title: Rapid and irreversible inactivation of protein tyrosine phosphatases PTP1B, CD45, and LAR by peroxynitrite.
abstract: Protein tyrosine phosphatases (PTPs) contain an essential thiol in the active site which may be susceptible to attack by nitric oxide-derived biological oxidants. We assessed the effects of peroxynitrite, nitric oxide, and S-nitrosoglutathione on the activity of three human tyrosine phosphatases in vitro. The receptor-like T-cell tyrosine phosphatase (CD45), the non-receptor-like tyrosine phosphatase PTP1B, and leukocyte-antigen-related (LAR) phosphatase were all irreversibly inactivated by peroxynitrite in less than 1 s with IC(50) values of </=0.9 microM. PTP inactivation was also seen with equivalent concentrations of peroxynitrite generated by SIN-1, indicating that bolus peroxynitrite and cogeneration of superoxide and nitric oxide were equipotent. Rate constants for peroxynitrite-mediated PTP inactivation were determined by competition with cysteine and were among the fastest rates yet seen for reaction of peroxynitrite with any biological molecules. The bimolecular reaction rates for CD45, LAR, and PTP1B were 2.0 x 10(8), 2.3 x 10(7), and 2.2 x 10(7) M(-1) s(-1), respectively. Inactivation by peroxynitrite was essentially irreversible as incubation with dithiothreitol (DTT) restored less than 10% of the original phosphatase activity. Prolonged treatment with 0.4 mM DETA NONOate, which generated a steady-state concentration of 2 microM nitric oxide, was only slightly inhibitory. S-Nitrosoglutathione (1.0 mM) inhibited PTPs by approximately 50% after 30 min and the inhibition was completely reversed by DTT. Nitrotyrosine immunoblots of peroxynitrite-treated PTP1B revealed that peroxynitrite completely inactivated PTP1B prior to the appearance of protein tyrosine nitration. Peroxynitrite anion is structurally similar to phosphate anion both in terms of molecular diameter and charge. Thus, the extreme vulnerability of these PTPs to peroxynitrite-mediated inactivation is consistent with attraction of peroxynitrite anion to the active site and subsequent oxidation of the essential thiolate. These findings suggest that any PTP possessing the CXXXXXR active-site sequence could potentially be inactivated by peroxynitrite in vivo resulting in a net increase in tyrosine phosphorylation and profound effects on phosphotyrosine-dependent signaling cascades.
authors.affiliation: Department of Anesthesiology and Reanimatology, Fukui Medical University, Fukui, Japan.
authors.forename: K
authors.initials: K
authors.lastname: Takakura
authors.forename: J S
authors.initials: JS
authors.lastname: Beckman
authors.forename: L A
authors.initials: LA
authors.lastname: MacMillan-Crow
authors.forename: J P
authors.initials: JP
authors.lastname: Crow
mesh_terms: D000814:Aniline Compounds / Q000494:pharmacology; D000838:Anions / Q000737:chemistry; D004229:Dithiothreitol / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005978:Glutathione / Q000031:analogs & derivatives / Q000494:pharmacology; D006801:Humans; D017493:Leukocyte Common Antigens / Q000737:chemistry / Q000187:drug effects; D008958:Models, Molecular; D008981:Molsidomine / Q000031:analogs & derivatives / Q000494:pharmacology; D009566:Nitrates / Q000737:chemistry / Q000494:pharmacology*; D009569:Nitric Oxide / Q000494:pharmacology; D009603:Nitroso Compounds / Q000494:pharmacology; D010755:Organophosphates / Q000737:chemistry; D009943:Organophosphorus Compounds / Q000494:pharmacology; D017027:Protein Tyrosine Phosphatases / Q000037:antagonists & inhibitors* / Q000737:chemistry; D054630:Receptor-Like Protein Tyrosine Phosphatases, Class 4; D011956:Receptors, Cell Surface; D026422:S-Nitrosoglutathione; D013482:Superoxide Dismutase / Q000494:pharmacology; D014443:Tyrosine / Q000737:chemistry / Q000187:drug effects
chemical_list: D000814:Aniline Compounds; D000838:Anions; D004791:Enzyme Inhibitors; D009566:Nitrates; D009603:Nitroso Compounds; D010755:Organophosphates; D009943:Organophosphorus Compounds; D011956:Receptors, Cell Surface; C094210:2,2'-(hydroxynitrosohydrazono)bis-ethanamine; C039642:peroxynitric acid; D009569:Nitric Oxide; D014443:Tyrosine; D026422:S-Nitrosoglutathione; C002385:linsidomine; C068798:4-aminophenylphosphate; D008981:Molsidomine; D013482:Superoxide Dismutase; D017493:Leukocyte Common Antigens; C516841:PTPRA protein, human; D017027:Protein Tyrosine Phosphatases; D054630:Receptor-Like Protein Tyrosine Phosphatases, Class 4; D005978:Glutathione; D004229:Dithiothreitol