Inhibition of membrane lipid-independent protein kinase Cα 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), PKC alpha, 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 alpha 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 alpha 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 4 beta-12-O-tetradecanoylphorbol-13-acetate (TPA) and also by the fluorescent analog, SAPD, opposite to its effect on membrane-associated PKC alpha. 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 alpha-TPA (4 alpha-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 alpha-TPA was only effective at displacing high affinity SAPD binding from nonmembrane-associated PHC. 4 alpha-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.