Based on marked differences in the enzymatic properties of diacylglycerols compared with phorbol esteractivated protein kinase C (PKC), we recently proposed that activation induced by these compounds may not be equivalent (Slater, S. J., Kelly, M. B., Taddeo, F. J., Rubin, E., and Stubbs, C. D. (1994) J. Biol. Chem. 269, 17160 -17165). In the present study, direct evidence is provided showing that phorbol esters and diacylglycerols bind simultaneously to PKC␣. Using a novel binding assay employing the fluorescent phorbol ester, sapintoxin-D (SAPD), evidence for two sites of high and low affinity was obtained. Thus, both binding and activation doseresponse curves for SAPD were double sigmoidal, which was also observed for dose-dependent activation by the commonly used phorbol ester, 4-12-O-tetradecanoylphorbol-13-acetate (TPA). TPA removed high affinity SAPD binding and also competed for the low affinity site. By contrast with TPA, low affinity binding of SAPD was inhibited by sn-1,2-dioleoylglycerol (DAG), while binding to the high affinity site was markedly enhanced. Again contrasting with both TPA and DAG, the potent PKC activator, bryostatin-I (B-I), inhibited SAPD binding to its high affinity site, while low affinity binding was unaffected. Based on these findings, a model for PKC activation is proposed in which binding of one activator to the low affinity site allosterically promotes binding of a second activator to the high affinity site, resulting in an enhanced level of activity. Overall, the results provide direct evidence that PKC␣ contains two distinct binding sites, with affinities that differ for each activator in the order: DAG > phorbol ester > B-I and B-I > phorbol ester > DAG, respectively.
Protein kinase C (PKC)1 comprises a family of isozymes that are pivotal in intracellular signal transduction (1-4). Activation of PKC requires association with the membrane and is induced by a number of activators and cofactors, the requirements for which differ for each isoform (2). Thus, with the exception of the so called atypical PKC isoforms, the activity of membrane-associated PKC is potentiated by the second messenger, diacylglycerol, a product of hormone receptor-phospholipase C-catalyzed phospholipid hydrolysis.In addition to the natural activators, including diacylglycerol, the enzyme is activated with high specificity by the tumorpromoting phorbol esters (5). For this reason, phorbol esters are often used in the study of the mechanism of PKC activation, based on the assumption that they compete directly with diacylglycerols for a common binding site on the enzyme (6, 7). Also it is commonplace in studies of the regulation of cellular processes to infer PKC involvement from a response elicited by phorbol esters (5). However, a number of studies have provided evidence indicating that the activated conformational forms of PKC induced by diacylglycerol compared with phorbol esters may not be equivalent based on observations of marked differences in the biological (8, 9) and enzymatic properties (10 -1...