Interaction of protein kinase C isozymes with Rho GTPases

Evidence is provided for direct protein-protein interactions between protein kinase C (PKC) alpha, betaI, beta II, gamma, delta, epsilon, and zeta and members of the Rho family of small GTPases. Previous investigations, based on the immunoprecipitation approach, have provided evidence consistent with a direct interaction, but this remained to be proven. In the study presented here, an in vitro assay, consisting only of purified proteins and the requisite PKC activators and cofactors, was used to determine the effects of Rho GTPases on the activities of the different PKC isoforms. It was found that the activity of PKC alpha was potently enhanced by RhoA and Cdc42 and to a lesser extent by Rad, whereas the effects on the activities of PKC betaI, -beta II, -gamma, -delta, -epsilon, and -zeta were much reduced. These results indicate a direct interaction between PKC alpha and each of the Rho GTPases. However, the Rho GTPase concentration dependencies for the potentiating effects on PKC alpha activity differed for each Rho GTPase and were in the following order: RhoA > Cdc42 > Rac1. PKC alpha was activated in a phorbol ester- and Ca2+-dependent manner. This was reflected by a substantial decrease in the phorbol ester concentration requirements for activity in the presence of Ca2+ which for each Rho GTPase was induced within a low nanomolar phorbol ester concentration range. The activity of PKC alpha also was found to be dependent on the nature of the GTP- or GDP-bound state of the Rho GTPases, suggesting that the interaction may be regulated by conformational changes in both PKC alpha and Rho GTPases. Such an interaction could result in significant cross-talk between the distinct pathways regulated by these two signaling elements.