Protein Kinases from Spinach Chloroplasts - The Journal of Biological

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Oct 25, 2015 - purified by ammonium sulfate precipitation, sucrose gradient centrifugation ... There have been reports on both soluble and membrane-bound ...

THEJOURNALOF BIOLOGICAL CHEMISTRY Vol. 257, No. 20, Issue of October 25, pp. 12153-12156, 1982 Printed in U.S.A.

Protein Kinasesfrom Spinach Chloroplasts I. PURIFICATION AND IDENTIFICATION OFTWO DISTINCT PROTEIN KINASES* (Receivedfor publication,April 9, 1982)

Zhe-Fu fin$, Hector A. Lucerog, and Efraim Racker From the Section of Biochemistry, Molecular dl Cell Biology, Division of Biological Sciences, Cornell University, Zthaca, New York 14853

EXPERIMENTAL PROCEDURES2’3 T w o protein kinases (chloroplast protein kinases 1 M U and 2 (ChlPK1 and ChlF’K2)) were isolated from spinach Enzyme-grade sucmle and u l t r a p u r e a m n i u n s u l f a t e wem purchased f- SchwarztMnn; chloroplasts. After solubilization of the chloroplasts sodium dadecylsulfate,acrylmrde, N,N’nethylene-biracrylanride, N,N,N’,N’.-tetrarethylenediamine. m n i m persulfate. and low range l n l e c u l a r w i g h t s t a n d a r d k i t f o r SOS-PAGE’ were with octylglucoside and cholate, these kinases were from BioRad. k t y l g l u c o n d e war purchased from Calbimha.Cholate frm Sigma was re70% ethanol ( 8 ) . [ Y - ~ ~ PATP ] 12,000 m c i l m l ) war purchased from herrham. c r y s t a l l i z e d fpurified by ammonium sulfate precipitation, sucrose Casein ( p a r t i a l l y h y d r o l y z e d and dephosphorylated). d i t h i o t h r e l t o l . r 3 i m ascorbate.Tricine. T r i s , MES, disodium ATP, 8-mercaptoethanol, pbrphorerine. phorphathreonlne, and T r i t o n X-100 gradient centrifugation, and hydroxylapatite chromawere f m Sigma. Sta h lococcu~ a u r e u ~Ve p m t e a l e was obtained f m MilesLabOratOPiel. EDTA was p u r c h a r e d f E E i i n and Bell.Arolectrnlcwde roVbean phospholipids) tography. ChlPKl traveled as a single band in polywas Obtained fmn AssociatedCancentratel, Yaodside, Nr. Phorphotymrine was prepared by M. Spector. [ T - ~ ~ P8-azido ] ATP (2.1 mCl1wmle) was a generous g‘ft frm Or. 8. Haley, Uniacrylamide gel electrophoresis corresponding to 25,000 v e r r i t y o f Uyoming. Preparations o f c a t a l y t i c s u b u n i t the cMP-dependent kinase were g l f t S fmm Dr. E. Fischer and Or. L . Pike.University Yarhington. daltons; ChlPKztraveled as a single band correspondPreparation of chloroplasts ing to 38,000 daltons. After exposureto 8-azid0-[y-~~P] ChlomplaltB were i s o l a t e d f m spinachleavesobtained fmm l o c a l m r k e t r by a m d i f l ATP, the radioactive bands appeared in the same poc a t i o n o f a proceduredescribedbyNelson st. (9) f o rl e t t u c ec h l o m p l a l t s . Spinach leaves 11.5 kg) were washed w i t h d i s t i l l e d watel, Cut into ma11 pieces and gmund i n a sitions revealed by Coomassie blue staining. However, Yarinblender i n 1.5 1 I medium containing 0.4 M wcmse. 10 1111 NaCl, 20 1111 TrlclneNaOH ?pH 7.8) (SNT buffer) and 50 nll sodium ascorbate. The hanogenate was f i l t e r e dt h m u g h a traceof ChlPKzwas detected in the ChlPKl preparafour layers cheesecloth and the f i l t r a t e was centrifuged at 1.400 xg for 1 min. The discarded and thesupernatant was centrifuged at 10,400 x g f o r 15 nin. The tion and a faint second lower molecular weight radio- pp ee ll ll ee tt was was washed t h r e e t i m e r , f i r s t w i t h 750 ml o f SNT buffer, then with 5 0 0 ml 150 1111 NaCl. 2 0 mM Tridne-NaOH (pH 7.8) and f i n a l l y w i t h 500 ml SNT buffer. Each the active band was seen in the ChlPKz preparations. Both c h l o m p l a r t r w w e redimented a t 10.4W xg f o l 10 min. The f i n a l p e l l e t was suspended in a m a ll volume o f SNT b u f f e r and the chlorophyll concentration was determined I p e c t m p b t o enzymes acted on casein or histone IIIS as substrate m e t r i c a l l y( 1 0 ) . and phosphorylated a serine residue. The proteolytic Extraction of pmtein kinases fmm C h l o m p l a s t l peptide maps, however, were clearly distinguishable in E x t r a c t i m o f p m t e i n k i n a l e s f m c h l o m p l a l t l was Carried Out f o l l o w i n g a m d i f i c a t i o n O S 1 (11). The c h l o m p l a r t suspension conofthepmceduredescribedforextnctionof autoradiograms, suggesting that different serine resitaining 4 mglml c h l o m p h y l l was incubated with 50 1111 d i t h i o t h r e i t o l f a r I S nin a t O°C withgentlestirring.Extraction c h l o m p l a l t l w i t h d e t e r g e n t s was accomplished by dues were phosphorylated by ChlPKl and ChlPK2. the dithiothreitol-treated fraction to 2 nglml chlorophyll in a d i m condilution Of Of

