Evidence is emerging that resveratrol (RV), a polyphenolic phytoaxelin present in dietary sources including red wine, may protect against atherosclerosis and cardiovascular disease by enhancing the integrity of the endothelium. In this study, the possibility that such beneficial effects of RV may arise from a modulation of protein kinase C (PKC)-mediated signaling was investigated by determining the effects of RV on the in vitro activities of PKC isozymes. It was found that the Ca(2+)-dependent activities of membrane-associated PKCalpha induced by either phorbol ester or diacylglycerol were potently inhibited by RV, each with an IC(50) of approximately 2 microM. The inhibitory effect of RV was also observed for conventional PKCbetaI, whereas the activities of novel PKC epsilon and atypical PKCzeta were each unaffected. The inhibition of PKCalpha activity was found to be competitive with respect to phorbol ester concentration but noncompetitive with respect to Ca(2+) and phosphatidylserine concentrations, suggesting that the RV may compete for phorbol ester-binding to the C1 domains. Supporting this, it was found that RV bound to a fusion peptide containing the C1A and C1B domains of PKCalpha. Similar to the effects of diacylglycerol and phorbol ester, the interaction of RV with the C1 domains induced the association of PKCalpha with membrane lipid vesicles, although this did not result in activation. Overall, the results suggest that the inhibitory effect of RV on PKC activity, and therefore on the associated signaling networks, may, in part, underlie the mechanism(s) by which this agent exerts its beneficial effects on endothelial and cardiovascular function. Furthermore, the effects of RV on these signaling networks are predicted to differ according to the cellular localization and the regulating PKC isozyme.

In this study, the role of interdomain interactions involving the C1 and C2 domains in the mechanism of activation of PKC was investigated. Using an in vitro assay containing only purified recombinant proteins and the phorbol ester, 4␤-12-O-tetradecanoylphorbol-13-acetate (TPA), but lacking lipids, it was found that PKC␣ bound specifically, and with high affinity, to a ␣C1A-C1B fusion protein of the same isozyme. The ␣C1A-C1B domain also potently activated the isozyme in a phorbol ester-and diacylglycerol-dependent manner. The level of this activity was comparable with that resulting from membrane association induced under maximally activating conditions. Furthermore, it was found that ␣C1A-C1B bound to a peptide containing the C2 domain of PKC␣. The ␣C1A-C1B domain also activated conventional PKC␤I, -␤II, and -␥ isoforms, but not novel PKC␦ or -⑀. PKC␦ and -⑀ were each activated by their own C1 domains, whereas PKC␣, -␤I, -␤II, or -␥ activities were unaffected by the C1 domain of PKC␦ and only slightly activated by that of PKC⑀. PKC activity was unaffected by its own C1 domain and those of the other PKC isozymes. Based on these findings, it is proposed that the activating conformational change in PKC␣ results from the dissociation of intra-molecular interactions between the ␣C1A-C1B domain and the C2 domain. Furthermore, it is shown that PKC␣ forms dimers via intermolecular interactions between the C1 and C2 domains of two neighboring molecules. These mechanisms may also apply for the activation of the other conventional and novel PKC isozymes.The 10 closely related isozymes that constitute the protein kinase C (PKC) 1 family of serine/threonine kinases each occupy critical nodes in the complex cellular signal transduction networks that regulate diverse cellular processes, including: secretion, proliferation, differentiation, apoptosis, permeability, migration, and hypertrophy (1-7). In common with many signaling proteins, the structure of PKC is modular, consisting of a C-terminal catalytic region containing the active site, and a regulatory region with conserved domains that mediate membrane association and activation. PKC isozymes are classified according to the structural and functional differences in these conserved domains (8, 9). In the case of the "conventional" PKC␣, -␤I/␤II, and -␥ isozymes, these include the activatorbinding C1 domains, and the Ca 2ϩ -binding C2 domain. The C1 domains consist of a tandem C1A and C1B arrangement, each of which can potentially bind the endogenous activator, diacylglycerol and exogenous activators including phorbol esters. The "novel" PKC␦, -⑀, -, -, and -isozymes, contain C2 domains that lack Ca 2ϩ binding ability, while retaining functional C1A and C1B domains. The "atypical" PKC, -, and -regulatory domains also lack a functional C2 domain and contain a single C1 domain that lacks the ability to bind activators, the function of which remains obscure. Each isozyme becomes catalytically competent by undergoing multiple serine/threonine and tyrosine phosphorylations that...

