FIGURE 5.

Interdependence of Y540 and Y541 phosphorylation and PRR1 function. (A) HEK293 cells were cotransfected with Lck and either THEMIS-Strep wt, an all tyrosine to Phe mutant (19YF) and swap mutants between wt and 19YF (residue 220 as swapping boundary). Tyrosine phosphorylation of THEMIS pull-downs was assessed by immunoblotting. (B) THEMIS-Strep wt or Y540 and Y541 to Phe mutant were isolated from Jurkat cells treated as indicated and probed for tyrosine phosphorylation, GRB2, and LAT association. (C) Peptide scanning array as in Fig. 1A with phosphorylation and substitutions at Y540 and Y541. (D) HEK293 cells were cotransfected with THEMIS-Strep wt or PRR1 mutant and increasing amounts of Lck. Precipitated THEMIS was probed for tyrosine phosphorylation and GRB2 association. (E) A total of 500 ng recombinant THEMIS-Strep was phosphorylated in vitro with 25 ng recombinant Lck. Where indicated, THEMIS-Strep was preincubated with recombinant GST-GRB2 (10 μM) for 1 h. THEMIS tyrosine phosphorylation was quantified by immunoblotting. (F) HEK293 cells were transduced with C-terminally Strep-tagged lentiviral constructs of either full-length wt and Y540/541F mutant THEMIS or truncation constructs consisting of CABIT domain 1 (aa 1–260) and CABIT domain 2 including the tail sequence (aa 261–641). After Streptactin pull downs, THEMIS constructs were assessed for GRB2 binding by immunoblotting. Data are representative of three independent experiments.