Biochemical analysis of activating mutations of the kit receptor tyrosine kinase

grantor: University of TorontoThe recent identification a number of activating Kit mutants in human cancers has increased the need and the importance of understanding the oncogenic properties and biochemical mechanisms of the oncogenically mutated Kit receptors. In this thesis, I provide biological evidence that expression of either Kit$\rm\sp{KDY}$ or Kit$\rm\sp{G559}$ in IC2 cells leads to the degradation of the protein tyrosine phosphastase, Shp-1 and the relocalization of many signalling proteins downstream of the Kit receptor to the cytoskeletal fraction of the cell. One of the aberrantly localized proteins is the adaptor protein Shc, which is involved in activation of the Map kinase pathway and is a potential substrate of Shp-1. In addition, I also provide biochemical evidence which suggests that tyrosine phosphorylation may be required for the relocalization of Shc to this insoluble fraction of the cell. I further demonstrate through immunofluoresence staining that Shc is co-localized with the Kit receptor to a "cap" like area of the cell membrane after Steel factor stimulation and that the majority of Shc and Kit are localized to this area in IC2/KDY and IC2/G559 cells. This data suggests that the relocalization of Shc and other proteins downstream of the Kit receptor may be involved in the transformation of IC2 cells expressing KDY and G559. Furthermore, these results help further elucidate the molecular mechanisms underlying the malignant transformation by both KDY and G559 and further demonstrate the importance of Shp-1 as an important attenuator of the Kit signalling pathway.M.Sc