A modified cysteinyl-labeling assay reveals reversible oxidation of protein tyrosine phosphatases in angiomyolipoma cells.
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.
Boivin
Benoit
B
Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724-2208, USA.
Zhang
Sheng
S
Arbiser
Jack L
JL
Zhang
Zhong-Yin
ZY
Tonks
Nicholas K
NK
D018207:Angiomyolipoma / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D000077214:Becaplermin; D020134:Catalytic Domain; D045744:Cell Line, Tumor; D003545:Cysteine / Q000737:chemistry; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007680:Kidney Neoplasms / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D010084:Oxidation-Reduction; D010982:Platelet-Derived Growth Factor / Q000235:genetics / Q000378:metabolism; D017027:Protein Tyrosine Phosphatases / Q000737:chemistry* / Q000378:metabolism*; D020574:Proto-Oncogene Proteins c-sis; D017382:Reactive Oxygen Species / Q000378:metabolism; D017479:Receptors, Platelet-Derived Growth Factor / Q000737:chemistry / Q000378:metabolism; D015398:Signal Transduction; D014170:Transformation, Genetic
D010982:Platelet-Derived Growth Factor; D020574:Proto-Oncogene Proteins c-sis; D017382:Reactive Oxygen Species; D000077214:Becaplermin; D006861:Hydrogen Peroxide; D017479:Receptors, Platelet-Derived Growth Factor; D017027:Protein Tyrosine Phosphatases; D003545:Cysteine