IDENTIFICATION AND CHARACTERIZATION OF PROTEIN-KINASE C-ZETA-IMMUNOREACTIVE PROTEINS

Two immunoreactive proteins (75 and 80 kDa) were detected in rat brain and rabbit aorta using a polyclonal peptide-directed antibody to the C terminus of the zeta isoenzyme of protein kinase C (PKC). The 75-kDa protein resembled authentic PKC zeta; it was recovered in cytosolic f ractions prepared in the presence or absence of Ca2+. The 80-kDa prote in, however, bound to the particulate fraction in a Ca2+-dependent and reversible manner, but phosphorylated a synthetic peptide derived fro m the autoinhibitory domain of PKC epsilon in a Ca2+-independent but p hospholipid- and diacylglycerol-dependent manner. Purification of the 80-kDa protein from rat brain, which separated two PKC zeta immunoreac tive proteins of 81.3 and 79.4 kDa, was achieved by EGTA extraction of the particulate fraction followed by chromatography on DEAE-Sephacel, phenyl-Sepharose, and hydroxylapatite. The 81.3-kDa protein copurifie d with PKC alpha, and the 79.4-kDa protein copurified with PKC beta an d PKC gamma. PKC alpha, -beta, and -gamma isoenzymes separated by hydr oxylapatite chromatography all cross-reacted with anti-PKC zeta. Using recombinant PKC isoenzymes, however, anti-PKC zeta was shown to recog nize rPKC alpha, rPKC beta(I), and rPKC beta(II), but not rPKC gamma. The regulatory properties of these isoenzymes differed from each other and depended on the particular substrate. PKC alpha was found to sepa rate into two peaks on hydroxylapatite chromatography. The first peak exhibited regulatory properties characteristic of PKC alpha, whereas t he second peak (PKC alpha') exhibited constitutive activity. PKC alpha ' appears to be derived from PKC alpha by irreversible oxidative modif ication.