Critical roles of c-Kit tyrosine residues 567 and 719 in stem cell factor–induced chemotaxis: contribution of src family kinase and PI3-kinase on calcium mobilization and cell migration

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Abstract
Stem cell factor (SCF) has crucial roles in proliferation, survival, and differentiation of hematopoietic stem cells and mast cells through binding to c-Kit receptor (KIT). Chemotaxis is another unique function of SCF. However, little is known about the intracellular signaling pathway of SCF/KIT-mediated cell migration. To investigate the signaling cascade, we made a series of 22 KIT mutants, in which tyrosine (Y) residue was substituted for phenylalanine (F) in the cytoplasmic domain, and introduced into BAF3 cells or 293T cells. On stimulation with SCF, BAF3 expressing KITWT(WT) showed cell migration and Ca2+ mobilization. Among 22 YF mutants, Y567F, Y569F, and Y719F showed significantly reduced cell migration and Ca2+ mobilization compared to WT. In Y567F, Lyn activation on SCF stimulation decreased and C-terminal Src kinase (Csk) suppressed KIT-mediated Ca2+ influx and cell migration, suggesting that Y567-mediated Src family kinase (SFK) activation leads to Ca2+ influx and migration. Furthermore, we found that p38 mitogen-activated protein kinase (p38 MAPK) and Erk1/2 were also regulated by Y567/SFK and involved in cell migration, and that p38 MAPK induced Ca2+ influx, thereby leading to Erk1/2 activation. In Y719F, the binding of phosphatidylinositol 3′-kinase (PI3K) to KIT was lost and KIT-mediated cell migration and Ca2+ mobilization were suppressed by PI3K chemical inhibitors or dominant-negative PI3K, suggesting the involvement of Y719-mediated PI3K pathway in cell migration. Combination of Csk and the PI3K inhibitor synergistically reduced cell migration, suggesting the cooperation of SFK and PI3K. Taken together, these results indicate that 2 major KIT signaling pathways lead to cell migration, one is Y567-SFK-p38 MAPK-Ca2+ influx-Erk and the other is Y719-PI3K-Ca2+ influx.