Protein kinase C
(PKC
) has been shown to contain two discrete activator sites with differingbinding 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 potentiatedlevel of activity [
Slater, S.
J., Ho, C., Kelly, M. B., Larkin, J. D., Taddeo, F. J., Yeager, M. D., andStubbs, C. D. (1996)
J. Biol. Chem. 271, 4627-4631]. In this study, the mechanism of this enhancementof activity was examined with respect to the Ca
2+ dependences of membrane association and accompanyingconformational changes that lead to activation. The association of PKC
with membranes containing12-
O-tetradecanoylphorbol 13-acetate (TPA) or 1,2-dioleoylglycerol (DAG), determined from tryptophanto dansyl-PE resonance energy transfer (RET) measurements, was found to occur at relatively low Ca
2+levels (
1
M). However, PKC
was found to be inactive even though membrane association was completeat these Ca
2+ levels and further titration of Ca
2+ to a concentration of ~100
M was required for activation.This increase in Ca
2+ concentration also led to a further increase in RET, which was due to a Ca
2+-induced activating conformational change, as verified by an accompanying increase in the PKC
tryptophanfluorescence anisotropy. Coaddition of DAG and TPA resulted in a reduction in the Ca
2+ levels requiredfor both the conformational change and enzyme activation. Also, it was found that incubation of theenzyme with TPA alone resulted in a time-dependent increase in the Ca
2+-independent PKC
activity,the rate and extent of which was further enhanced upon coaddition with DAG.
he results suggest thatthe enhanced level of activity induced by coaddition of DAG and TPA involves both Ca
2+-dependent andCa
2+-independent activating conformational changes which result in active conformers of PKC
distinctfrom those formed by interaction with either activator separately.