Mast cells are granulocytes that require signaling from the receptor protein-tyrosine kinase Kit and its ligand stem cell factor (SCF) for their maturation and function. In addition to providing growth and survival signals, the Kit receptor is involved in crosstalk to β1 integrins leading to mast cell adhesion, spreading, and migration on fibronectin (FN). Previous studies reported the involvement of the non-receptor protein-tyrosine kinases Fps/Fes and Fer in signaling downstream of the high affinity IgE receptor in mast cells and identified cell migration defects in Fer-deficient bone marrow-derived mast cells (BMMCs). Fps/Fes also becomes phosphorylated downstream of the Kit receptor in BMMCs, and this involves the action of the Src family kinase Fyn as an upstream activator of Fps/Fes. In this study, the Fps/Fes SH2 domain was observed to bind the phosphorylated Kit receptor in vitro, suggesting that the SH2 domain plays a role in the activation mechanism of Fps/Fes. To investigate the function of Fps/Fes in Kit signaling, BMMCs were generated from wild-type and Fps/Fes-null mice. Analysis of downstream effectors revealed that Fps/Fes is required for maximal p38 MAPK signaling. Further examination of Fps/Fes-deficient BMMCs revealed increases in adhesion, spreading, and a defect in cell polarization on full-length FN (a ligand for multiple β1 integrins), compared to wild-type BMMCs. Similar phenotypes are observed using an α5β1 integrin-specific FN fragment (9-11) as the matrix. Reduced phosphorylation of the putative Fps/Fes substrate HS1 (a cortactin homologue involved in actin regulation) is observed in Fps/Fes-deficient BMMCs, compared to control cells, and this may contribute to the observed cytoskeletal defects. Restoring Fps/Fes expression in Fps/Fes-deficient BMMCs by retroviral transduction results in a rescue of cell spreading, polarization, and chemotaxis defects to levels similar to those of wild-type cells. This thesis provides novel insights into the potential mode of Fps/Fes activation downstream of the Kit receptor, and a role for Fps/Fes in regulating crosstalk between Kit and α5β1 integrins to promote cytoskeletal reorganization and motility of mast cells. / Thesis (Master, Biochemistry) -- Queen's University, 2009-01-28 15:05:04.056

The fps proto-oncogene encodes a 92 kDa cytoplasmic tyrosine kinase. Previous studies have shown that Fps expression in the mammary gland changes with development, and Fps has a suppressor function in mammary tumorigenesis. The aim of my thesis was to elucidate the role of the Fps tyrosine kinase in regulating mammary gland development and function. We have shown that the expression of the Fps kinase in the mammary gland increased during pregnancy and reached its maximum during lactation. The level of Fps tyrosine phosphorylation paralleled the expression pattern. Pups reared by fps-null females gained weight more slowly than those reared by wild-type females. Epithelial cells were the primary source of Fps expression. Milk protein and fat content were not affected by the absence of Fps. Similarly, no differences in mammary gland structure were observed with whole mount or histological analysis. Fps was shown to be in a multi-protein complex with E-cadherin, β-catenin and p120-catenin. A strong co-localization signal was observed for Fps and E-cadherin. Immunofluorescence analysis indicated that the localization of E-cadherin and β-catenin was disorganized and less concentrated at sites of cell-cell contacts in the fps-null glands. The interactions between the different adherens junction components were altered in the fps-null tissue. Specifically, less E-cadherin and β-catenin was associated with p120-catenin in the fps-null glands. Suprisingly, no phosphotyrosine differences were detected for the adherens junction components. Conditions were established to grow primary murine epithelial cell cultures that could be used to investigate the function of Fps. Fps expression was up-regulated in these cells in response to lactogenic hormones. A lentiviral system encoding a murine p53 shRNA sequence was used to increase the growth potential of the primary cells. Continual growth of the infected and uninfected primary epithelial cell mixture resulted in the establishment of an immortalized cell line. Immunofluorescent and immunoblot analyses revealed that the cells have undergone an epithelial-to-mesenchymal transition. With the transduction of a myc-epitope tagged Fps into the cells, we have generated cell lines with the appropriate genetic backgrounds to study the function of the Fps kinase in the mammary gland, specifically as it relates to tumorigenesis. / Thesis (Ph.D, Pathology & Molecular Medicine) -- Queen's University, 2008-07-03 11:53:01.135