The overall objectives of this proposal is to investigate the role of PI 3- kinase in c-kit receptor mediated processes in vivo and in vitro. The proto-oncogene c-kit, encodes a receptor tyrosine kinase (RTK) which belongs to a family of receptors that includes the platelet- derived growth factor (PDGF). The ligand of the c-kit receptor, designated KL, encodes a cell surface molecule which may be processed to produce a soluble product. Whereas the c-kit gene is allelic with the white spotting locus (W) in the mouse, kit-ligand (KL) is allelic with the steel locus (Sl) in the mouse. W and Sl mutations affect various aspects of hematopoiesis, including the stem cell compartment, gametogenesis during embryonic development and in the adult animal, causing macrocytic anemia, mast cell deficiency anemia, mast cell deficiency, spotting, male and female sterility and megacolon. Based on mutant phenotypes the cellular responses mediated by the c- kit receptor are quite diverse and include: cell proliferation, cell survival, cell adhesion, cell migration, secretion of mediators, cell differentiation and other post mitotic functions. The elucidation of the mechanisms of these different responses is of fundamental importance. Similar to other RTK's Kit mediates its action through a variety of cytoplasmic signal transducers including PI 3-kinase in KIT mediated adhesion of BMMC to a fibronectin matrix and potentiation of IgE mediated secretion in BMMC. To study Kit/PI 3-kinase mediated processes in other cell systems we will modify the c-kit gene in the germ line to mutate the PI 3-kinase binding site in the Kit receptor by using a cre-lox based strategy and derive mice homozygous for this mutation. We then will study the effect of the mutation on various targets of W and SI mutations, the dynamics of hematopoietic cell populations and their behavior in situations that require cell adhesion and migration, i.e. homing to the marrow compartment and mobilization of stem cells into the periphery. We will also modify tyrosine residues in the juxtamembrane region of the Kit receptor in ES cells which affect KL mediated cell proliferation but not suppression of apoptosis and investigate the consequences of this mutation in vivo. Finally, we will investigate the mechanism of Kit mediated suppression of radiation and factor deprivation induced apoptosis in mast cells.
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