Mechanism of activation of human c-KIT kinase by internal tandem duplications of the juxtamembrane domain and point mutations at aspartic acid 816

The proto-oncogene c-Kit receptor has been implicated as an essential component in the activation of leukemic cells. The internal tandem duplication (ITD) of c-Kit has also been identified as a predominant cause of acute myeloid leukemia (AML), although its role in the activation process is still unclear. To investigate the biological mechanisms of c-Kit activation, we generated a c-Kit receptor bearing two different immunological tags, HA and Flag tags. In this study, we demonstrated that the mutant (Mt)-ITD and Asp816 (D816Y) c-Kit receptors spontaneously formed dimers and that these Mt-ITD forms of c-Kit displayed high levels of phosphorylation and increased cellular tyrosine phosphorylation. The amount of wild-type homodimers increased following the addition of the c-Kit ligand, while the level of mutant homodimers was less affected by the addition of the c-Kit ligand. Furthermore, we demonstrated that Mt-ITD and activating point mutations of D816Y induced constitutive activation of c-Kit kinase in the absence of ligand in COS-1 cells. These data suggest a novel mechanism for the regulation of cell growth autonomy. Overall, our study suggests that c-Kit activation might have significant effects on hematopoietic cells and might help to improve our understanding of the pathogenesis of systemic mast cell disease, gastrointestinal stromal tumors and AML and potentially lead to the development of novel therapeutic approaches.