Reversible Oxidation and Inactivation of Protein Tyrosine Phosphatases In Vivo

Abstract

We have investigated the regulation of protein tyrosine phosphatases (PTPs) by reactive oxygen species (ROS) in a cellular environment. We demonstrate that multiple PTPs were reversibly oxidized and inactivated following treatment of Rat-1 cells with H(2)O(2) and that inhibition of PTP function was important for ROS-induced mitogenesis. Furthermore, we show transient oxidation of the SH2 domain containing PTP, SHP-2, in response to PDGF that requires association with the PDGFR. Our results indicate that SHP-2 inhibits PDGFR signaling and suggest a mechanism by which autophosphorylation of the PDGFR occurs despite its association with SHP-2. The data suggest that several PTPs may be regulated by oxidation and that characterization of this process may define novel links between specific PTPs and particular signaling pathways in vivo.

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

“…Thiol-specific alkylating reagents such as iodoacetic acid (IAA) or N-ethyl maleamide (NEM) which irreversibly bind to sulfhydryls, while not affecting oxidized cysteines [29,[217][218][219] have opened the field to inquiry. The subsequent addition of reductant coupled to the use of a thiol-specific labeling agent such as biotinylated IAA or biotinylated NEM will then identify the oxidized Cys (Figure 3, top panel) [47].…”

“…The subsequent addition of reductant coupled to the use of a thiol-specific labeling agent such as biotinylated IAA or biotinylated NEM will then identify the oxidized Cys (Figure 3, top panel) [47]. This methodology, also referred to as "the thiol trapping technique" [220] has been successfully applied to cells in culture and demonstrated reversible oxidation and inactivation of tyrosine phosphatases in response to stimulation with growth factor receptors [29,116,117,219]. A similar strategy has been developed to detect Snitrosylated proteins, widely known as the "biotin switch method" (Figure 3, middle panel) [56,221].…”

“…Amino acids that are targets for reversible oxidation include cysteines with a low pKa sulhydryl group (4)(5), which causes unique susceptibility to oxidation compared to the typical pKa of non-reactive cysteines (8.5) [29]. In addition, methionine, tryptophan, and tyrosine residues are also prone to oxidative modification, although the functional impact of those events in physiological signal transduction remains to be established.…”

“…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

“…In that case, the modifications of the protein kinases and phosphatases must be reversible. Reversible inactivation of several different protein tyrosine phosphatases has been shown in relevant cell types stimulated by PDGF, EGF, insulin, and B cell receptor ligands (40,(72)(73)(74).…”