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.
Highlights
Phosphotyrosine phosphatases (PTPs)1 compose a superfamily of phosphatases that hydrolyze phospho-Tyr residues in proteins critically involved in several cell-signaling pathways [1,2,3]
We showed that radiation-induced activation of the ERK1/2 signaling pathway in CHO cells as revealed by measurements of enzyme activity and Tyr phosphorylation of ERK1/2 could be blocked by nitric-oxide synthase (NOS) inhibitors such as L-NAME or expression of a dominant negative mutant of NOS-1
We proposed that one mechanism for this activation might be inhibition of a counteracting PTP acting at one step of the ERK1/2 activation pathway by S-nitrosylation of the PTP active site Cys
Summary
Phosphotyrosine phosphatases (PTPs) compose a superfamily of phosphatases that hydrolyze phospho-Tyr residues in proteins critically involved in several cell-signaling pathways [1,2,3] Their active sites are characterized by the consensus sequence (I/V)HCXAGXXR(S/T). Several investigators have provided evidence suggesting that ligand-stimulated generation of ROS activates growth factor receptor Tyr kinase-dependent signal transduction pathways by inhibiting counteracting PTPs (1, 2, 6 –12). Key evidence in these studies has included the use of fluorescent dyes to qualitatively measure the ROS generated. Using a combination of chemical inhibitors and genetic manipulation of NOS-1 activity, we were able to show that the radiation-induced transient activation of ERK1/2 signaling in these cells was dependent on NOS activity and RNS generation
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