CHARACTERIZATION OF A CA-2+- AND PHOSPHOLIPID-DEPENDENT ATPASE REACTION CATALYZED BY RAT-BRAIN PROTEIN KINASE-C

Protein kinase C (PKC) consists of a family of Ca2+- and phospholipid-dependent protein kinases that catalyze the transfer of the γ-phosphate of ATP to phosphoacceptor serine or threonine residues of protein and peptide substrates. In this report, we demonstrate that purified, autophosphorylated rat brain PKC catalyzes a Ca2+- and phospholipid-dependent ATPase reaction, that appears to represent the bond-breaking step of its phosphotransferase reaction. The histone kinase and ATPase activities of PKC each had a Kmapp of 6 µM for ATP, and their metal ion cofactor requirements were similar. The rate of the Ca2+- and phospholipid-dependent PKC-catalyzed ATPase reaction was approximately 5 times slower than the rate of histone phosphorylation, but the basal rates of the PKC-catalyzed ATPase and histone kinase activities differed by less than a factor of 2. The mechanism of the ATPase reaction could entail either direct hydrolysis of ATP by water or formation of a stable phosphoenzyme (PKC-P) followed by its hydrolysis (PKC + Pi). The latter mechanism appears unlikely since [γ-32P]ATP failed to label autophosphorylated PKC. Furthermore, the PKC preparation did not contain contaminating protein phosphatases, excluding the possibility that the ATPase activity represented dephosphorylation of contaminating PKC substrates. Therefore, our results suggest that water may effectively compete with protein substrates of PKC for the 7-phosphate of ATP. Using PKC inhibitors and activators, we found that the ATPase and protein kinase activities of PKC were regulated analogously, providing evidence that allosteric activation of PKC involves facilitation of the bond-breaking step of the phosphotransferase reaction. Use of the ATPase reaction in the analysis of PKC catalysis may shed further light on the active-site chemistry of PKC and on the allosteric regulation of its protein kinase activity. © 1990, American Chemical Society. All rights reserved.