Competitive binding of protein kinase Calpha to membranes and Rho GTPases.
Previously, we have shown that protein kinase C (PKC) 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 PKCalpha involves an initial translocation from cytosolic to membrane compartments, the present study investigates the interdependence between the direct protein-protein interaction of PKCalpha with the Rho GTPase, Cdc42, and the protein-lipid interactions of PKCalpha with membranes. It was hypothesized that the interaction of PKCalpha 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 PKCalpha, whereas the association of PKCbetaI, PKCdelta, PKCepsilon, and PKCzeta was each unaffected. The presence of membrane-bound Cdc42 resulted in a rightward shift in both the PS- and Ca2+-concentration response curves for PKCalpha 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 PKCalpha undergoes a isozyme-specific interaction with membrane-bound Cdc42 to form a PKCalpha-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 PKCalpha. Consistent with this, it was found that the interaction of PKCalpha with membrane-bound Cdc42 was accompanied by the physical dissociation of the PKCalpha-Cdc42 complex from membranes. Thus, the study provides a novel mechanism by which the membrane association and activation of PKCalpha and Cdc42 may be regulated by competing protein-protein and protein-lipid interactions.
Cook
Anthony C
AC
Department of Pathology and Cell Biology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
Ho
Cojen
C
Kershner
Jennifer L
JL
Malinowski
Steve A
SA
Moldveen
Heath
H
Stagliano
Brigid A
BA
Slater
Simon J
SJ
D000818:Animals; D001667:Binding, Competitive; D002118:Calcium / Q000494:pharmacology; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D011485:Protein Binding; D051571:Protein Kinase C-alpha / Q000235:genetics / Q000378:metabolism*; D018411:Spodoptera; D020349:Surface Plasmon Resonance; D020764:cdc42 GTP-Binding Protein / Q000378:metabolism; D020741:rho GTP-Binding Proteins / Q000378:metabolism*
D051571:Protein Kinase C-alpha; D020764:cdc42 GTP-Binding Protein; D020741:rho GTP-Binding Proteins; D002118:Calcium