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.
Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, IA 50011, U.S.A.
R J
RJ
Mallis
J E
JE
Buss
J A
JA
Thomas
D016475:3T3 Cells; D000818:Animals; D003545:Cysteine / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D051379:Mice; D009603:Nitroso Compounds / Q000378:metabolism*; D015689:Oncogene Protein p21(ras) / Q000737:chemistry / Q000378:metabolism*; D010084:Oxidation-Reduction; D011233:Precipitin Tests; D013438:Sulfhydryl Compounds / Q000378:metabolism*
D009603:Nitroso Compounds; D013438:Sulfhydryl Compounds; D015689:Oncogene Protein p21(ras); D003545:Cysteine