Synergistic Activation of Protein Kinase Cα, -βI, and -γ Isoforms Induced by Diacylglycerol and Phorbol Ester:  Roles of Membrane Association and Activating Conformational Changes

Protein kinase Cα (PKCα) has been shown to contain two discrete activator sites with differing binding affinities for phorbol esters and diacylglycerols. The interaction of diacylglycerol with a low-affinity phorbol ester binding site leads to enhanced high-affinity phorbol ester binding and to a potentiated level of activity [Slater, S. J., Ho, C., Kelly, M. B., Larkin, J. D., Taddeo, F. J., Yeager, M. D., and Stubbs, C. D. (1996) J. Biol. Chem. 271, 4627−4631]. In this study, the mechanism of this enhancement of activity was examined with respect to the Ca2+ dependences of membrane association and accompanying conformational changes that lead to activation. The association of PKCα with membranes containing 12-O-tetradecanoylphorbol 13-acetate (TPA) or 1,2-dioleoylglycerol (DAG), determined from tryptophan to dansyl-PE resonance energy transfer (RET) measurements, was found to occur at relatively low Ca2+ levels (≤1 μM). However, PKCα was found to be inactive even though membrane association was complete at these Ca2+ levels and further titration of Ca2+ to a concentration of ∼100 μM was required for activation. This increase in Ca2+ concentration also led to a further increase in RET, which was due to a Ca2+-induced activating conformational change, as verified by an accompanying increase in the PKCα tryptophan fluorescence anisotropy. Coaddition of DAG and TPA resulted in a reduction in the Ca2+ levels required for both the conformational change and enzyme activation. Also, it was found that incubation of the enzyme with TPA alone resulted in a time-dependent increase in the Ca2+-independent PKCα activity, the rate and extent of which was further enhanced upon coaddition with DAG. Τhe results suggest that the enhanced level of activity induced by coaddition of DAG and TPA involves both Ca2+-dependent and Ca2+-independent activating conformational changes which result in active conformers of PKCα distinct from those formed by interaction with either activator separately.