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t a i n i n g i n a f i n a l c o n c e n t r a t i o n 0.1% a r o l e c t i n . 0.4 W m n i m sulfate. 50 1111 d i t h i o t h r e i t o l . 0 . 5 1 cholate and 0.71 octylglucoside. After incubation for 15 min at O’C with gentle S t i F r i n g t h e rurpenrian was centrifuged at 2W.wO rg for 90 m i ” a t 0% The yellowish supernatant was c o l l e c t e d and 1.25 m1 Of saturated m n i m s u l f a t e was added per nl o f supernatant. The suspension *dl incubated f o r 1 0 m i ” a t O’C, centrifuged a t 22.WO xg for 10 min and the p e l l e t rerurpended i n II -11 v o l m l a b u t one-tenth of tk extract)of I b u f f e r containing101glycerol and 20 1111 T r i c i n (pH 8.0). A l i q u a t r of 0 . 5 ml Containing15-20 “9 p m t e i n were i m d i a t e l y frozen i n l i q u i d n i t m g e n and kept a t -7O’C. Under these conditions the pmtein kinase activity was preserved for several mnthr.

Protein kinases catalyze the transfer of the y-phosphate of ATP or other trinucleotides to serine, threonine, or tyrosine Sedimentation i n I Iucmle gradient residues in proteins (1,2).Although the literature is abundant The m n i m s u l f a t e f r a c t i o n 1100-120 mg o f p m t e l n ) uas q u i c k l y 2 2 - d and d i l u t e d with descriptions of protein kinases from animals, relatively t o 1 0 ml w i t h a r e d i m r e s u l t i n g i n a f i n a l c o n c e n t r a t i o n 30 1111 Trir-succinate (pH 6.51, 0.1 1111 ATP, 0 . 5 1111 EDTA. 0.332 T r i t o n X-lM, 0.12 Cholate and 0.12 alOlectin.Aliquatr little is known about the properties and function of protein o f 1.6 ml o f t h i s m i x t u r e were applied to the top of a 10-301 d w sucmle gradient 111.4 ml) f o d i n t h e d i m used f o r t h e d i l u t i o n of t h e m n i m w l f a t e p r e c i p i t a t e e x c e p t t h a t I Beckman Sy11 m t o r a t 1 6 0 . W xg f o r T r i t o n x-100 was omitted. After centrifugation in kinases in plants. There have been reports on both soluble 10-12 h a t O°C, a l i q u o t s 0.7 a1 r e m w i t h d r a m fm t h e t o p thecentrifugetuber. and protein, pmtein kinase activity, and 32Pi-ATP exchange a c t i v i t y were determired I S and membrane-bound kinases in higher plants and cultured described under ‘ “ E x p r i n e n t a l Pmceduml.” U S U l l y . 4.2 nl fmn t h e U P each tube were conbined. d i v i d e d i n t o 1 ml a l i q w t l , f m z e n i n l i q u i d n i t m g e n and s t o r e d a t -7OY. plant cells (3-5, cfi Ref. 6 ) , but no extensive purification and C h m t o s r a p b w i t h h y d r o x y l a p a t i t e characterizationof these proteins have been recorded.Protein s i x g p m s of hydroxylapatite (810-gel HTP frm BioRad) *re suspended i n 1 W nl of 10 mI4 kinase activity from spinach chloroplasts was solubilized with Trlcine-NaOH (pH 8 . 