Compensatory mutation analysis in the T212R mutant. (A) The threonine of residue 212 (green) is located near the interface between SH2 and the kinase domain's N-terminal lobe in the active conformation of ABL (PDB entry 1OPL, molecule B).24 (B) In the T212R mutant, arginine is in position to form an electrostatic interaction with E275 in the kinase domain (gray). The kinase domain is shown in gray with bound inhibitor in yellow. Stick format atoms are color-coded for nitrogen (dark blue), oxygen (red), and sulfur (orange). The figure was created using PyMol.22 (C) Single-, double-, and triple-mutant ABL expression constructs with T212R, ABL PP (P223E/P230E), and mutations at the E275 position were transiently transfected in HEK293 cells, lysed 40 hours later, and total protein extracts analyzed by anti-ABL and antiphosphotyrosine immunoblotting. (D) These ABL mutant proteins were next immunoprecipitated and assayed for activity by in vitro kinase assays with an optimal substrate peptide. The graphs show catalytic activity relative to ABL for 2 experiments done in duplicate (mean ± SD). (E) The E275K and S154N mutants were tested in native and T212R BCR-ABL by Ba/F3 cell proliferation assays, as before. E275K displayed substantial imatinib resistance alone, and this effect was additive to that of T212R in the double mutants. T212R/E275A had the same sensitivity to that of the T212R single mutant. In the T212R/S154N double mutant, the sensitivity of imatinib was reverted back to that of WT BCR-ABL.
