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

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.