0 ) and heated in a m t e r bath I t 90°C f o r 15 n i n w i t h OccasiOna1 shaking. and -Val thefinestheslurry l d s pured After sedimentation at man t - m u r e octylglucoside and cholate and partially purified (7). i n t o a c o l m 10.6 x 26 on). The o u t l e t o f tk colunn u s conne;ted t o a p e r i s t a l t i c PUP and t h e s l u r r y packed t o y i e l d a f l o w rate 1 nllnin. Fimlly. the calm was placed In this paper we show that in spinach chloroplasts both i n t h e c o l d morn a t 2-C and e q u i l i b r a t e d w i t h a buffer containing 10 1111 T r i C i N (pH 8.0). 0.1 1111 ATP, 10 1111 octylglucoride, 0.11 cholateand 5 1111 e-mrcaptoethawl. Equilibration was c o n t m l l e d by measuring c o n d u c t i v i t y and Optical density at 260 111. Five 11 16 “9) of serine and threonine residuesof proteins are phosphorylated the protein kinase purified by sucmse gradient centrifugation !+as dialyzed for 3 h a t P C against100 ~ l morf t h e b u f f e r used f o r t h e e q u i l i b r a t i o n t h e m l m . The r m p l e was in the light on addition of [Y-~~PIATP. Serine residues were then diluted to the conductivity tk calm buffer. T k Ianple M I placed on t h e column which was t k n washed w i t h 50 m1 equilibrationbuffer. The p m t e i n kimse 4 c t i v i t i e l heavily phosphorylated also in the dark, whereas phosphorywere e l u t e d a t a flo) r a t e 0.5 m l l n i n w i t h a l i n e a r g r a d i n t o b t a i n e d by n i x i n g 120 nl o f t h e e q u i l i b r a t i o n b u f f e r w i t h 120 nl t h e same l u f f e l c o n t a i n i n g UHI 1111 YPi. Three nl lations of threonine residueswere comparatively much lower. fractions were c o l l e c t e d and dialyzedovernightat 2’C against Mo v o l u r r of10TridneNaaMI (pH 8.0). We have isolated two distinct and highly purifiedserine protein kinases from spinach. A third threonine-phosphory‘The “ExperimentalProcedures”arepresentedinminiprint as lating protein kinase was solubilized from chloroplasts and prepared by the authors. Miniprint is easily read with the aid of a recovered in crude fractions as will be shown in the accom- standard magnifying glass. Full size photocopies are available from panying paper.’ The Journal of Biological Chemistry, 9650 RockvillePike, Bethesda, Of

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* This investigationwassupported by Grant PCM80-21201, awarded bythe National Science Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be herebymarked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ Supported by the Chinese government. 8 Recipient of a Fellowship from Consejo Nacional de Investigaciones Cientificasy Tecnicas from Argentina. H. A. Lucero, Z.-F. Lin, and E. Racker (1982)J.Biol. Chem. 257, 12157-12160.

MD20814. Request Document No. 82M-931, cite the authors, and include a check or money order for $1.60 per set of photocopies. Full sizephotocopiesarealsoincludedin the microfilmedition of the Journal that is available from Waverly Press. The abbreviations used are: SDS, sodium dodecylsulfate;PAGE, polyacrylamide gel electrophoresis; ChlPKl, chloroplast protein kinase 1; ChlPK2, chloroplast protein kinase 2; DCCD, N,N’-dicyclohexylcarbodiimide;DSA, DCCD-sensitiveATPase; CFl, couplingfactor 1 from chloroplasts; 8-azido-ATP, 8-azido adenosine 5”triphosphate); BSA, bovine serum albumin; SNT, buffer containing 0.4 M sucrose, 10 mM NaCl, 20 mM Tricine/NaOH (pH 7.8).

