Spectroscopy LC/MS and LC/MS/MS

W. Schröder, Η. Ε. Meyer, Τ. Covey, Κ. Buchner, Κ. Eckart and F. Hucho

Α Sensitive Colorimetric Assay for Protein Phosphatase Activity 85 Τ. P. Geladopoulos, Τ. G. Sotiroudis and Α. E. Evangelopoulos

II I . STRUCTURE - FUNCTION RELATIONSHIP

Protein Kinase Structure and Function: 91 cAMP-Dependent Protein Kinase

S. S. Taylor, W. Yonemoto, W. R. G. Dostmann, D. L. Knighton, J . M. Sowadski, F. W. Herberg, J . A. Buechler and Y. Ji-Buechler

Expression in E. Coli of Mutated R Subunits of the cAMP- 107 Dependent Protein Kinase From Dictyostelium Discoideum

M.-N. Simon, S. Gazeau, O. Pellegrini and M. Veron

Expression of the -Subunit of Phosphorylase b 113 Kinase in E. Coli

S. Cox and L. N. Johnson

Conformational and Shape Changes Associated with 119 cAMP-Dependent Protein Kinase

F. W. Herberg and S. S. Taylor

Crystallographic Studies of the Catalytic Subunit 125 of cAMP-Dependent Protein Kinase

D. R. Knighton, J . Zheng, V. A. Ashford, S. S. Taylor, N.-H. Xuong and J . M. Sowadski

6-Phosphofructo 2-Kinase/Fructose 2,6-Bisphosphatase: 131 Kinetic Changes Induced by Phosphorylation

F. Ventura, J . L. Rosa, S. Ambrosio, J . Gil, A. Tauler and R. Bartrons

IX

Enzymes Involved in the Reversible Phosphorylation 137 in Microvessels of the Brain

U. Dechert, M. Weber-Schaueffelen, S. Lang-Heinrich and E. Wollny

Covalent Modification of Creatine Kinase by ATP; 143 Evidence for Autophosphorylation

W. Hemmer, S. J . Glaser, G. R. Hartmann, Η. Μ. Eppenberger and Τ. Wallimann

Two Adjacent Phosphoserines in Bovine, Rabbit and Human 149 Cardiac Troponin I

K. Mittmann, Κ. Jaquet and L. M. G. Heilmeyer, Jr .

Characterisation of Thylakoid Membrane Protein Kinase 159 by Affinity and Immunological Methods

I. R. White, M. Hodges and P. A. Millner

IV. CA 2 + AND CYCLIC NUCLEOTIDE-INDEPENDENT PHOSPHORYLATION

A Diversity of Elements in the Protein Kinase C 167 Signal Transduction Pathway

P. J . Parker

"Independent" Protein Kinases: A Challenge to Canons 179 L. A. Pinna

Biphasic Activation of the S6 Kinase: 195 Identification of Signalling Pathways

G. Thomas

Changes on the Electrophoretic Mobility of CD5 209 Molecules Induced by PKC-mediated Phosphorylation

J . Alberola-Ila, L. Places, J . Vives and F. Lozano

Opposing Effects of Protein Kinase C in IgE-dependent 215 Exocytosis and InsP3 Formation

G. Gat-Yablonski and R. Sagi-Eisenberg

Proteinphosphorylation in Platelets-Evidence for Increased 221 Protein Kinase C Activity in Essential Hypertension

C. Lindschau, Η. Haller, P. Quass and A. Distler

χ

Protein Phosphorylation in Luteal Membrane Fraction 227 L. T. Budnik and A. K. Mukhopadhyay

Purification of Bovine Brain Protein Kinase C 233 Employing Metal Ion Dependent Properties

C. Block and D. Beyersmann

Cardiac Protein Kinase C Isoenzymes: Phosphorylation of 239 Phospholamban in Longitudinal and Junctional Sarcoplasmic Reticulum

B. G. Allen and S. Katz

Characterization of the Phosphorylation Sites 245 of 40S Ribosomal Protein S6

H. R. Bandi, S. Ferrari , Η. E. Meyer and G. Thomas

V. CYCLIC NUCLEOTIDE-DEPENDENT SIGNALLING

G Protein Oncogenes 253 H. R. Bourne

A New Role for β -Subunits of G-Proteins 261 E. J . M. Helmreich

Conditions Favouring Phosphorylation Inhibit the Activation 271 of Adenylate Cyclase in Human Platelet Membranes

