title: Quantitative phosphoproteomic analysis reveals a role for serine and threonine kinases in the cytoskeletal reorganization in early T cell receptor activation in human primary T cells.
abstract: Protein phosphorylation-dephosphorylation events play a primary role in regulation of almost all aspects of cell function including signal transduction, cell cycle, or apoptosis. Thus far, T cell phosphoproteomics have focused on analysis of phosphotyrosine residues, and little is known about the role of serine/threonine phosphorylation in early activation of the T cell receptor (TCR). Therefore, we performed a quantitative mass spectrometry-based analysis of the global phosphoproteome of human primary T cells in response to 5 min of TCR activation with anti-CD3 antibody. Combining immunoprecipitation with an antiphosphotyrosine antibody, titanium dioxide phosphopeptide enrichment, isobaric tag for the relative and absolute quantitation methodology, and strong cation exchange separation, we were able to identify 2814 phosphopeptides. These unique sites were employed to investigate the site-specific phosphorylation dynamics. Five hundred and seventeen phosphorylation sites showed TCR-responsive changes. We found that upon 5 min of stimulation of the TCR, specific serine and threonine kinase motifs are overrepresented in the set of responsive phosphorylation sites. These phosphorylation events targeted proteins with many different activities and are present in different subcellular locations. Many of these proteins are involved in intracellular signaling cascades related mainly to cytoskeletal reorganization and regulation of small GTPase-mediated signal transduction, probably involved in the formation of the immune synapse.
authors.affiliation: Department of Pharmaceutical Chemistry, Mass Spectrometry Facility, School of Pharmacy, University of California San Francisco, San Francisco, California 94158, USA.
authors.forename: Patricia
authors.initials: P
authors.lastname: Ruperez
authors.forename: Ana
authors.initials: A
authors.lastname: Gago-Martinez
authors.forename: A L
authors.initials: AL
authors.lastname: Burlingame
authors.forename: Juan A
authors.initials: JA
authors.lastname: Oses-Prieto
mesh_terms: D000595:Amino Acid Sequence; D000961:Antilymphocyte Serum / Q000502:physiology; D017252:CD3 Complex / Q000276:immunology; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D016000:Cluster Analysis; D016384:Consensus Sequence; D003599:Cytoskeleton / Q000378:metabolism*; D006801:Humans; D008213:Lymphocyte Activation; D010446:Peptide Fragments / Q000737:chemistry; D010750:Phosphoproteins / Q000302:isolation & purification / Q000378:metabolism*; D061251:Primary Cell Culture; D060066:Protein Interaction Maps; D017346:Protein Serine-Threonine Kinases / Q000502:physiology*; D020543:Proteome / Q000302:isolation & purification / Q000378:metabolism; D040901:Proteomics; D011948:Receptors, Antigen, T-Cell / Q000378:metabolism*; D015398:Signal Transduction; D013601:T-Lymphocytes / Q000276:immunology / Q000378:metabolism*
chemical_list: D000961:Antilymphocyte Serum; D017252:CD3 Complex; D010446:Peptide Fragments; D010750:Phosphoproteins; D020543:Proteome; D011948:Receptors, Antigen, T-Cell; D017346:Protein Serine-Threonine Kinases