12153

from Chloroplasts Spinach

Kinases Protein

12154 P m t e i n k i n a s e assax

R e c o n s t ~ t u t i o nO f DSA i n t o l i p r o m e r and '2Pi-ATPexchange described by P i c k and Racker

n.

P m t e i n k i n a s e a c t i v i t y was narured by a m d i f i c a t i o n o f t h e filter paper m t b d O f Corbin and Reimaan (12). I n a f i n a l wlo f 110 "1. 2 to 1 0 ug o f p m t e i n k 1 M w were incubated i n I &{urn c o n t a i n i n g 50 nll PSS-TII (pH 8.0) (IF (pH 6.5). IO m la^. 50 m 500 ug casein (Sips). 250 YM [ Y - ' ~ P I ATP (200 c p l / p a l ) and 1 0 nll MgCl,. A f t e r 30 min a t TO(XD t e w m a t u r e ( e x c e p t * h m s p e c i f i e d o t h e r w i s e ) t h e r e a c t i o n was terminated by the a d d i t i o n O f c o l d ATP a t a f i n a l c o n c e n t r a t i o n o f 20 *I and i m d i a t e l y Bo e l a l i q u o t r w en r p t t e d on Yhatmn 3 Iw paper (1.6 x 1.6 cn) I h i c h were t k n dmpped i n t o 10% t r i c h l o m a c e t i c acid (10 nllfllter paper). Afterthreechanger Of 10% t r i c h l o m a c e t i c a c i d ( e v e r y 1 5 min) the papers were Mashed o v e r n i g h t . F i n a l l y . t h e papers were washed f o r 1 5 mln w i t h 951 ethanol. then for 15 nln w i t h e t h y l e t h e r . and f i n a l l y d r i e d andcounted i n a k c h n S c i n t i l l . 3 t i a n Counter.

on.

P r o t e i n c a n c e n t r a t m *as determined according t o k n r a d o u n and Y e i n s t e i n ( 1 3 ) . Sodium d o d e c y ls u l f a t ep o l y a c r y l a m i d eg e le l e c t m p h a r e r i r discontinuous buffer system describedby L a m l i w i t h stackinggel.

Separation of Two Protein Kinase Activities from Spinach Chloroplasts-The protein kinase preparation from chloroplastsdescribedpreviously (7) wasshowntocontaintwo distinct protein kinase activities that could be separated by sucrose gradient centrifugation and hydroxylapatite chromatography. The startingmaterialwas the partiallypurified ammonium sulfate precipitate of chloroplast extract described under "Experimental Procedures." As can be seen from Fig. 1, protein kinaseactivity was fwst separated fromthe chloroplast DCCD-sensitiveATPase complex by centrifugation in a sucrose gradient.The fractions containing protein kinaseactivity were combined and placed on an hydroxylapatite column. Elution with aPi gradient yielded several peaks containing protein kinase activity (Fig. 2). The most consistent and prominent activities were eluted at around 165 and 225 rrm Pi. Maximal resolution was obtained when no more than 14 mg of protein of the fraction obtained by sucrose gradient

P h o t o l a b e l l i n g w i t h 8 - a z l d o ATP

o f i r r a d i a t i o n was appmximately 1303 w l m z .

