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.

The second messenger hydrogen peroxide is required for optimal activation of numerous signal transduction pathways, particularly those mediated by protein tyrosine kinases. One mechanism by which hydrogen peroxide regulates cellular processes is the transient inhibition of protein tyrosine phosphatases through the reversible oxidization of their catalytic cysteine, which suppresses protein dephosphorylation. Here we describe a structural analysis of the redox-dependent regulation of protein tyrosine phosphatase 1B (PTP1B), which is reversibly inhibited by oxidation after cells are stimulated with insulin and epidermal growth factor. The sulphenic acid intermediate produced in response to PTP1B oxidation is rapidly converted into a previously unknown sulphenyl-amide species, in which the sulphur atom of the catalytic cysteine is covalently linked to the main chain nitrogen of an adjacent residue. Oxidation of PTP1B to the sulphenyl-amide form is accompanied by large conformational changes in the catalytic site that inhibit substrate binding. We propose that this unusual protein modification both protects the active-site cysteine residue of PTP1B from irreversible oxidation to sulphonic acid and permits redox regulation of the enzyme by promoting its reversible reduction by thiols.

Many studies have illustrated that the production of reactive oxygen species (ROS) is important for optimal tyrosine phosphorylation and signaling in response to diverse stimuli. Protein-tyrosine phosphatases (PTPs), which are important regulators of signal transduction, are exquisitely sensitive to inhibition after generation of ROS, and reversible oxidation is becoming recognized as a general physiological mechanism for regulation of PTP function. Thus, production of ROS facilitates a tyrosine phosphorylation-dependent cellular signaling response by transiently inactivating those PTPs that normally suppress the signal. In this study, we have explored the importance of reversible PTP oxidation in the signaling response to insulin. Using a modified ingel PTP assay, we show that stimulation of cells with insulin resulted in the rapid and transient oxidation and inhibition of two distinct PTPs, which we have identified as PTP1B and TC45, the 45-kDa spliced variant of the T cell protein-tyrosine phosphatase. We investigated further the role of TC45 as a regulator of insulin signaling by combining RNA interference and the use of substrate-trapping mutants. We have shown that TC45 is an inhibitor of insulin signaling, recognizing the ␤-subunit of the insulin receptor as a substrate. The data also suggest that this strategy, using ligand-induced oxidation to tag specific PTPs and using interference RNA and substrate-trapping mutants to illustrate their role as regulators of particular signal transduction pathways, may be applied broadly across the PTP family to explore function.The reversible phosphorylation of tyrosyl residues in proteins, catalyzed by the coordinated actions of protein tyrosine kinases and protein-tyrosine phosphatases (PTPs), 1 is of paramount importance to the control of such fundamental physiological functions as cell proliferation, differentiation, survival, metabolism, and motility (1, 2). The phosphorylation of a target protein alters its function, including changes in enzymatic activity or its ability to associate with other proteins. In response to a stimulus, such as a growth factor or hormone, multiple phosphorylation and dephosphorylation reactions are coordinated in signal transduction cascades that culminate in the physiological response (3, 4). A characterization of the enzymes responsible for the regulation of protein tyrosine phosphorylation in vivo will be essential for an understanding of the control of signal transduction under normal and pathophysiological conditions and would be expected to identify important new targets for therapeutic intervention in human disease. We are focusing on this process from the perspective of the PTP family of enzymes.A substantial body of information has been accumulated to describe the role of protein tyrosine kinases in the regulation of signal transduction. In contrast, we are only now beginning to appreciate in mechanistic detail the role of some members of the PTP family in fine-tuning the signaling response to extracellular stimuli. Analysis ...

Androgen plays a critical role in regulating the growth and differentiation of normal prostate epithelia, as well as the initial growth of prostate cancer cells. Nevertheless, prostate carcinomas eventually become androgen-unresponsive, and the cancer is refractory to hormonal therapy. To gain insight into the mechanism involved in this hormone-refractory phenomenon, we have examined the potential role of the androgen receptor (AR) in that process. We have investigated the expression of AR and two prostate-specific androgen-responsive antigens, prostatic acid phosphatase (

