\u3ci\u3eIn Vivo\u3c/i\u3e Regulatory Phosphorylation Site in C4-Leaf Phosphoenolpyruvate Carboxylase from Maize and Sorghum (original) (raw)
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PLANT PHYSIOLOGY, 1991
Reversible seryl-phosphorylation contributes to the light/dark regulation of C4-leaf phosphoenolpyruvate carboxylase (PEPC) activity in vivo. The specific regulatory residue that, upon in vitro phosphorylation by a maize-leaf protein-serine kinase(s), leads to an increase in catalytic activity and a decrease in malatesensitivity of the target enzyme has been recently identified as Ser-15 in 32P-phosphorylated/activated dark-form maize PEPC
Archives of Biochemistry and Biophysics, 1989
A reconstituted system composed of purified phosphoenolpyruvate carboxylase (PEP-Case) and a soluble protein kinase (PK) from green maize leaves was developed to critically assess the effects of in vitro protein phosphorylation on the catalytic and regulatory (malate sensitivity) properties of the target enzyme. The PK was partially purified from light-adapted leaf tissue by ammonium sulfate fractionation (O-69% saturation fraction) of a crude extract and blue dextran-agarose affinity chromatography. The resulting preparation was free of PEPCase. This partially purified protein kinase activated PEP-Case from dark-adapted green maize leaves in an ATP-, Mgs'-, time-, and temperaturedependent fashion. Concomitant with these changes in PEPCase activity was a marked decrease in the target enzyme's sensitivity to feedback inhibition by L-malate. The PKmediated incorporation of "P from [y-32P]ATP into the protein substrate was directly correlated with these changes in PEPCase activity and malate sensitivity. The maximal molar 32P-incorporation value was about 0.25 per lOO-kDa PEPCase subunit (i.e., 1 per holoenzyme). Phosphoamino acid analysis of the 32P-labeled target enzyme by two-dimensional thin-layer electrophoresis revealed the exclusive presence of phosphoserine. These in vitro results, together with our recent studies on the light-induced changes in phosphorylation status of green maize leaf PEPCase in vivo (J.-A. Jiao and R. Chollet (1988) Arch. Biochem. Biophys. 261,409-41'7), collectively provide the first unequivocal evidence that the seryl-phosphorylation of the dark-form enzyme by a soluble protein kinase is responsible for the changes in catalytic activity and malate sensitivity of C, PEPCase observed in vivo during dark/light transitions of the parent leaf tissue. o 1989 Academic Press, Inc.
\u3ci\u3eIn Vitro\u3c/i\u3e Phosphorylation of Maize Leaf Phosphoenolpyruvate Carboxylase
1986
Autoradiography of total soluble maize (Zea mays) leaf proteins incubated with 32P-labeled adenylates and separated by denaturing electrophoresis revealed that many polypeptides were phosphorylated in vitro by endogenous protein kinase(s). The most intense band was at 94 to 100 kilodaltons and was observed when using either [γ -32P]ATP or [β-32P]ADP as the phosphate donor. This band was comprised of the subunits of both pyruvate, Pi dikinase (PPDK) and phosphoenolpyruvate carboxylase (PEPCase). PPDK activity was previously shown to be dark/light-regulated via a novel ADP-dependent phosphorylation/Pi-dependent dephosphorylation of a threonyl residue. The identity of the acidstable 94 to 100 kilodalton band phosphorylated by ATP was established unequivocally as PEPCase by two-dimensional gel electrophoresis and immunoblotting. The phosphorylated amino acid was a serine residue, as determined by two-dimensional thin-layer electrophoresis. While the in vitro phosphorylation of PEPCase f...
Light/dark regulation of maize leaf phosphoenolpyruvate carboxylase by in vivo phosphorylation
Archives of Biochemistry and Biophysics, 1988
Phosphoelzolpyruvate carboxylase (PEPCase) from light-and dark-adapted maize leaves was rapidly purified in the presence of L-malate and glycerol to apparent electrophoretic homogeneity by ammonium sulfate fractionation, hydroxylapatite chromatography, and fast-protein liquid chromatography on Mono Q. The resulting preparations were totally devoid of pyruvate,orthophosphate dikinase protein based on immunoblot analysis. Throughout the purification, both forms of PEPCase retained their different enzymatic properties. The specific activity of the light enzyme was consistently about twice that of the dark form when assayed at suboptimal (but physiological) pH (pH '7.0-7.3), and the former was also less sensitive to feedback inhibition by L-malate than that from darkened leaves under various conditions. Covalently bound phosphate and high-performance liquid chromatography-based phosphoamino acid analyses showed that both forms of purified PEPCase were phosphorylated exclusively on serine residues, but the degree of phosphorylation was about 50% greater in the light enzyme. Notably, incubation of purified PEPCase in vitro with exogenous alkaline phosphatase led to an increase in malate sensitivity and a decrease in specific activity of the light form enzyme to levels observed with the dark form, which was essentially not affected by phosphatase treatment. These results with the puri3ed enzyme from light-and dark-adapted maize leaves indicate that the light-induced changes in activity and malate sensitivity of C, PEPCase are related, at least in part, to the degree of covalent seryl phosphorylation of the protein in viva.