Analysis of peptide mps Histone 111s (20 r g ) MIp u r i f i e d by suspending it i n e q u i l i b r a t i o n buffel' c o n t a i n i n g 80 mH T r i d n e (pH 8 . 0 ) and 50 nW KC1 and passage t h m u g h I Sephadex G-50 COlUm (28 m X 0.7 cm). The p m t e i n was e l u t e d M i t h t h e e q u i l i b r a t i o n b u f f e r a t a f l o w r a t e o f 0.25 m l l m i n and 0 . 5 ml f n C t i O n S * r e c o l l e c t e d . The p r o t e i n peak appearing I n the void y o l m e (ca 4 ~ 1 ) MIused f o rp e p t i d e map i n g ChlPK1 and ChlPK ( 5 ug each) were i n c u b a t e dw i t h and Without y l c o n t a i n i n g 1 0 mM MgC12. 50 nn WESp u r i f i e d h i s t o n e (235 "97 i n ' a f i n a l v a l m o f Trir b u f f e r a t pH 6.5 f a r ChlPK1 and a t pH 8 . 0 f o r ChlPK i n t h e presence o f [y-"P] ATP (1500 c m l p ~ o l ) . A f t e r 30 mln a t moln temperature. 25 p f O f 1 M SOS. 62.5 u l O f T r i r - C 1 i n a l 0.051 103% 50 ul of yl of b m p h e n o l b l u e *re added t o a f (pH 6.87, g l y c e r n l .1 a t mom volume O f 5W u l . The smngler lare heated f a r 2 .in a t l W o and a l l a e d to c m l temperaturefar 20 m i " . p m t e a r e (42 u l o f 0.3 m g h l ) was added a n dt h em i l t w ei n c u b a t e d f o r 10 rnin a t 37OC. The r e a c t i o n was stoppedby adding 124 u l o f 10% SDS, 1 5 u l O f 8(30 u l a l i q u o t s ) were l e p a m t e d mercaptaethanol and t a i l i n g f o r 2 min.Phorphapeptides i n SDS-PAGE and detected i n autoradiograms as d e s c r i b e d l a t e r under "ExperimntalProcedures."

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Phorphoamino a c i d a n a l y s i s P r o t e i n p h o s p m r y l a t i o n was c a r r i e d Out a t 37'c f o r 30 n i n i n 150 y l O f a m e d i m cont a i n i n g 50 nW d i t h i o t h r e i t o l , 10 nW N a f . 50 1111 M a r - T r i r (pH 8.01, 500 yg p m t e i n s u b s t r a t e . 5 uH [Y-~'P] ATP contarning15W-2000cp"Ip831 and 10 mW NgCl The a m u n to fp r n t e i n kinase was as i n d i c a t e di nt h ea p p r o p r i a t ef i g u r el e g e n d .T h i ' r e a c t i o n was terminatedby a d d i t i o n O f 2.5 u l O f 10% BSA. 100 y l HI0 and 250 "1 501 t r i c h l o r o a c e t i c a c i d and t h e i n c u f o r 10 m?n. Sampler were c e n t r i f u g e d a t 3000 xg f a r 10 min b a t l o n was c o n t i n u e d a t O'C and t h e p e l l e t s were washed t h r e e t i m e s M i t h 5W y l 25% t T i C h l O m a c e t i c a c i d , once w i t h 500 u l 95% ethanol and once w i t he t h y 1 e t h e r : e t h a n o l( 1 : l ) .A f t e re v a p o r a t i o no ft h e samples were dispersed I n 200 "1 6N HCl. The tubes solventunder a n l t m g e ns t r e a mt h e were sealed under vacuum and heated a t O l C ' for 1.5 h.The HCl was r m v e d by l y o p h y l l m t l o n O v e r n i g h t and t h e s m p l e ~*ere wsuspended i n 25 y l Of pH 3.5 e l e c t m p h o r e s i s b u f f e r H 0 10:1:89), and 3 y l O f a mixtureContaining phospho( g l a c l a la c e t i ca c i dp y r i d i n e threonine. phosphare:ine and p i a r b h o t y r o r i n e (each 5 mglml). The sampler were spotted on Yhatmn 3 HM paper (62.5 crn x 45 m i ) and e l e c t m p h o r e s e d f o r 8 0 "in a t 3,000 Y i t 2 Y C . Phorphoamina a c i d s were d e t e c t e d w i t h n i n h y d r i n and r a d i o a c t i v e s p t s w r e detected by a Cornex i n t e n l l f y i n g screen. autoradiography On Kodak XAR-5 x - r a y f i l m w i t h

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RESULTS AND DISCUSSION

Sampler c o n t a i n i n g p m t e i n k i n a s e (5-50 ug p m t e i n ) a t d i f f e r e n t s t a g e r Of p u r i f i c a t i o n were lncubdted i n a m d l m c o n t a i n i n g 5 1111 Trir-HC1 (pH 7.5). 5 1111 MClt and 5 YW [ r - 3 2 P l 8 - a r i d a ATP a t O'C for 15 5 . The r a p l e r were p l a c e d a t 1 5 cm from a Short wave l a w (254 m) and i r r a d i a t e d fm t h e t o p a t C'O f o r 1 ain. Under t h e s ec o n d i t i o n st h e eneWY

