An analysis of steel factor-stimulated receptor trafficking, survival signals and mitogenesis, understanding the role of c-Kit-associated signaling proteins

grantor: University of TorontoReceptor tyrosine kinases, upon binding to their respective ligands, undergo dimerization, activation of intrinsic kinase domains and phosphorylation of intrinsic tyrosine residues. This results in the recruitment and activation of a number of intracellular signaling molecules. In the case of the receptor tyrosine kinase c-Kit, activation by its ligand Steel Factor (SLF) is important for a number of developmental systems including hematopoiesis, gametogenesis and germ cell formation. SLF stimulation of c-Kit induces the recruitment of a number of SH2-containing proteins including Phosphatidylinositol-3-kinase (PI3-kinase) and Phospholipase C gamma (PLC-ã). PI3-kinase generates D3 phosphoinositide second messengers. In the case of PLC-ã activation, hydrolysis of phosphatidylinositol bisphosphate into inositol trisphosphate and diacylglycerol leads to Ca2+ mobilization and activation of Protein Kinase C. In this thesis, using c-Kit positive mast cells and myelomonocytic cell lines I have examined the involvement of these two signaling enzymes in SLF-stimulated survival and mitogenic signals as well as SLF-stimulated receptor internalization. The mechanism of SLF-stimulated c-Kit internalization is examined and a requirement for both Ca2+ influx and PI3-kinase activity in this process is identified. In addition, I have determined that mitogenic stimulation of c-Kit positive cells by membrane-bound SLF requires the activity of PLC-ã. Finally, I have identified an important role for PLC-ã-mediated Ca2+ influx in c-Kit-mediated survival signals and characterized a SLF-mediated apoptotic mechanism involving Ca2+ influx blockade. Therefore, the use of both c-Kit receptor mutants as well as specific inhibitors has allowed the identification of important roles for these signaling proteins and their second messengers in SLF-stimulated cellular processes. The relevance of these c-Kit associated signaling molecules 'in vivo' will be discussed.Ph.D