The reversible regulation of protein tyrosine phosphatase is an important mechanism in processing signal transduction and regulating cell cycle. Recent reports have shown that the active site cysteine residue, Cys215, can be reversibly oxidized to a cysteine sulfenic derivative (Denu and Tanner, 1998; Lee et al., 1998). We propose an additional modification that has implications for the in vivo regulation of protein tyrosine phosphatase 1B (PTP1B, EC 3.1.3.48): the glutathionylation of Cys215 to a mixed protein disulfide. Treatment of PTP1B with diamide and reduced glutathione or with only glutathione disulfide (GSSG) results in a modification detected by mass spectrometry in which the cysteine residues are oxidized to mixed disulfides with glutathione. The activity is recovered by the addition of dithiothreitol, presumably by reducing the cysteine disulfides. In addition, inactivated PTP1B is reactivated enzymatically by the glutathione-specific dethiolase enzyme thioltransferase (glutaredoxin), indicating that the inactivated form of the phosphatase is a glutathionyl mixed disulfide. The cysteine sulfenic derivative can easily oxidize to its irreversible sulfinic and sulfonic forms and hinder the regulatory efficiency if it is not converted to a more stable and reversible end product such as a glutathionyl derivative. Glutathionylation of the cysteine sulfenic derivative will prevent the enzyme from further oxidation to its irreversible forms, and constitutes an efficient regulatory mechanism.

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 siteexhibits 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 phosphorylated proteins in non-phagocytic cells. This elevation can be achieved by the activation of protein-tyrosine kinases (PTKs) 1 and/or inactivation of protein-tyrosine phosphatases (PTPs). The latter is particularly important, since PTPs exhibit much higher specific activity relative to that of PTKs (7) . , is the more efficient oxidant in regulating PTP-1B during signaling, we investigated the kinetics of PTP-1B inactivation by each of these oxidants and examined the reversibility of the oxidatively inactivated PTP-1B. Based on the results, a regulatory mechanism was proposed and verified by data obtained from an in vivo study. EXPERIMENTAL PROCEDURESMaterials and Cell Culture-30% hydrogen peroxide was supplied by Fisher. Chelex 100 Resin (200 -400 mesh, sodium form) was from Bio-Rad. Glutathione, manganese superoxide dismutase (Escherichia coli) (MnSOD), DTT, and xanthine (X) were from Sigma. Xanthine oxidase (bovine) (XO) was from Roche Molecular Biochemicals. The synthetic peptide DADEpYLIPQQG (corresponding to EGFR 988 -998 ) was from Alpha Diagnostic International (San Antonio, TX). Recombinant PTP-1B was purified as described previously (16). Human A431 epidermoid carcinoma cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum under 5% CO 2 . Anti-PTP-1B was supplied by Upstate Biotechnology.Assay of PTP-1B Activity-The activity of PTP-1B was monitored using a continuous spectrofluorometric assay described previously (17). Briefly, the peptide substrate was incubated at 25°C in 50 mM imidazole, 1...

Cu,Zn-superoxide dismutase (SOD) is known to be a locus of mutation in familial amyotrophic lateral sclerosis (FALS). Transgenic mice that express a mutant Cu,Zn-SOD, Because the levels of total Cu,Zn-SOD activity in these transgenic mice are greater than normal, the plausible interpretation of the ALS symptoms in these mice is that the mutant is in some way cytotoxic. The nature of this cytotoxic gain-offunction of the FALS mutants is yet to be identified.In addition to the usual superoxide dismutation activity, Cu,Zn-SOD has a peroxidative function that utilizes its own dismutation product, H202, as a substrate (17)(18)(19). During this reaction, the enzyme becomes inactivated in a relatively short period of time. However, this inactivation can be prevented by various anionic radical scavengers (18)(19)(20). Previous investigations from our laboratory (18,19) have shown that, during these reactions, free radicals, such as the hydroxyl radical ('OH), and scavenger-derived radicals are generated and some of these free radicals can escape from the active channel to the bulk solution. Because the enzymic activity is protected by small anions against H202 inactivation and concomitant generation of the anion-derived radicals, Cu,Zn-SOD behaves as an enzyme that catalyzes the formation of free radicals using anionic scavengers and H202 as substrates (18,19). These anionic scavengers, which become free radicals in this peroxidative reaction, include some neurotransmitters such as glutamate and taurine (19).We investigated whether there are any differences in the free radical-generating function between the wild-type and FALS mutant Cu,Zn-SOD. For this purpose, we cloned the wild-type and G93A cDNA of human Cu,Zn-SOD, overexpressed them in insect cells (Sf9), purified proteins, and measured their enzymic activities. Our results revealod-thatthe G93A and the wild-type Cu,Zn-SOD have identical dismutation activities. However, the free radical-generating function of the G93A mutant measured by the spin trapping method is enhanced relative to that of the wild-type enzyme. We found that this enhancement is due to a small decrease in the value of Km for H202. This gain-of-function, in part, may provide an explanation for the association of the FALS Cu,Zn-SOD mutants to ALS (21).