PURIFICATION AND CHARACTERIZATION OF PROTEIN KINASE-C-EPSILON FROM RABBIT BRAIN

Protein kinase C-epsilon was chromatographically purified from rabbit brain to electrophoretic homogeneity. We identified the enzyme as the epsilon-species of novel-type protein kinase C (nPKC-epsilon), originally discovered and defined by cDNA cloning [Ohno, S., et al. (I 988) Cell 53, 731-741], on the basis of the following observations: (i) the enzyme reacts specifically with an antipeptidic antiserum to nPKC-epsilon but not with antisera to any of the other molecular species of PKC thus far known; (ii) it exhibits enzymatic behavior essentially identical to that of a recombinant nPKC-epsilon purified from transfected COS cells [Konno, Y., et al. (1989) J. Biochem. 106, 673-678] and distinct from that of conventional PKC (alpha, beta-I/II, and gamma) in its dependence on magnesium concentration and cofactors such as phospholipids, calcium, and phorbol ester; and (iii) it has an apparent molecular weight of 95.7K +/- 0.4K on SDS-PAGE, significantly greater than the other conventional and novel PKCs thus far identified. Notably, calcium exhibits a complex effect, both positive and negative, on the kinase activity of epsilon depending on the kind of substrate and the coexisting phospholipid, calling for a modification of the current notion that epsilon is a kinase unresponsive to calcium. The amount of epsilon-species in the brain was estimated to be comparable to that of each conventional species, indicating that epsilon stands as one of the major PKC family members in brain. Furthermore, the enzyme shows a broader substrate spectrum than conventional PKC when examined with endogenous substrates, implying that it may cover a wider or different range of physiological functions. As is the case with the conventional PKC species, nPKC-epsilon is also proteolytically converted to an active form with no cofactor requirement by the calcium-dependent proteases mu- and m-calpain. This first demonstration that an nPKC member is a calpain substrate predicts that the whole family of PKC, the conventional and the novel, may be under proteolytic regulation by calpain, and thus by calcium, in cells.