Vol. 155, No. 2, 1988

September 15, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Pages 835-840

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'e Gadal*

*Laboratoire de Physiologie V~g~tale Mol~,culaire, UA CNRS 1128, Universit~ de Paris-S ud, Centre d'Orsay, B~ttiment 430, F 91405, Orsay Cedex, France

~Institutde Physiolo.cle V~g~tale, ONRS, F 91190 Gif/Yvette, F r a n c e

# Centre de Physiologie V~g~tale, UA CNRS 241, Universit6 Paul Sabatier, 118 Route de Narbonne, F 31002 Toulouse Cedex, France

Received August 3, 1988

Regulation ofthe ,,n J.'~ phosphorylation process ofthe photosynthetic form (G form) of S~lh~'cn leaf Phosphoenolpyruvate carboxylase (PEPC: EC 4.1 .I .31) was studied. Results established that: I) PEPC was efficiently phosphorylated on seryl residues in crude leaf exlr'act 2) Pyruvate, orthophosphate dikinase (E¢ 2.7.9.1 .) which has been supposed to interfere with tlle process, was found not to be significantly phosphorylated 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 nullified itand Ca ++ alone was found to reverse the inhibitory effect exerted by both chelators 5) addition of 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 results established that the photosynthetic form or "G-form" of PEPO

from S~Thtcn leaf undergo in J.YJ.~ga reversible phosphorylation/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 inhibition by

malate than the dephosphorylated form (2). It was also demonslrated that enzyme

phosphorylation displayed significant variations through a day-light alternaUon which

suggested a light control of the process (1,2). Therefore, the light effect may 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'-tetraacetic acid, KF: Potassium fluoride, IgG: Immunoglobulin G, S DS-PAG: Sodium dodecyl sulphate polyacrylamide gel electrophoresis, PBS: Phosphate buffer saline, CaM: calmoduline

835

0006-291X/88 $1.50 Copyright © 1988 by Academic Press, Inc.

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Vol. 155, No. 2, 1988 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

by post-lr'anslational modifications of PEPC which result in differential regulatory

properties of the enzyme.

The present work was an aZempt to unravel some of the components of the

Iransduction system which is involved in the stimulus/response coupling. It reports on

the fact that the phosphorylation cascade of PEPO from S~Thtrn leaves involves a

calcium-calmodulin dependent protein kinase to suggest that calcium is most likely a

second messenger in the photocontrol of PEPC.

MATERIAL AND METHODS

Plant rnaterial: 5'~Th,,cn seeds ~ghum yulgare vat. Tamaran) were cultivated from germination during 10-15 days as previously described (I). Leaf samples were collected during the night and used for enzyme exlraction and 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 activity and definition of enzyme unit was as described (4). Protein .ph...osphorylation assa, y~: Protein phosphorylation v~.s carried out in 150 I~I of crude exlract (0.5 units of PEPO) at 30 ° O, in the presence of 6 rnM MgCI2, 0.5 mM

CaOl2, and 3 i~Oi of 32p ATP (3-I 00i/mmole). After a 30 rain incubation, the reaction

was stopped by addition of I% SDS, and half of the medium was heated for 2 min at g0 ° O, and submiZed to denaturing PAGE (I 0% acrylamide) according to Laemmli (5). Gel processing: The gel was stained by Coomassie blue, destained in methanol- acetic 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). Equilibrating buffer 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), redissolved in PBS and the buffer was changed as required by using gel fillration through small Sephadex G25 columns. Western blot and Immunoprecipitation experimenl~ were as previously described (6,3) respectively.

RESULTS AND DISCUSSION

Since PEPO occurs in its low phosphorylated form in dark-grown plants, all the

standard experiments were done with proteins exl~acted during the night period of the

culture. In these conditions, supplementing the protein exit-acts with (32p) ATP resulted

in the phosp horylatio n of a few n umber of polypeptides (fig. I ). The 94 k Da spec ies was

immunoprecipitated by antibodies raised against PEPO and remained labelled after

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Vol. 155, No. 2, 1 9 8 8 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

A B

(3

A B A B C

94kd PE Pc -*-

IgG

94kd

52kd

94kd

-- ÷ -- ÷

® ® Figure I:/n J.:~ phosphorylation of 5"t~;,hzz'nleaf exlrac~ ob~ined during the night: Exlraction was performed as described in Method section. The supernal;ant of cenlrifugation (10 min, 14000xg) was used for phosphorylation. Protein phosphorylation was carried out in 150 ml (0.5 units of 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.