1.2

w t i v i t r w s measured a s

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40 so FRACTION NUMBER

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FIG. 2. Purificationof protein kinases with hydroxylapatite. The upper fraction (fractions 1-6, Fig. 1) from the sucrose gradient centrifugationwas subjected to chromatographywith hydroxylapatite as described under "ExperimentalProcedures,"except that the total amount of protein placed on the column was 14 mg. The peaks of protein kinase activity that eluted at 165 mM KPi (fractions 29-53) and at 225 m~ KPi (fractions 55-68) were dialyzed as described under "ExperimentalProcedures"and concentrated 10 times against a buffer containing 30%polyethyleneglycol and 20 mM Tricine/NaOH, pH 7.8. Samples were divided into small aliquots and stored frozen at -70 "C. TABLEI

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4 6 8 1014 12 FRACTION NUMBER

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FIG. 1. Purification of protein kinases by centrifugation in a sucrose gradient.Isolation of spinach thylakoids, extraction with detergents, and ammonium sulfate precipitation were carried out as described under "ExperimentalProcedures."The ammonium sulfate precipitate (45 mg ofprotein) was diluted to 10 mg of protein/ml with a buffer containing 20 mM Tris/succinate (pH 6.5), 0.1 mM ATP, 0.5 mM EDTA, 0.33% Triton X-100,0.1% cholate, and 0.1% soybean phospholipids. The solution (1.6 m l ) was applied to the top of 11.4 ml of a IO-30% continuous sucrose gradient in the above buffer. Centrifugation was carriedout in an SW-41 rotor at 160,OOO X g for 10-12 h. Aliquots (700~1)from the top of the tube were withdrawnand protein, protein kinase, and "PI-ATP exchange activities were measured as described under "ExperimentalProcedures."

Purification ofprotein kinases by sucrose gradient centrifugation and hydroxylapatite chromatography Experimental details were as described under "Experimental Procedures" exceut that urotein kinase activitv was measured at 37 'C. Purification step

Total protein

mg

Total activity units"

4500 Ammonium sulfate 45 precipitate 230 3220 Upper fractions from 14 72 sucrose gradient Hydroxylapatite chromatography ChlPKl 1.5 1056 ChlPKz 0.6246 148

Specific activity units/mg

Yield

100

100

%

Purification -fold

1

2.3

704

Picomoles of 32Pincorporated into casein per min.

