Fig. 1

Csk and Chk exhibit different efficiencies in phosphorylating the Csk/Chk optimal peptide, Src (K295M) and Src. a Parameters of kinetic analysis of phosphorylation of Csk/Chk optimal peptide by Csk and Chk. The residues in red font were substrate specificity determinants of Chk and Csk identified by arrayed peptide library screen (Additional file 4: Figure S3). In the phosphorylation reaction, Csk or Chk (0.25 μM) was used to phosphorylate the substrate peptide at concentrations ranging from 0 to 0.5 mM. The initial velocities were plotted against the substrate peptide concentrations. The data points were fitted into Michaelis-Menten equation and transformed to Lineweaver-Burk plots (Additional file 4: Figure S3) to yield the V_max and K_M values. b Specific enzymatic activities of Csk and Chk in phosphorylation of the kinase-dead Src (K295M) mutant. Src (K295M) (0.08 μM to 4.3 μM) was incubated with Chk or Csk (0.03 μM), [γ-³²P] ATP (250 μM) and assay buffer. The reaction mixtures containing Src (K295M) only were the negative controls. Gel slices containing the phosphorylated Src (K295M) were excised and [³²P] phosphate associated with the slices was determined by scintillation counting for calculation of the specific enzymatic activities. c Left panel: Stoichiometry of phosphorylation of Src at both Tyr-416 and Tyr-527 in total in the presence and absence of increasing concentrations of Csk or Chk. The red arrows indicate the experimental data points corresponding to the phosphopeptide maps shown in the right panels. Right panels: The reaction mixtures contain Chk (1.04 μM) or Csk (0.75 μM), Src (0.6 μM) [γ-³²P] ATP (250 μM) and assay buffer. Two-dimensional phosphopeptide maps of Src phosphorylated by Csk or Chk. The locations of the origin and phosphopeptides containing phospho-Tyr-416 or phospho-Tyr-527 in the maps are similar to those indicated in Additional file 6: Figure S5B