Fig 2: Rasal1 inhibits in vitro anti-CD3 and dendritic cell-peptide induced T-cell activation. a Antigen (OVA323-39)-dependent activation and proliferation of DO11.10 CD4 + cells expressing Flag-tagged Rasal1 compared with control cells at different OVA peptide concentrations (5–10 ng). CTLA-Ig was used as a control. Upper inset shows expression of Flag-tagged Rasal using anti-Flag in blotting (n = 3). b Inhibition of T-cell responses by Rasal1 to increasing anti-CD3 concentrations. Upper inset: effects of Rasal1 at 48 and 72 h post-anti-CD3 activation (n = 3). c Anti-CD3-driven NFATc1/AP1 promoter activity in Jurkat T cells was inhibited by the presence of Flag-tagged Rasal1 (n = 3). d siRNA (esiRNA) knockdown of Rasal1 enhances T-cell proliferation. esiRNA were tested for their knockdown efficiency by western blot (top right panel), esiRNA1 and 2 correspond to 500 nM and 1000 nM concentrations (n = 3). e Contact time between D011.10 CD4+T-cells expressing Flag-Rasal1 (red) or empty vector (blue) and OVA peptide on bone marrow dendritic cells (BMDCs). Dot blot of T cell/DC contact duration time in presence of OVA was unaffected by Flag-tagged Rasal1 or vector transfection (n = 3). Representative plots show mean with ± SD. p-value was calculated by unpaired t test. *p < 0.05, **p < 0.01, ***p < 0.001

Fig 3: Rasal1 binds to the kinase domain of ZAP-70 and inhibits ZAP-70 kinase activity. a Deletion mutants of ZAP-70 that include the V5-tagged N-terminal SH2 domain plus the intervening region, the N-terminal SH2 domain plus the intervening region and the C-terminal-oriented SH2 domain, the N-terminal SH2 domain plus the intervening region and the C-terminal-oriented SH2 domain plus the intervening I-B region and the kinase domain of ZAP-70. Right panel: PCR of ZAP-70 fragments. b The Rasal1 binds to the kinase domain of ZAP-70. Each of the proteins were expressed in 293T cells in the presence of Flag-tagged Rasal1 (left panel). Anti-Rasal1 precipitation resulted in the co-precipitation of full-length ZAP-70 and the kinase domain of ZAP-70 (right panel) (n = 2). c Rasal1 inhibits the kinase activity of ZAP-70 and the phosphorylation of LAT. Recombinant active ZAP-70 from baculovirus was incubated with Flag-LAT from 293T cells as a substrate in the presence of increasing concentrations of GST-Rasal1 and ?-32P ATP for 30 min at 37 °C. Increasing concentrations of GST-Rasal1 inhibited ZAP-70 autophosphorylation and the phosphorylation of LAT (lanes 3–6). Lower panels: input levels of GST-ZAP-70 and LAT were assessed by western blotting (n = 2). d Rasal1 inhibits the kinase activity of ZAP-70 and the phosphorylation of LAT. Recombinant active ZAP-70 from baculovirus was incubated with Flag-LAT from 293T cells as a substrate in the presence of increasing concentrations of GST-Rasal1 and cold ATP for 30 min at 37 °C. Increasing concentrations of GST-Rasal1 inhibited ZAP-70 autophosphorylation and the phosphorylation of LAT (n = 2). Lower panels show blots for Flag-LAT and GST-Rasal1 expression. e Rasal1, but not Bora, inhibits the kinase activity of ZAP-70 and the phosphorylation of LAT. Recombinant active ZAP-70 from baculovirus was incubated with GST-Bora or GST-Rasal1 with ?-32P ATP for 30 min at 37 °C. Increasing concentrations of GST-Rasal1, but not GST-Bora, inhibited ZAP-70 autophosphorylation. GST-Bora (lanes 2–5); GST-Rasal1 (lanes 6–9). f LAT phosphorylation of Y-171 is reduced in the presence of Rasal1 in Jurkat T-cells. Jurkat cells were transfected with Rasal1 and ligated for 5 min with anti-CD3. Rasal1 transfection (lanes 3, 4); resting (anti-IgG) (lanes 1, 3); anti-CD3 (lanes 3, 4) (n = 3). Right panel: histogram showing the quantification of phospho-LAT normalized to total LAT levels in the blot

Fig 4: Rasal1 inhibits the ERK1/2 pathway, but not p38 pathway. a Expression of Rasal1 in T cells inhibits ERK1,2 phosphorylation. Upper and lower panels: two independent experiments where primary mouse T cells were transfected with Flag-tagged Rasal1 and were ligated with anti-CD3 for 5 min followed by blotting with anti-phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204); anti-ERK1/2, anti-ZAP-70 or anti-Flag. Flag-PLK expression served as a negative control (top panel) (n > 2). Upper right panel shows endogenous levels of Rasal1 in activated primary T cells. Lower right panel: histogram showing the inhibition of pERK relative to ERK expression. b Rasal1 expression reduces on ERK, but not p38 activation in activated T cells. Anti-CD3-stimulated (0-15 min) cells were gated on Rasal1-HA+ cells and analyzed. p38 activation analyzed by phospho-p38 staining in response to anti-CD3 stimulation for 0–15 min (lower panel). The data are ± SD and p-value calculated by Student's t test (n > 2). c Rasal1 expression reduces on the phospho-activation of Raf1 and MEK1 in T cells. The phosphorylation of proximal activators of Raf and MEK-1 on phospho-activation sites Raf-Ser338 and MEK-Thr286 were examined. In both cases, anti-CD3 increased phosphorylation in mock transfected cells (lane 2 vs 1). By contrast, the transfection with Flag-Rasal1 reduced the phosphorylation of both mediators (lane 4 vs 3)

Fig 5: TCR complex purification and Rasal1 association with ZAP-70 in the TCR complex. a Analytic gel from tandem affinity purification (TAP) of TCR plus associated proteins captured from TCR-constituted cells (TCR complex) versus TCR-null negative control cells (control) (left panel). Chart showing the list of TCR-associated proteins identified by MS analysis (right upper panel). The structure of Rasal1 (right lower panel). b Co-precipitation of ZAP-70 with TCR from 3A9 T cells. 3A9 cells were stimulated for 10 min with anti-CD3 followed by immunoprecipitation with anti-TCR zeta and blotting with anti-ZAP-70 or anti-zeta. Lane 1: resting T cells; lane 2: anti-CD3-stimulated T cells. c Rasal1 expression is induced with anti-CD3 activation. Relative fold change expression of Rasal1 versus Rasal3 upon anti-CD3 stimulation of CD4+ and CD8 + peripheral T cells. 18 s rRNA served as a control. d Anti-CD3 induces Rasal1 translocation to the membranes of T cells. Stimulation-dependent association of Rasal1 with the membrane (upper panel) versus cytosolic Rasal1 (lower panel). Anti-actin served as a loading control. LAT was used as a marker of membrane enrichment fractionation (n = 3)