Protein kinase is an isozyme family, that plays key roles in cell growth and differentiation and in chemical carcinogenesis. Potent and selective activation of PKC is achieved by phorbol-ester tumor promoters, and dithiothreitol (DTT)- reversible oxidative modifications induced in PKC by oxidant tumor promoters profoundly alter its activity. The nature of the oxidative modifications has not been established. Protein S-thiolation, intramolecular disulfide bridge formation, and stable conversion to sulfenic acid are the major reversible oxidative modifications of Cys residues of cellular proteins. S-thiolation and intramolecular disulfide bridge formation are endogenous antioxidant mechanisms that protect reactive Cys residues of cellular proteins from permanent oxidative modifications. The objective of the proposed studies is to determine if these antioxidant mechanisms are operative in the regulation of PKC isozymes. Such mechanisms could serve as a defense against tumor-promoting oxidative insults. We recently reported that Cys-bearing synthetic peptide-substrate analogs oxidatively inactivate PKC lsozymes in a DTT-sensitive manner in association with covalent modification of the active site. We have also found that glutathione disulfide oxidatively inactivates PKC. The proposed studies will test two hypotheses. The first hypothesis is that PKC is subject to active-site S-thiolation by the inactivator peptides. The second hypothesis is that S-thiolation and/or intramolecular disulfide bridge formation are endogenous reversible mechanisms of regulation of PKC activity in mammalian cells that protect PKC isozymes from permanent oxidative modifications.
Specific Aims are: I) To define the mechanism of PKC isozyme inactivation by the Cysbearing peptide-substrate analogs; 2) To determine whether PKC isozymes are protected from permanent oxidative damage in cells exposed to oxidant tumor promoters by S- thiolation and/or intramolecular disulfide bridge formation; and 3) To determine if the respiratory burst induces oxidative modifications of PKC isozymes in human neutrophils that protect the isozymes against permanent oxidative damage and/or modify their function.
