Figure 4.

SFK inhibitors can modulate C-terminal tail phosphorylation. (A) Experimental schematic of SFK Tyr527 phosphorylation. Src or Hck^Y416F was incubated with Csk^DR in the presence of inhibitor. The concentration of each inhibitor is such that >85% of Src or Hck^Y416F is bound to inhibitor and Csk^DR is inhibited by <13%. ATP was added to the mixture to initiate Csk^DR-mediated phosphorylation of the SFK–inhibitor complex. After 1 h of incubation, pTyr527 levels were determined by performing SDS-PAGE, followed by immunoblotting with an α-phospho-527 Src antibody for Src or α-pTyr antibody for Hck^Y416F. All values are normalized to that of the SFK–1 complex. (B) Quantification of Src and Hck^Y416F pTyr527 levels in the presence of inhibitors 1, 2, or 5. All values are normalized to that of the SFK–1 complex (mean ± SEM, n = 3). (C) Chemical genetic method for generating covalent SFK–inhibitor complexes to measure SFK Tyr527 phosphorylation. Covalent SFK–ligand complexes were generated by incubating SFK^S245C with ligand 6 or 7. The resulting SFK–ligand substrate was purified and then incubated with Csk and γ³²P-ATP. Phosphorylation levels were measured at 0.5, 1, and 1.5 h. (D) Structures of electrophile-containing analogues. (E) Quantification of Src^S245C pTyr527 levels in the presence of inhibitors 6 or 7 (mean ± SEM, n = 3). (F) Quantification of Hck^245C pTyr527 levels in the presence of inhibitors 6 or 7 (mean ± SEM, n = 3).