In breast cancer, tumorigenesis occurs through an accumulation of genetic and epigenetic changes in epithelial cells involving both the activation of oncogenes and the inactivation of tumor suppressor genes. The Syk protein-tyrosine kinase, known best for its roles in the immune system, has recently been identified as a tumor suppressor in breast cancer; its expression being inversely correlated with malignant cell growth and metastasis. This is an unusual property for a tyrosine kinase, enzymes more typically found as the products of oncogenes. Thus, an understanding of how Syk functions to suppress the malignant phenotype of breast cancer cells is of considerable importance to our understanding of the critical pathways involved in growth control in breast epithelial cells and to the identification of possible, novel therapeutic targets. Preliminary studies have led to the hypothesis that Syk regulates two important aspects of of epithelial cell function: 1) cellular responses to tumor necrosis factor (TNF), a factor that regulates cell death/cell survival decisions and 2) cell adhesion and motility. These hypotheses are based on the identification of Syk- interacting proteins that participate in each of these pathways. The research proposed in this project will 1) characterize the physical and functional interactions between Syk and components of the TNF-signaling pathway with an emphasis on the characterization of a novel Syk-interacting protein thought to participate in this pathway; 2) characterize the critical structural features and mechanisms by which Syk, through its interacting partners, regulates cell adhesion and motility; and 3) characterize through phosphoproteomics studies the substrates and binding partners important for Syk's regulatory functions in breast epithelial cells. Methodologies to be employed include 1) genetic, biochemical and microscopic evaluations of protein- protein interactions and the consequences of their disruption and 2) state-of-the-art analyses of the phosphoproteome of Syk-expressing cells.