The phosphorylation of Sorghum leaf phosphoenolpyruyate carboxylase is a Ca++-calmodulin dependent process

The phosphorylation of Sorghum leaf phosphoenolpyruyate carboxylase is a Ca++-calmodulin dependent process

Vol. 155, No. 2, 1988 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 835-840 September 15, 1988 THE PHOSPHOFIYLATION OF ~ LEAF PHOSPHOE...

852KB Sizes 0 Downloads 14 Views

Vol. 155, No. 2, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 835-840

September 15, 1988

THE PHOSPHOFIYLATION OF ~

LEAF PHOSPHOENOLPYIqUYATE

CARBOXYLASE IS A C~++- CALMODULIN DEPENDENT PROCESS

Cristina Ecllevarria*£, Jean Vidal*,Pierre Le Mar~cllal*,Jeanne Brulfert~, Raoul Ranjeva #, and Pien'eGadal* *Laboratoire de Physiologie V~g~tale Mol~,culaire,UA CNRS 1128, Universit~de Paris-Sud, Centre d'Orsay, B~ttiment430, F 91405, Orsay Cedex, France ~InstitutdePhysiolo.cleV~g~tale, ONRS, F 91190 Gif/Yvette,

France

# Centre de Physiologie V~g~tale, UA CNRS 241, Universit6Paul Sabatier, 118 Route de Narbonne, F 31002 Toulouse Cedex, France Received August 3, 1988

Regulation ofthe ,,nJ.'~ phosphorylation process ofthe photosyntheticform (G form) of S~lh~'cn leaf Phosphoenolpyruvate carboxylase (PEPC: EC 4.1 .I.31) was studied. Results established that: I) PEPC was efficientlyphosphorylated on seryl residues in crude leaf exlr'act2) Pyruvate, orthophosphate dikinase (E¢ 2.7.9.1.) which has been supposed to interfere with tlle process, was found not to be significantlyphosphorylated in our experimental conditions 3) KF, as v~ll as both Oa ++ and Mg ++ ions increased the radioactive signal detected 4) addition of EDTA or EGTA nullifieditand Ca ++ alone was found to reverse the inhibitoryeffectexerted by both chelators 5) additionof anti-Calmodulin antibodies to the medium also abolished the PEPC phosphorylation. Present data demonstrated that tlle post-Izanslational modification of the C4-plant photosyntl~eUc PEPC is a Ca++/Calmodulin dependent process, ~ ~8 ~o~:~ ~. . . . . ::~. Previous resultsestablished thatthe photosynthetic form or "G-form" of PEPO from S~Thtcn leaf undergo in J.YJ.~gareversiblephosphorylation/dephosphorylation process at seryl residues (I). Data obtained by otllers on the maize leaf enzyme suggested that phosphorylated PEPO was less sensitive to feed-back inhibitionby malate than the dephosphorylated form (2). Itwas also demonslrated that enzyme phosphorylation displayed significantvariations through a day-lightalternaUon which suggested a lightcontrolof the process (1,2).Therefore, the lighteffectmay be sensed £ To whom correspondence should be addressed. Abbreviations: AP5A: PIP5 di (adenosine-5') pentaphosphate, EDTA: Ethylene diamine telraacetic acid, EGTA: Ethylene glycol bis (8-aminoethyl ether) N,N,N',N'-tetraaceticacid, KF: Potassium fluoride,IgG: Immunoglobulin G, S DS-PAG: Sodium dodecyl sulphate polyacrylamide gel electrophoresis, PBS: Phosphate buffer saline, CaM: calmoduline 0006-291X/88 $1.50 835

Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol. 155, No. 2, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

by post-lr'anslationalmodifications of PEPC which result in differentialregulatory propertiesof the enzyme. The present work was an aZempt to unravel some of the components of the Iransductionsystem which is involved in the stimulus/responsecoupling. Itreportson the factthatthe phosphorylation cascade of PEPO from

S~Thtrn leaves involves a

calcium-calmodulin dependent protein kinase to suggest thatcalcium is most likelya second messenger inthe photocontrolof PEPC.

