Journal
BIOCHEMICAL JOURNAL
Volume 355, Issue -, Pages 145-153Publisher
PORTLAND PRESS
DOI: 10.1042/bj3550145
Keywords
glutathione; MAP kinase; protein oxidation; S-nitroso cysteine
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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 (H2O2), 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 H2O2 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 san 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 an normally targeted for lipid-modification reactions.
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