I. A. Wadman, R. W. Farndale and B. R. Martin

VI. PROTEIN TYROSINE PHOSPHORYLATION

Regulation of Tyrosine Kinases by Tyrosine Phosphorylation 279 J . A. Cooper, A. MacAuley and A. Kazlauskas

pp75: A Novel Tyrosine Phosphorylated Protein 289 That Heralds Differentiation of HL-60 Cells

I. Bushkin, J . Roth, D. Heffetz and Y. Zick

Preliminary Biochemical Studies of a Drosophila 295 Homolog of p 6 0 c " s r c

S. J . Kussick and J . A. Cooper

XI

Phosphoinositide Kinases and EGF Receptor Activation 301 in Plasma Membranes of Tyrosine from A431 Cells

B. Payrastre, M. Plantavid, M. Breton, E. Chambaz and H. Chap

Phosphorylation of Synaptophysin by the c-src Encoded 307 Protein Tyrosine Kinase p p 6 0 c " s r c

A. Barnekow

Altered Thymocyte Development Induced by Augmented 313 Expression of ρ 56*°^

Κ. Μ. Abraham, S. D. Levin, J . D. Marth, Κ. Α. Forbush and R. Μ. Perlmutter

VTI. PROTEIN PHOSPHATASES

Regulation and Regulatory Role of the Inactive ATP, 321 Mg-dependent Protein Phosphatase (pp-11)

J . R. Vandenheede, P. Agostinis and J . Van Lint

Okadaic Acid from Laboratory Cultures of a 331 Dinoflagellate Alga: Effects on Protein Phosphorylation in C3H10T1/2 Fibroblasts

H. Angel Manjarrez Hernandez, L. A. Sellers and A. Aitken

Regulation of Proto-oncogene Expression and Rate of 337 Protein Synthesis by the Tumor Promoter Okadaic Acid

A. Schönthal

The Use of β Cyclodextrin in the Purification of 343 Protein Phosphatase G from Rat L iver

S. Wera, M. Bollen and W. Stalmans

Characterization of a Human T -ce l l Protein Tyrosine 349 Phosphatase Expressed in the Baculovirus System

N. F. Zander, J . A. Lorenzen, G. Daum, D. E. Cool and Ε. H. Fischer

Inhibition of Tyrosine Protein Phosphatases from Muscle 355 and Spleen by Nucleic Acids and Polyanions

C. Stader, S. Dierig, N. Tidow, S. Kirsch and H. W. Hofer

XII

VIII. CONTROL OF CELLULAR PROCESSES

Biochemical Regulation of the CDC2 Protein Kinase 363 G. Draetta

Molecular and Biochemical Characterization of the Mitogen- 375 activated S6 Kinase

G. Thomas

Protein Phosphorylation in the Nervous System 389 M. W. Kilimann

Mitotic Control in Mammalian Cells, Positive and 397 Negative Regulation by Protein Phosphorylation

A. Fernandez and N. Lamb

Regulation of Eukaryotic Translation by Protein 411 Phosphorylation

H. Trachsel

Regulation of Microfilament Assembly 421 B. Schoepper, C. Weigt and A. Wegner

Stimulation of Human DNA Topoisomerase I by 429 Protein Kinase C

J . Alsner, E. Kjeldsen, J . Q. Svejstrup, K. Christiansen and 0. Westergaard

Protein Phosphorylation in Partially Synchronized Cel l 435 Suspension Culture of Alfalfa

L. Bako, L. Bögre and D. Dudits

Phosphorylation of Elongation Factor Tu in vitro 441 and in vivo

C. Lippmann, C. Lindschau, Κ. Buchner and V. Α. Erdmann

About a Controversy Concerning the Existence of a Mitogen- 447 responsive S6-Kinase (Late-eluting from DEAE-Sephacel)

Ο. H. W. Martini, A. Lawen and M. Burger

Expression of Ferr it in Messenger RNA in an in vitro Model of Human CD3-mediated T-Lymphocyte Activation

L. de Oliveira and M. de Sousa

453

XIII

Relaxation of Smooth Muscle at High Levels of Myosin 459 Light Chain (MLC) Phosphorylation

G. Pfitzer and S. Katoch

Evidence for Site- and Domain-specific Phosphorylation 465 of the 145 kDa Neurofilament Subunit in vivo

R. K. Sihag and R. A. Nixon

Tumor Necrosis Factor-induced Gene Expression and 471 Cytotoxicity Share a Signal Transduction Pathway

