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 fractions prepared in the presence or absence of Ca2+. The 80-kDa protein, however, bound to the particulate fraction in a Ca(2+)-dependent and reversible manner, but phosphorylated a synthetic peptide derived from the autoinhibitory domain of PKC epsilon in a Ca(2+)-independent but phospholipid- and diacylglycerol-dependent manner. Purification of the 80-kDa protein from rat brain, which separated two PKC zeta-immunoreactive 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 copurified with PKC alpha, and the 79.4-kDa protein copurified with PKC beta and PKC gamma. PKC alpha, -beta and -gamma isoenzymes separated by hydroxylapatite chromatography all cross-reacted with anti-PKC zeta. Using recombinant PKC isoenzymes, however, anti-PKC zeta was shown to recognize rPKC alpha, rPKC beta 1, 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 separate into two peaks on hydroxylapatite chromatography. The first peak exhibited regulatory properties characteristic of PKC alpha, whereas the second peak (PKC alpha') exhibited constitutive activity. PKC alpha' appears to be derived from PKC alpha by irreversible oxidative modification.
Department of Medical Biochemistry, Faculty of Medicine, University of Calgary, Alberta, Canada.
B G
BG
Allen
J E
JE
Andrea
M P
MP
Walsh
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D001921:Brain / Q000201:enzymology; D002853:Chromatography, Liquid; D008969:Molecular Sequence Data; D009131:Muscle, Smooth, Vascular / Q000201:enzymology; D010766:Phosphorylation; D011493:Protein Kinase C / Q000302:isolation & purification / Q000378:metabolism*; D011817:Rabbits; D051381:Rats; D000099221:Protein Kinase C zeta
D011493:Protein Kinase C; D000099221:Protein Kinase C zeta