Evidence is provided for direct protein-protein interactions between protein kinase C (PKC) alpha, betaI, betaII, gamma, delta, epsilon, and zeta and members of the Rho family of small GTPases. Previous investigations, based on the immunoprecipitation approach, have provided evidence consistent with a direct interaction, but this remained to be proven. In the study presented here, an in vitro assay, consisting only of purified proteins and the requisite PKC activators and cofactors, was used to determine the effects of Rho GTPases on the activities of the different PKC isoforms. It was found that the activity of PKCalpha was potently enhanced by RhoA and Cdc42 and to a lesser extent by Rac1, whereas the effects on the activities of PKCbetaI, -betaII, -gamma, -delta, -epsilon, and -zeta were much reduced. These results indicate a direct interaction between PKCalpha and each of the Rho GTPases. However, the Rho GTPase concentration dependencies for the potentiating effects on PKCalpha activity differed for each Rho GTPase and were in the following order: RhoA > Cdc42 > Rac1. PKCalpha was activated in a phorbol ester- and Ca(2+)-dependent manner. This was reflected by a substantial decrease in the phorbol ester concentration requirements for activity in the presence of Ca(2+), which for each Rho GTPase was induced within a low nanomolar phorbol ester concentration range. The activity of PKCalpha also was found to be dependent on the nature of the GTP- or GDP-bound state of the Rho GTPases, suggesting that the interaction may be regulated by conformational changes in both PKCalpha and Rho GTPases. Such an interaction could result in significant cross-talk between the distinct pathways regulated by these two signaling elements.

PKC is also known to interact with both cytoskeletal and nuclear proteins; however, less is known concerning the mode of activation of this non-membrane form of PKC. By using the fluorescent phorbol ester, sapintoxin D (SAPD), PKC␣, alone, was found to possess both low and high affinity phorbol ester-binding sites, showing that interaction with these sites does not require association with the membrane. Importantly, a fusion protein containing the isolated C1A/C1B (C1) domain of PKC␣ also bound SAPD with low and high affinity, indicating that the sites may be confined to this domain rather than residing elsewhere on the enzyme molecule. Both high and low affinity interactions with native PKC␣ were enhanced by protamine sulfate, which activates the enzyme without requiring Ca 2؉ or membrane lipids. However, this "non-membrane" PKC activity was inhibited by the phorbol ester 4␤-12-O-tetradecanoylphorbol-13-acetate (TPA) and also by the fluorescent analog, SAPD, opposite to its effect on membrane-associated PKC␣. Bryostatin-1 and the soluble diacylglycerol, 1-oleoyl-2-acetylglycerol, both potent activators of membrane-associated PKC, also competed for both low and high affinity SAPD binding and inhibited protamine sulfate-induced activity. Furthermore, the inactive phorbol ester analog 4␣-TPA (4␣-12-O-tetradecanoylphorbol-13-acetate) also inhibited non-membrane-associated PKC. In keeping with these observations, although TPA could displace high affinity SAPD binding from both forms of the enzyme, 4␣-TPA was only effective at displacing high affinity SAPD binding from nonmembrane-associated PKC. 4␣-TPA also displaced SAPD from the isolated C1 domain. These results show that although high and low affinity phorbol ester-binding sites are found on non-membrane-associated PKC, the phorbol ester binding properties change significantly upon association with membranes. Protein kinase C (PKC)1 constitutes a group of isozymes that are central in cellular signaling pathways that regulate numerous cellular processes, including cell growth, differentiation, and metabolism (1). Each isoform can be classified into one of three major classes according to the cofactor and activator requirements. The "conventional" PKC␣, -␤I, -␤II, and -␥ isoforms are Ca 2ϩ -and anionic phospholipid-dependent, whereas the "novel" PKC␦, -⑀, -, and -and "atypical" PKC andisozymes retain a phospholipid dependence but lack a Ca 2ϩ requirement (2). In addition, the activities of all PKC isoforms, except atypical PKC, are potentiated by the lipid second messenger, diacylglycerol, derived from the receptor-G-protein and phospholipase-catalyzed hydrolysis of phosphatidylinositides and phosphatidylcholines (3) and also by the potent tumorpromoting phorbol esters (4).The Ca 2ϩ and phospholipid requirements for PKC activity differ according to the lysine and arginine content of the substrate (5). Thus, the PKC-catalyzed phosphorylation of the lysine-rich protein, histone H1, requires the presence of both Ca 2ϩ and phospholipid, whereas the phosphorylation of t...

Protein kinase Calpha (PKCalpha) 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 PKCalpha 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 (