The human protein tyrosine phosphatase TCPTP exists as two forms: an endoplasmic reticulum-targeted 48-kDa form (TC48) and a nuclear 45-kDa form (TC45). Although targeted to the nucleus, TC45 can exit in response to specific stimuli to dephosphorylate cytoplasmic substrates. In this study, we investigated the downregulation of insulin receptor (IR) signaling by TCPTP. In response to insulin stimulation, the TC48-D182A and TC45-D182A "substrate-trapping" mutants formed stable complexes with the endogenous tyrosinephosphorylated IR ␤-subunit in 293 cells. Moreover, in response to insulin stimulation, the TC45-D182A mutant accumulated in the cytoplasm of cells overexpressing the IR and in part colocalized with the IR ␤-subunit at the cell periphery. These results indicate that the IR may serve as a cellular substrate for both TC48 and TC45. In immortalized TCPTP ؊/؊ murine embryo fibroblasts, insulin-induced IR ␤-subunit tyrosine phosphorylation and protein kinase PKB/Akt activation were enhanced relative to the values in TCPTP ؉/؉ cells. Importantly, the expression of TC45 or TC48 to physiological levels suppressed the enhanced insulininduced signaling in TCPTP ؊/؊ cells. These results indicate that the differentially localized variants of TCPTP may dephosphorylate the IR and downregulate insulin-induced signaling in vivo.Insulin binding to its cell surface transmembrane receptor stimulates intrinsic protein tyrosine kinase activity, autophosphorylation, and subsequent tyrosyl phosphorylation of insulin receptor substrate (IRS) proteins and adapter proteins such as Shc and Cbl/CAP (reviewed in references 5, 40, 54, and 55). Tyrosyl phosphorylation of IRS and adapter proteins generates docking sites for src homology 2 (SH2) domain-containing signaling proteins such as the p85 subunit of phosphatidylinositol 3-kinase. Collectively, these signaling proteins mediate the biological effects of insulin, which include antilipolysis, glucose uptake, glycogen synthesis, and cell growth (reviewed in references 5, 40, 54, and 55).Protein tyrosine phosphatases (PTPs) have a prominent role in the control of insulin receptor (IR) signaling. PTPs dephosphorylate the IR and its substrates and thus serve to inactivate the IR and terminate signaling. The IR and IRS proteins are rapidly and transiently phosphorylated in response to insulin, with phosphotyrosine (pTyr) content in the IR returning to basal levels within minutes of stimulation (10, 16). It is probable that numerous PTPs participate in the dephosphorylation of the IR and the multiple downstream tyrosine-phosphorylated signaling proteins. Several PTPs have been implicated in the negative regulation of the IR, including the endoplasmic reticulum-associated PTP1B (6,8,49,50) and the transmembrane PTPs LAR (34) and PTP␣ (30,41). PTP␣ has been shown to inhibit insulin-induced prolactin gene expression without altering the phosphorylation status of the IR, IRS-1, or Shc (26). Similarly, LAR may not dephosphorylate the IR in vivo but may downregulate insulin signaling by dephos...

Protein S-nitrosylation mediated by cellular nitric oxide (NO) plays a primary role in executing biological functions in cGMPindependent NO signaling. Although S-nitrosylation appears similar to Cys oxidation induced by reactive oxygen species, the molecular mechanism and biological consequence remain unclear. We investigated the structural process of S-nitrosylation of protein-tyrosine phosphatase 1B (PTP1B). We treated PTP1B with various NO donors, including S-nitrosothiol reagents and compound-releasing NO radicals, to produce sitespecific Cys S-nitrosylation identified using advanced mass spectrometry (MS) techniques. Quantitative MS showed that the active site Cys-215 was the primary residue susceptible to S-nitrosylation. The crystal structure of NO donor-reacted PTP1B at 2.6 Å resolution revealed that the S-NO state at Cys-215 had no discernible irreversibly oxidized forms, whereas other Cys residues remained in their free thiol states. We further demonstrated that S-nitrosylation of the Cys-215 residue protected PTP1B from subsequent H 2 O 2 -induced irreversible oxidation. Increasing the level of cellular NO by pretreating cells with an NO donor or by activating ectopically expressed NO synthase inhibited reactive oxygen species-induced irreversible oxidation of endogenous PTP1B. These findings suggest that S-nitrosylation might prevent PTPs from permanent inactivation caused by oxidative stress.

Cellular PAcP can down-regulate prostate cancer cell growth, at least partially by dephosphorylating c-ErbB-2/neu. Therefore, decreased cellular PAcP expression in cancer cells may be involved in prostate cancer progression.

The oxidation and inactivation of protein tyrosine phosphatases is one mechanism by which reactive oxygen species influence tyrosine phosphorylation‐dependent signaling events and exert their biological functions. In the present study, we determined the redox status of endogenous protein tyrosine phosphatases in HepG2 and A431 human cancer cells, in which reactive oxygen species are produced constitutively. We used mass spectrometry to assess the state of oxidation of the catalytic cysteine residue of endogenous PTP1B and show that this residue underwent both reversible and irreversible oxidation to high stoichiometry in response to intrinsic reactive oxygen species production. In addition, our data show that the oxidation of PTP1B is specific to the active site Cys, with the other Cys residues in the protein remaining in a reduced state. Treatment of these cells with diphenyleniodonium, an inhibitor of NADPH oxidases, decreased reactive oxygen species levels. This resulted in inhibition of protein tyrosine phosphatase oxidation, concomitant with decreased tyrosine phosphorylation of cellular proteins and inhibition of anchorage‐independent cell growth. Therefore, our data also suggest that the high level of intrinsic reactive oxygen species may contribute to the transformed phenotype of HepG2 and A431 cells via constitutive inactivation of cellular protein tyrosine phosphatases.