MATERIAL A N D M E T H O D S

Plant rnaterial:5'~Th,,cnseeds ~ghum yulgare vat.Tamaran) were cultivatedfrom germination during 10-15 days as previously described (I). Leaf samples were collectedduring the nightand used forenzyme exlractionand assays. Exl~ction of PEPC: One g fresh leaves was ground in 3ml of 100 mM Tris(Cl-)buffer, pH 8, containing:20% Glycerol, 10 mM D-L Malate, 20 mM KF, I rnM EDTA, 14 rnM l~-Mercaptoethanol. The brei was cenlrifuged at 14000 g for 10 rain and the superna~nt was used for phosphorylation experiments. Where indicated,PEPO was sieved on Sephadex G25 equilibrated with exlraction buffer.Immunoprecipil~tion experimen~ were performed as already described (3) using a highly specific immuneserum. Enzyme activityand definitionof enzyme unitwas as described (4). Protein.ph...osphorylationassa,y~: Protein phosphorylation v~.s carriedout in 150 I~Iof crude exlract(0.5 unitsof PEPO) at 30° O, in the presence of 6 rnM MgCI2, 0.5 mM CaOl2, and 3 i~Oiof 32p ATP (3-I00i/mmole). Aftera 30 rainincubation,the reaction was stopped by addition of I% SDS, and halfof the medium was heated for 2 min at g0° O, and submiZed to denaturing PAGE (I0% acrylamide)according to Laemmli (5). Gel processing: The gel was stained by Coomassie blue, destained in methanolacetic acid (4/I/5)solution,then immersed in autofadiographic enhancer (Enligl~tning nom NEN) dried and fluOrographed at-80°C. General methods: Anti-Calmodulin antibodies were affini~ purified on either Calmodulin-Sepharose (Pharmacia) or Protein A Sepharose columns (Phamlacia). Equilibratingbuffer was PBS; IgG eluted by washing with 200 mM civic acid-cilrate buffer,pH 2.6 were recovered from the eluate by ammonium sulphate precipitation (50% saturation),redissolvedin PBS and the bufferwas changed as required by using gel fillration through small Sephadex G25 columns. Western blot and Immunoprecipitationexperimenl~ were as previouslydescribed (6,3)respectively.

RESULTS A N D DISCUSSION

Since PEPO occurs in its low phosphorylated form in dark-grown plants,all the standard experiments were done with proteinsexl~acted during the nightperiod of the culture.Inthese conditions,supplementing the proteinexit-actswith(32p) ATP resulted inthe phosp horylation of a few n umber ofpolypeptides (fig.I).The 94 k Da spec ieswas immunoprecipitated by antibodies raised against PEPO and remained labelledafter 836

Vol. 155, No. 2, 1 9 8 8

BIOCHEMICAL AND BIOPHYSICALRESEARCHCOMMUNICATIONS A

AB

A

B

BC 94kd

94kd PE Pc -*-

IgG

94kd

52kd --

(3

®

÷

--

÷

®

Figure I:/n J.:~ phosphorylationof 5"t~;,hzz'nleaf exlrac~ ob~ined duringthe night: Exlraction was performed as described in Method section. The supernal;antof cenlrifugation (10 min, 14000xg) was used for phosphorylation. Protein phosphorylationwas carriedout in 150 ml (0.5 unitsof PEPC) of crude exlract,30" C, for 30 min, in the presence of 6ram MgCl 2, 0.5 mM CaCI2, and g32p ATP (3-10 Cilmmol), 3 l~Ci. SDS-PAGE(10% acrylamide).(A) Coomassie blue staining,(B) fluorography. Figure2: Identification of PEPC, ExlTacl~were preparedfrom leavescollectedduringthe night./nvl#'ophosphorylatlon and subsequent SDS-gel elec~ophoresiswere as in figure I. (A) Coomassie blue sl:aining,(AI) protein patternfrom crude exl~act, (A2) immunoprecipiLatedprotein using specificPEPC antibodies, (B) corresponding Western experiment using the immune serum and (C) fluorography. Aden y.latekinase Flgure 3: Effectof APSA ,(pip5di(.adenosine-5:)_pentaphosphate: inhibitor)on the /n J.'/&~phosphorylationof PEPC: The night forrnof PEPC was phosphorylated in the presence (+)or absence(-)of the inhibitor(0.25raM).Proteins were submilted to SDS-gel eleclrophoresis.(A) Coomassie blue staining,(B) Fluorography.

Western blotting and fluorogtaphy (fig.2).Similarly to what had been previously

checked in in J'iJ'~,~studies (1), possible interference with PPDK was examined. PPDK is a plant protein, i-already shown to undergo ADP-dependent phosphorylation on threonyl residues (7), ii-relatively abundanL and iii- whose subunit molecular weight is very close to that of PEPC. As depicted in Figure 3, addition of APSA (wtlich inhibits Adenylate kinase activib/and therefore prevents the formation of ADP ~om ATP) (8) to the reaction medium did not alter the radioacUve signal of the 94 Kd band. Moreover, acid hydrolysis of the immunopurified protein, alter in J , ~ phosphorylaUon, revealed the presence of a single phosphoaminoacid identified as phosphoserine (not shown). The immunological properties of the peptide and the identification of phosphoserine insteadt of phosphothreonine as the modified aminoacid esl;ablished that the 94 kDa radioactive band is a subunit of PEPC. 837

