Role and Regulation of the Proteasome in Epithelial Cell Adhesion and Migration

Epithelial cells disassemble their adherens junctions and 'scatter' during processes such as tumor cell invasion, as well as some stages of embryonic development. As migrating epithelia do not always exhibit changes in the composition of cadherin-catenin complexes, it is likely that post-transcriptional regulation of cellular processes can impinge on the assembly/disassembly of adherens junctions and contribute to the loss of cell polarity and the acquisition of a migratory potential, a process known as epithelial to mesenchymal transition (EMT). Control of actin polymerization is a powerful mechanism for regulating the strength of cell-cell adhesion. In this regard, studies have shown that sustained activation of Rac1, a well known regulator of actin dynamics, results in the accumulation of polymerized actin at cell-cell contacts and an increase in E-cadherin-mediated adhesion in epithelia. This study describes a link between the activity of the proteasome, the actin cytoskeleton and growth factor stimulated adherens junction disassembly. The disassembly of the adherens junction complex has been documented to be accompanied by a transient decrease in the cellular levels of Rac1-GTP. In this study, we demonstrate that Rac1-GTP (but not Rac1-GDP) is ubiquitinated and subject to proteasome-mediated degradation during the early steps of epithelial cell scattering. These findings delineate a mechanism for the down-regulation of Rac1 in the disassembly of epithelial cell-cell contacts and support the emerging theme that ubiquitin proteasome system (UPS)-mediated degradation of the Rho family GTPases may serve as an efficient mechanism for GTPase deactivation in the sustained presence of Dbl-exchange factors. Furthermore, we provide insight into the control of proteasome activity during E-cadherin-mediated adhesion. We show that arfaptin 2, an ARF interacting protein, regulates the disassembly of cell contacts via its effect on proteasome activity. Using cell-based assays, we show that expression of wild type arfaptin 2 inhibits proteasome activity and hence, the disruption of cell-cell contacts in MDCK cells despite the presence of scatter factor. Depletion of arfaptin 2 using small interfering RNA, results in the disorganization of E-cadherin-based contacts and increased cell volume. We have also begun to elucidate a role for the GTPase, Rap1, in the regulation of E-cadherin membrane traffic during adherens junction turnover in epithelial cells. Thus, this dissertation delineates a novel mechanism involved in the disassembly of cell-cell contacts during epithelial cell scattering and documents some key regulators of this process.