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

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 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...

Targeted disruption of the glutaredoxin 1 gene does not sensitize adult mice to tissue injury induced by ischemia/reperfusion and hyperoxia

“…7A). S-glutathionylation can inactivate the catalytic function of many proteins such as transcription factors NF-κB and NF-1 [59,60], protein tyrosine phosphatase 1B (PTP-1B) [61,62], protein kinase C-α [63], etc. However, the activities of a number of other proteins, such as HIV-1 protease, glutathione S-transferase, and Ras, are enhanced by Sglutathionylation [64][65][66].…”

Activation of the Raf-1/MEK/ERK cascade by bile acids occurs via the epidermal growth factor receptor in primary rat hepatocytes

“…Reactive oxygen species have been reported to inhibit protein tyrosine phosphatases as these enzymes have a sensitive cysteine (e.g., Cys-215 in SHP1) residue within their active site. 31 Indeed, our preliminary data indicate that cell extracts prepared from hepatocytes treated with DCA had a decreased ability to dephosphorylate the EGFR (unpublished data). Because protein phosphatases generally have much higher turnover num- Fig.…”

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

“…Those proteins are included here as the same critical cysteine(s) can be modified by either ROS or RNS and very likely by HNE as well when the cysteine is in the thiolate form. Among the signaling proteins (and their critical cysteines) that have been well characterized are the PTPs, PTP1B (cys 215 ) (26,27,(34)(35)(36), low molecular weight PTP (cys12,17) (37-39) and Srchomology 2 domain-containing PTP (SHP-2) (40), the small G protein, Ras (cys 118 ) (41)(42)(43)(44), the large G proteins, G i (cys 267 ) and G o (cys 326 ) (45) and the lipid phosphatase, phosphatase and tensin homolog deleted on chromosome 10 (PTEN). In addition, as mentioned above, in its reduced form Trx binds and inhibits ASK1 and possibly other signaling proteins as well (46)(47)(48)(49)(50)(51)(52)(53).…”