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Methods to monitor classical protein-tyrosine phosphatase oxidation - PubMed

Review

Methods to monitor classical protein-tyrosine phosphatase oxidation

Robert Karisch et al. FEBS J. 2013 Jan.

Abstract

Reactive oxygen species (ROS), particularly hydrogen peroxide (H(2)O(2)), act as intracellular second messengers in many signaling pathways. Protein-tyrosine phosphatases (PTPs) are now believed to be important targets of ROS. PTPs contain a conserved catalytic cysteine with an unusually low pK(a). This property allows PTPs to execute nucleophilic attack on substrate phosphotyrosyl residues, but also renders them highly susceptible to oxidation. Reversible oxidation, which inactivates PTPs, is emerging as an important cellular regulatory mechanism and might contribute to human diseases, including cancer. Given their potential toxicity, it seems likely that ROS generation is highly controlled within cells to restrict oxidation to those PTPs that must be inactivated for signaling to proceed. Thus, identifying ROS-inactivated PTPs could be tantamount to finding the PTP(s) that critically regulate a specific signaling pathway. This article provides an overview of the methods currently available to identify and quantify PTP oxidation and outlines future challenges in redox signaling.

© 2012 The Authors Journal compilation © 2012 FEBS.

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Figures

Figure 1
Figure 1. Redox regulation of PTPs

(A) Model for PTP redox regulation. RTK activation results in the tranisent and localized production of H2O2 by NOX enzymes. Due to the intrinsic sensitivity of their catalytic cysteinyl residues, PTPs are reversibly oxidized (PTP-SOH) and inactivated in the presence of H2O2, leading to increased tyrosyl phosphorylation and downstream signaling. As signaling continues, NOX enzymes are inactivated, resulting in decreased H2O2 levels, PTP re-activation (PTP-S−) by reducing agents (i.e., glutathione peroxidases) and a reduction in tyrosyl phosphorylation/signal transmission. (B) Schematic depicting PTP catalysis and oxidation. In the active (PTP-S−) state, PTPs can dephosphorylate phosphotyrosyl substrates; however, in the presence of physiological levels of H2O2, PTPs are reversibly oxidized to the sulfenic acid (PTP-SOH) state and thereby inactivated. This state is labile and, in different PTP family members, rapidly rearranges to form a intramolecular sulfenylamide or a disulfide bond with a nearby cysteine residue. The sulfenylamide and disulfide states help to prevent hyper-oxidation to the biologically irreversible sulfinic (PTP-SO2H) and sulfonic (PTP-SO3H) acid states. Figure adapted from [87].

Figure 2
Figure 2. Indirect methods to monitor PTP oxidation

(A) The state (S− or SOH) monitored after the indicated method is shown. Summary of the two general categories of indirect approaches to quantify PTP oxidation. In negative techniques (B), cells are lysed in the presence of a labelled (e.g., radioactive) alkylating agent, and PTP oxidation is reflected by decreased detection of the probe. In positive approaches (C), cells also are lysed in the presence of an alkylating agent; however, oxidized PTPs are then converted to the active state (using a reducing agent) and reacted with a radiolabelled substrated (modified in-gel PTPase assay; i), IAP-Biotin (ii), BBP- Biotin (iii), PVSN-N3 (iv) or pervanadate (v). (C) Structures of IAP-Biotin, BBP-Biotin and PVSN-N3 are shown.

Figure 3
Figure 3. Activity-based PTP probes

(A) Schematic of PTP catalysis. Mechanism for the reaction of a PTP with the activity-based probes BBP-Biotin (B) and PVSN-N3 (C). Figures adapted from [54, 55]. (D) Example of a Cu-catalyzed azide-alkyne cycloaddition click chemistry reaction.

Figure 4
Figure 4. qPTPome and q-oxPTPome

Schematic of qPTPome and q-oxPTPome, respectively. For q-oxPTPome, basal or ligand stimulated PTP oxidation (PTP-SOH) is monitored by lysing cells in the presence of NEM, which irreversibly alkylates active PTPs (PTP-S-NEM). Excess NEM is removed by gel filtration, and oxidized PTPs are reduced with DTT (PTP-S−). A second buffer exchange is applied to remove DTT, and reduced PTPs (representing PTPs that were initially oxidized) are hyper-oxidized to the sulfonic acid (PTP-SO3H) state by PV treatment. For qPTPome, the alkylation step is omitted, cells are lysed in the presence of DTT and all PTPs initially present in cells are hyper-oxidized to the sulfonic acid state by PV treatment. In both methods, hyper-oxidized PTPs can then be monitored by immunoblotting or processed for MS. For MS analysis, PV-treated lysates are digested with trypsin, and the hyper-oxidized active site peptides of classical PTPs are immunoprecipitated with the oxPTP Ab and detected using either “discovery”-based proteomics by LC-MS/MS or in “monitoring”-mode by LC-MS/SRM. Note that PTP catalytic domains are represented as ribbon diagrams. Figure adapted from [66].

Figure 5
Figure 5. Dimedone-based probes

(A) Reaction of dimedone with a reversibly oxidized protein. (B) Structures of the indicated dimedone-based probes.

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