Regulation of PTP1B via Glutathionylation of the Active Site Cysteine 215

Abstract

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.

Redox-based regulation of signal transduction: Principles, pitfalls, and promises

“…Inactivation of tyrosine phosphatases by H 2 O 2 has been demonstrated in a number of settings, including physiological scenarios, such as stimulation of cells with the growth factor, epidermal growth factor, (EGF), or PDGF [17]. Reversible inactivation of the tyrosine phosphatases, PTP-1B [116], PTEN [117], and SHP-2 [29] also have been reported, and a formation of a sulfenic acid intermediate was reported as the oxidative event, although the formation of sulfenyl amide species (Cys-S-N-R) and glutathionylated species also have been shown [118,119]. Elegant biochemical analyses were done to demonstrate the sulfenic and sulfenyl amide forms of PTP1B in vitro, and the glutathionylated form of PTP1B has been shown in intact cells.…”

The chemistry of cell signaling by reactive oxygen and nitrogen species and 4-hydroxynonenal

“…The oxidative reaction of cysteine with H 2 O 2 may change structure and function of protein mediating response to changes of redox state in the cell (61,62). An important protein effector by which H 2 O 2 signaling occurs is the family of PTPs (34,(36)(37)(38)63,64). PTPs constitute a large family of important regulatory enzymes that play a key role in several physiological functions.…”

“…As described above, glutathionylation (formation of a mixed protein-glutathione disulfide) is one of the post-translational modifications that occurs in cells in response to oxidative stress that could prevent overoxidation (76); but, it also has been proposed in cell signaling (36,63,77). During this process, a critical cysteine sensitive to oxidation by H 2 O 2 or other hydroperoxide may be oxidized and can then form a disulfide bond with glutathione (GSH), resulting in altered activity.…”

“…During this process, a critical cysteine sensitive to oxidation by H 2 O 2 or other hydroperoxide may be oxidized and can then form a disulfide bond with glutathione (GSH), resulting in altered activity. Several signaling proteins have been shown to undergo glutathionylation, including PTPs and transcription factors (36,63,(77)(78)(79)(80). Although the mechanism by which glutathionylation occurs is not agreed upon, this process has been shown occur in vivo in relevant cell types (81)(82)(83)(84)(85)(86).…”

Disulfide relays and phosphorylative cascades: partners in redox-mediated signaling pathways

“…Protein tyrosine phosphatases (PTPases) activity is highly regulated by oxidation/reduction reactions involving the cysteine residue required for catalysis. 38 PTPases share a conserved 230-amino-acid domain containing the cysteine residue that catalyzes the hydrolysis of phosphate from phospho-tyrosine residues by the formation of a cysteinyl-phosphate intermediate. PTPases are specifically involved in the regulation of reversible tyrosine phosphorylation in the insulin action pathway.…”