Oxidation Of Protein Tyrosine Phosphatases Research Articles

Differential Oxidation of Protein-tyrosine Phosphatases

Oxidation is emerging as an important regulatory mechanism of protein-tyrosine phosphatases (PTPs). Here we report that PTPs are differentially oxidized, and we provide evidence for the underlying mechanism. The membrane-proximal RPTPalpha-D1 was catalytically active but not readily oxidized as assessed by immunoprobing with an antibody that recognized oxidized catalytic site cysteines in PTPs (oxPTPs). In contrast, the membrane-distal RPTPalpha-D2, a poor PTP, was readily oxidized. Oxidized catalytic site cysteines in PTP immunoprobing and mass spectrometry demonstrated that mutation of two residues in the Tyr(P) loop and the WPD loop that reverse catalytic activity of RPTPalpha-D1 and RPTPalpha-D2 also reversed oxidizability, suggesting that oxidizability and catalytic activity are coupled. However, catalytically active PTP1B and LAR-D1 were readily oxidized. Oxidizability was strongly dependent on pH, indicating that the microenvironment of the catalytic cysteine has an important role. Crystal structures of PTP domains demonstrated that the orientation of the absolutely conserved PTP loop arginine correlates with oxidizability of PTPs, and consistently, RPTPmu-D1, with a similar conformation as RPTPalpha-D1, was not readily oxidized. In conclusion, PTPs are differentially oxidized at physiological pH and H(2)O(2) concentrations, and the PTP loop arginine is an important determinant for susceptibility to oxidation.

An antibody-based method for monitoring in vivo oxidation of protein tyrosine phosphatases

Regulation of protein tyrosine phosphatases (PTPs) through reversible oxidation of the active site cysteine is emerging as a general, yet poorly characterized, mechanism for control of the activity of this important group of enzymes. This regulatory mechanism was initially described after in vitro treatment of PTPs with oxidizing agents. However, accumulating evidence has substantiated the notion that this mechanism is also operating in vivo, e.g., in association with the transient increase in H 2O 2 production which occurs after activation of receptor tyrosine kinases. A novel generic antibody-based method for monitoring of PTP oxidation is described. The sensitivity of this strategy has been validated by the demonstration of oxidation of endogenously expressed PTPs after stimulation of cells with growth factors. The method was also instrumental in providing the first evidence for intrinsic differences between PTP domains with regard to sensitivity to oxidation.

Regulation of Insulin Signaling through Reversible Oxidation of the Protein-tyrosine Phosphatases TC45 and PTP1B

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 beta-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.

UVA inactivates protein tyrosine phosphatases by calpain-mediated degradation.

UV irradiation causes inflammatory and proliferative cellular responses. We have proposed previously that these effects are, to a large extent, caused by the ligand-independent activation of several receptor tyrosine kinases due to the inactivation of their negative control elements, the protein tyrosine phosphatases (PTPs). We examined the mechanism of this inactivation and found that, in addition to reversible oxidation of PTPs, UV triggers a novel mechanism: induced degradation of PTPs by calpain, which requires both calpain activation and substrate PTP oxidative modification. This as yet unrecognized effect of UV is irreversible, occurs predominantly with UVA and UVB, the range of wavelengths in sunlight that reach the skin surface, and at physiologically relevant doses.

Redox regulation of PTEN and protein tyrosine phosphatases in H 2O 2-mediated cell signaling

Protein tyrosine phosphatase (PTP) is a family of enzymes important for regulating cellular phosphorylation state. The oxidation and consequent inactivation of several PTPs by H 2O 2 are well demonstrated. It is also shown that recovery of enzymatic activity depends on the availability of cellular reductants. Among these redox-regulated PTPs, PTEN, Cdc25 and low molecular weight PTP are known to form a disulfide bond between two cysteines, one in the active site and the other nearby, during oxidation by H 2O 2. The disulfide bond likely confers efficiency in the redox regulation of the PTPs and protects cysteine-sulfenic acid of PTPs from further oxidation. In this review, through a comparative analysis of the oxidation process of Yap1 and PTPs, we propose the mechanism of disulfide bond formation in the PTPs.

Preferential oxidation of the second phosphatase domain of receptor-like PTP-alpha revealed by an antibody against oxidized protein tyrosine phosphatases.

Protein tyrosine phosphatases (PTPs) constitute a large enzyme family with important biological functions. Inhibition of PTP activity through reversible oxidation of the active-site cysteine residue is emerging as a general, yet poorly characterized, regulatory mechanism. In this study, we describe a generic antibody-based method for detection of oxidation-inactivated PTPs. Previous observations of oxidation of receptor-like PTP (RPTP) alpha after treatment of cells with H(2)O(2) were confirmed. Platelet-derived growth factor (PDGF)-induced oxidation of endogenous SHP-2, sensitive to treatment with the phosphatidylinositol 3-kinase inhibitor LY294002, was demonstrated. Furthermore, oxidation of RPTPalpha was shown after UV-irradiation. Interestingly, the catalytically inactive second PTP domain of RPTPalpha demonstrated higher susceptibility to oxidation. The experiments thus demonstrate previously unrecognized intrinsic differences between PTP domains to susceptibility to oxidation and suggest mechanisms for regulation of RPTPs with tandem PTP domains. The antibody strategy for detection of reversible oxidation is likely to facilitate further studies on regulation of PTPs and might be applicable to analysis of redox regulation of other enzyme families with active-site cysteine residues.

