Fig. 3
Chk but not Csk efficiently inhibits and tightly binds Hck (2PA-YEEI). a A schematic representation of the possible conformation adopted by Hck (2PA-YEEI) revealed in our previous studies [39]. The mutant is constitutively phosphorylated at the C-terminal YEEI motif. Upon phosphorylation, it binds tightly with the SH2 domain. The 2PA mutations which replace the two key proline residues in the SH2-kinase linker with alanine prevent the linker from binding to the SH3 domain to adopt the closed inactive conformation. Consequently, the mutant is autophosphorylated at consensus site (YA) in the activation loop and remains constitutively active. b Enzymatic activity of Hck (2PA-YEEI) in the presence and absence of varying concentrations of Chk or Csk. c-d Sensorgrams of surface plasmon resonance spectroscopy showing the kinetics of interactions of Csk (c) and Chk (d) with Hck (2PA-YEEI) immobilised on sensor chips. Biosensor response is recorded as response units (RU). 100 μg/ml of Hck (2PA-YEEI) was immobilised onto the sensorchips. Designated concentrations of Csk and Chk were allowed to flow through the respective channels at a rate of 10 μl/min. At 250 s (red arrow), HBS buffer was injected to initiate dissociation of the protein complexes formed by Csk and Hck (2PA-YEEI) or Chk and Hck (2PA-YEEI). e Molecular binding activities (M.B.As) of Csk and Chk at all concentrations determined from the responses of Csk and Chk binding to the immobilized Hck (2PA-YEEI), and the amount of Hck (2PA-YEEI) immobilized on the sensor chip. f The association and dissociation rate constants (k ON and k OFF) and equilibrium constants of dissociation (KD) of interactions between Csk or Chk with the immobilized Hck (2PA-YEEI)