Vol. 155, No. 2, 1 9 8 8

BIOCHEMICAL AND BIOPHYSICALRESEARCHCOMMUNICATIONS

A

B

--~--

f

~,,r

- -

-

PEPc..,~

A

94kd

B

94kd

PEI~:-~

QI

2

3 4 5

1

2

3 4

5

(,~

123

I

23

Figure4: Factorsinfluencing/nJ,'/~o phosphon/la.tion. ProteinphosphorylaUonwas carriedout in150 ulofcrude exlractpreparedinabsence of KF (laneI);Additionof(lane2) 20 m M KF;(lane3) 20 m M KF, 0.5m M CaCl2,6 m M MgOl2; (lane4-5)completemedium + I m M and 4 m M EDTA respectively.SDS-gel elecl:rophoresis. (A) Coomassle bluestalnlng,(B)fluorography. Figure 5: Calcium effecton in J,~ phosphop/.lationof PEPO. Phosphorylationwas conductedon partiallypurified(G25) exlractusingtheoptimizedconditionsdescribed underfigureI.(laneBI) conlzol;additionsof:(laneB2) 5raM EGTA, (laneB3) 5 rnM EGTA + 20 m M OaOl 2. SDS-gel elec~ophoresis.(A) Ooornassiebluesl~ining,(B) fluorography. The phosphorylaUon process of PEPC was studied furtherby examining the effect of various potential effectors (fig.4).Thus, when added separately or in combination, KF, Mg 2+ and/or calcium increased the label into PEPC. Potassium fluoride by itselfmay stabilize the phosphoenzyme by inhibiUng endogenous phosphatases. More surprisingly,0.5 m M calcium, in the presence of 20mM KF, was enough to stimulate the phosphorylation of PEPC to suggest that a very low concentrationof free calcium was enough to elicitthe process.The calcium elTectwas abolished by EGTA and recovered on adding calcium back (fig.5)but was not replaced by magnesium (notshown). When the protein extract,~rasdepleted of tlleacidic calcium-bindingprotein calmodulin by immunoprecipitation, it turned out that the 32p label decreased dramatically with the amounts of added antibodies wllereas the amount of P E P C-proteinremained esse ntiallyco nstant(fig.6). From all these data, itappears that PEPC from ,~Izhz~ was phosphorylated in a calcium-calmodulindependent manner. In thisway, PEPC is one of the few plant proteinsthatare photoregulatedand conl~olledby a complex cascade of events (9,10). Such a situationis invaluable inplant biochemisby where lightissuspected to conlyol 838

Vol. 155, No. 2, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS A

B

!~i iii ¸¸

94kd

PEP,

1A2

3

4 B

1

2

3

4

r

PEPc..~

~lkd

II i23

123

Figure 6: Immunoinhibitionof PEPO.phosphorylationby.anti-Calmodulinantibodies.. Phosphorylationwas performedfrom eitherpartiallypurifiedexlract(I)or crude exlract {II) by using experimental conditions described under Figure I SDS-gel eleclrophoresis(A) Coomassie blue staining,(B)fluorography. I- (lanel) conlrol,(lanes 2,3,4)additionof 75, 150, 300 I~ of Sheep Calmodulin antibodiespurifiedby alTinitychromatographyon Calmodulin-Sepharosecolumns. II-(lanel)conlrol,(lanes 2,3) additionof antibodies(400 ~ ) partiallypurifiedon ProteinA-Sepharose.

the functioning of calcium channels (I I) and stimulate the production of messenger such as inositolIriphospl~ate(I 2). Therefore, tllePEPC system isa good candidate for a bel~erunders~nding of the stimulus/response coupling in higher plants.

ACKNOWLEDGMENTS: Thanks are due to M. Weimbaum for photographs, E. Keryer forexcellent technicalassistance, Pr M, Kluge, DrO. Queiroz, C. Cretin,F, Gil and A. De la Tortes for helpfuldiscussion; this wod< was supported by a concerted research program betcceen Spain and france MECIMRES. REFERENCES

I. Guidici-Orticoni,M.T., Vidal,J., Le Mar~chal, P., Thomas, M. and Gadal, P. (I 988) Biochimie, In press. 2. Nimmo, G. A.,Mcnaughton, G.A,L., Fewson, C.A.,Wilkins,M.B. and Nimmo, H.G.(1987) FEBs Left.,213(I), 18-22. 3. Vidal,J.,Godbillon, G. and Gadal, P. (I 983) Physiol.Plant.,57, 124-I 28. 4. Vidal,J. and Gadal, P. (I 983). Physiol.Plant.,57, 119-I 23. 5. Laemmli, U.K. (1970) Nature 227, 680-685. 6. Thomas, M., CrY'tin,C., Keryer, E.,Vidal,J.,Gadal, P., Bidart,J.M. and Bohuon, C, (I 987) Biochem. Biophys. IRes.Commun., 143 (I),170-I 77. 839

Vol. 155, No. 2, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

7. Ashton,A.R. and Hatch,M,D. (I983) Biochem. Biophys. Res. Commun., 115 (I), 53-60. 8. Budde, R.J.A.and Chollet,R. (I986) PlantPhysiol.,82, 1107-I 114. 9. Ranjeva,R. and Boudet, A.M. (1987) Ann. Rev. Plant.Physiol. 38, 73-93. 10. Budde, R.J,A.and Cbollet,R. (I988) Physiol.Plant.72, 435-439. 1 I. Das, R. and Sopory, S.K.(I985) Biochem. Biophys. Res. Commun., 128, 1455-1460. 12. Morse, M.J.,Crain,R.C. and Saller,R.L.(I987) Proc.Natl.Acad. Sci.USA. 84, 7075-7078.

840