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. T lymphocytes are able to recognize specific antigenic peptides presented by molecules of the major histocompatibility complex on the surface of other cell types. This interaction is mediated by a dimeric specialized molecule called T cell receptor (TCR), 1 which is part of a larger membrane complex in association with CD3 ␥, ␦, , and chains. The binding between TCR and the major histocompatibility complex-antigen is of relatively low affinity, and it is stabilized by the association with co-receptors (CD4 or CD8). All of these molecules in turn recruit, via their intracellular domains, different polypeptides to carry out signal transduction. In addition to antigen recognition, coactivation by CD28 is required to trigger full activation of the T cell, which expresses then different cell surface molecules and releases soluble mediators (cytokines) that promote changes in the activity of different target cell types (1).During the TCR-major histocompatibility complex-antigen recognition, T cells undergo considerable membrane and cytoskeletal rearrangements that lead to the formation of the immunological synapse (IS). During this maturation, precise molecular reorganizations occur at the interface between T cells and an antigen presenting cell. Cell motility, polarization, and receptor relocalization events are dependent on the lymphocyte cytoskeleton and are necessary for the maturation of the IS. TCR, co-receptors, intracellular signaling molecules, and adhesion receptors polarize to the IS and form small aggregates known as microclusters (2, 3), processes all dependent on functional microtubule and actin cytoskeleton. This results in the stabilization and functional maturation of the signaling complexes.Protein phosphorylation...

Microtubule interfering agents (MIAs) are anti-tumor drugs that inhibit microtubulearrest and cell death [3,5]. Nowadays, the most used MIAs for 49 cancer treatment are vinca alkaloids (VAs) and taxanes [6]. 50VAs are drugs derived from the periwinkle Catharanthus Gel spots were subjected to in-gel digestion (http://msfacility. 161ucsf.edu/ingel.html) with trypsin (porcine, side-chain removed using a Y-10 microcone (Millipore), proteins were (Fig. 1a). induced forms are phosphorylated (Fig. 2). Spot x also disappeared 246 after λ-PPase treatment and therefore, this form is also con-247 sidered as phosphorylated (Fig. 2) (Fig. 3a). 260These phosphorylations were also confirmed by Western blot 261 analysis (see Fig. 1 of supplementary material). As expected, 262the flow cytometry analysis of these cells showed that all the 263 compounds used induced G2/M arrest and cell death (Fig. 3b). 264Furthermore, a Western blot analysis using antibodies against 265 the molecular marker phospho-histone H3 indicated that the 266 G2/M arrest induced by these agents is at M stage (Fig. 3c). (Fig. 4b) and M phase arrest (Fig. 4c). More interestingly, (Fig. 4a) (Fig. 5a). This downregulation is coupled to a reduction in the 296 number of M phase cells (Fig. 5c) Aphidicolin was added 24 h prior to MIA treatment. Then, cell 312 extracts were subjected to 2D-PAGE followed by Western blot. (Fig. 7b). This drug also induces the spots x, y and z which 337 correspond to processed forms of the protein (Fig. 7b). 338Roscovitine treatment induced p54 nrb processing but did not 339 affect its phosphorylation (Fig. 7b). Interestingly, the addition of (Fig. 8a). In addition, a Western blot analysis using antibodies 354 against phospho-histone H3 indicated that MIAs also cause M 355 arrest in this cell line (Fig. 8b). The KSP inhibitor STLC also 356 induced the phosphorylation of this nuclear factor (see Fig. 2 357 of supplementary material). 4. Discussion