Fig 2: PSD-95 enhances the binding and tyrosine phosphorylation of DHHC5 by Fyn kinase.(a–d) Fourteen DIV hippocampal neurons were stimulated, lysed at the indicated time points, immunoprecipitated with anti- (a) DHHC5 or (c) Fyn and blots probed with the indicated antibodies. (a,b) DHHC5 tyrosine phosphorylation (phY; P=0.007, F6,14=7.749) and DHHC5/Fyn interactions (P<0.001, F6,14=11.06) are decreased transiently, whereas DHHC5/AP2µ interactions are increased transiently following cLTP (P=0.0076, F6,14=7.78). (c,d) Non-phosphoryated tyrosine 420 Fyn levels (P=0.0001, F6,14=11.24) and STEP61/Fyn interactions (P=0.004, F6,14=5.549) are transiently increased following cLTP. (e–i) HEK293T cells were transfected with the indicated HA-DHHC5, PSD-95-GFP or Fyn constructs for 36 h. (e,f) PSD-95 enhances Fyn/DHHC5 WT but not Fyn/DHHC5 ?PDZb interactions (P=0.0014, F2,6=24.11). (e,g) PSD-95 enhances Fyn-mediated phosphorylation of DHHC5 WT but not DHHC5 ?PDZb (P=0.0104, F2,6=10.75). (h,i) Lysates were biotinylated and immunoprecipitated with neutravidin-coated beads and blots probed with the indicated antibodies. (i) Normalized intensity of DHHC5 in the surface fraction (P<0.001, F7,16=43.23). (b,d,f,g,i) n=3 blots from 3 separate cultures. All graphs display mean±s.e.m. */#P<0.05, **/##P<0.01, ***P<0.001; one-way analysis of variance; Tukey's post-hoc test. Asterisks and cross-hatches above data points indicate significance relative to unstimulated controls for (b) DHHC5 phY or AP2µ, and Fyn or (d) Y420-Fyn and STEP61, respectively. (a,c,e,h) Five per cent of whole-cell lysates were loaded as inputs and (a,c) are from the same blots but with different exposure times. Full-length blots are presented in Supplementary Fig. 5.

Fig 3: Phosphorylation of DHHC5 regulates its association with endocytic proteins and its subcellular localization.(a) Schematic depiction of DHHC5 constructs N-terminally tagged with GFP or HA (not shown here) and illustrating the approximate localization of transmembrane domains (grey boxes), the DHHC motif, a putative Fyn-binding site (dashed line; RLLPTGP), a putative AP2µ-binding site (dashed line; YDNL) and the PDZ-binding motif required for binding PSD-95 (solid line; EISV). (b–e) HEK293T cells were transfected with the indicated HA-DHHC5 and Fyn constructs for 36 h, lysates immunoprecipitated with an HA antibody and blots probed with the indicated antibodies. (b,c) Fyn binding of the DHHC5 P520,3A mutant is reduced, but not for for the Y533E mutant (P=0.0153, F2,6=9.07). (b,d) Fyn-mediated tyrosine phosphorylation is attenuated in DHHC5 P520,3A and Y533E mutants (P<0.001, F5,12=20.04). (b,e) Fyn decreases AP2µ association with DHHC5 WT, but not P520,3A or Y533E mutants (P=0.018, F5,12=6.29). n=3 blots from 3 separate cultures. (f) Confocal images of 14 DIV neurons transfected with the indicated GFP–DHHC5 construct and immunostained for TfR and VGluT1. Scale bar, 5 µm. (g) DHHC5 P520,3A increases and Y533E decreases co-localization with TfR (P<0.001, F2,58=24.05), and (h) decreases and increases co-localization with VGlut1, respectively (P<0.001, F2,58=23.5). n=22 (WT), 18 (P520,3A) and 21 (Y533E) cells from 3 cultures. Co-localized puncta are denoted by white arrowheads. All graphs display mean±s.e.m. *P<0.05, **P<0.01, ***P<0.001; one-way analysis of variance; Tukey's post-hoc test. (b) Five per cent of whole-cell lysates were loaded as inputs. Full-length blots are presented in Supplementary Fig. 5.

Fig 4: Coagonist pMHC enhance the recruitment of pCD3?, pLck, and pZap70 into the immunological synapse. Different concentrations of E183-A2 monomer or GAG-A2 monomer were added to the lipid bilayers in a glass chamber slide. Totally, 105 E183 CTL were added into each well of the chamber and stimulated for 5 min. The CTL were fixed, permeabilized and stained with different antibodies to check the recruitment of pCD3? (a, b), pLck (c, d), and pZap70 (e, f) into the immunological synapse by TIRF microscopy. Anti-pSrc (Y416) antibody was used to detect active Lck (p-Y394). Statistical significance was determined by using unpaired two-sided Student’s t test and shown as mean ± s.d. (ns nonsignificant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). Data are representative of three independent experiments

Fig 5: Analog amplifier model for the mechanism of coagonism. Nonstimulatory pMHC molecules recruit CD8-Lck into the immunological synapse. Preconcentrated Lck further phosphorylate the ITAMs of TCR-CD3? which are engaged with specific antigenic pMHC. Enhanced pCD3? provide more docking sites for Zap70 to be phosphorylated. Enhanced pZap70 signal amplifies the downstream TCR signaling pathway including the phosphorylation of Erk. pErk translocates into the nucleus and enhances the effector functional readouts of T-cell activation