CHARACTERIZATION OF PHORBOL ESTER BINDING TO PROTEIN-KINASE-C ISOTYPES

A mixed micellar assay was used to study the in vitro binding of [H-3] phorbol-12,13-dibutyrate ([H-3]PDBu) to pure recombinant protein kinas e C (PKC)-alpha, -beta(1), -beta(2), -gamma, -delta, -epsilon, and -ze ta isotypes expressed in the baculovirus/insect cell system. Scatchard analysis revealed that all isotypes except PKC-zeta were able to spec ifically bind PDBu, with K-d values ranging from 1.6 to 18 nM in the p resence of calcium. In the absence of calcium PKC-alpha, -beta(1), -be ta(2), and -delta were observed to have a 2-3-fold drop in affinity, a lthough B-max values remained unchanged, at a stoichiometry of 1.4-2.8 mol of PDBu/mol of enzyme. Competition with specific [3H]PDBu binding was assessed for the phorbol esters PDBu, 12-tetradecanoylphorbol-13- O-acetate, 12-deoxyphorbol-13-O-phenylacetate, 12-deoxyphorbol-13-O-ph enylacetate-20-acetate, thymeleatoxin, resiniferatoxin, and sapintoxin A. Resiniferatoxin and 12-deoxyphorbol-13-O-phenylacetate-20-acetate were found to compete effectively only with PDBu bound to the PKC-beta (1) and -beta(2) isotypes and were the least potent of the phorbol est ers tested (IC50, > 5 mu M). The phorbol esters sapintoxin A, 12-deoxy phorbol-13-O-phenylacetate, 12-tetradecanoylphorbol-13-O-acetate, and PDBu (in order of potency) competed for binding to all isotypes (IC50 values ranging from 2 to 70 nM), with unchanged or slightly decreased potency when calcium was replaced by ethylene glycol bis(beta-aminoeth yl ether)-N,N,N',N'-tetraacetic acid. Thymeleatoxin, which was similar in other respects to these potent phorbol esters, was found to be les s able to compete with binding to PKC-alpha and -epsilon isotypes (IC5 0, 3-5 mu M). It appears that, whereas the binding of phorbol esters t o PKC depends primarily on the C20 substituent, other areas of the mol ecule have an influence on this interaction and the PKC isotypes thems elves display heterogeneity in their phorbol ester-binding characteris tics.