Oxidative modification of H-ras: S-thiolation and S-nitrosylation of reactive cysteines

The reactive cysteines in H-ras are subject to oxidative modifications that potentially alter the cellular function of this protein. In this study, purified H-ras was modified by thiol oxidants such as hydrogen peroxide (H 2 O 2 ), S -nitrosoglutathione, diamide, glutathione disulphide (GSSG) and cystamine, producing as many as four charge-isomeric forms of the protein. These results suggest that all four reactive cysteines of H-ras are potential sites of regulatory modification reactions. S -nitrosylated and S -glutathiolated forms of H-ras were identified by protocols that depend on separation of alkylated proteins on electrofocusing gels. S -nitrosoglutathione could S -nitrosylate H-ras on four cysteine residues, while reduced glutathione (GSH) and H 2 O 2 mediate S -glutathiolation on at least one cysteine of H-ras. Either GSSG or diamide S -glutathiolated at least two cysteine residues of purified H-ras. Iodoacetic acid reacts with three cysteine residues. In intact NIH-3T3 cells, wild-type H-ras was S -glutathiolated by diamide. Similarly, cells expressing a C118S mutant or a C181S/C184S double mutant of H-ras were S -glutathiolated by diamide. These results suggest that H-ras can be S -glutathiolated on multiple thiols in vivo and that at least one of these thiols is normally lipid-modified. In cells treated with S -nitrosocysteine, evidence for both S -nitrosylated and S -glutathiolated H-ras was obtained and S -nitrosylation was the predominant modification. These results show that oxidative modification of H-ras can be extensive in vivo , that both S -nitrosylated and S -glutathiolated forms may be important, and that oxidation may occur on reactive cysteines that are normally targeted for lipid-modification reactions.