Oxidation of protein tyrosine phosphatases as a pharmaceutical mechanism of action: a study using 4-hydroxy-3,3-dimethyl-2H-benzo[g]indole-2,5(3H)-dione.

Growth factor and insulin signal transduction comprise series of protein kinases and protein phosphatases whose combined activities serve to propagate the growth factor signal in a regulated fashion. It was shown previously that such signaling cascades generate hydrogen peroxide inside cells. Recent work has implied that one function of this might be to enhance the feed-forward signal through the reversible oxidation and inhibition of protein tyrosine phosphatases (PTPs). We identified compound 4-hydroxy-3,3-dimethyl-2H-benzo[g]indole-2,5(3H)-dione (BVT.948) as an agent that is able to inhibit PTP activity in vitro noncompetitively, a mechanism involving oxidation of the catalytic cysteine residue. We investigated the pharmaceutical utility of this compound by examining its effects in a series of in vitro cellular and in vivo assays. Results showed that BVT.948 was able to enhance insulin signaling in cells, although it did not increase tyrosine phosphorylation globally. Furthermore, the compound was active in vivo, enhancing insulin tolerance tests in ob/ob mice, therefore apparently enhancing insulin sensitivity. BVT.948 was able to inhibit several other PTPs tested and also was efficient at inhibiting several cytochrome P450 (P450) isoforms in vitro. The data suggest that inhibitors of PTPs that display noncompetitive kinetics must be viewed with caution because they may oxidize the enzyme irreversibly. Furthermore, although such compounds display interesting biological effects in vitro and in vivo, their general pharmaceutical utility may be limited due to undesired effects on P450 enzymes.

In vivo by reversible oxidation" chap="23">Analysis of the Regulation of Protein Tyrosine Phosphatases in Vivo by Reversible Oxidation

The chapter presents analysis of the regulation of protein tyrosine phosphatases (PTPs) in vivo by reversible oxidation. Consistent with an important function as regulators of signal transduction, the activity of the PTPs is tightly controlled in vivo. Various mechanisms of regulation are described, from ligand binding to the receptor-like PTPs and targeting of nontransmembrane PTPs to defined subcellular locations, to regulation of PTP function by covalent modification, such as phosphorylation and proteolysis. It has become apparent that the production of ROS in nonphagocytic cells is important for propagating an optimal response to stimulation by growth factors, cytokines, and hormones. The unique features of the active site of the PTPs and their mechanism of catalysis have drawn attention to their potential for regulation by reversible oxidation. In order to promote the anaerobic environment and ensure that the oxidation status of the PTPs in the lysate reflects accurately that of the intact cell, degassed buffers should be used for these studies. The chapter describes the measurement of PTP activity using the in-gel assay and the stimulus-induced oxidation of PTPs.

Phosphatases Feel the Oxidative Stress

H 2 O 2 inhibits protein tyrosine phosphatase (PTP) activity in vitro because of the oxidation of a critical cysteine residue in the catalytic domain. However, it has not been clear whether this is a physiologically relevant regulatory mechanism. Meng et al. now show that when Rat-1 cells were exposed to H 2 O 2 , several PTPs were oxidized and inactivated in vivo. These were quickly reduced once exogenous H 2 O 2 was removed. Treatment of cells with platelet-derived growth factor (PDGF), a mitogen that stimulates intracellular reactive oxygen species (ROS) production, also caused PTP oxidation, including SHP-2. Recruitment of cytoplasmic SHP-2 to activated PDGF receptors was required for SHP-2 oxidation. Receptor association may induce a conformational change in SHP-2 that facilitates oxidation of the cysteine residue and inactivates the enzyme. This would allow recruitment of downstream signaling to phosphorylated receptors. Blanchetot et al. also show that receptor-type PTPs (RPTPs) are inhibited by oxidative stress. Most RPTPs contain two intracellular enzymatic domains (D1 and D2). By expressing various deletion mutants of RPTPα in cultured cells, the authors show that a region in the cytoplasmic domain containing D2 is in a closed conformation and is unable to bind to RPTPα monomers. However, treatment of cells with H 2 O 2 induced a conformational change in the cytoplasmic region in vivo that was dependent on a cysteine residue in D2. The conformational change correlated with increased RPTPα dimerization and inactivation of phosphatase activity. Inactivation by oxidation and subsequent dimer stabilization may be a general regulatory mechanism for RPTP activity. C. Blanchetot, L. G. J. Tertoolen, J. den Hertog, Regulation of receptor protein-tyrosine phosphatase a by oxidative stress. EMBO J. 21 , 493-503 (2002). [Abstract] [Full Text] T.-C. Meng, T. Fukuda, N. K. Tonks, Reversible oxidation and inactivation of protein tyrosine phosphatases in vivo. Molec. Cell 9 , 387-399 (2002). [Online Journal]