Journal Article

PKC isozyme <i>S</i>-cysteinylation by cystine stimulates the pro-apoptotic isozyme PKCδ and inactivates the oncogenic isozyme PKCε

Feng Chu, Nancy E. Ward and Catherine A. O’Brian

in Carcinogenesis

Volume 24, issue 2, pages 317-325
Published in print February 2003 | ISSN: 0143-3334
Published online February 2003 | e-ISSN: 1460-2180 | DOI: http://dx.doi.org/10.1093/carcin/24.2.317
PKC isozyme S-cysteinylation by cystine stimulates the pro-apoptotic isozyme PKCδ and inactivates the oncogenic isozyme PKCε

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Protein kinase C (PKC) is a family of ten isozymes that play distinct and in some cases opposing roles in cell growth and survival. We recently reported that diamide, a diazene carbonyl derivative which oxidizes thiols to disulfides through addition/displacement reactions at the diazene bond, induces potent GSH-dependent inactivation of several PKC isozymes, including the oncogenic isozyme PKCε, via S-glutathiolation. PKCδ, a pro-apoptotic isozyme, was distinguished by its resistance to inactivation. In this report, we show that PKC-regulatory S-thiolation modifications produced by physiological disulfides elicit opposing effects on PKCδ and PKCε activity. We report that PKCδ is stimulated 2.0–2.5 fold by GSSG, (Cys–Gly)2 and cystine, under conditions where PKCγ and PKCε are fully inactivated by cystine, and PKCα activity is affected marginally or not at all by the disulfides. Focusing on cystine, we show that DTT quenches cystine-induced PKCδ stimulation and PKCγ and PKCε inactivation, indicative of oxidative regulation. By analyzing DTT-reversible isozyme radiolabeling by [35S]cystine, we demonstrate that PKCγ, PKCδ and PKCε are each [35S] S-cysteinylated in association with the concentration-dependent regulation of isozyme activity by cystine. The restricted reactivity of cystine, together with the effects of DTT and thioredoxin on cystine-induced PKC isozyme regulation reported here, indicate that the cystine-induced PKC-regulatory effects entail isozyme S-cysteinylation. We recently hypothesized that antagonism of tumor promotion/progression by small cellular thiols may involve PKC regulation via oxidant-induced S-thiolation reactions with PKC isozymes. The findings of cystine-induced PKC isozyme regulation by S-cysteinylation reported here offer correlative support to the hypothetical model. Thus, PKCδ, a potent antagonist of DMBA–TPA-induced tumor promotion/progression in mouse skin, is stimulated by S-cysteinylation, PKCε, an important mediator of the tumor promotion/progression response, is inactivated by S-cysteinylation, and PKCα, which is not influential in DMBA–TPA-induced tumor promotion/progression, is not regulated by cystine. Furthermore, PKCγ has oncogenic activity, and S-cysteinylation inactivated PKCγ and PKCε similarly. These findings provide evidence that S-cysteinyl acceptor-sites in PKC isozymes may offer attractive targets for development of novel cancer preventive agents.

Keywords: (Cys–Gly)2, Cys–Gly disulfide; DAG, sn-1,2-dioleoylglycerol; DTT, dithiothreitol; GSH, glutathione; GSSG, glutathione disulfide; PKC, protein kinase C; [Ser25]PKC(19–31), RFARKGALRQKNV

Journal Article.  7347 words.  Illustrated.

Subjects: Clinical Cytogenetics and Molecular Genetics

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