Superoxide-induced stimulation of protein kinase C via thiol modification and modulation of zinc content.
We investigated the effects of mild oxidation on protein kinase C (PKC) using the xanthine/xanthine oxidase system of generating superoxide. Exposure of various PKC preparations to superoxide stimulated the autonomous activity of PKC. Similarly, thiol oxidation increased autonomous PKC activity, consistent with the notion that superoxide stimulates PKC via thiol oxidation. The superoxide-induced stimulation of PKC activity was partially reversed by reducing agents, suggesting that disulfide bond formation contributed to the oxidative stimulation of PKC. In addition, superoxide increased the autonomous activity of the alpha, beta(II), epsilon, and zeta PKC isoforms, all of which contain at least one cysteine-rich region. Taken together, our observations suggested that superoxide interacts with PKC at the cysteine-rich region, zinc finger motif of the enzyme. Therefore, we examined the effects of superoxide on this region by testing the hypothesis that superoxide stimulates PKC by promoting the release of zinc from PKC. We found that a zinc chelator stimulated the autonomous activity of PKC and that superoxide induced zinc release from an PKC-enriched enzyme preparation. In addition, oxidized PKC contained significantly less zinc than reduced PKC. Finally, we have isolated a persistent, autonomously active PKC by DEAE-cellulose column chromatography from hippocampal slices incubated with superoxide. Taken together, these data suggest that superoxide stimulates autonomous PKC activity via thiol oxidation and release of zinc from cysteine-rich region of PKC.
Knapp
L T
LT
Department of Neuroscience and the Center for the Neural Basis of Cognition, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, USA.
Klann
E
E
D000818:Animals; D001921:Brain / Q000201:enzymology; D002614:Chelating Agents / Q000494:pharmacology; D004220:Disulfides / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D006624:Hippocampus / Q000201:enzymology; D066298:In Vitro Techniques; D007527:Isoenzymes / Q000187:drug effects / Q000378:metabolism; D008297:Male; D010084:Oxidation-Reduction; D011493:Protein Kinase C / Q000187:drug effects* / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D019163:Reducing Agents / Q000494:pharmacology; D013347:Subcellular Fractions / Q000201:enzymology; D013438:Sulfhydryl Compounds / Q000378:metabolism; D013481:Superoxides / Q000494:pharmacology*; D015032:Zinc / Q000378:metabolism*
D002614:Chelating Agents; D004220:Disulfides; D007527:Isoenzymes; D019163:Reducing Agents; D013438:Sulfhydryl Compounds; D013481:Superoxides; D011493:Protein Kinase C; D015032:Zinc