Roles of Superoxide Radical Anion in Signal Transduction Mediated by Reversible Regulation of Protein-tyrosine Phosphatase 1B

doi:10.1074/jbc.274.49.34543

Roles of Superoxide Radical Anion in Signal Transduction Mediated by Reversible Regulation of Protein-tyrosine Phosphatase 1B*

From the ^‡Laboratory of Biochemistry and the ^§Laboratory of Biophysical Chemistry, NHLBI, National Institutes of Health, Bethesda, Maryland 20892 and the ^¶Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York 10461

Abstract

Growth factors induce intracellular production of reactive oxygen species in non-phagocytic cells and elevation of their phosphorylated protein tyrosine level. The latter can be achieved by activating protein-tyrosine kinases and/or inactivating protein-tyrosine phosphatases (PTPs). A highly abundant PTP, PTP-1B, is known to be inactivated by oxidation of its catalytic site Cys-215. We show that O·̄₂ is kinetically more efficient and chemically more specific oxidant than H₂O₂ for inactivating PTP-1B. The second-order rate constant for the O·̄₂- and H₂O₂-mediated inactivation is 334 ± 45 m ⁻¹ s⁻¹ and 42.8 ± 3.8 m ⁻¹ s⁻¹, respectively. PTP-1B oxidized by H₂O₂ exhibits significantly more oxidized methionine residues and shows a lower degree of reversibility. The initial oxidative product, the Cys-215 sulfenic derivative, can easily be oxidized further to its irreversible sulfinic and sulfonic derivatives. This step is prevented by glutathionylation of the sulfenic derivative to form aS-glutathionylated PTP-1B, which can be reactivated by dithiothreitol or thioltransferase. Thus, a signal transduction mechanism mediated by the O·̄₂ and the participation of glutathione is proposed for the regulation of PTP-1B. This mechanism is supported by the in vivo demonstration that glutathionylated PTP-1B at Cys-215 is formed in A431 cells when they were treated with epidermal growth factor.

Footnotes

↵* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵‖ Both authors supported by National Institutes of Health Grant CA69202.
↵** To whom correspondence should be addressed: LB, NHLBI, NIH, Bldg. 3, Rm. 204, 3 Center Dr., MSC-0342, Bethesda, MD 20892-0342. Tel.: 301-496-2073; Fax: 301-496-0599; E-mail: pbc@helix.nih.gov.
Abbreviations:

PTK(s)

protein-tyrosine kinase(s)

PTP(s)

protein-tyrosine phosphatase(s)

ROS

reactive oxygen species

X

xanthine

XO

xanthine oxidase

MnSOD

manganese superoxide dismutase

NBD-Cl

7-chloro-4-nitrobenzo-2-oxa-1,3-diazole

LC/MS/MS

liquid chromatography/tandem mass spectrometry

EGF

epidermal growth factor

DTT

dithiothreitol

DTPA

diethylenetriaminepentaacetic acid

Cat

catalase
- Received September 1, 1999.
- Revision received September 29, 1999.
The American Society for Biochemistry and Molecular Biology, Inc.