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 (PKC alpha, -betaI/II, and -gamma) isozyme activities are potentiated by 1,2- diacyl-sn-glycerol. This has been attributed to a ''cooperative'' interaction of the two activa tors with two discrete sites termed the low- and high-affinity phorbol este r 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., Yeage r, M. D., and Stubbs, C. D. (1999) Biochemistry 38, 3804-3815]. Here, we re port that the 1-O-alkyl ether diglyceride, 1-O-hexadecyl-2-acetyl-sn-glycer ol (HAG), like its 1,2-diacyl counterpart, 1-oleoyl-2-acetyl-sn-glycerol (O AG), also potentiated PKC alpha, -betaI/II, and -gamma activities induced b y the phorbol ester 4 beta -12-O-tetradecanoylphorbol-13-acetate (TPA). Sim ilar to GAG, HAG was found to bind to the low-affinity phorbol ester bindin g site and to enhance high-affinity phorbol ester binding, and to decrease the level of Ca2+ required for phorbol ester-induced activity, while being without effect on the Ca2+ dependence of membrane association. Thus, simila r to GAG, HAG may also potentiate phorbol ester-induced activity by interac ting with the low-affinity phorbol ester binding site, leading to a reduced level of Ca2+ 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 GAG-induced activ ity. 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.