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

Persson, C.; Sjöblom, Tobias; Groen, Arnoud; Kappert, Kai; Engström, Ulla; Hellman, Ulf; Heldin, Carl‐Henrik; Hertog, Jeroen den; Östman, Arne

doi:10.1073/pnas.0304403101

Cited by 124 publications

(115 citation statements)

References 31 publications

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“…The strategy also involves cell lysis in the presence of IAA, to alkylate reduced Cys residues, and reduction of any reversibly oxidized PTPs back to the active state. However, in a separate step, these PTPs are then terminally oxidized to the sulfonic acid form by treatment with pervanadate and identified by immunoblotting with the oxPTP antibodies (25). This approach has been used successfully to distinguish the susceptibility of different PTPs to oxidation (26), in particular demonstrating that the second, membrane-distal PTP domain of RPTP␣ is more sensitive to oxidation than the membrane-proximal catalytic domain and may serve as a redox sensor (26).…”

Section: Discussionmentioning

confidence: 99%

“…Other potential challenges include the specificity and sensitivity of the antibody. Most of the studies to date that have used this approach have examined the oxidation of ectopically expressed classical PTPs (25,26). It remains to be established whether this antibody is able to recognize all members of the PTP superfamily, in particular the DSPs, and to what extent it has the sensitivity to measure oxidation of PTPs at physiological levels of expression.…”

Section: Discussionmentioning

confidence: 99%

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A modified cysteinyl-labeling assay reveals reversible oxidation of protein tyrosine phosphatases in angiomyolipoma cells

Boivin

Zhang

Arbiser

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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The production of reactive oxygen species (ROS) exerts an additional tier of control over tyrosine phosphorylation-dependent signal transduction by transiently inhibiting the catalytic activity of specific protein tyrosine phosphatases (PTPs). Hence, the ability to detect reversible oxidation of PTPs in vivo is critical to understanding the complex biological role of ROS in the control of cellular signaling. Here, we describe an assay for identifying those PTPs that are reversibly oxidized in vivo, which utilizes the unique chemistry of the invariant catalytic Cys residue in labeling the active site with biotinylated small molecules under mildly acidic conditions. We have applied this cysteinyl-labeling assay to the study of platelet-derived growth factor (PDGF) receptor signaling in an angiomyolipoma cell model. Doing so has allowed us to detect reversible oxidation of several proteins in response to sustained PDGF stimulation. As in other cell systems, we have observed the reversible oxidation of the classical PTP SHP2 and the tumor suppressor phosphatase PTEN in response to PDGF stimulation. Furthermore, we detected reversible oxidation of members of two other subclasses of PTPs, the receptor PTP LAR and the dual-specificity phosphatase MKP1. These data demonstrate the broad selectivity of the assay, allowing us to detect representatives of all of the major subgroups of the PTP superfamily. We anticipate that this cysteinyl-labeling enrichment strategy can be applied broadly to study reversible oxidation as a mechanism of harnessing PTP catalytic activity in a variety of signaling pathways.reactive oxygen species ͉ signal transduction ͉ tyrosine phosphorylation ͉ dual specificity phosphatases ͉ cancer R egulated and localized generation of reactive oxygen species (ROS) creates an oxidative microenvironment important for optimal tyrosine phosphorylation-dependent signal transduction. Previous studies have shown that ROS-mediated transient inhibition of the catalytic activity of members of the protein tyrosine phosphatase (PTP) superfamily plays a crucial role in facilitating signal transduction (1-3). The PTP superfamily consists of Ϸ100 genes encoding structurally diverse enzymes, which are characterized by a conserved catalytic domain and which all perform phosphoryl hydrolysis by using a cysteinylbased nucleophilic core within a conserved signature motif. The PTP signature motif, HC(X) 5 R(S/T), creates a unique environment for the catalytic Cys residue. In this setting, the pK a of the Cys residue, located at the base of a cleft formed by the extremity of ␤-sheet 12 and ␣-helix 4 [features named for the structure of PTP1B (4)], is lowered by the presence of the conserved Arg residue, in addition to the helix dipole of ␣-4 and microdipoles generated by backbone atoms in the signature motif (4, 5). Hence, the Cys residue of the signature motif displays an unusually low pK a , making it both a good nucleophile at neutral pH in addition to being highly susceptible to oxidation (6). The sensitivity of PTPs to i...

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

A modified cysteinyl-labeling assay reveals reversible oxidation of protein tyrosine phosphatases in angiomyolipoma cells

Boivin

Zhang

Arbiser

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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“…Interestingly, oxidation may also underlie a mechanism for regulation of receptor PTPs. In RPTP␣, the cysteine of the signature motif from the second, membrane-distal PTP domain (RPTP␣-D2) is more susceptible to oxidation than the membrane-proximal catalytic domain (39). Oxidation of RPTP␣-D2 inside the cell leads to a change in the conformation of the extracellular segment of RPTP␣ (40), suggesting that the membrane-distal PTP domain may serve as an oxygen sensor and may underlie inside-out signaling through RPTP␣.…”

Section: Discussionmentioning

confidence: 99%

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

Meng

Buckley

Galić

et al. 2004

Journal of Biological Chemistry

251

220

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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 ...

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“…Because of the presence of an invariant catalytic cysteine residue within PTPs, which possesses an unusually low pK a due to the presence of vicinal basic residues, these enzymes are uniquely susceptible to oxidative inactivation, and a number of PTPs or other cysteine-containing phosphatases have been shown to be similarly subjected to oxidative inactivation in relation to cytokine and/or growth factor signaling due to the activation of NOX/DUOX enzymes, including PTP1B, low molecular weight (LMW-) PTP, SHP-1 and SHP-2, and PTEN (3,(41)(42)(43)(44)(45)(46)(47) (Fig. 3).…”

Section: Targets For Nox-derived Ros: Protein Cysteine Residuesmentioning

confidence: 99%

NADPH oxidases in lung biology and pathology: Host defense enzymes, and more

Vliet

2008

Free Radical Biology and Medicine

187

192

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The deliberate production of reactive oxygen species (ROS) by phagocyte NADPH oxidase is widely appreciated as a critical component of antimicrobial host defense. Recently, additional homologs of NADPH oxidase (NOX) have been discovered throughout the animal and plant kingdoms, which appear to possess diverse functions in addition to host defense, including cell proliferation, differentiation, and regulation of gene expression. Several of these NOX homologs are also expressed within the respiratory tract, where they participate in innate host defense as well as in epithelial and inflammatory cell signaling and gene expression, and fibroblast and smooth muscle cell proliferation, in response to bacterial or viral infection and environmental stress. Inappropriate expression or activation of NOX/DUOX during various lung pathologies suggests their specific involvement in respiratory disease. This review summarizes the current state of knowledge regarding the general functional properties of mammalian NOX enzymes, and their specific importance in respiratory tract physiology and pathology.

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

Cited by 124 publications

References 31 publications

A modified cysteinyl-labeling assay reveals reversible oxidation of protein tyrosine phosphatases in angiomyolipoma cells

A modified cysteinyl-labeling assay reveals reversible oxidation of protein tyrosine phosphatases in angiomyolipoma cells

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

NADPH oxidases in lung biology and pathology: Host defense enzymes, and more

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