Inhibition of protein-tyrosine phosphatases by mild oxidative stresses is dependent on S-nitrosylation

Daniel M. Barrett, Stephen M. Black, Horia Todor, Rupert K. Schmidt-Ullrich, Kathryn S. Dawson, Ross B. Mikkelsen

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112 Scopus citations


Previous studies have shown that a Ca2+-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 H2O2. S-Nitrosylation of total cellular PTP and immunopurifiecl 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 2O2 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 H2O2 stimulated a transient increase in cytoplasmic free [Ca2+]. Radiation, H2O2, and the Ca2+ ionophore, ionomycin, also stimulated NOS activity, and this was associated with an enhanced S-nitrosylation of the active site Cys453 determined by isolation of S-nitrosylated wild type but not active site Cys453 → 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.

Original languageEnglish (US)
Pages (from-to)14453-14461
Number of pages9
JournalJournal of Biological Chemistry
Issue number15
StatePublished - Apr 15 2005

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology


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