Abstract
Oxidative inactivation of cysteine-dependent Protein Tyrosine Phosphatases (PTPs) by cellular reactive oxygen species (ROS) plays a critical role in regulating signal transduction in multiple cell types. The phosphatase activity of most PTPs depends upon a ‘signature’ cysteine residue within the catalytic domain that is maintained in the de-protonated state at physiological pH rendering it susceptible to ROS-mediated oxidation. Direct and indirect techniques for detection of PTP oxidation have been developed (Karisch and Neel, 2013). To detect catalytically active PTPs, cell lysates are treated with iodoacetyl-polyethylene glycol-biotin (IAP-biotin), which irreversibly binds to reduced (S-) cysteine thiols. Irreversible oxidation of SHP-1 after treatment of cells with pervanadate or H2O2 is detected with antibodies specific for the sulfonic acid (SO3H) form of the conserved active site cysteine of PTPs. In this protocol, we describe a method for the detection of the reduced (S-; active) or irreversibly oxidized (SO3H; inactive) form of the hematopoietic PTP SHP-1 in thymocytes, although this method is applicable to any cysteine-dependent PTP in any cell type.
Keywords: Reactive oxygen species, Protein tyrosine phosphatase, Catalytic activity
Background
Reactive oxygen species (ROS) are generated by cellular NADPH oxidases and mitochondria. Most Protein Tyrosine Phosphatases (PTPs) contain a conserved catalytic cysteine with a low dissociation constant (pKa) that is highly susceptible to oxidation by ROS (Rudyk and Eaton, 2014). ROS-inactivation of PTPs plays an important role in regulating tyrosine-kinase-mediated signaling responses in numerous cell types. PTPs are rapidly oxidized in cells treated with the ROS H2O2 or the PTP inhibitor pervanadate (Huyer et al., 1997; Choi et al., 2017). Iodoacetyl-polyethylene glycol-biotin (IAP-biotin) selectively and irreversibly reacts with de-protonated (S-) cysteine thiols (Rudyk and Eaton, 2014). To label basal reduced (active) cellular PTPs, IAP-biotin is added at the time of cell lysis. PTP active site cysteines can be oxidized by ROS to the sulfenic acid form (-SOH), which can then be converted into either sulfenylamide (-SN-) or cysteine disulfide (S-S) forms. Sulfenic acid, sulfenylamide and disulfide oxidized PTPs can be ‘re-activated’ by treatment with thiol reducing agents such as dithiothreitol (DTT) which convert the catalytic cysteine to the active (SH) state. PTP oxidation can be detected indirectly by a three-step method (irreversible alkylation of reduced active site cysteines with an alkylating agent followed by reduction of reversibly oxidized (SOH) active site cysteines with a reducing agent, then finally labeling of the newly formed cysteine thiols) (Weibrecht et al., 2007). This method for direct detection of reversibly oxidized PTPs is not widely used due to the fact that the sulfenic acid state is labile and transient (Karisch and Neel, 2013). PTP catalytic cysteines can also be progressively irreversibly ‘hyper-oxidized’ to the sulfinic (-SO2H) followed by the sulfonic (-SO3H) forms by higher concentrations of ROS or prolonged exposure to ROS. The availability of monoclonal antibodies specific for the sulfonic acid (-SO3H) form of the conserved active site of PTPs has provided a straightforward way to detect oxidized PTPs following treatment of cells with oxidizing agents (Persson et al., 2004). The susceptibility of PTPs in different cell populations to oxidation can be assessed by treatment of cells with oxidizing agents (varying the concentration of oxidizing agent and/or treatment time) followed by blotting of proteins from cell lysates after separation by SDS-PAGE with anti-oxidized-PTP mAb. Here, we describe methods used to detect either reduced (S-; active) or irreversibly oxidized (SO3H; inactive) SHP-1 in thymocytes.
Materials and Reagents
Equipment
Software
Procedure
Data analysis
For quantitation of band density, we used MultiGauge (Fujifilm software). At least three, preferably 4-6 independent experiments were performed for statistical analysis. For statistical analysis, we used GraphPad Prism (GraphPad software). Typically, we provide a representative blot that was used for statistical analysis in the body of the paper and provide all additional blots used for statistical analysis in the supplemental section. Since band densities are normalized to control bands (e.g., total SHP-1 blots), all data should be usable and included unless there were technical problems with a particular gel or blot. Normalization to control total protein bands is considered essential especially if separate blots are included in the statistical analysis. We have also found that the marginal lanes on gels (first and last) can be problematic, so we try to avoid using those two lanes for critical samples. See Choi et al. (2017) for an example of published data from these experiments.
Notes
Recipes
Acknowledgments
This work was supported by the Intramural Research Program of the Eunice Kennedy Shriver, National Institute of Child Health and Human Development (1ZIAHD001803 to P.E.L.). The protocol has been adapted from Choi et al. (2017) Nature Immunology 18, 433-441. The authors have no conflicts of interests or competing interests relating to this publication.
References
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