Fig 2: Inhibiting Csk increases the magnitude of ligand-induced TCR signaling and reduces the threshold for TCR activation.(A) Purified CskAS (AS) or wildtype (WT) CD4+ T cells stimulated for 2 min with 1 µg/ml, 5 µg/ml or 10 µg/ml anti-CD3e antibody in the presence of DMSO or 10 µM 3-IB-PP1 were analyzed by immunoblotting for the phosphorylation of the activation loop tyrosine of Lck and Fyn (Src pY416 antibody) and the inhibitory tyrosine of Lck (Lck pY505), phosphorylated ZAP-70 (ZAP70 pY319), LAT (LAT pY132) and PLC-?1 (PLC?1 pY783), as well as total actin (loading control). Data are representative of at least three independent experiments. (B) Purified total CskAS (AS) or wildtype (WT) T cells stimulated with the indicated dose of anti-CD3e antibody for 2 min in the presence of DMSO or 5 µM, 1 µM or 0.4 µM 3-IB-PP1 were analyzed for phosphorylated ERK (p-ERK) by phosphoflow. Histograms were gated on CD4+ cells. Data are representative of at least three independent experiments for AS cells and two independent experiments for WT cells.DOI: http://dx.doi.org/10.7554/eLife.08088.003

Fig 3: Inhibiting Csk reduces the threshold for TCR activation and prolongs signaling induced by pMHC engagement.(A) Purified CskAS;OTII CD4+ T cells stimulated for 3 min with 5 µg/ml bead-bound control pMHC tetramer or 5 µg/ml, 2.5 µg/ml or 1.25 µg/ml bead-bound OVA pMHC tetramer in the presence of DMSO or 10 µM 3-IB-PP1 were analyzed by immunoblotting for the phosphorylated ZAP-70, LAT and PLC-?1, as well as total actin (loading control). Data are representative of three independent experiments. (B) Purified CskAS;OTII CD4+ T cells stimulated with 5 µg/ml bead-bound control-pMHC tetramer or 2.5 µg/ml bead-bound OVA pMHC tetramer for 2, 5 or 10 min in the presence of DMSO or 5 µM 3-IB-PP1 were analyzed for phosphorylated ERK (p-ERK) by phosphoflow. Histograms were gated on CD4+ Va2+ cells. Data are representative of three independent experiments.DOI: http://dx.doi.org/10.7554/eLife.08088.005

Fig 4: PLC?1 cooperates with Grb2 to regulate LAT microcluster formation. (A) TIRF microscopy revealed that PLC?1 regulates LAT microcluster formation in a nonmonotonic manner. Physiologically relevant concentrations of proteins were used in the assay: LAT at 300 molecules/µm2, Grb2 at 3 µM, Sos1 at 0.3 µM, and PLC?1 at 50 nM. LAT was labeled with Alexa Fluor 488, PLC?1 (SH2-2-3 domains) was labeled with DY547, and Sos1 was labeled with Alexa Fluor 647. Scale bar, 5 µm. (B) Quantification of LAT clustering, membrane recruitment of Sos1. Shown are mean ± SD; n = 3 independent experiments. Unpaired two-tailed t test was used. **, P < 0.01. (C) PLC?1 accelerates LAT cluster formation. TIRF microscopy revealed the time course of LAT microcluster formation in the presence or absence of PLC?1. LAT–Alexa Fluor 488 at 1,000 molecules/µm2 was incubated with 1,000 nM Grb2 and 500 nM Sos1 and/or 50 nM PLC?1 at time 0. Shown are mean ± SEM; n = 3 independent experiments.

Fig 5: PLC?1 protects LAT from dephosphorylation by CD45. (A) Reduced phosphorylation at LAT Y132 in PLC?1-null cells. Cells as indicated were stimulated with 2 µg/ml anti-CD3 and anti-CD28 antibodies for 2 min, lysed, and applied for Western blot analysis. The level of indicated proteins, after being normalized to the level of GAPDH, was quantified. Shown are mean ± SD; n = 3 independent experiments. Unpaired two-tailed t test was used. **, P < 0.01. MW, molecular weight. (B) PLC?1 prevents LAT Y132 from being dephosphorylated. Cells as indicated were pretreated with 0.1 mM vanadate (pan-phosphatase inhibitor) before being stimulated with 2 µg/ml anti-CD3 and anti-CD28 antibodies for 2 min, lysed, and applied for Western blot analysis. The level of indicated proteins, after being normalized to the level of GAPDH, was quantified. Shown are mean ± SD; n = 3 independent experiments. Unpaired two-tailed t test was used. **, P < 0.01; ns, not significant. (C) Schematics of the in intro dephosphorylation assay. (D) PLC?1 prevents LAT Y132 from being dephosphorylated by CD45 in vitro. pLAT, at 1,000 molecules/µm2, was incubated with 1 µM Grb2, 0.5 µM Sos1, and/or 100 nM full-length PLC?1 for 0.5 h. CD45 was then added to dephosphorylate pLAT for 5 min. The reaction was terminated by adding SDS-PAGE loading buffer with 2 mM vanadate. The level of phosphorylated LAT, after being normalized to total LAT, was quantified. Shown are mean ± SD; n = 3 independent experiments.