Additional file 1: Figure S1. of Csk-homologous kinase (Chk) is an efficient inhibitor of Src-family kinases but a poor catalyst of phosphorylation of their C-terminal regulatory tyrosine
Hypothetical conformations of the SFK and mutants used in this study. The two SFK members Src and Hck were chosen for our analyses. The N-terminal unique domain of both Src and Hck mutants is replaced by poly-His (His6) and Flag tags. For Src (K295M), Lys-295 critical to binding ATP is replaced by Met, hence the mutant is inactive. For Hck (2PA-YEEI), two conserved prolines in the SH2-kinase linker are replaced by alanines (referred to as the 2PA mutation), and the C-terminal YQQQP motif is replaced by the YEEIP motif (referred to as the YEEI mutation). The 2PA mutation prevents the Hck mutant from adopting the “closed” inactive conformation because the two conserved prolines are critical for intramolecular interactions between the PQKP motif with the SH3 domain. The mutant is therefore constitutively active. The YEEI mutation converts the motif around the C-terminal tail tyrosine into YEEIP motif which is an optimal phosphorylation sequence of SFKs. The constitutively active mutant undergoes autophosphorylation at both the conserved autophosphorylation site (YA) and the C-terminal tail tyrosine (YT). Upon phosphorylation, the pYEEIP motif can bind to the SH2 domain of the mutant with high affinity. Based upon the results of the structural and biochemical analyses of the Src and Hck mutants presented by Lerner, et al. [40] and Cowan-Jacob, et al. [82], the predicted conformations of the Src and Hck mutants are depicted in the left column. (TIFF 623 kb
