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

Abstract

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...

Redox-Directed Cancer Therapeutics: Molecular Mechanisms and Opportunities

“…The role of ROS-dependent redox dysregulation in tumor progression has been studied recently in much detail in human melanoma where overexpression of Akt converts radial (noninvasive) to vertical (invasive) growth phase tumors with increased generation of superoxide originating from NOX4 upregulation, preferential glycolytic energy metabolism, and VEGFdependent angiogenesis (115,134). In this context, it should also be mentioned that the antagonist of phosphoinositidedependent Akt activation and tumor suppressor PTEN and other members of the protein tyrosine phosphatase superfamily are established molecular targets of ROS signaling, chemically inactivated by ROS-dependent oxidation of essential cysteine residues facilitating tumorigenic tyrosine kinase receptor signaling (36,214,215,247,387).…”

12/15-lipoxygenase–derived lipid peroxides control receptor tyrosine kinase signaling through oxidation of protein tyrosine phosphatases

“…Consequently, alterations in expression levels or the specific activity of PTPs will affect the cellular response to ligands of RTKs. With regard to the PDGF β-receptor, a series of PTPs, including T cell protein tyrosine phosphatase (TC-PTP), density enhanced phosphatase-1 (DEP-1), SHP-2, and PTP-1B, have been found to modulate receptor signaling (4)(5)(6)(7). Detailed analyses of the consequences of deletion of individual PTPs have shown that individual PDGF receptorantagonizing PTPs preferentially dephosphorylate particular phospho-tyrosine residues of the receptor and thereby are able to modulate the signaling output (4,(8)(9)(10).…”

Dusp-5 and Snrk-1 coordinately function during vascular development and disease

“…24,25 Our studies suggest that the pathogenesis of vascular malformations may result from aberrant dusp-5 and snrk-1 signaling. Reactive oxygen species has been reported to inhibit the catalytic activity of specific tyrosine phosphatases (dusp-1) in an angiomyolipoma cell model 26 and treatment using nicotinamide adenine dinucleotide phosphate (reduced form) oxidase (Nox) inhibitors abolished hemangioma growth in vivo via blockage of angiopoietin-2 production. 27 It makes sense that mutations in dusp-5 and snrk-1 play an integral role in these conditions because both genes work together to maintain vascular precursors.…”