Analysis of the redox regulation of protein tyrosine phosphatase superfamily members utilizing a cysteinyl-labeling assay.
The catalytic activity of protein tyrosine phosphatase (PTP) superfamily members is regulated by the reversible oxidation of their invariant catalytic Cys residue in vivo. Transient and specific regulation of PTP activity by reactive oxygen species (ROS) attenuates dephosphorylation and, thereby, promotes phosphorylation, hence facilitating signal transduction. We have recently developed a modified cysteinyl-labeling assay [Boivin, B., Zhang, S., Arbiser, J. L., Zhang, Z. Y., and Tonks, N. K. (2008). Proc. Natl. Acad. Sci. USA105, 9959-9964.] that showed broad selectivity in detecting reversible oxidation of members from different PTP subclasses in platelet-derived growth factor (PDGF)-BB overexpressing cells. Herein, we applied this assay, which utilizes the unique chemistry of the invariant catalytic Cys residue to enrich and identify PTPs that are reversibly oxidized upon acute growth factor stimulation. Performing the cysteinyl-labeling assay with Rat-1 fibroblasts enabled us to capture both PTEN and SHP-2 as a consequence to acute PDGF-BB stimulation. Given the ability of this assay to detect reversible oxidation of a broad array of members of the PTP family, we anticipate that it should permit profiling of the entire ROS-regulated PTPome in a wide array of signaling paradigms.
Boivin
Benoit
B
Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, USA.
Tonks
Nicholas K
NK
D000818:Animals; D001681:Biological Assay / Q000379:methods*; D003545:Cysteine / Q000737:chemistry*; D006801:Humans; D010084:Oxidation-Reduction; D017027:Protein Tyrosine Phosphatases / Q000032:analysis* / Q000737:chemistry / Q000145:classification; D013194:Staining and Labeling / Q000379:methods*
D017027:Protein Tyrosine Phosphatases; D003545:Cysteine