title: Revisiting the Timing of Action of the PAG Adaptor Using Quantitative Proteomics Analysis of Primary T Cells.
abstract: The protein tyrosine kinase LCK plays a key role in TCR signaling, and its activity is dynamically controlled by the tyrosine kinase C-terminal Src kinase (CSK) and the tyrosine phosphatase CD45. CSK is brought in contiguity to LCK via binding to a transmembrane adaptor known as phosphoprotein associated with glycosphingolipid-enriched microdomains (PAG). The lack of a blatant phenotype in PAG-deficient mice has impeded our understanding of the mechanisms through which PAG exerts its negative-regulatory role in TCR signaling. We used quantitative mass spectrometry and both thymocytes and CD4(+) T cells from mice in which a tag for affinity purification was knocked in the gene coding for PAG to determine the composition and dynamics of the multiprotein complexes that are found around PAG over 5 min of activation. Most of the high-confidence interactions that we observed were previously unknown. Using phosphoproteomic analysis, PAG showed low levels of tyrosine phosphorylation in resting primary mouse CD4(+) T cells; the levels of tyrosine phosphorylation increased and reached a maximum 2 min after stimulation. Analysis of the dynamics of association of the protein tyrosine phosphatase PTPN22 and lipid phosphatase SHIP-1 with PAG following T cell activation suggests that both cooperate with CSK to terminate T cell activation. Our findings provide a model of the role for PAG in mouse primary CD4(+) T cells that is consistent with recent phosphoproteomic studies of the Jurkat T cell line but difficult to reconcile with former biochemical studies indicating that PAG is constitutively phosphorylated in resting T cells and rapidly dephosphorylated once the TCR is engaged.
authors.affiliation: Centre d'Immunologie de Marseille-Luminy, Aix Marseille Université UM2, INSERM, U1104, CNRS UMR 7280, 13288 Marseille, France;
authors.forename: Kavita
authors.identifier: http://orcid.org/0000-0003-1530-5934
authors.initials: K
authors.lastname: Reginald
authors.affiliation: Institut de Pharmacologie et de Biologie Structurale, Université de Toulouse, CNRS UMR 5089, 31077 Toulouse Cedex, France;
authors.forename: Karima
authors.initials: K
authors.lastname: Chaoui
authors.affiliation: Centre d'Immunologie de Marseille-Luminy, Aix Marseille Université UM2, INSERM, U1104, CNRS UMR 7280, 13288 Marseille, France;
authors.forename: Romain
authors.initials: R
authors.lastname: Roncagalli
authors.affiliation: Institut de Pharmacologie et de Biologie Structurale, Université de Toulouse, CNRS UMR 5089, 31077 Toulouse Cedex, France;
authors.forename: Mathilde
authors.initials: M
authors.lastname: Beau
authors.affiliation: Centre d'Immunologie de Marseille-Luminy, Aix Marseille Université UM2, INSERM, U1104, CNRS UMR 7280, 13288 Marseille, France;
authors.forename: Marisa
authors.initials: M
authors.lastname: Goncalves Menoita
authors.affiliation: Institut de Pharmacologie et de Biologie Structurale, Université de Toulouse, CNRS UMR 5089, 31077 Toulouse Cedex, France;
authors.forename: Bernard
authors.initials: B
authors.lastname: Monsarrat
authors.affiliation: Institut de Pharmacologie et de Biologie Structurale, Université de Toulouse, CNRS UMR 5089, 31077 Toulouse Cedex, France;
authors.forename: Odile
authors.initials: O
authors.lastname: Burlet-Schiltz
authors.affiliation: Centre d'Immunologie de Marseille-Luminy, Aix Marseille Université UM2, INSERM, U1104, CNRS UMR 7280, 13288 Marseille, France;
authors.forename: Marie
authors.initials: M
authors.lastname: Malissen
authors.affiliation: Institut de Pharmacologie et de Biologie Structurale, Université de Toulouse, CNRS UMR 5089, 31077 Toulouse Cedex, France;
authors.forename: Anne
authors.initials: A
authors.lastname: Gonzalez de Peredo
authors.affiliation: Centre d'Immunologie de Marseille-Luminy, Aix Marseille Université UM2, INSERM, U1104, CNRS UMR 7280, 13288 Marseille, France; Centre d'Immunophénomique, Aix Marseille Université UM2, INSERM, US012, CNRS UMS3367, 13288 Marseille, France bernardm@ciml.univ-mrs.fr.
authors.forename: Bernard
authors.identifier: http://orcid.org/0000-0003-1340-9342
authors.initials: B
authors.lastname: Malissen
mesh_terms: D000818:Animals; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology*; D000081247:CSK Tyrosine-Protein Kinase; D002478:Cells, Cultured; D053595:Embryonic Stem Cells; D004789:Enzyme Activation; D055879:Gene Knock-In Techniques; D000072181:Inositol Polyphosphate 5-Phosphatases; D036341:Intercellular Signaling Peptides and Proteins; D008213:Lymphocyte Activation / Q000276:immunology; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck) / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D046912:Multiprotein Complexes / Q000235:genetics / Q000378:metabolism*; D000072183:Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism; D010766:Phosphorylation; D061251:Primary Cell Culture; D054596:Protein Tyrosine Phosphatase, Non-Receptor Type 22 / Q000378:metabolism; D040901:Proteomics; D011948:Receptors, Antigen, T-Cell / Q000276:immunology; D053719:Tandem Mass Spectrometry; D060168:Thymocytes / Q000276:immunology*; D019061:src-Family Kinases / Q000378:metabolism*
chemical_list: D036341:Intercellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D046912:Multiprotein Complexes; C408344:Pag protein, mouse; D010750:Phosphoproteins; D011948:Receptors, Antigen, T-Cell; D000081247:CSK Tyrosine-Protein Kinase; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D019061:src-Family Kinases; D010744:Phosphoric Monoester Hydrolases; D054596:Protein Tyrosine Phosphatase, Non-Receptor Type 22; C516766:Ptpn22 protein, mouse; D000072181:Inositol Polyphosphate 5-Phosphatases; C000606433:Inpp5d protein, mouse; D000072183:Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases