Fig 2: S-acylation of ZAP-70 is required for T cell activation.A, IL-2 production by P116 (ZAP-70 -/-) Jurkat T cells stably rescued with WT or acylation-deficient C564R ZAP-70. IL-2 concentrations were measured by ELISA in supernatants from resting cells or cells stimulated for 24 h with plate-bound anti-CD3 antibody. Data shown are representative of three independent biological repeats and represented as mean ± SEM. B and C, Expression of CD25 and CD69 T cell surface activation markers by P116 stably rescued with WT or C564R ZAP-70. Cells were stimulated for 24 h with plate-bound anti-CD3 antibody and analyzed by flow cytometry. D and E, Quantification of CD25 and CD69 expression measured by flow cytometry. Data shown are pooled from three independent biological repeats and represented as mean ± SEM.

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 binds to the immune cell tyrosine kinase ZAP-70. a Proximity ligation assay (PLA) of Rasal1 and ZAP-70 in Jurkat T-cells stained with DAPI, anti-Rasal1, and anti-ZAP-70. Positive PLA signal (fluorescent dots) indicates a distance of 30–40 nm between Rasal1 and ZAP-70 in anti-CD3-activated T cells, indicating their close proximity (n = 2). b Anti-Rasal1 co-precipitates ZAP-70 from primary mouse T cells. Lane 1–2 loading controls, lanes 3-4: anti-Rasal1 IP: lanes 2 and 4. Cells were stimulated with anti-CD3 ligation for 5 min vs IgG control ligation in lanes 1 and 3. c Anti-Rasal1 co-precipitates ZAP-70 from transfected Jurkat T cells. Lanes 1: nonligated (i.e., IgG); lane 2: anti-CD3 ligated T cells for 5 min. Lower panel shows similar level of ZAP-70 expression in lysates of (n = 2)

Fig 5: S-acylation of ZAP-70 is required for proximal TCR signaling.A, Phosphorylation of proximal TCR signaling proteins in P116 (ZAP-70 -/-) Jurkat T cells stably rescued with WT or acylation-deficient C564R ZAP-70. Cells were stimulated with anti-CD3 antibody for the indicated time points, and phosphorylation of TCR signaling proteins was assayed by immunoblotting. Total protein levels shown as the loading control. Data shown are representative of three independent biological repeats. B, Quantifications showing robust increase in phosphorylated ERK1/2 (pERK1/2) in cells stably rescued with WT ZAP-70 in response to TCR stimulation. ZAP-70-deficient cells as well as cells stably rescued with S-acylation-deficient C564R ZAP-70 failed to show activation of ERK1/2 in response to TCR stimulation. Data shown are representative of three independent biological repeats and represented as mean ± SEM. C, TCR-dependent calcium release in P116 cells stably rescued with WT or C564R ZAP-70. Shown are representative single-cell responses measured by Fura-2 imaging. D, Quantification of peak calcium influx in response to anti-CD3 stimulation. Data shown are representative of three independent biological repeats and represented as mean ± SEM.