Cysteine S-Nitrosylation Protects Protein-tyrosine Phosphatase 1B against Oxidation-induced Permanent Inactivation

Yi-Yun Chen,Hsing-Mao Chu,Kuan-Ting Pan,Chun-Hung Teng,Danny-Ling Wang,Andrew H.-J Wang,Kay-Hooi Khoo,Tzu-Ching Meng

doi:10.1074/jbc.m805287200

Abstract

Protein S-nitrosylation mediated by cellular nitric oxide (NO) plays a primary role in executing biological functions in cGMP-independent NO signaling. Although S-nitrosylation appears similar to Cys oxidation induced by reactive oxygen species, the molecular mechanism and biological consequence remain unclear. We investigated the structural process of S-nitrosylation of protein-tyrosine phosphatase 1B (PTP1B). We treated PTP1B with various NO donors, including S-nitrosothiol reagents and compound-releasing NO radicals, to produce site-specific Cys S-nitrosylation identified using advanced mass spectrometry (MS) techniques. Quantitative MS showed that the active site Cys-215 was the primary residue susceptible to S-nitrosylation. The crystal structure of NO donor-reacted PTP1B at 2.6 A resolution revealed that the S-NO state at Cys-215 had no discernible irreversibly oxidized forms, whereas other Cys residues remained in their free thiol states. We further demonstrated that S-nitrosylation of the Cys-215 residue protected PTP1B from subsequent H(2)O(2)-induced irreversible oxidation. Increasing the level of cellular NO by pretreating cells with an NO donor or by activating ectopically expressed NO synthase inhibited reactive oxygen species-induced irreversible oxidation of endogenous PTP1B. These findings suggest that S-nitrosylation might prevent PTPs from permanent inactivation caused by oxidative stress.

Highlights

Both NADPH oxidases and nitric oxide (NO) synthases (NOSs) can be activated in signaling response to a number of extracellular stimuli [3,4,5,6]
It has been proposed that various signaling modulators are targeted by reactive oxygen species (ROS) and reactive nitrogen species (RNS) [9, 10, 43], there has been limited direct in vivo evidence to delineate the underlying mechanism of free radicals-mediated signal transduction
Structural analysis confirmed the selective S-nitrosylation of Cys-215, whereas Cys-32 and Cys-92, both of which are surface-exposed residues, and other Cys residues situated in the interior space (Cys-121, -226, and -231) remained in their reduced form under the condition applied

Summary

Introduction

Both NADPH oxidases and NOSs can be activated in signaling response to a number of extracellular stimuli [3,4,5,6]. Studies have described how ROS is involved in regulating cell signaling through oxidation-dependent, reversible inactivation of PTPs in response to various physiological stimuli [16]. Recent studies have demonstrated that the same active site Cys residue is irreversibly oxidized to sulfinic (Cys-SO2H) or sulfonic (Cys-SO3H) acid states [17, 18], rendering PTPs permanently inactive under certain pathological conditions (e.g. when certain cancer cells produce high levels of ROS) [17]. Recent studies using indirect measurements and enzymatic activity assays suggest that a number of cellular PTPs, including PTP1B [20, 25, 26], PTEN [21], SHP1 [22], and SHP2 [22], may be inactivated through S-nitrosylation in response to stress stimulation. We investigated whether an increased level of cellular NO prior to exposure of cells with ROS could inhibit permanent oxidation and inactivation of endogenous PTP1B

Methods

Results

Conclusion