Effects of ethanol on protein kinase C activity induced by filamentous actin.

Protein kinase C (PKC) can be activated by interaction with filamentous actin (F-actin) in the absence of membrane lipids (S.J. Slater, S.K. Milano, B.A. Stagliano, K.J. Gergich, J.P. Curry, F.J. Taddeo and C.D. Stubbs, Biochemistry 39 (2000) 271-280). Here, the effects of ethanol on the F-actin-induced activities of a panel of PKC isoforms consisting of 'conventional' (cPKC) alpha, betaI, gamma, 'novel' (nPKC) delta, epsilon and 'atypical' (aPKC) zeta were investigated using purified PKC and F-actin. Ethanol was found to inhibit the Ca2+- and phorbol ester-dependent activities of cPKCalpha and betaI, and the Ca2+- and phorbol ester-independent activity of cPKCgamma, whereas the activities of nPKCdelta, epsilon and aPKCzeta were unaffected. Although the activities of cPKCalpha and betaI induced by saturating levels of phorbol ester were inhibited by ethanol, the binding of these isozymes to F-actin was unaffected within the same phorbol ester concentration range. Conversely, within submaximal levels of phorbol ester, cPKCalpha and betaI activities were unaffected by ethanol whereas binding to F-actin was inhibited. The potency of the inhibition of F-actin-induced cPKCbetaI activity increased with n-alkanol chain length up to n-hexanol, after which it declined. The results indicate that PKC activities associated with F-actin, and therefore cellular processes involving the actin cytoskeleton, are potential targets for ethanol action. The effects of ethanol on these processes may differ according to the particular regulating PKC isoform, its intracellular localization and the presence of activators and cofactors.

Low- and high-affinity phorbol ester and diglyceride interactions with protein kinase C: 1-O-alkyl-2-acyl-sn-glycerol enhances phorbol ester- and diacylglycerol-induced activity but alone does not induce activity.

Phorbol ester-induced conventional protein kinase C (PKCalpha, -betaIota/IotaIota, and -gamma) isozyme activities are potentiated by 1,2-diacyl-sn-glycerol. This has been attributed to a "cooperative" interaction of the two activators with two discrete sites termed the low- and high-affinity phorbol ester binding sites, respectively [Slater, S. J., Milano, S. K., Stagliano, B. A., Gergich, K. J., Ho, C., Mazurek, A., Taddeo, F. J., Kelly, M. B., Yeager, M. D., and Stubbs, C. D. (1999) Biochemistry 38, 3804-3815]. Here, we report that the 1-O-alkyl ether diglyceride, 1-O-hexadecyl-2-acetyl-sn-glycerol (HAG), like its 1,2-diacyl counterpart, 1-oleoyl-2-acetyl-sn-glycerol (OAG), also potentiated PKCalpha, -betaI/II, and -gamma activities induced by the phorbol ester 4beta-12-O-tetradecanoylphorbol-13-acetate (TPA). Similar to OAG, HAG was found to bind to the low-affinity phorbol ester binding site and to enhance high-affinity phorbol ester binding, and to decrease the level of Ca(2+) required for phorbol ester-induced activity, while being without effect on the Ca(2+) dependence of membrane association. Thus, similar to OAG, HAG may also potentiate phorbol ester-induced activity by interacting with the low-affinity phorbol ester binding site, leading to a reduced level of Ca(2+) required for the activating conformational change. However, HAG was found not to behave like a 1,2-diacyl-sn-glycerol in that alone it did not induce PKC activity, and also in that it enhanced OAG-induced activity. The results reveal HAG to be a member of a new class of "nonactivating" compounds that modulate PKC activity by interacting with the low-affinity phorbol ester binding site.

Interaction of protein kinase C with filamentous actin: isozyme specificity resulting from divergent phorbol ester and calcium dependencies.

The mechanism of activation of protein kinase C isoforms by filamentous actin (F-actin) was investigated with respect to isozyme specificity and phorbol ester and Ca(2+) dependencies. It was found that the "conventional" (cPKC), alpha, betaI, betaII, and gamma, "novel" (nPKC) delta and epsilon, and "atypical" (aPKC) zeta isoforms were each activated by F-actin with varying potencies. The level of activity along with the affinity for binding to F-actin was further potentiated by the phorbol ester 4beta-12-O-tetradecanoylphorbol 13-acetate (TPA), the potency of which again varied for each isoform. By contrast to the other cPKC isoforms, the level of cPKC-gamma activity was unaffected by TPA, as was also the case for aPKC-zeta. It was found that whereas in the absence of F-actin the soluble form of cPKC-betaI contained two phorbol ester binding sites of low and high affinity, respectively, as previously reported for cPKC-alpha [Slater et al. (1998) J. Biol. Chem. 273, 23160-23168], the F-actin-bound form of the isozyme contained only a single site of relatively low affinity. The level of TPA required to induce cPKC-alpha, -betaI, and -betaII activity and the binding of these isozymes to F-actin was reduced in the presence of Ca(2+). By contrast, the activity of cPKC-gamma was unaffected by Ca(2+), as were the activities of nPKC-delta and -epsilon and aPKC-zeta, as expected. Thus, the interaction with F-actin appears to be a general property of each of the seven PKC isozymes tested. However, isoform specificity may, in part, be directed by differences in the phorbol ester and Ca(2+) dependences, which, with the notable exception of cPKC-gamma, appear to resemble those observed for the activation of each isoform by membrane association. The observation that cPKC isoforms may translocate to F-actin as well as the membrane as a response to an elevation of Ca(2+) levels may allow for the functional coupling of fluctuations of intracellular Ca(2+) levels through cPKC to F-actin cytoskeleton-mediated processes.

Synergistic activation of protein kinase Calpha, -betaI, and -gamma isoforms induced by diacylglycerol and phorbol ester: roles of membrane association and activating conformational changes.

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 (

Inhibition of membrane lipid-independent protein kinase Calpha activity by phorbol esters, diacylglycerols, and bryostatin-1.

The activity of membrane-associated protein kinase C (PKC) has previously been shown to be regulated by two discrete high and low affinity binding regions for diacylglycerols and phorbol esters (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). 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), PKCalpha, 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 PKCalpha 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 PKCalpha were enhanced by protamine sulfate, which activates the enzyme without requiring Ca2+ or membrane lipids. However, this "non-membrane" PKC activity was inhibited by the phorbol ester 4beta-12-O-tetradecanoylphorbol-13-acetate (TPA) and also by the fluorescent analog, SAPD, opposite to its effect on membrane-associated PKCalpha. 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 4alpha-TPA (4alpha-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, 4alpha-TPA was only effective at displacing high affinity SAPD binding from non-membrane-associated PKC. 4alpha-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.