A Ca(2+)-dependent protein kinase with characteristics of protein kinase C in leaves and mesophyll cell protoplasts from Digitaria sanguinalis: possible involvement in the C(4)-phosphoenolpyruvate carboxylase phosphorylation cascade.
In mesophyll cells (MC) of Digitaria sanguinalis, the C(4)-phosphoenolpyruvate carboxylase (C(4)-PEPC) initiating the photosynthetic pathway is controlled by a complex light-dependent phosphorylation process. We showed previously that the transduction cascade involves the phosphoinositide pathway and a Ca(2+)-dependent step, which precedes the upregulation of the PEPC kinase (PEPCk). We have now further characterized the cascade component requiring Ca(2+). A Ca(2+)-dependent protein kinase that shows several characteristics of the conventional type of mammalian protein kinase C (PKC) was detected in protein extracts from mesophyll cell protoplasts (MCPs). It catalyzed the in vitro phosphorylation of the C1-peptide PKC substrate and was markedly inhibited by a PKC-specific pseudosubstrate domain. However, it was only modestly activated by the phospholipids phosphatidylserine and lysophosphatidylcholine, while choline, oleyl acetylglycerol, phosphatidylinositol, and the phorbol ester phorbol 12-myristate 13-acetate did not show any effect. Nevertheless, its activity was found to be associated with a polypeptide of 75kDa that was recognized by a PKC antibody raised against the C-terminus of rabbit PKCbeta II. In addition, this protein kinase was also inhibited by the Ca(2+)-dependent protein kinase (CDPK)/PKC inhibitors W7, H7, and staurosporine. Surprisingly, it was found to be phosphorylated in dark-adapted MCPs, albeit to a low extent, and this did not change during protoplast induction by light. W7, H7, and staurosporine were shown to markedly inhibit C(4)-PEPC phosphorylation in light-treated MCPs. These results support the view that this protein kinase is a good candidate to represent the Ca(2+)-activated component of the C(4)-PEPC phosphorylation cascade.
Osuna
Lidia
L
Laboratorio de Fisiologia Vegetal, Facultad de Biologia, Universidad de Sevilla, Avda., Reina Mercedes, Sevilla, Spain.
Coursol
Sylvie
S
Pierre
Jean-Noël
JN
Vidal
Jean
J
D000818:Animals; D001671:Biochemistry / Q000379:methods; D015153:Blotting, Western; D001921:Brain / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D031746:Digitaria / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D008027:Light; D010455:Peptides / Q000737:chemistry; D010730:Phosphoenolpyruvate Carboxylase / Q000378:metabolism*; D010766:Phosphorylation; D010936:Plant Extracts / Q000378:metabolism*; D018515:Plant Leaves / Q000378:metabolism*; D011493:Protein Kinase C / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D064546:Protein Kinase C beta; D017434:Protein Structure, Tertiary; D011523:Protoplasts / Q000378:metabolism; D011817:Rabbits; D051381:Rats; D015398:Signal Transduction; D015854:Up-Regulation
D004791:Enzyme Inhibitors; D010455:Peptides; D010936:Plant Extracts; D011493:Protein Kinase C; D064546:Protein Kinase C beta; D010730:Phosphoenolpyruvate Carboxylase; D002118:Calcium