A RP-UFLC Assay for Protein Tyrosine Phosphatases: Focus on Protein Tyrosine Phosphatase Non-Receptor Type 2 (PTPN2).
Fiche publication
Date publication
juin 2015
Journal
Scientific reports
Auteurs
Membres identifiés du Cancéropôle Est :
Dr GUIDEZ Fabien
Tous les auteurs :
Duval R, Bui LC, Berthelet J, Dairou J, Mathieu C, Guidez F, Dupret JM, Cools J, Chomienne C, Rodrigues-Lima F
Lien Pubmed
Résumé
Protein tyrosine phosphatases (PTPs) are involved in numerous signaling pathways and dysfunctions of certain of these enzymes have been linked to several human diseases including cancer and autoimmune diseases. PTPN2 is a PTP mainly expressed in hematopoietic cells and involved in growth factor and JAK/STAT signaling pathways. Loss of function analyses in patients with mutation/deletion of the PTPN2 gene and knock-out mouse models indicate that PTPN2 acts as a tumor suppressor in T-cell malignancies and as a regulator of inflammation and immunity. The use of sensitive and quantitative assays is of prime importance to better characterize the biochemical properties of PTPN2 and its biological roles. We report a highly sensitive non-radioactive assay that allows the measurement of the activity of purified PTPN2 and of endogenous PTPN2 immunoprecipitated on agarose beads. The assay relies on separation and quantitation by reverse-phase ultra fast liquid chromatography (RP-UFLC) of a fluorescein-labeled phosphotyrosine peptide substrate derived from the sequence of STAT1. The applicability and reliability of this approach is supported by kinetic and mechanistic studies using PTP inhibitors. More broadly, our PTPN2 assay provides the basis for the design of flexible methods for the measurement of other PTPs.
Mots clés
Cell Line, Tumor, Chromatography, Liquid, methods, Chromatography, Reverse-Phase, methods, Enzyme Activation, drug effects, Enzyme Assays, methods, Enzyme Inhibitors, pharmacology, Humans, Hydrogen Peroxide, pharmacology, Kinetics, Peptides, metabolism, Phosphorylation, Protein Tyrosine Phosphatase, Non-Receptor Type 2, metabolism, Recombinant Proteins, Substrate Specificity, Sulfones, pharmacology, Vanadates, pharmacology
Référence
Sci Rep. 2015 06 4;5:10750