Characterization of a Maize Ca2+-Dependent Protein Kinase Phosphorylating Phosphoenolpyruvate Carboxylase (original) (raw)
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A Ca2+-dependent protein kinase phosphorylates phosphoenolpyruvate carboxylase in maize
FEBS Letters, 1992
In C, plants the activity of phosphoenolpyruvate carboxylase (PEPC; EC 4.1. I .3 1) is regulated by phosphorylation/dephosphorylation which is mediated by light/dark signals. The study using protein kinasc inhibitors showed that the inhibition pattern of maize PEPC-protein kinasc (PEPC-PK) is similar to that of myosin light chain kinase, a Ca"-calmodulin-dependent PK. The kinasc activity was also inhibited by EGTA and the inhibition was relieved by Ca"'. These results suggest that PEPC-PK is Ca!*-dependent in contrast with previous observations by other research groups.
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...
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.
Archives of Biochemistry and Biophysics, 1997
vested from plants subjected to stem girdling or prolonged darkness. Furthermore, the kinase activity in Phosphoenolpyruvate carboxylase (PEPC) kinase nodules was controlled reversibly by illumination and was partially purified about 3000-fold from soybean extended darkness pretreatments of the parent plants, root nodules by a fast-protein liquid chromatography suggesting that photosynthate supply from the shoots protocol. This protein-serine kinase has an apparent is likely responsible for these striking changes in native molecular mass of about 30,000 as estimated by PEPC kinase activity observed in planta in the legume size-exclusion chromatography. Following electrophonodule. ᭧ 1997 Academic Press resis of this partially purified PEPC-kinase prepara-Key Words: PEPC kinase; phosphoenolpyruvate cartion in a denaturing gel containing dephospho maize boxylase (PEPC); protein kinase; protein phosphorylaleaf PEPC as substrate, the in situ renaturation and tion; root nodule; soybean (Glycine max). assay of protein kinase activity revealed two, PEPCdependent kinase polypeptides with molecular masses of about 32 and 37 kDa. The Ç32-kDa polypeptide was significantly more active than the Ç37-kDa catalytic Increasing evidence indicates that reversible protein subunit. The activity of this partially purified PEPC phosphorylation is an important mechanism for regukinase, and a less purified sample, was Ca 2/-insensilating plant phosphoenolpyruvate carboxylase (PEPC 3 ; tive. This protein kinase preparation was able to phos-EC 4.1.1.31) in vivo (1-4). It is well documented that phorylate purified PEPCs from soybean nodules, phosphorylation of a specific, plant-invariant serine maize leaves, and a sorghum recombinant C 4 PEPC. In residue near the N-terminus of the C 4 leaf enzyme recontrast, this PEPC kinase was unable to phosphorysults in an increased catalytic activity and a reduced late a phosphorylation-site mutant form of sorghum C 4 PEPC (S8Y), two other soybean nodule phosphopro-sensitivity to feedback inhibition by L-malate when asteins [nodulin-26 and nodulin-100 (sucrose synthase)], sayed at suboptimal but near-physiological conditions bovine serum albumin, and histone III-S. Following in (1, 2, 4). The phosphorylation state of the photosynvitro phosphorylation of purified dephospho soybean thetic PEPC isoforms in C 4 and Crassulacean acid menodule PEPC from stem-girdled plants by the partially tabolism (CAM) leaves is controlled largely by the acpurified nodule PEPC kinase, the former's activity and tivity of a Ca 2/-independent protein-Ser/Thr kinase sensitivity to L-malate inhibition increased and de-(1-3, 5, 6). In C 4 leaves, PEPC kinase and, thus, its creased, respectively. Notably, the Ca 2/-independent cytosolic target enzyme are activated by illumination PEPC kinase activity in nodules from illuminated and inactivated by darkness or inhibition of photosynplants was markedly greater than that in nodules har
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[
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.