Figure 1.
Schematic representation of the Bcr-Abl tyrosine kinase.
Tyrosine kinases, such as Bcr-Abl, are phosphoryl transferases that transfer phosphate from ATP to Tyr residues on specific substrate proteins. These enzymes have an ATP-binding site that is independent of the catalytic site; when bound to ATP, they become activated and exert their activity. (A) The catalytic and ATP-binding sites of Bcr-Abl are located in the Abl domain. (B) The 3D structure of the Abl domain with imatinib (red) bound to the ATP cleft (PDB: 3k5v); imatinib occludes the ATP binding and locks the enzyme in the inactive conformation.
Figure 2.
(A) Map of plasmid pBAD33-eCPX-abltide (left) and schematic representation of abltide fused to eCPX expressed on the cell surface of an E. coli cell transformed with pBAD33-eCPX-abltide (right). The plasmid carries the arabinose promoter pBAD and resistance to chloramphenicol (CMr). The recognition sites for the restriction enzyme SfiI and annealing region are show. (B) Schematic representation of the BacKin assay: 1. E. coli cells transformed with eCPX-substrate plasmid are incubated at 37°C and shaken to grow until mid log phase; 2. Substrate (e.g. abltide) expression and display on the bacteria surface is induced with arabinose; 3. Substrate phosphorylation, cells are incubated with kinase (e.g. Abl kinase) and an excess of ATP; 4. Phosphorylated substrate is labeled by incubation of cells with biotinylated-anti-phosphotyrosine antibody (biotin-anti-PY) followed by incubation with streptavidin-phycoerythrin (SAPE); 5. Mean fluorescence of kinase-treated cells is quantified by flow cytometry and compared with mean fluorescence of kinase untreated cells.
Figure 3.
Validation of BacKin assay examined with flow cytometry.
(A) Dotplot of side scatter and fluorescence emission intensity at 585/42 nm for eCPX-displaying cells and abltide-displaying cells. After treatment, the percentage of fluorescent cells (shown in green) is ∼0.1% in eCPX-displaying cells and > 96% in abltide-displaying cells. (B) Representative fluorescence intensity histograms for eCPX-displaying and abltide-displaying cells. The incubation time with kinase is given in brackets. All samples were fluorescently-labeled by incubation with biotin-anti-PY and SAPE. F is the mean fluorescence signal obtained for 100,000 cells analyzed.
Figure 4.
Characterization of Abl kinase using the BacKin assay.
Mean fluorescence was converted into fraction of phosphorylation, assuming maximum response as 100% of abltide phosphorylation. Each data point is the average of three independent experiments±SD (A) Abltide phosphorylation catalyzed by increasing concentrations of Abl kinase in the presence of 500 µM ATP and incubated for 30 min at 37°C. The kinase concentration required to achieve half of the maximum response, KD±SD, was calculated by fitting a nonlinear sigmoidal curve. (B) ATP concentration effect on abltide phosphorylation catalyzed by 0.5 U/mL Abl Kinase for 30 min at 37°C. The ATP concentration required to achieve half of the maximum response, KD±SD, was calculated by fitting a nonlinear sigmoidal curve. (C) Time-course of abltide phosphorylation catalyzed by 0.5 U/mL Abl kinase in the presence of 500 µM ATP at 37°C obtained with BacKin assay (circles) or with LC-MS assay (squares). The time required to phosphorylate half of the abltide molecules, t1/2±SD, was calculated by fitting a pseudo-first order association kinetics.
Figure 5.
Kinase inhibition study using the BacKin assay.
Inhibition of surface-displayed abltide phosphorylation by (A) imatinib or (B) soluble abltide. Reaction was catalyzed with 0.5 U/mL Abl kinase in the presence of 50 (squares) or 500 µM ATP (circles) and incubated for 30 min at 37°C. Mean fluorescence was converted into percentage of inhibition. Each data point represent the average of three independent experiments±SD and data were fit with a sigmoidal curve. The abltide analogue [Y4F,F8Y] is shown in black circles and was tested with 500 µM ATP, average of two replicates are shown.
Table 1.
Kinase inhibition parameters.[a]