A modified cysteinyl-labeling assay reveals reversible oxidation of classical and dual specificity phosphatases in angiomyolipoma cells

Boivin, B., Zhang, S. H., Arbiser, J. L., Zhang, Z.-Y., Tonks, N. K. (July 2008) A modified cysteinyl-labeling assay reveals reversible oxidation of classical and dual specificity phosphatases in angiomyolipoma cells. Proc Natl Acad Sci U S A, 105 (29). pp. 9959-9964.

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URL: http://www.ncbi.nlm.nih.gov/pubmed/18632564
DOI: 10.1073/pnas.0804336105

Abstract

The production of reactive oxygen species (ROS) exerts an additional tier of control over tyrosine phosphorylation-dependent signal transduction by transiently inhibiting the catalytic activity of specific protein tyrosine phosphatases (PTPs). Hence, the ability to detect reversible oxidation of PTPs in vivo is critical to understanding the complex biological role of ROS in the control of cellular signaling. Here, we describe an assay for identifying those PTPs that are reversibly oxidized in vivo, which utilizes the unique chemistry of the invariant catalytic Cys residue in labeling the active site with biotinylated small molecules under mildly acidic conditions. We have applied this cysteinyl-labeling assay to the study of platelet-derived growth factor (PDGF) receptor signaling in an angiomyolipoma cell model. Doing so has allowed us to detect reversible oxidation of several proteins in response to sustained PDGF stimulation. As in other cell systems, we have observed the reversible oxidation of the classical PTP SHP2 and the tumor suppressor phosphatase PTEN in response to PDGF stimulation. Furthermore, we detected reversible oxidation of members of two other subclasses of PTPs, the receptor PTP LAR and the dual-specificity phosphatase MKP1. These data demonstrate the broad selectivity of the assay, allowing us to detect representatives of all of the major subgroups of the PTP superfamily. We anticipate that this cysteinyl-labeling enrichment strategy can be applied broadly to study reversible oxidation as a mechanism of harnessing PTP catalytic activity in a variety of signaling pathways.

Item Type: Paper
Uncontrolled Keywords: reactive oxygen species signal transduction tyrosine phosphorylation dual specificity phosphatases cancer
Subjects: bioinformatics > genomics and proteomics > genetics & nucleic acid processing
bioinformatics > genomics and proteomics
bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification
bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification > protein types > enzymes
bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification > protein types
bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification > protein types > enzymes > protein tyrosine phosphatase
CSHL Authors:
Communities: CSHL labs > Tonks lab
Depositing User: Matt Covey
Date: 22 July 2008
Date Deposited: 26 Feb 2013 16:12
Last Modified: 08 Nov 2017 21:32
PMCID: PMC2481340
Related URLs:
URI: http://repository.cshl.edu/id/eprint/27573

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