INHIBITION OF MEMBRANE LIPID-INDEPENDENT PROTEIN-KINASE-C-ALPHA ACTIVITY BY PHORBOL ESTERS, DIACYLGLYCEROLS, AND BRYOSTATIN-1

The activity of membrane-associated protein kinase C (PKC) has previou sly been shown to be regulated by two discrete high and low affinity b inding 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 know n to interact with both cytoskeletal and nuclear proteins; however, le ss is known concerning the mode of activation of this non-membrane for m of PKC. By using the fluorescent phorbol ester, sapintoxin D (SAPD), PKC alpha, alone, was found to possess both low and high affinity pho rbol ester-binding sites, showing that interaction with these sites do es not require association with the membrane. Importantly, a fusion pr otein containing the isolated C1A/C1B (C1) domain of PKC alpha also bo und SAPD with low and high affinity, indicating that the sites may be confined to this domain rather than residing elsewhere on the enzyme m olecule. Both high and low affinity interactions with native PKC alpha were enhanced by protamine sulfate, which activates the enzyme withou t requiring Ca2+ or membrane lipids. However, this ''non-membrane'' PK C activity was inhibited by the phorbol ester 4 beta-12-O-tetradecanoy lphorbol-13-acetate (TPA) and also by the fluorescent analog, SAPD, op posite to its effect on membrane-associated PKC alpha. Bryostatin-1 an d the soluble diacylglycerol, 1-oleoyl-2-acetylglycerol, both potent a ctivators of membrane-associated PKC, also competed for both low and h igh affinity SAPD binding and inhibited protamine sulfate-induced acti vity. Furthermore, the inactive phorbol ester analog 4 alpha-TPA (4 al pha-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-associa ted PKC, the phorbol eater binding properties change significantly upo n association with membranes.