B Stelte

GD Sabato

H Sze

J Soll

LE Hokin

R Serrano

English

Soll, Jürgen

Steidl, A.

Schröder, Inge

Open Access LMU

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 Berlin Heidelberg NewYork London Paris Tokyo Hong Kong Barcelona Budapest ¥_ 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. 

Dynamic phosphorylation of a small chloroplast protein exhibiting so far undescribed labelling properties

Universität München: Elektronischen Publikationen

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 
Berlin Heidelberg NewYork London Paris Tokyo 
Hong Kong Barcelona Budapest 
¥_ 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 
chromato­
graphy 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 phospho­
r 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. 


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J. Soll

A. Steidl

I. Schröder

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

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Dynamic phosphorylation of a small chloroplast protein exhibiting so far undescribed labelling properties

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