23 3

7

2.5

Protein Kinases from Spinach Chloroplasts

12155

centrifugation was applied to the column as described in the plasts in the Lighta n d Dark-It was shown by Bennett (16) legend of Fig. 2. Although the relative contribution of the two that a threonine residue of protein is phosphorylated in chloenzymes to the total protein kinase activity varied with dif- roplasts from peas. With spinach chloroplasts exposed to light, ferent batches, the patternof elution of ChlPKl eluting at 165 we detected both serine and threonine phosphate after acid m~ Pi and ChlPK2 eluting at 225 mM Pi remained fairly hydrolysis of the protein as shown in Fig. 5. Threonine phosconstant. A summary sheet of the yield and purification of the phorylation was preferentially increased in the light, as in the two kinases is given in Table I. It should be stressed that the case of pea chloroplasts (16). Quantitative measurement of values of yield and -fold purification are atbest approximate, the radioactive spot from the electrophoresis paper yielded since the exact distribution of ChlPK, and ChlPK2 activities (in the dark) 558 cpm for threonine and 1392 cpm for serine in the ammonium sulfate precipitate are unknown. In any (a ratio of threonineserine of 0.4). In the same experiment case, it is apparent that thespecific activities of the purified (exposed to light), the threonine spot had1450 cpm, the serine fractions are quite low when compared to the activity of 1854 cpm (ratio of 0.8). Identification of Phosphoamino Acid Residuesand Analprotein kinases from animal sources (1). It should be mentioned here that following extraction of chloroplasts with ysis of Peptide Maps of Purified Enzymes-With casein as detergents as described in this paper, the residue contains A. insignificant protein kinase activity. It thus seems that chloI 2 3 4 5 S 0 ' 1 2 3 4 5 6 roplasts contain much less total protein kinase activity than, for example, mitochondria from bovine heart (15).Since prokaryotes are known to be even less endowed with protein kinase activities, it seems likely that control mechanisms by "ChlPK2 protein phosphorylation were increasingly used in the course of evolution. In Fig. 3, the analyses of crude and purified fractions in SDS-PAGE are shown. The M , values for ChlPKl andChlPK2 were estimated to be 25,000 and 38,000, respectively. Although it is apparent thata high degree of purification was achieved from the crude ammonium sulfate fraction (lane 3) to the FIG. 4. Labeling of the ChlPKl and ChlPKs with I-azido-[yh a l preparations (lanes 5 and 6), it should be emphasized that we do not claim, based on the Coomassie blue staining, "P]ATP. The upper fraction from the sucrose gradient was dialyzed against 100 volumes of 20 n" Tricine (pH 7.8) for 8 h with several that these preparationsare homogenous. However, the assign- changes of the buffer. The dialyzed fraction (61pg), 5 pg of ChlPKI, ments of 25,000 and 38,000 daltons for the protein kinase were and 6 pg of C W K 2were separately incubated ina medium containing corroborated by experiments with 8-azido-ATP. Purified prep- 5 rn Tris-HCI (pH 7 3 , 5 n" MgClz, and 5 p~ 8-a~ido-[y-~P]ATP arations of ChlPKl and ChlPK2 were exposed to 8-azido-[y at 0 "C for 15 s and thenexposed for 1 min to ultraviolet radiation as 32P]ATPin the absence and presence of uv light. It can be described under "Experimental Procedures." Controls were treated seen from Fig. 4 that whereas numerous proteins were pho- in the dark for 1 min under the same conditions. The samples were then subjected to SDS-PAGE. Odd-numbered lunes contained samtolabeled in the sucrose gradient fraction, the major photola- ples that were exposed to uv light. Even-numbered lanes contained beled bands in the purified preparations corresponded to the samples that were kept in the dark. Lunes 1 and 2, sucrose gradient bands which stained with Coomassie brilliant blue. A second fractions; lunes 3 and 4, ChlPK,; lunes 5 and 6, ChlPK,. A, Coomassie band weakly reactive with 8-azido-ATP in the light was seen blue stain, B, autoradiograph. in the ChlPK2 preparation. The ChlPKlpreparation con1 2 1 2 3 tained a trace of ChlPK2. We should mention here that we -p-tyrOsim, -P-lymk have noted in other experiments that interaction with 8-azidoATP may reveal impurities that cannot be detected by Coomassie blue staining even in gels overloaded with proteins. Identification of Phosphoamino Acid Residues inChloro"P-threonine "

I

"p-lmom

1

2

3

4

5

6

68,m--

-?-uk*

"

43.m- 0

21,000-

0

- -

14.300"

-P-aerh

FIG. 3. Analysis of crude and purified fractions of chloroplast protein kinases in SDS-PAGE. The peptide composition of fractions a t different stages of purification was determined as described under "Experimental Procedures." Lune l , molecular weight standards, 40 pg; lune 2, CF,, 45 pg; lune 3, ammonium sulfate fraction, 40 pg; lune 4, upper sucrose gradient fraction (fractions 1-6, Fig. l), 20 pg; lune 5,ChlPK, (fractions 29-53, Fig. 2), 3 p g ; lune 6,ChlPK, (fractions 55-58, Fig. 2),3 pg.

FIG. 5 (Zeft). Phosphoamino acid analysis of chloroplasts labeled with [ys2P]ATP. Spinach chloroplasts were prepared as described under "Experimental Procedures" except that 5 n" MgCh was included in SNT buffer. Chloroplasts containing 200 pg of chlorophyll were exposed to [Y-~'P]ATPin 500 pl of a medium containing SNT buffer (pH 7.8), 5 n" MgC12, and 50 PM [y3'P]ATP (350cpm/ pmol) for 10 min a t 26 "C in dark or light (8 X 105 ergs/cm*/s). After addition of 5 ml of SNT buffer containing 10 mM EDTA and 20 mM ATP, thesamples were c6intrifugeda t 3000 X g for 10 min. The pellets were washed twice with 5 ml of 80% acetone and finally resuspended in 400 plof H20. Hydrolysis of samples of this suspension and phosphoamino acid analysis were carried out as described under "Experimental Procedures." Lune I , thylakoids treated in the dark; lane 2, light-treated chloroplasts. FIG. 6 (right). Identification of the amino acid residue phosphorylated by ChlPKl and ChlPKz. Phosphorylation of casein by chloroplast protein kinases or catalytic subunit of the CAMP-dependent protein kinase and phosphoamino acid analysis were carried out as described under "Experimental Procedures." Lune 1, catalytic subunit of protein kinase (2.5pg); lune 2, ChlPKl (25 p g ) ; lune 3, ChlPKz (6pg).

