ABSTRACT Protein tyrosine kinases (PTKs) phosphorylate tyrosine in proteins involved in cell signaling pathways that stimulate cell migration, proliferation and oncogenic transformation. Phosphotyrosine (pY) proteins may be inactivated by dephosphorylation or by proteasomal degradation following ubiquitylation by pY-specific ubiquitin E3 ligases. We are focusing on the multi-subunit Cullin 5-RING ligase (CRL5), which interacts with pY proteins through suppressors of cytokine signaling (SOCS) adaptor proteins. Our past studies have shown that various CRL5-SOCS complexes are critical in normal epithelial cells to repress an oncogenic PTK, Src. When we inhibit expression of Cullin 5 or specific SOCS genes, Src is activated and cell migration, proliferation and transformation are stimulated. Increased Src activity is necessary but not sufficient for the migration and proliferation of Cullin 5-deficient cells, suggesting that CRL5 has additional substrates. Our past studies have identified two such substrates that are critical for cell migration. Here we propose to extend these studies, to understand how CRL5-SOCS regulates epithelial cell migration. Our scientific premise is that the identification of key CRL5-SOCS substrates and functions in migration is critical for understanding normal and transformed cell biology. During the last funding period, we used innovative live imaging, optogenetics and mutational analysis to uncover the molecular mechanism by which CRL5-SOCS6 regulates the signaling scaffold, Cas (Crk- associated substrate), within focal adhesion sites at the leading edge of migrating epithelial cells. We also used quantitative proteomics to identify other CRL5 substrates and test their roles in cell migration. Specifically, we identified BCAR3 (breast cancer anti-estrogen resistance 3) as a second CRL5-SOCS6 substrate that regulates cell migration. Finally, we found that SOCS2, like SOCS6, localizes to focal adhesion sites and regulates cytoskeletal dynamics. We propose three broad aims to extend these observations and understand the molecular mechanisms. First, we will understand the importance of BCAR3 phosphorylation in BCAR3 stability and focal adhesion dynamics. Second, BCAR3 binds and cooperates with Cas: each protein stabilizes the other from degradation. We will understand the mechanism and importance of BCAR3-Cas mutual stabilization. Third, we will determine the mechanism by which SOCS2 regulates focal adhesion dynamics. Together, these aims will extend our understanding of how protein-tyrosine phosphorylation and ubiquitylation cooperate to regulate cell migration.