Oxidation of PTPs in Tyrosine Phosphorylation Dependent Signaling

Reactive oxygen species (ROS) are produced in response to a wide variety of cellular stimuli [52]. A substantial body of data emphasizes the importance of ROS production as a mechanism for fine-tuning tyrosine-phosphorylation-dependent signaling through the transient oxidation and inactivation of members of the PTP family [53]. In the unique environment of the PTP active site, the invariant Cys residue of the signature motif, which displays an unusually low pKa, is present predominantly as the thiolate anion [9,10]. This not only enhances its nucleophilic properties but also renders it susceptible to oxidation. Oxidation can yield a stable, single-oxidized sulfenic acid modification of the Cys (Cys-SOH), which inhibits activity because the oxidized Cys can no longer function as a nucleophile. This modification is reversible and thus can form the basis for a mechanism of reversible regulation of PTP activity. Glutathionylation of the sulfenic acid form of PTPs has been reported [54] which may not only promote reduction back to the active state but also prevent further, irreversible oxidation by the addition of two (sulfinic acid) or three (sulfonic acid)

oxygens to the active site Cys. Treatment of both PTPs and DSPs with H2O2 in vitro results in oxidation and inactivation [55]. More importantly, oxidation and inactiva-tion of PTPs have now been demonstrated in response to physiological stimuli. For example, PTP1B is oxidized and inactivated in response to growth factors, such as epidermal growth factor (EGF) [56], and hormones, such as insulin [57].

In order to examine this issue further, we developed a modified "in-gel" PTP assay to visualize the oxidation of PTPs in response to a stimulus in a cellular context. We observed the reversible oxidation of multiple PTPs in response to treatment of Rat 1 cells with H2O2 and demonstrated that this oxidation was required for the mitogenic effects of H2O2 [53]. We also demonstrated that stimulation of Rat 1 fibroblasts with platelet-derived growth factor (PDGF) led to the production of reactive oxygen species, which induced the rapid and reversible oxidation of the PTP SHP2 [53]. Ligand-induced autophosphorylation of the PDGF receptor (PDGFR) generates docking sites for various signaling proteins, including SHP2. We showed that mutant forms of the PDGFR that were unable to bind to SHP2 displayed enhanced autophosphorylation and enhanced activation of MAP kinase. Thus, SHP2 appears to recognize the PDGFR as a substrate and functions as an inhibitor of PDGFR signaling. Interestingly, it was only the population of SHP2 that was bound to the PDGFR that was susceptible to reversible oxidation and inhibition. We propose that PDGF stimulation induces localized production of ROS, leading to the rapid oxidation of the pool of SHP2 that has been recruited into a complex with the PDGFR. This augments autophosphoryla-tion of the receptor and initiation of the signaling response.

Figure 2 Regulation of protein tyrosine phosphatase (PTP) activity by reversible oxidation. Ligand-dependent activation of a receptor protein tyrosine kinase (RTK) triggers the activity of a Rac-dependent NADPH oxidase leading to production of reactive oxygen species (ROS). ROS oxidize the active site Cys residue of members of the PTP family, converting it from a thiolate ion (the active form) to sulfinic acid. Oxidation results in inhibition of PTP activity, thereby promoting tyrosine phosphorylation. However, due to the action of glutathione or thioredoxin, oxidation of the PTPs is transient. Restoration of PTP activity following reduction back to the thiolate form of the active site Cys residue terminates the tyrosine-phosphorylation-dependent signal. A variety of growth factors, hormones, and cytokines induce ROS production and stimulate tyrosine phosphoryla-tion. We are developing methods to identify the PTPs that become oxidized in response to a physiological stimulus as a way of establishing links between particular PTPs and the regulation of defined signaling pathways.

Figure 2 Regulation of protein tyrosine phosphatase (PTP) activity by reversible oxidation. Ligand-dependent activation of a receptor protein tyrosine kinase (RTK) triggers the activity of a Rac-dependent NADPH oxidase leading to production of reactive oxygen species (ROS). ROS oxidize the active site Cys residue of members of the PTP family, converting it from a thiolate ion (the active form) to sulfinic acid. Oxidation results in inhibition of PTP activity, thereby promoting tyrosine phosphorylation. However, due to the action of glutathione or thioredoxin, oxidation of the PTPs is transient. Restoration of PTP activity following reduction back to the thiolate form of the active site Cys residue terminates the tyrosine-phosphorylation-dependent signal. A variety of growth factors, hormones, and cytokines induce ROS production and stimulate tyrosine phosphoryla-tion. We are developing methods to identify the PTPs that become oxidized in response to a physiological stimulus as a way of establishing links between particular PTPs and the regulation of defined signaling pathways.

The transient nature of the oxidation ensures reduction and reactivation of the pool of SHP2, which promotes dephos-phorylation of the PDGFR and termination of the signal. These data illustrate how ligand-induced production of ROS may augment tyrosine-phosphorylation-dependent signaling in general through inactivation of PTPs (Fig. 2).

The production of ROS is observed in response to a wide variety of stimuli, including growth factors, hormones, cytokines, and activators of G-protein-coupled receptors, leading to PTK activation. The operating principle is that the stimulus enhances tyrosine phosphorylation directly by activation of a PTK or indirectly by inactivation of a PTP. Thus, one function of ROS produced following agonist stimulation is transient inactivation of the critical PTP that provides the inhibitory constraint upon the system, thus facilitating initiation of the signaling response to that stimulus. We propose that stimulus-induced oxidation may be used as a means of "tagging" and identifying those PTPs that are integral to the regulation of the signaling events triggered by that stimulus. It is hoped that this will provide further insights into the physiological function of members of the PTP family.

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