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In Vitro Activity Assays to Quantitatively Assess the Endogenous Reversible Oxidation State of Protein Tyrosine
Avinash D Londhe1, Syed H M Rizvi1, Benoit Boivin1
1Department of Nanobioscience, SUNY Polytechnic Institute, Albany, New York.
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.
Insights
Protein tyrosine phosphatases (PTPs) are reversibly inactivated by oxidation, a process crucial for cellular signaling. New assays measure PTP activity changes in cells, detailing methods to avoid artifactual oxidation during analysis.
Area of Science:
- Biochemistry
- Cellular Signaling
- Enzymology
Background:
- Protein tyrosine phosphatases (PTPs) regulate cellular processes by removing phosphate groups from tyrosine residues.
- Reversible oxidation of a catalytic cysteine residue in PTPs leads to their transient inactivation.
- This inactivation is a key mechanism for enabling phospho-dependent signaling pathways in response to cellular oxidants.
Purpose of the Study:
- To present novel assays for measuring the endogenous activity of specific PTPs.
- To assess the transient inactivation and reactivation of PTPs under cellular oxidative stress.
- To provide methods that avoid post-lysis oxidation artifacts when studying PTP inactivation.
Main Methods:
- Development and description of assays to measure endogenous PTP activity.
- Cellular extraction under anaerobic conditions to preserve PTP activity.
- Protocols for PTP enrichment, activity assays, and direct cysteinyl labeling for active PTP measurement.
Main Results:
- Demonstration of transient PTP inactivation in cells exposed to growth factors.
- Validation of methods to accurately assess PTP inactivation and reactivation.
- Identification of critical steps to prevent artifactual oxidation during sample preparation.
Conclusions:
- Oxidative inactivation of PTPs is a critical regulatory mechanism in signal transduction.
- The presented assays enable accurate measurement of PTP activity changes in vivo.
- Proper methodology is essential to study PTP regulation by cellular oxidants.
