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J Immunol. 2015 Dec 1;195(11):5472-81. doi: 10.4049/jimmunol.1501300. Epub 2015 Oct 28.

Revisiting the Timing of Action of the PAG Adaptor Using Quantitative Proteomics Analysis of Primary T Cells.

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Centre d'Immunologie de Marseille-Luminy, Aix Marseille Université UM2, INSERM, U1104, CNRS UMR 7280, 13288 Marseille, France;
Institut de Pharmacologie et de Biologie Structurale, Université de Toulouse, CNRS UMR 5089, 31077 Toulouse Cedex, France;
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


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.

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