Protein Kinase Cα Contains Two Activator Binding Sites That Bind Phorbol Esters and Diacylglycerols with Opposite Affinities

Abstract

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...

Lateral diffusion of peripheral membrane proteins on supported lipid bilayers is controlled by the additive frictional drags of (1) bound lipids and (2) protein domains penetrating into the bilayer hydrocarbon core

“…Note that parameters involving cube or square roots must be recalculated by taking the cube or square root of the term for each domain, then summing over all domains in the construct. References for number of bound lipids per domain: antibody binds one or two target lipids(10); each PH domain binds one PIP 3 lipid (11,12,44); cPLA2 C2 domain has no specific lipid binding site (22); PKCa C2 domain binds two PS lipids, or one PS and one PIP2 (19,21,45–47); C1A, C1B each bind one PS and one DAG (26,33,48–50). …”

Mechanisms Revealed Through General Anesthetic Photolabeling

“…“Conventional” and “novel” PKC isoforms contain four domains, termed C1–C4. The C1 domain consists of conserved subdomains, C1A and C1B, which each bind diacyglycerol and phorbol esters (PE) with varying affinities [67]. Diacylglycerol and PE sufficiently activate novel isoforms while conventional PKCs also require calcium, which binds the C2 domain.…”

Interactions of Protein Kinase C-α C1A and C1B Domains with Membranes: A Combined Computational and Experimental Study

“…In particular, many typical C1 domains of PKCs bind DAG or PMA preferentially, while others have comparable affinities to both activators. 13 There also exist atypical C1 domains that bind neither DAG nor PMA. 4 These differences in binding affinities and preferences are challenging to explain on the basis of the currently available, limited structural data alone.…”

“…32 Other results suggest that, in the case of PKCα, both domains bind to activators 28,29 yet with opposite affinities. 13,33 It is generally believed that the PKCα C1A domain has a higher affinity for DAG than the C1B domain, while the latter has a higher affinity for PMA. 14,33,34 Regarding mutations of equivalent or ionic residues, more pronounced impacts appeared in the C1A domain than in the C1B domain on PKCα membrane binding and activation.…”