A modified cysteinyl-labeling assay reveals reversible oxidation of protein tyrosine phosphatases in angiomyolipoma cells

Benoit Boivin, Sheng Zhang, Jack L. Arbiser, Zhong Yin Zhang, Nicholas K. Tonks

Research output: Contribution to journalArticle

71 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)9959-9964
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume105
Issue number29
DOIs
StatePublished - Jul 22 2008

Keywords

  • Cancer
  • Dual specificity phosphatases
  • Reactive oxygen species
  • Signal transduction
  • Tyrosine phosphorylation

ASJC Scopus subject areas

  • Genetics
  • General

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