title: Inhibition of protein-tyrosine phosphatases by mild oxidative stresses is dependent on S-nitrosylation.
abstract: Previous studies have shown that a Ca(2+)-dependent nitric-oxide synthase (NOS) is activated as part of a cellular response to low doses of ionizing radiation. Genetic and pharmacological inhibitor studies linked this NO signaling to the radiation-induced activation of ERK1/2. Herein, a mechanism for the radiation-induced activation of Tyr phosphorylation-dependent pathways (e.g. ERK1/2) involving the inhibition of protein-Tyr phosphatases (PTPs) by S-nitrosylation is tested. The basis for this mechanism resides in the redox-sensitive active site Cys in PTPs. These studies also examined oxidative stress induced by low concentrations of H(2)O(2). S-Nitrosylation of total cellular PTP and immunopurified SHP-1 and SHP-2 was detected as protection of PTP enzymatic activity from alkylation by N-ethylmaleimide and reversal by ascorbate. Both radiation and H(2)O(2) protected PTP activity from alkylation by a mechanism reversible by ascorbate and inhibited by NOS inhibitors or expression of a dominant negative mutant of NOS-1. Radiation and H(2)O(2) stimulated a transient increase in cytoplasmic free [Ca(2+)]. Radiation, H(2)O(2), and the Ca(2+) ionophore, ionomycin, also stimulated NOS activity, and this was associated with an enhanced S-nitrosylation of the active site Cys(453) determined by isolation of S-nitrosylated wild type but not active site Cys(453) --> Ser SHP-1 mutant by the "biotin-switch" method. Thus, one consequence of oxidative stimulation of NO generation is S-nitrosylation and inhibition of PTPs critical in cellular signal transduction pathways. These results support the conclusion that a mild oxidative signal is converted to a nitrosative one due to the better redox signaling properties of NO.
authors.affiliation: Department of Radiation Oncology, Virginia Commonwealth University, Richmond, Virginia 23298-0058, USA.
authors.forename: Daniel M
authors.initials: DM
authors.lastname: Barrett
authors.forename: Stephen M
authors.initials: SM
authors.lastname: Black
authors.forename: Horia
authors.initials: H
authors.lastname: Todor
authors.forename: Rupert K
authors.initials: RK
authors.lastname: Schmidt-Ullrich
authors.forename: Kathryn S
authors.initials: KS
authors.lastname: Dawson
authors.forename: Ross B
authors.initials: RB
authors.lastname: Mikkelsen
mesh_terms: D000818:Animals; D001205:Ascorbic Acid / Q000494:pharmacology; D001665:Binding Sites; D001710:Biotin / Q000737:chemistry; D016466:CHO Cells; D002118:Calcium / Q000737:chemistry / Q000378:metabolism; D045744:Cell Line, Tumor; D006224:Cricetinae; D003593:Cytoplasm / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004229:Dithiothreitol / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D005799:Genes, Dominant; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D015151:Immunoblotting; D047908:Intracellular Signaling Peptides and Proteins; D015759:Ionomycin / Q000494:pharmacology; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D009154:Mutation; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D009569:Nitric Oxide / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000378:metabolism; D009584:Nitrogen / Q000737:chemistry*; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D010100:Oxygen / Q000378:metabolism; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6; D017027:Protein Tyrosine Phosphatases / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000378:metabolism; D015398:Signal Transduction; D013997:Time Factors
chemical_list: D047908:Intracellular Signaling Peptides and Proteins; D009569:Nitric Oxide; D015759:Ionomycin; D001710:Biotin; D006861:Hydrogen Peroxide; D019001:Nitric Oxide Synthase; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; C516724:PTPN11 protein, human; C509249:PTPN6 protein, human; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6; D017027:Protein Tyrosine Phosphatases; D009584:Nitrogen; D005033:Ethylmaleimide; D001205:Ascorbic Acid; D010100:Oxygen; D002118:Calcium; D004229:Dithiothreitol; D019331:NG-Nitroarginine Methyl Ester