Fig. 3.

Characterization of the Abl N368S mutation. (A) Sequence alignment of human protein kinases represented as weblogo. The size of the letters reflects the degree of conservation, and numbering is according to Abl1A numbering. Asparagine 368 is as highly conserved as the catalytic His-Arg-Asp (HRD) and the DFG motif. (B) Structural overview of Abl N368 in the active conformation (PDB entry 2G2I). The sidechain of N368 is near the nucleotide and is positioned to form hydrogen bonds with the catalytic aspartate D363 and D381 in the DFG motif. (C) Kinase activity of purified Abl N368S is undetectable in the absence of activation loop phosphorylation (designated as pAbl). (D) The Michaelis–Menten constant for ATP of purified pAbl N368S is twofold higher than that of pAbl wt. (E) The affinity of purified Abl N368S for imatinib determined by a spectroscopic-binding assay is slightly weaker than for Abl wt. (F) BaF3 cells expressing Bcr-Abl wt, T315I, or N368S. BaF3 cells expressing N368S are equally sensitive to imatinib as cells expressing Bcr-Abl wt. (G) BaF3 cells expressing imatinib resistance mutations T315I or N368S exhibit a faster doubling time in the absence of imatinib. ns: P > 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.