12156

Kinases Protein

from Chloroplasts Spinach

A. B. 1 2 3 4 1 2 3 4

FIG.7. Analysis of peptide maps. Phosphorylation of purified histone 111s by ChlPKl or ChlPK2 and proteolytic digestion of the samples was carried out as described under “Experimental Procedures.” Samples containing 10-12 pg of protein were electrophoresed with SDS-PAGE as described. Lane I, digested ChlPKI; lane 2, digested ChlPK2; lane 3, digested phosphorylated histoneby ChlPKI; lane 4, digested phosphorylated histone by ChlPK2. A, Coornassie blue stain; E , autoradiograph. substrate, the purified preparations of CMPKl and ChlPK2 phosphorylated a serine residue (Fig. 6). As shown in Fig. 7, the peptide maps of histone 111s phosphorylated by ChlPKl and CMPK2 were clearly different, indicating that the phosphorylated serine residues are in different positions in the histone substrate. It is clear from previous work in our (6.7)and several other laboratories (16-19) that thelight-dependent phosphorylation is a complex process probably involving a plastoquinone oxi-

dation-reduction system which preferentially phosphorylates a threonine residue (see Fig. 5). A reduced plastoquinone regenerating system markedly stimulated the phosphorylation of chloroplast proteins in the dark and addition of phospholipids also enhanced protein kinase activity (6).It is therefore possible that future work on the isolation of the threoninespecific protein kinase may require a reconstitution assay with the appropriate reductants, protein substrates, and lipids. REFERENCES 1. Krebs, E.G., and Beavo, J. A. (1979)Annu. Rev. Biochem. 48, 923-959 2. Collett, M. S., Purchio, A. F., and Erikson, R. L. (1980)Nature 285, 167-169 3. Ralph, R. K., McCornbs, P. J. A., Tener, G., and Wojcik, S. J. (1972)Biochem. J. 130,901-911 4. Keates, R. A. B. (1973)Bwchem. Bwphys. Res. Commun. 54, 655-661 5. Nakaya, N., Sugano, N., Nishi, A., and Tsukada, K. (1975) Biochim. Bwphys. Acta 410,273-278 6. Alfonzo, R. (1980)thesis, Graduate School of Cornell University 7. Alfonzo, R., Nelson, N., and Racker, E. (1980)Plant Physiol. 65, 730-734 8. Kagawa, Y.,and Racker, E. (1971)J. B i d . Chem. 246,5477-5487 9. Nelson, N., Eytan, E., Notsani, B. E., Sigrist, H., Sigrist-Nelson, K., and Gitler, C. (1977)Proc. Natl. Acad. Sci. U.S. A . 74, 2375-2378 10. Amon, D. I. (1949)Plant Physwl. 24, 1-15 11. Pick, U.,and Racker, E. (1979)J. Biol. Chem. 254,2793-2799 12. Corbin, J. D., and Reimann, E. M. (1974)Methods Enzymol. 38, 287-290 13. Benadoun, A., and Weinstein, D. (1976)Anal.Bbchem. 70, 241-250 14. Laemmli, U. K. (1970)Nature (Lond.)227,680-685 15. Kitagawa, Y.,and Racker, E. (1982)J. Bwl. Chem. 257,4547-4551 16. Bennett* J . (1977)Nature 2699 344-346 17. Bennett, J. (1979)FEBS Lett. 103,342-344 18. Horton, P., Allen, J. F., Black, M. T., and Bennett, J. (1981) FEES Lett. 125,193-196 19. Millner, P. A., Widger, W.R., Abbott, M. S., Cramer, W.A., and Dilley, R.A. (1982)J. Biol. Chem. 257, 1736-1742

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