Potent inactivation of representative members of each PKC isozyme subfamily and PKD via S-thiolation by the tumor-promotion/progression antagonist glutathione but not by its precursor cysteine.

We recently established that S-glutathiolation of cPKCalpha fully inactivates the isozyme, at a stoichiometry of approximately 1 mol GSH/mol cPKCalpha. In this report we demonstrate that, in addition to cPKCalpha, six other PKC isozymes that are representative of the three subfamilies within the PKC family (cPKCbeta1, cPKCbeta2 and cPKCgamma, nPKCdelta and nPKCepsilon and aPKC-zeta) are subject to inactivation by S-glutathiolation induced by the thiol-specific oxidant diamide, which induces disulfide bridge formation. Among PKD and the seven PKC isozymes examined in this report only nPKCdelta has been directly implicated as an antagonist of tumor promotion/progression, while several of the kinases have been implicated in the mediation of tumor promotion/progression. We report that of the kinases examined nPKCdelta was the most resistant to inactivation by diamide-induced S-glutathiolation. In the absence of GSH only nPKCdelta activity exhibited a biphasic response to diamide, with low diamide concentrations oxidatively enhancing nPKCdelta activity and higher concentrations inactivating the isozyme; the other seven kinases were subject to monophasic, concentration-dependent, oxidative inactivation by diamide to various extents. The results provide evidence that at least some pro-oxidant environments may support the potent inactivation of nPKCepsilon and other PKC isozymes implicated in tumor promotion/progression by the mechanisms of S-glutathiolation and, in some cases, disulfide bridge formation among the isozyme thiols, without inducing substantial nPKCdelta inactivation. The results also show that neither the seven PKC isozymes examined nor PKD are inactivated by S-cysteinylation under conditions that support potent inactivation by S-glutathiolation. This indicates that the protection that the tumor promotion/progression antagonist GSH may afford against oxidative tumor promotion/progression mechanisms by S-thiolating and inactivating PKC isozymes and PKD cannot be afforded by the metabolic GSH precursor cysteine. These observations support a role for PKC inactivation via S-glutathiolation in the mechanism of tumor promotion/progression antagonism by GSH in pro-oxidant environments.