Figure 2: Identification of PEPC, ExlTacl~ were prepared from leaves collected during the night./n vl#'o phosphorylatlon and subsequent SDS-gel elec~ophoresis were as in figure I. (A) Coomassie blue sl:aining, (AI) protein pattern from crude exl~act, (A2) immunoprecipiLated protein using specific PEPC antibodies, (B) corresponding Western experiment using the immune serum and (C) fluorography.

Flgure 3: Effect of APSA ,(pip5 di(.adenosine-5:)_pentaphosphate: Aden y.late kinase inhibitor) on the /n J.'/&~ phosphorylation of PEPC: The night forrn of PEPC was phosphorylated in the presence (+) or absence(-) of the inhibitor (0.25 raM). 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.

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Vol. 155, No. 2, 1 9 8 8 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

f

PEPc..,~

Q I

A B - - ~ - - ~ , , r - - -

94kd A B

PEI~:-~

2 3 4 5 1 2 3 4 5 ( , ~

Figure 4: Factors influencing/n J,'/~o phosphon/la.tion.

94kd

1 2 3 I 2 3

Protein phosphorylaUon was carried out in 150 ul of crude exlract prepared in absence of KF (lane I); Addition of(lane 2) 20 mM KF; (lane 3) 20 mM KF, 0.5 mM CaCl 2, 6 mM MgOl2; (lane 4-5) complete medium + I mM and 4 mM EDTA respectively. SDS-gel elecl:rophoresis. (A) Coomassle blue stalnlng, (B) fluorography.

Figure 5: Calcium effect on in J,~ phosphop/.lation of PEPO. Phosphorylation was conducted on partially purified (G25) exlract using the optimized conditions described under figure I. (lane BI) conlzol; additions of: (lane B2) 5raM EGTA, (lane B3) 5 rnM EGTA + 20 mM OaOl 2. SDS-gel elec~ophoresis. (A) Ooornassie blue sl~ining, (B) fluorography.

The phosphorylaUon process of PEPC was studied further by 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 itself may stabilize the phosphoenzyme by inhibiUng endogenous

phosphatases. More surprisingly, 0.5 mM calcium, in the presence of 20mM KF, was

enough to stimulate the phosphorylation of PEPC to suggest that a very low

concentration of free calcium was enough to elicit the process. The calcium elTect was

abolished by EGTA and recovered on adding calcium back (fig.5) but was not replaced

by magnesium (not shown).

When the protein extract ,~ras depleted of tlle acidic calcium-binding protein

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-protein remained esse ntia l ly co nsta nt (fig. 6).

From all these data, it appears that PEPC from ,~Izhz~ was phosphorylated

in a calcium-calmodulin dependent manner. In this way, PEPC is one of the few plant

proteins that are photoregulated and conl~olled by a complex cascade of events (9,10).

Such a situation is invaluable in plant biochemisby where light is suspected to conlyol

838

Vol. 155, No. 2, 1988 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

PEP,

A B

! ~ i i i i ¸¸ 94kd

r

PEPc..~

1 A 2 3 4 B 1 2 3 4

~lkd

II i23 123

Figure 6: Immunoinhibition of PEPO.phosphorylation by. anti-Calmodulin antibodies.. Phosphorylation was performed from either partially purified exlract (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) addition of 75, 150, 300 I~ of Sheep Calmodulin antibodies purified by alTinity chromatography on Calmodulin-Sepharose columns. II- (lanel) conlrol, (lanes 2,3) addition of antibodies (400 ~ ) partially purified on Protein A-Sepharose.

the functioning of calcium channels (I I) and stimulate the production of messenger

such as inositol Iriphospl~ate (I 2). Therefore, tlle PEPC system is a good candidate for

a bel~er unders~nding of the stimulus/response coupling in higher plants.

ACKNOWLEDGMENTS: Thanks are due to M. Weimbaum for photographs, E. Keryer for excellent technical assistance, Pr M, Kluge, DrO. Queiroz, C. Cretin, F, Gil and A. De la Tortes for helpful discussion; 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.

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Vol. 155, No. 2, 1988 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

7. Ashton, A.R. and Hatch, M,D. (I 983) Biochem. Biophys. Res. Commun., 115 (I), 53-60.

8. Budde, R.J.A. and Chollet, R. (I 986) Plant Physiol., 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. (I 988) Physiol. Plant. 72, 435-439. 1 I. Das, R. and Sopory, S.K. (I 985) Biochem. Biophys. Res. Commun., 128,

1455-1460. 12. Morse, M.J., Crain, R.C. and Saller, R.L. (I 987) Proc.Natl. Acad. Sci. USA. 84,

7075-7078.

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