Fig 2: Phosphorylation of Y416 in the closed, repressed conformation is driven by core kinase domains. A. Confocal micrographs of c-Src expressed as fusions to Emerald in AD293 cells in either full length or truncated form lacking the first 63 residues (?1–63), which contains the unique domain and myristoylation sequences. Cells were co-transfected with mKate2-F, which is a fluorescent protein containing a farnesylation signal to localize it to membranes. Turquoise shows c-Src-Em, red shows mKate2-F. Scale bar, 10 µm. Images are representative of three independent experiments. B. Western Blots of AD293 cells transfected with the indicated constructs for 24 h with 10 µg total protein lysate. Phospho-Y416 was detected with two different antibodies: #1, cat. PK1109 from Calbiochem; #2, cat. 2101 from Cell Signaling. Key: Em, Emerald fluorescent protein; closed, Q528E, P529E, G530I mutant of c-Src; open, Y527F mutant of c-Src; ß-gal, ß -galactosidase in same vector as c-Src constructs. Experiments were performed at least twice, which showed consistent results.

Fig 3: Src activated MyD88 by phosphorylating 58 tyrosine residue in macrophages. (A) p-Tyr MyD88 in whole-cell lysates of RAW264.7 cells treated with LPS (1 µg/mL) for the indicated time was detected by immunoprecipitation and western blot analysis. (B) p-Tyr MyD88 and Flag in whole-cell lysates of RAW264.7 cells transfected with empty (pcDNA), Flag-MyD88, Flag-MyD88(?ITAM) or Flag-MyD88(Y58F) plasmids for 48 h were detected by immunoprecipitation and western blot analysis. (C) p-Tyr MyD88 in whole-cell lysates of RAW264.7 cells co-transfected with Flag-MyD88 and empty (pcDNA), Src, Fyn, Mal, or TLR4 for 48 h was detected by immunoprecipitation and western blot analysis. (D) Src and p-Src in whole-cell lysates of RAW264.7 cells treated with LPS (1 µg/mL) for the indicated time were detected by western blot analysis (E) p-Tyr MyD88 in whole-cell lysates of RAW264.7 cells co-transfected with Flag-MyD88 and empty (pcDNA), Src, or Src(?KD) was detected by immunoprecipitation and western blot analysis. (F) p-Tyr MyD88 in whole-cell lysates of RAW264.7 cells treated with LPS (1 µg/mL) for the 1 min with absence or presence of PP2 (20 µM) was detected by immunoprecipitation and western blot analysis (G) p-Tyr MyD88 in whole-cell lysates of RAW264.7 cells co-transfected with Src and either empty (pcDNA), Flag-MyD88, or Flag-MyD88(Y58F) plasmids for 48 h were detected by immunoprecipitation and western blot analysis.

Fig 4: LckY192E displays an impaired ability to phosphorylate ZAP70 independently of Y505 phosphorylation. a HEK 293T cells (–) or HEK 293T cells stably expressing either Lckwt or LckY192E were transiently transfected with either an empty vector (Bos) or Myc-tagged ZAP70 (75 kDa). The phosphorylation of ZAP70 on Y319 (used as a read-out for Lck activity) as well as the phosphorylation of Lck on Y505 were monitored using phosphospecific antibodies. One representative experiment is shown (n = 5). b Parental TCR/CD3+ HEK 293T cells (–) or TCR/CD3+ HEK 293T cells stably expressing Lckwt, or LckY192E were transiently co-transfected with either a Myc-tagged ZAP70 or an empty vector (Bos). Subsequently, cells were lysed and the phosphorylation of pY319 of ZAP70 and pY505 of Lck were assessed using phosphospecific antibodies. One representative experiment is shown (n = 6). c, d The densiometric mean values of the phosphosignal pY319 in a and b were normalized against the value of the total ZAP70 signal. The bar graph shows the calculated mean + SEM values of the normalized ZAP70 phosphorylation in HEK 293T (c) and the TCR/CD3+ HEK 293T (d) cell systems. One dot represents one experiment. e Photo-caged Lck (WT, Y394F and Y192E) and ZAP70 were transiently transfected in TCR/CD3+ HEK 293T cells. Lck kinase activity was initiated by global illumination of the cells and the kinetics of ZAP70 phosphorylation by Lck was measured at defined time points by quantitative fluorescent Western blot analysis. Data were fit using a three-parameter logistic function and are presented as mean ± SEM (n = 3)

Fig 5: Y192 influences ligand-induced closed proximity between Lck and the TCR. a Technical PLA controls in J.Lck cells reconstituted with Lckwt stimulated with a CD3 antibody at 37 °C for 5 min. PLA was performed with both primary antibodies (left), with only the anti-Lck primary antibody (middle) or with only the anti-CD3e primary antibody (right). In all cases, both secondary antibodies were used. The quantification of two independent experiments was analyzed using One-way ANOVA test (bar diagram). Mean values ± SEM are shown. ****p < 0.0001. b Biological PLA controls in Jurkat cells expressing surface TCR and Lck (left), Jurkat cells lacking Lck expression by CRISPR/Cas9 gene editing (J.Lck, middle) and Jurkat cells lacking CD3e by CRISPR/Cas9 gene editing (J.CD3eKO, right). The PLA was performed between the TCR (CD3e) and Lck at 37 °C in unstimulated conditions. The quantification of two independent experiments is shown (bar diagram). Statistical analysis was performed as in a. c In situ PLA between the TCR (CD3e) and Lck was performed; a red fluorescent signal indicates a distance between Lck and the TCR smaller than 80 nm. J.Lck cells were transfected to stably express an empty vector, Lckwt or LckY192E. Cells were either left unstimulated, stimulated with a TCR antibody (C305) or stimulated with pervanadate at 37 °C for 5 min. Nuclei were stained with DAPI. Data of one representative experiment performed in technical duplicates is shown (upper bar diagram). In order to pool independently performed experiments and to normalize for Lck expression, the fold induction between unstimulated and stimulated samples per experiment was calculated (lower bar diagram). An unpaired Student’s t test on pooled data of 6–7 independent experiments was analysed. Mean values ± SEM are shown, *p < 0.1; **p < 0.01