STIMULATION OF THE ATPASE ACTIVITY OF RAT-BRAIN PROTEIN-KINASE-C BY PHOSPHO ACCEPTOR SUBSTRATES OF THE ENZYME

We recently reported that autophosphorylated rat brain protein kinase C (PKC) catalyzes a Ca2+- and phosphatidylserine- (PS-) dependent ATPase reaction. The Ca2+- and PS-dependent ATPase and histone kinase reactions of PKC each had a K(m app)(ATP) of 6-mu-M. Remarkably, the catalytic fragment of PKC lacked detectable ATPase activity. In this paper, we show that subsaturating concentrations of protein substrates accelerate the ATPase reaction catalyzed by PKC and that protein and peptide substrates of PKC induce ATPase catalysis by the catalytic fragment. At subsaturating concentrations, histone III-S and protamine sulfate each accelerated the ATPase activity of PKC in the presence of Ca2+ and PS by as much as 1.5-fold. At saturating concentrations, the protein substrates were inhibitory. Poly(L-lysine) failed to accelerate the ATPase activity, indicating that the acceleration observed with histone III-S and protamine sulfate was not simply a result of their gross physical properties. Furthermore, histone III-S induced the ATPase activity of the catalytic fragment of PKC, at both subsaturating and saturating histone concentrations. The induction of ATPase activity was also elicited by the peptide substrate Arg-Arg-Lys-Ala-Ser-Gly-Pro-Pro-Val, when the peptide was present at concentrations near its K(m app). The induction of the ATPase activity by the nonapeptide provides strong evidence that the binding of phospho acceptor substrates to the active site of PKC can stimulate ATP hydrolysis. Taken together, our results indicate that PKC-catalyzed protein phosphorylation is inefficient, since it is accompanied by P(i) production. Our results also provide support for a model of PKC catalysis in which the binding of the phospho acceptor substrate to the active site of PKC enhances the rate of phospho donor substrate hydrolysis.