G. Haegeman, V. Vandevoorde and W. Fiers

Protein Phosphorylation Is Involved in the Recognition 477 of Pathogen-derived Signals by Plant Cells

D. G. Grosskopf, G. Felix and Th. Boiler

Effect of Membrane Modifiers on Polyphosphoinositide 483 Synthesis in Rat Heart Sarcolemma

N. Mesaeli and V. Panagia

Phosphorylation and Acylation of the Growth-related 489 Murine Small Stress Protein P25

S. Oesterreich, R. Benndorf, G. Reichmann and H. Bielka

INDEX 495

DYNAMIC PHOSPHORYLATION OF A SMALL CHLOROPLAST PROTEIN EXHIBITING SO FAR UNDESCRIBED LABELLING PROPERTIES

J . S o i l , A. S t e i d l , I . Schröder F a c h r i c h t u n g Botan ik der Univers ität des Saa r l andes D-6600 Saarbrücken

A 19 kDa phosphoprote in from mixed envelope membranes of s p i n ­ach c h l o r o p l a s t s w i t h extreme l a b e l l i n g k i n e t i c s has been c h a r a c t e r i z e d . I t s l o c a l i z a t i o n between the i nne r and the outer enve lope membrane can be deduced by the d i f f e r e n t i a l l a b e l l i n g between i n t a c t and broken c h l o r o p l a s t s (Tab le 1 ) , ( S o i l and Bennett 1988, S o l l e t a l . 1989 ) .

Table 1 D i f f e r e n t i a l l a b e l l i n g of p r o t e i n s from i n t a c t and l y s e d c h l o r o p l a s t s . Phospho ry l a t i on was done a t 10 nM ATP a t 4 °C for 3 0 s e c . Va lue s a re expressed i n dpm μg c h l o r o p h y l l χ min"" 1

i n t a c t c h l o r o p l a s t s

l y s e d c h l o r o p l a s t s

r a t i o

thy lako id/LHCP 53 125 0.424 stroma a 12 110 0.109 stroma b 11 100 0.11 outer envelope (86 kDa) 63 16 3.9 intermembrane space (64 kDa) 98 54 1.8 19 kDa p r o t e i n 2340 375 6.24

I f the 19 kDa p r o t e i n i s indeed l o c a l i z e d i n the envelope l u ­men, i n t a c t and broken c h l o r o p l a s t s should d i f f e r i n t h e i r l a ­b e l l i n g k i n e t i c s . I n t a c t c h l o r o p l a s t s s t i l l c o n t a i n r e s i d u a l , endogenous ATP, t h i s means t h a t dur ing l a b e l l i n g of i n t a c t c h l o r o p l a s t s two ATP poo ls e x i s t , w i th d i f f e r e n t s p e c i f i c a c ­t i v i t y which i s encountered by envelope membrane p r o t e i n s and another w i t h low s p e c i f i c a c t i v i t y which i s encountered by p r o t e i n s i n s i d e the c h l o r o p l a s t . I n i n t a c t c h l o r o p l a s t s the outer envelope 86 kDa p r o t e i n , the 64 kDa and the 19 kDa p r o -

Ν Α Τ Ο A S I Series, Vo l . Η 56 Cel lular Regulat ion by Protein Phosphorylat ion Edi ted by L . M . G . Hei lmeyer, Jr. © Springer-Verlag Ber l in Heide lberg 1991

74

t e i n were l a b e l l e d much e a r l i e r and s t r onge r than the s t roma l and t h y l a k o i d phosphoprote ins .

The f u n c t i o n of the i n t e r enve l ope space i s unknown; the dyna ­m i c a l l y phosphory la ted 19 kDa p r o t e i n has been p u r i f i e d from mixed envelope membranes (Table 2) and c h a r a c t e r i z e d . I t seems r easonab l e t h a t i t p a r t i c i p a t e s i n a s i g n a l t r a n s d u c t i o n p r o ­c e s s . The f i r s t p u r i f i c a t i o n s t ep was a m i l d s o n i c a t i o n f o l ­lowed by anion exchange chromatography on DEAE c e l l u l o s e of the supernatant (Table 2 ) . Most of the p r o t e i n e l u t e d a t 12 5 mM NaCl . A c t i v e p r o t e i n f r a c t i o n s were pooled and p u r i f i e d f u r t h e r on a h y d r o x y l a p a t i t e column from which i t cou ld be e l u t e d a t 60 mM phosphate bu f fe r pH 7 . 6 .

