Regulation of PTP1B via glutathionylation of the active site cysteine 215

The reversible regulation of protein tyrosine phosphatase is an important m echanism in processing signal transduction and regulating cell cycle. Recen t reports have shown that the active site cysteine residue, Cys215, can be reversibly oxidized to a cysteine sulfenic derivative (Denu and Tanner, 199 8; Lee et al., 1998). We propose an additional modification that has implic ations for the in vivo regulation of protein tyrosine phosphatase 1B (PTP1B , EC 3.1.3.48): the glutathionylation of Cys215 to a mixed protein disulfid e. Treatment of PTP1B with diamide and reduced glutathione or with only glu tathione disulfide (GSSG) results in a modification detected by mass spectr ometry 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 PT P1B is reactivated enzymatically by the glutathione-specific dethiolase enz yme thioltransferase (glutaredoxin), indicating that the inactivated form o f the phosphatase is a glutathionyl mixed disulfide. The cysteine sulfenic derivative can easily oxidize to its irreversible sulfinic and sulfonic for ms and hinder the regulatory efficiency if it is not converted to a more st able and reversible end product such as a glutathionyl derivative. Glutathi onylation of the cysteine sulfenic derivative will prevent the enzyme from further oxidation to its irreversible forms, and constitutes an efficient r egulatory mechanism.