Fig 2: 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 3: 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)

Fig 4: 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 γ-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 γ-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 5: Heat maps of mathematical mean (average) peptide phosphorylation in lymphocytes from untrained (UT) and endurance-trained (ET) participants. (a) The heat map visualizes the mean phosphorylation of all quality controlled peptides on PTK chips which serve as substrates for all tyrosine kinases. (b) Selections of peptides (out of the complete list of phosphorylable peptides displayed in Fig. 2a) which were used to separately predict the increased upstream kinase activity for FGFR1, FGFR2, FGFR3, FGFR4, and ZAP70 in lymphocytes from ET subjects. (c) The heat map visualizes the mean phosphorylation of all quality controlled peptides on STK chips which serve as substrates for all serine/threonine kinases. (d) Selections of peptides (out of the complete list of phosphorylable peptides displayed in Fig. 2c) which were used to separately predict the decreased upstream kinase activity for IKKα, PKCα, PKG1, PKAα, and p70s6Kβ in lymphocytes from ET subjects. AU: Arbitrary units.