EVIDENCE FOR DISCRETE DIACYLGLYCEROL AND PHORBOL ESTER ACTIVATOR SITES ON PROTEIN-KINASE-C - DIFFERENCES IN EFFECTS OF 1-ALKANOL INHIBITION, ACTIVATION BY PHOSPHATIDYLETHANOLAMINE AND CALCIUM CHELATION

Stimulation of protein kinase C (PKC) activity is achieved in vivo by diacylglycerol but can also be obtained with tumor-promoting phorbol e sters. Evidence is presented indicating that these two classes of acti vator may interact at different regions of the enzyme. The activity of a calcium-dependent PKC isoform (PKC-I) preparation was determined us ing 1,2-dioleoylglycerol (DOG) together with the phorbol ester 4 beta- 12-O-tetradecanoylphorbol-13-acetate (TPA). The resulting PKC activity was in excess of that attained with either activator alone, each bein g at a maximum concentration for activation. A similar result was obta ined with purified PKC-alpha and -epsilon isoforms, indicating that th e additive effect was not due to sites being on distinct enzyme molecu les. Support for two dissimilar activator sites came from the observat ion that the inactive phorbol ester 4 alpha-TPA competed for TPA but n ot for DOG in PKC activation. Other differences were observed between TPA and DOG-activated PKC. It was found that 1-butanol inhibited DOG-a ctivated PEG-I, while being without effect on stimulation by TPA. Also , the inclusion of phosphatidylethanolamine in the lipid vesicles led to a potentiation of PKC-I activity which was greater when activation was achieved by DOG compared to TPA. Further, the calcium- and DOG dep endent active conformational change of PKC was fully reversible upon c alcium chelation, while that stimulated by TPA was only partially reve rsible. These experiments taken together suggest that diacylglycerols and phorbol esters bind with different affinities and at different sit es on PKC, and induce distinct activated conformational forms of the e nzyme.