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Competitive Binding of Protein Kinase Cα to Membranes and Rho GTPases

2006; American Chemical Society; Volume: 45; Issue: 48 Linguagem: Inglês

10.1021/bi0612420

1943-295X

Anthony Cook, Cojen Ho, Jennifer L. Kershner, Steve A. Malinowski, Heath Moldveen, Brigid A. Stagliano, Simon J. Slater,

Ion channel regulation and function

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.