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

Content Provider	PubMed Central
Author	Boivin, Benoit Zhang, Sheng Arbiser, Jack L. Zhang, Zhong-yin Tonks, Nicholas K.
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.
Related Links	http://dx.doi.org/10.1073/pnas.0804336105
Ending Page	9964
Page Count	6
Starting Page	9959
File Format	PDF
ISSN	00278424
e-ISSN	10916490
Journal	Proceedings of the National Academy of Sciences of the United States of America
Issue Number	29
Volume Number	105
Language	English
Publisher	National Academy of Sciences
Publisher Date	2008-07-22
Access Restriction	Open
Rights Holder	National Academy of Sciences
Subject Keyword	General Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Multidisciplinary

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