In Vitro Activity Assays to Quantitatively Assess the Endogenous Reversible Oxidation State of Protein Tyrosine Phosphatases in Cells

Abstract

The reversible oxidation of protein tyrosine phosphatases (PTPs) impairs their ability to dephosphorylate substrates in vivo. This transient inactivation of PTPs occurs as their conserved catalytic cysteine residue reacts with cellular oxidants thereby abolishing the ability of this reactive cysteine to attack the phosphate of the target substrate. Hence, in vivo, the inhibition of specific PTPs in response to regulated and localized rises in cellular oxidants enables phospho‐dependent signaling. We present assays that measure the endogenous activity of specific PTPs that become transiently inactivated in cells exposed to growth factors. Here, we describe the methods and highlight the pitfalls to avoid post‐lysis oxidation of PTPs in order to assess the inactivation and the reactivation of PTPs targeted by cellular oxidants in signal transduction. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Cell transfection (optional) Support Protocol: Preparation of degassed lysis buffers Basic Protocol 2: Cellular extraction in anaerobic conditions Basic Protocol 3: Enrichment and activity assay of specific PTPs Alternate Protocol: Measurement of active PTPs via direct cysteinyl labeling

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“…For PTP oxidation, ventricles were pulverized under liquid N 2 in an argon-filled hypoxic glove box. The powder was resuspended using a Potter-Elvehjem tissue grinder (10 strokes), in 1 ml of ice-cold degassed lysis buffer [25 mM Hepes (pH 7.45), 100 mM NaCl, 0.25% (v/v) deoxycholic acid, 1% Surfact-Amp NP-40, superoxide dismutase (100 U/ml), catalase (100 U/ml), and cOmplete protease inhibitor cocktail] ( 65 ) . Lysis was completed by mixing for 30 min at 4°C on a clinical rotator.…”