The overall objective of this proposal is to continue our investigations into the mechanisms of Kit receptor signaling in vitro and in vivo with emphasis on hematopoiesis. Furthermore, we will develop mouse models to investigate roles for Kit in oncogenesis. The Kit receptor tyrosine kinase encoded at the murine W locus functions in gametogenesis, hematopoiesis and melanogenesis Normal Kit receptor mediated functions include cell proliferation, cell survival, cell adhesion, cell migration, secretory response and differentiation. In human neoplasia oncogenic activation of Kit is thought to have roles in mastocytosis/mast cell leukemia, acute myelogenous leukemia, gastro intestinal stromal tumors (GlST) and germ cell tumors. Kit receptor functions are mediated by kinase activation, receptor autophosphorylation and association with various signaling molecules, We had investigated the role of PI 3-kinase and Src kinases in Kit mediated cell proliferation, suppression of apoptosis, cell adhesion and secretory responses. Analysis of Kit-/- BMMC expressing mutant Kit receptors indicated that both pathways contribute to the proliferative and the cell survival responses and that elimination of both pathways abolishes them. These studies also revealed that the P1 3-kinase and Src kinase signaling pathways converge to activate Raci and JNK. Moreover, recruitment and activation of Fl 3-kinase was shown to play a critical role in mediatingn cell adhesion and secretory responses. To investigate the consequences in vivo of blocking Kit mediated Fl 3-kinase activation we have mutated tyrosine 719 in the mouse c-kit gene (KitY719F), a known binding site for the p85 subunit of Fl 3-kinase. Analysis of homozygous mutant KitY719F/KitY719F mice indicated essential roles for Kit induced PIl 3-Kinase activity in mouse gametogenesis. In hematopoiesis phenotypes were minimal showing an effect on peritoneal mast cell numbers and no other phenotypes. These findings emphasize the importance of the cellular context for Kit receptor signaling in vivo. The purpose of this application is twofold: 1) to investigate more precisely the mechanism and the consequences of Kit mediated Fl 3-kinase and Kit mediated src signaling in vivo in hematopoiesis, and 2) to construct mouse models for investigating the role of Kit in oncogenesis (hematopoietic malignancies and gastrointestinal stromal tumors) and to elucidate the mechanisms of signaling by oncogenically activated Kit receptors.
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