Table 2 P u r i f i c a t i o n of s p inach envelope 19 kDa p r o t e i n

Step Volume

ml

P r o t e i n T o t a l

u n i t s

S p e c i f i c a c t i v i t y units/mg

Reco ­v e ry

%

P u r i f i ­c a t i o n f o l d

Envelope membranes 1 6950 63.2 9 .1 100 1

S o n i c a t i o n supernatant 0.89 1510 55 36.4 87 4 .6

DEAE chromato­graphy 5.6 8.9 28 .5 3200 45 352

Hyd roxy l ­a p a t i t e 4 .5 0.54 4.4 8161 7 897

1 u n i t egua l s 1 fmol _p i nco rpo ra t ed i n t o the 19 kDa p r o t e i n from [ 7 - 2 P ] - A T P χ min 1

The 19 kDa p r o t e i n shows an extreme a f f i n i t y fo r ATP and GTP as demonstrated by the low K m v a l u e s of 8 nM and 5 nM for ATP and GTP r e s p e c t i v e l y ( F i g . 1 A ) . The phospho ry l a t i on , t h a t i s t r i c h l o r a c e t i c a c i d or acetone p r e c i p i t a b l e , i s dependent on the presence of d i v a l e n t c a t i o n s (Mg 2 + and Mn 2 + ) ( F i g . 1 B ) .

75

The c a t i o n C a 2 + has no e f f e c t . ADP and GDP i n h i b i t phospho­r y l a t i o n ( F i g . 1 D ) . The opt imal pH fo r phospho ry l a t i on i s i n the range between pH 7 and pH 9 ( F i g . 1 C ) . The p i of the phosphory la ted enzyme has the va lue 6 . 2 , whereas the p i of the non phosphory la ted enzyme i s 6 . 3 .

ι Γ

o o

/ ο - o / o

/ y

/ Y % :-«>

1«

> / ' -o ' t

Km«SnM ATP

-1 L

01 02 03 04 OS V ISUM) - '

A T P [ n M ]

[ A D P ] AJM

F i g . 1) C h a r a c t e r i z a t i o n of the phosphory l a t i on r e a c t i o n of the p a r t i a l l y p u r i f i e d 19 kDa p r o t e i n . A) Dete rminat ion of the Km v a l u e for ATP. B) I n f l u e n c e of d i v a l e n t c a t i o n s . C) pH dependence. D) The phosphory l a t i on i s i n h i b i t e d by ADP.

The mo l ecu l a r weight of the phosphory la ted p r o t e i n was e s t i m a ­ted by SDS g e l e l e c t r o p h o r e s i s and found to be 18.8 kDa ( F i g . 2 A ) . The phosphory l tu rnover i s ext remely r a p i d , as deduced from a p u l s e - c h a s e exper iment . I f the p r o t e i n was l a b e l l e d i n the p resence of 8 nM [ 7~ 3 2 P ] -ATP fo r 60 s e c and 10 μΜ c o l d ATP was added a t t h i s t ime p o i n t , 90 % of the l a b e l l e d phospho ry l -groups i n the p r o t e i n a re turned over w i t h i n 15 s e c .

The de t e rmina t i on of the phosphory la ted amino a c i d r e s i d u e de ­monstrated t h a t no hydroxy la ted amino a c i d was phosphory la ted , f i r s t l y the phosphate bond was l a b i l e to a c i d i c c o n d i t i o n s ;

76

secondly a f t e r a c i d h y d r o l y s i s of the p r o t e i n and h i g h vo l t age e l e c t r o p h o r e s i s no r a d i o a c t i v i t y was d e t e c t a b l e i n P - S e r , P -Thr or P -Ty r ( S o l l e t a l . 1989 ) . E x t r a c t i o n of the phospho-r y l a t e d p r o t e i n by chloroform methanol a t pH 1 r e s u l t e d i n no d e t e c t a b l e l a b e l i n the o r gan i c s o l v e n t phase , but t h e t o t a l r a d i o a c t i v i t y was s t i l l bound to the p r o t e i n . Exposure of the phosphoprotein to hydroxylamine or p y r i d i n e bu f f e red i n a c e ­t a t e showed a c o n c e n t r a t i o n dependent base c a t a l y z e d enhance ­ment of the h y d r o l y s i s r a t e ( F i g . 2 B) and exc luded most l i k e ­l y a s p a r t a t e and g lutamate as phosphorylgroup a c c e p t o r , as those a re not s u s c e p t i b l e to p y r i d i n e t rea tment (Sabato and Jencks 1961,Hokin e t a l . 1965 ) . The l a b e l was a l s o l a b i l e a t a l k a l i n e pH ( S t e l t e and W i t z e l 1986 ) . At the moment i t seems most l i k e l y t h a t we d e a l w i th a l y s i n e or h i s t i d i n e phosphate . The phosphory l a t i on of the 19 kDa p r o t e i n i s i n h i b i t e d by TNP-ATP and by e r y t h r o s i n ( F i g . 2 C ,D ) .

