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Revisiting the Timing of Action of the PAG Adaptor Using Quantitative Proteomics Analysis of Primary T Cells
- Source :
- Journal of Immunology, Journal of Immunology, 2015, 195 (11), pp.5472-5481. ⟨10.4049/jimmunol.1501300⟩, Journal of Immunology, Publisher : Baltimore : Williams & Wilkins, c1950-. Latest Publisher : Bethesda, MD : American Association of Immunologists, 2015, 195 (11), pp.5472-5481. ⟨10.4049/jimmunol.1501300⟩
- Publication Year :
- 2015
- Publisher :
- The American Association of Immunologists, 2015.
-
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.
- Subjects :
- CD4-Positive T-Lymphocytes
Proteomics
[SDV]Life Sciences [q-bio]
T cell
Primary Cell Culture
Immunology
Receptors, Antigen, T-Cell
Protein tyrosine phosphatase
Biology
Lymphocyte Activation
Jurkat cells
CSK Tyrosine-Protein Kinase
Mice
chemistry.chemical_compound
Tandem Mass Spectrometry
medicine
Animals
Immunology and Allergy
Gene Knock-In Techniques
Phosphorylation
Cells, Cultured
Embryonic Stem Cells
ComputingMilieux_MISCELLANEOUS
Thymocytes
Inositol Polyphosphate 5-Phosphatases
T-cell receptor
Membrane Proteins
Protein Tyrosine Phosphatase, Non-Receptor Type 22
Tyrosine phosphorylation
Phosphoproteins
Molecular biology
Phosphoric Monoester Hydrolases
Enzyme Activation
Mice, Inbred C57BL
src-Family Kinases
medicine.anatomical_structure
nervous system
chemistry
Lymphocyte Specific Protein Tyrosine Kinase p56(lck)
Multiprotein Complexes
Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases
Intercellular Signaling Peptides and Proteins
Tyrosine kinase
Proto-oncogene tyrosine-protein kinase Src
Subjects
Details
- ISSN :
- 15506606 and 00221767
- Volume :
- 195
- Database :
- OpenAIRE
- Journal :
- The Journal of Immunology
- Accession number :
- edsair.doi.dedup.....4f25b9846c09f19d8ddf94fec8075051