Abstract
Based on marked differences in the enzymatic properties of diacylglycerols compared with phorbol ester-activated 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 alpha. 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 dose-response curves for SAPD were double sigmoidal, which was also observed for dose-dependent activation by the commonly used phorbol ester, 4beta-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 PKCalpha 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.
Highlights
Protein kinase C (PKC)1 comprises a family of isozymes that are pivotal in intracellular signal transduction [1,2,3,4]
In order to determine activator binding to PKC␣, advantage was taken of the fluorescence properties of the 2-(N-methylamino)benzoyl fluorophore of SAPD, which has an excitation band centered at 362 nm that overlaps with the emission band of 333 nm measured for the PKC␣ tryptophans (Fig. 1A)
Addition of SAPD, at a concentration sufficient for occupation of both high and low affinity phorbol ester binding sites, in the presence of Ca2ϩ resulted in resonance energy transfer (RET), observed as a marked decrease in tryptophan fluorescence intensity and corresponding increase in the SAPD emission maxima (425 nm)
Summary
Protein kinase C (PKC) comprises a family of isozymes that are pivotal in intracellular signal transduction [1,2,3,4]. We recently showed that the level of activation induced by saturating concentrations of both diacylglycerol and phorbol ester together, in the same assay, was greater than that achievable by saturating concentrations of either activator in isolation [15] This observation is not consistent with a model where activators compete for a single activator binding site or for two equivalent sites. While a single Cys subdomain has been shown to be sufficient for high affinity phorbol ester binding using peptide fragments [21], both Cys-1 and Cys-2 subdomains may be capable of binding phorbol esters [17, 21, 25] This is consistent with the existence of discrete diacylglycerol and phorbol ester binding sites as proposed in our recent study [15]. The data provide direct evidence that diacylglycerols, phorbol esters, and B-I bind to two discrete sites on PKC␣
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