F i g . 2) A. Dete rminat ion of the mo lecu la r weight of t h e phosphory lated form of the p u r i f i e d 19 kDa p r o t e i n by SDS-PAGE B. Time course of h y d r o l y s i s of the phosphate bond i n 1 Μ a c e t a t e bu f fe r pH 5.5 i n the presence of 0 .1 Μ hydroxy lamin or P y r i d i n . C. I n h i b i t i o n of the 3 2 P - i n c o r p o r a t i o n by TNP-ATP. D. I n h i b i t i o n of 19 kDa p r o t e i n phosphory l a t i on by e r y t h r o s i n e

77

The p u r i f i e d 19 kDa p r o t e i n d i d not show s i g n i f i c a n t ATPase a c t i v i t y (not shown). These f i n d i n g s a r e co r robora ted by r e ­s u l t s ( Tab l e 3) , which demonstrate the e f f e c t of v a r i o u s ATPase i n h i b i t o r s on the phosphory l a t i on of the 19 kDa p r o t e i n (Sze e t a l . 1987, Serrano 1988, Sze and R a n d a l l 1987 ) . From f u r t h e r exper iments i t seems l i k e l y t h a t 3 2 p - i n c o r p o r a t i o n i n t o the 19 kDa p r o t e i n i s due t o au tophosphory l a t i on .

Table 3 I n h i b i t i o n of 19 kDa p r o t e i n phospho ry l a t i on by v a r i o u s sub ­s t r a t e s . The p u r i f i e d p r o t e i n was phosphory la ted by [ γ - 3 P ] -ATP i n t h e p r e sence of d i f f e r e n t e f f e c t o r s . A minimum of f i v e d i f f e r e n t e f f e c t o r c o n c e n t r a t i o n s was used i n every c a s e .

e f f e c t o r max . concent ra t i on % i n h i b i t i o n

NaN3 10 mM 0 NaF 20 mM 0 N a F / A l C l 3 10 mM/50 μΜ 0 ortho vanadate 0 .5 mM 29 molybdate 2.0 mM 0 n i t r a t e 10 mM 0 o l i gomyc in 0 .5 mM 0 DCCD 1) 0 .5 mM 0 ouabain 12 5 μΜ 0 d ihydroxyacetonephohate 2 mM 60 NaCl 150 mM 50

1) The p u r i f i e d p r o t e i n was p re incuba ted w i t h DCCD for to 2h.

Acknowledgements: T h i s s tudy was supported by the DFG (SFB 184 ) .

Literature Hokin, L . E . , S a s t r y , P . S . , Ga lsworthy , P .R. & Yoda, A.

( 1 9 6 5 ) P r o c . N a t l . A c a d . S e i . USA 54, 177-184. Sabato, G.D. & J e n c k s , W.P. (1961) J.Am.Chem.Soc. 83,

4393 -4400 . Se r rano , R. (1988) B ioch im.B iophys . Ac ta 947, 1-28. S o l l , J . & Bennet t , J . (1988) E u r . J . B i o c h e m . 175, 301 -307 . S o l l , J . , Be rge r , V. & Bennett , H . J . (1989) P l a n t a ( B e r l . )

177, 3 9 3 - 4 0 0 . S t e l t e , B. & W i t z e l , H. (1986) E u r . J . B i o c h e m . 155, 121-124. Sze , Η., R a n d a l l , S . K . , Kae s tne r , K .H . & L a i , S . (1987) i n

P l a n t Membranes: S t r u c t u r e , F u n c t i o n B i o g e n e s i s , pp. 195 -207 , e d s . C . L e a v e r & H. Sze , A lan L i s s I n c .

Sze , H. & R a n d a l l , S .K . (1987) J . B i o l . C h e m . 262, 7135-7141.