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 cPKC alpha fully inactiva tes the isozyme, at a stoichiometry of similar to1 mol GSH/mol cPKC alpha. In this report we demonstrate that, in addition to cPKC alpha, six other PK C isozymes that are representative of the three subfamilies within the PKC family (cPKC beta (1), cPKC beta (2) and cPKC gamma, nPKC delta and nPKC ep silon and aPKC-zeta) are subject to inactivation by S-glutathiolation induc ed by the thiol-specific oxidant diamide, which induces disulfide bridge fo rmation. Among PKD and the seven PKC isozymes examined in this report only nPKC delta has been directly implicated as an antagonist of tumor promotion /progression, while several of the kinases have been implicated in the medi ation of tumor promotion/progression. We report that of the kinases examine d nPKC delta was the most resistant to inactivation by diamide-induced S-gl utathiolation. In the absence of GSH only nPKC delta activity exhibited a b iphasic response to diamide, with low diamide concentrations oxidatively en hancing nPKC delta activity and higher concentrations inactivating the isoz yme; the other seven kinases were subject to monophasic, concentration-depe ndent, oxidative inactivation by diamide to various extents. The results pr ovide evidence that at least some pro-oxidant environments may support the potent inactivation of nPKC epsilon and other PKC isozymes implicated in tu mor promotion/progression by the mechanisms of S-glutathiolation and, in so me cases, disulfide bridge formation among the isozyme thiols, without indu cing substantial nPKC delta inactivation. The results also show that neithe r the seven PKC isozymes examined nor PKD are inactivated by S-cysteinylati on under conditions that support potent inactivation by S-glutathiolation. This indicates that the protection that the tumor promotion/progression ant agonist GSH may afford against oxidative tumor promotion/progression mechan isms by S-thiolating and inactivating PKC isozymes and PKD cannot be afford ed by the metabolic GSH precursor cysteine. These observations support a ro le for PKC inactivation via S-glutathiolation in the mechanism of tumor pro motion/progression antagonism by GSH in pro-oxidant environments.