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 H 2 O 2 for

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... g PTP-1B. The second-order rate constant for the O 2 . -and H 2 O 2 -mediated inactivation is 334 ؎ 45 M ؊1 s ؊1 and 42.8 ؎ 3.8 M ؊1 s ؊1 , respectively. PTP-1B oxidized by H 2 O 2 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 a Sglutathionylated 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. Cyclic cascades, which include reversible tyrosine phosphorylation step(s), play a pivotal role in regulating cell cycles and signal transduction due, in part, to their enormous capacity for integrating biological information and for signal amplification (1, 2). Recent findings indicated that treatment with growth factor (3, 4) or H 2 O 2 (5, 6) induces an elevation of tyrosine 1 The abbreviations used are: 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/