SPECIFIC AND REVERSIBLE INACTIVATION OF PROTEIN-TYROSINE PHOSPHATASESBY HYDROGEN-PEROXIDE - EVIDENCE FOR A SULFENIC ACID INTERMEDIATE AND IMPLICATIONS FOR REDOX REGULATION

Protein tyrosine phosphatases (PTPs) catalyze the hydrolysis of phosph otyrosine from specific signal-transducing proteins. Although regulato ry mechanisms for protein kinases have been described, no general mech anism for controlling PTPs has been demonstrated. Numerous reports hav e shown that cellular redox status plays an important role in tyrosine phosphorylation-dependent signal transduction pathways. This study ex plores the proposal that PTPs may be regulated by reversible reduction /oxidation involving cellular oxidants such as hydrogen peroxide (H2O2 ) Recent reports indicated that H2O2 is transiently generated during g rowth factor stimulation and that H2O2 production is concomitant with relevant tyrosine phosphorylation. By use of recombinant enzymes, the effects of H2O2 on three PTPs [PTP1, LAR (leukocyte antigen-related), and VHR (vaccinia Hi-related)] and three distinct serine/threonine pro tein phosphatases (PPs: PP2C alpha, calcineurin, and lambda phosphatas e) were determined. Hydrogen peroxide had no apparent effect on PP act ivity. In contrast, PTPs were rapidly inactivated (k(inact) = 10-20 M- 1 s(-1)) with low micromolar concentrations of H2O2 but not with large alkyl hydroperoxides. PTP inactivation was fully reversible with glut athione and other thiols. Because of the slower rate of reduction, mod ification occurred even in the presence of physiological thiol concent rations. By utilization of a variety of biochemical techniques includi ng chemical modification, pH kinetic studies, and mutagenesis, the cat alytic cysteine thiolate of PTPs was determined to be the selective ta rget of oxidation by H2O2. By use of the electrophilic reagent 7-chlor o-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl), it was shown that a cystein e sulfenic acid intermediate (Cys-SOH) is formed after attack of the c atalytic thiolate on H2O2 A chemical mechanism for reversible inactiva tion involving a cysteine sulfenic acid intermediate is proposed.