The Plant Journal, 2000
The phosphoenolpyruvate carboxylase (PEPC) isozyme involved in C 4 photosynthesis is known to undergo reversible regulatory phosphorylation under illuminated conditions, thereby decreasing the enzyme's sensitivity to its feedback inhibitor, L-malate. For the direct assay of this phosphorylation in intact maize leaves, phosphorylation state-speci®c antibodies to the C 4-form PEPC were prepared. The antibodies were raised in rabbits against a synthetic phosphorylated 15-mer peptide with a sequence corresponding to that¯anking the speci®c site of regulatory phosphorylation (Ser15) and subsequently puri®ed by af®nity-chromatography. Speci®city of the resulting antibodies to the C 4-form PEPC phosphorylated at Ser15 was established on the basis of several criteria. The antibodies did not react with the recombinant root-form of maize PEPC phosphorylated in vitro. By the use of these antibodies, the changes in PEPC phosphorylation state were semi-quantitatively monitored under several physiological conditions. When the changes in PEPC phosphorylation were monitored during the entire day with mature (13-week-old) maize plants grown in the ®eld, phosphorylation started before dawn, reached a maximum by mid-morning, and then decreased before sunset. At midnight dephosphorylation was almost complete. The results suggest that the regulatory phosphorylation of C 4-form PEPC in mature maize plants is controlled not only by a light signal but also by some other metabolic signal(s). Under nitrogen-limited conditions the phosphorylation was enhanced even though the level of PEPC protein was decreased. Thus there seems to be some compensatory regulatory mechanism for the phosphorylation.
FEBS Letters, 1997
In order to mimic regulatory phosphorylation of the Ser-15 of maize C 4-form phosphoewo/pyruvate carboxylase (PEPC), we replaced Ser-15 and Lys-12 with Asp (S15D) and Asn (K12N), respectively, by site-directed mutagenesis. Although both mutant enzymes were catalytically as active as the wild-type PEPC, they showed much less sensitivity to malate, an allosteric inhibitor, similarly to the phosphorylated wild-type PEPC. A maize protein kinase of 30 kDa which is known to be specific to PEPC (PEPC-PK), phosphorylated K12N as well as the wildtype PEPC but not S15D. The phosphorylation of K12N further diminished the sensitivity to malate. Thus, a positive charge of the conserved Lys-12 is not required for the recognition by PEPC-PK but contributes to the intrinsic sensitivity to malate inhibition.
In Vitro Phosphorylation of Maize Leaf Phosphoenolpyruvate Carboxylase
Springer eBooks, 1987
Autoradiography of total soluble maize (Zea mays) leaf proteins incubated with 32P-labeled adenylates and separated by denaturing electrophoresis revealed that many polypeptides were phosphorylated in vitro by endogenous protein kinase(s). The most intense band was at 94 to 100 kilodaltons and was observed when using either y'-32P]ATP or 1[
Plant and Cell Physiology, 1998
Phosphoe/io/pyruvate carboxylase (PEPC) [EC 4.1.1.31] of plants undergoes regulatory phosphorylation in response to light or nutritional conditions. However, the nature of protein kinase(s) for this phosphorylation has not yet been fully elucidated. We separated a Ca 2+-requiring protein kinase from Ca 2+-independent one, both of which can phosphorylate maize leaf PEPC and characterized the former kinase after partial purification. Several lines of evidence indicated that the kinase is one of the characteristic Ca 2+-dependent but calmodulin-independent protein kinase (CDPK). Although the M, of native CDPK was estimated to be about 100 kDa by gel permeation chromatography, in situ phosphorylation assay of CDPK in a SDS-polyacrylamide gel revealed that the subunit has an M r of about 50 kDa suggesting dimer formation or association with other protein(s). Several kinetic parameters were also obtained using PEPC as a substrate. Although the CDPK showed an ability of regulatory phosphorylation (Ser-15 in maize PEPC), no significant desensitization to feedback inhibitor, malate, could be observed presumably due to low extent of phosphorylation. The kinase was not specific to PEPC but phosphorylated a variety of synthetic peptides. The possible physiological role of this kinase was discussed.
FEBS Letters, 1990
Cd-leaf phosphoenolpyruvate carboxylase (PEPC; EC 4,1,1.3 1) undergoes reversible, light-induced increases in its activity-seryl phosphorylationstatus in vivo. We now report that the PEPC-protein kinase activity in desalted crude extracts of light-adapted maize leaves is several-fold greater than that from the corresponding dark tissue when in vitro phosphorylation assays are performed with either endogenous or purified dark-form maize PEPC as substrate, both in the absence or presence of okadaic acid, a potent inhibitor of the PEW type 2A protein phosphatase(s): These and related results indicate that the PEPC protein-scrine kinase(s) per se is reversibly light activated in vivo by either covalent modification, protein turnover or, less likely, a tight-binding effector.
In vitro phosphorylation of purified tobacco-leaf phosphoenolpyruvate carboxylase
FEBS Letters, 1993
C,-leaf phosphoenolpyruvate (PEP) carboxylase (PEPC) was purified about l,OOO-fold from tobacco and displayed a final specific activity of 35 ~mol/minlmg protein, an apparent K,,, (total PEP) of 95 mM (both at pH 8.0, 30°C). and an 1,&L-malate) value of 0.14 mM at pH 7.3, 0.2 mM PEP. The rapid, 5-step protocol involved polyethylene glycol fractionation and sequential FPLC on hydroxylapatite, phenyl-Sepharose. Mono Q and Superose 12. The electrophoretically pure protem and purified &leaf PEPC were phosphorylated in vitro in a reconstituted system with PEPC-kinase isolated from illummated tobacco and maize leaves. These reciprocal phosphorylation experiments (i) indicate that Ser" of tobacco PEPC is the likely target residue, sttuated in the plant-invariant Cm/Asp-LyslArg-X-X-Ser phosphorylation motif near the N-terminus, and (ii) lend support to the recent hypothesis that C-leaf PEPC is subject to&gulatory phosphorylation in vivo.