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 phosphomositide pathway and a Ca2+-dependent step, which precedes the upregulation of the PEPC kinase (PEPCk). We have now further characterized the cascade component requiring Ca2+. A Ca2+-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 Cl-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 75 kDa 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 Ca2+-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 C4-PEPC phosphorylation in light-treated MCPs. These results support the view that this protein kinase is a good candidate to represent the Ca2+-activated component of the C4-PEPC phosphorylation cascade. (C) 2003 Elsevier Inc. All rights reserved.