title: A RP-UFLC Assay for Protein Tyrosine Phosphatases: Focus on Protein Tyrosine Phosphatase Non-Receptor Type 2 (PTPN2).
abstract: 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.
authors.affiliation: Université Paris Diderot, Sorbonne Paris Cité, Unité de Biologie Fonctionnelle et Adaptative, CNRS UMR 8251, 75013, Paris, France.
authors.forename: Romain
authors.initials: R
authors.lastname: Duval
authors.affiliation: Université Paris Diderot, Sorbonne Paris Cité, Unité de Biologie Fonctionnelle et Adaptative, CNRS UMR 8251, 75013, Paris, France.
authors.forename: Linh-Chi
authors.initials: LC
authors.lastname: Bui
authors.affiliation: Université Paris Diderot, Sorbonne Paris Cité, Unité de Biologie Fonctionnelle et Adaptative, CNRS UMR 8251, 75013, Paris, France.
authors.forename: Jérémy
authors.initials: J
authors.lastname: Berthelet
authors.affiliation: Université Paris Diderot, Sorbonne Paris Cité, Unité de Biologie Fonctionnelle et Adaptative, CNRS UMR 8251, 75013, Paris, France.
authors.forename: Julien
authors.initials: J
authors.lastname: Dairou
authors.affiliation: Université Paris Diderot, Sorbonne Paris Cité, Unité de Biologie Fonctionnelle et Adaptative, CNRS UMR 8251, 75013, Paris, France.
authors.forename: Cécile
authors.initials: C
authors.lastname: Mathieu
authors.affiliation: Université Paris Diderot, Sorbonne Paris Cité, INSERM UMR_S1131, Institut Universitaire d'Hématologie, 75010 Paris, France.
authors.forename: Fabien
authors.initials: F
authors.lastname: Guidez
authors.affiliation: Université Paris Diderot, Sorbonne Paris Cité, Unité de Biologie Fonctionnelle et Adaptative, CNRS UMR 8251, 75013, Paris, France.
authors.forename: Jean-Marie
authors.initials: JM
authors.lastname: Dupret
authors.affiliation: VIB Center for the Biology of Disease, Leuven, Belgium.
authors.forename: Jan
authors.initials: J
authors.lastname: Cools
authors.affiliation: 1] Université Paris Diderot, Sorbonne Paris Cité, INSERM UMR_S1131, Institut Universitaire d'Hématologie, 75010 Paris, France [2] Service de Biologie Cellulaire, Assistance Publique des Hôpitaux de Paris (AP-HP), Hôpital Saint Louis, 75010 Paris, France.
authors.forename: Christine
authors.initials: C
authors.lastname: Chomienne
authors.affiliation: Université Paris Diderot, Sorbonne Paris Cité, Unité de Biologie Fonctionnelle et Adaptative, CNRS UMR 8251, 75013, Paris, France.
authors.forename: Fernando
authors.initials: F
authors.lastname: Rodrigues-Lima
mesh_terms: D045744:Cell Line, Tumor; D002853:Chromatography, Liquid / Q000379:methods*; D056148:Chromatography, Reverse-Phase / Q000379:methods*; D004789:Enzyme Activation / Q000187:drug effects; D057075:Enzyme Assays / Q000379:methods*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007700:Kinetics; D010455:Peptides / Q000378:metabolism; D010766:Phosphorylation; D054578:Protein Tyrosine Phosphatase, Non-Receptor Type 2 / Q000378:metabolism*; D011994:Recombinant Proteins; D013379:Substrate Specificity; D013450:Sulfones / Q000494:pharmacology; D014638:Vanadates / Q000494:pharmacology
chemical_list: D004791:Enzyme Inhibitors; D010455:Peptides; D011994:Recombinant Proteins; D013450:Sulfones; C512310:phenyl vinyl sulfone; D014638:Vanadates; D006861:Hydrogen Peroxide; D054578:Protein Tyrosine Phosphatase, Non-Receptor Type 2