Competitive Binding of Protein Kinase Cα to Membranes and Rho GTPases

Previously, we have shown that protein kinase Cα (PKCα) forms a direct high-affinity, isozyme-specific and membrane lipid-independent interaction with Rho GTPases [Slater, S. J., Seiz, J. L., Stagliano, B. A., and Stubbs, C. D. (2001) Biochemistry 40, 4437−4445]. Since the cellular activation of PKCα involves an initial translocation from cytosolic to membrane compartments, the present study investigates the interdependence between the direct protein−protein interaction of PKCα with the Rho GTPase, Cdc42, and the protein−lipid interactions of PKCα with membranes. It was hypothesized that the interaction of PKCα with membrane-bound Cdc42 would contribute to the overall membrane-binding affinity of the kinase by providing an additional anchor. However, it was found that the incorporation of isoprenylated Cdc42 into membranes resulted in an apparent decrease in the membrane-binding affinity of PKCα, whereas the association of PKCβI, PKCδ, PKCε, and PKCζ was each unaffected. The presence of membrane-bound Cdc42 resulted in a rightward shift in both the PS- and Ca2+-concentration response curves for PKCα membrane association and for the ensuing activation, whereas the maximal levels of binding and activation attained at saturating PS and Ca2+ concentrations were in each case unaffected. Overall, these findings suggest that PKCα undergoes a isozyme-specific interaction with membrane-bound Cdc42 to form a PKCα−Cdc42 complex, which possesses a membrane-binding affinity that is reduced relative to that of the individual components due to competition between Cdc42 and PS/Ca2+ for binding to PKCα. Consistent with this, it was found that the interaction of PKCα with membrane-bound Cdc42 was accompanied by the physical dissociation of the PKCα−Cdc42 complex from membranes. Thus, the study provides a novel mechanism by which the membrane association and activation of PKCα and Cdc42 may be regulated by competing protein−protein and protein−lipid interactions.