Rho GTPases are members of the Ras superfamily of GTP binding proteins that function as molecular switches. In their GTP-bound, active state, Rho proteins are most well known for their ability to modulate the actin cytoskeletal network. In addition, Rho GTPases control signaling pathways that regulate diverse cellular functions such as cell shape, motility, and proliferation. When misregulated in cancer cells, Rho proteins influence aberrant growth, invasion and metastasis. Misregulation of Rho GTPases themselves or their regulators is associated with human cancers, making them attractive pharmacological targets for molecularly targeted cancer therapy. The atypical Rho GTPase Wrch-1 was initially discovered as a Wnt-1 responsive gene that, when activated, phenocopies Wnt-morphological transformation of mouse mammary epithelial cells. Like other Rho GTPases, Wrch-1 modulates actin cytoskeletal organization, but little is known about its downstream effectors and signaling pathways that may contribute to cellular transformation. Therefore, in this dissertation I discuss the characterization of a downstream effector of Wrch-1 that contributes to Wrch-1 normal biological function and its ability to mediate cellular transformation. Wrch-1 distributed along the apical and basolateral membranes in polarized MDCK epithelial cells and bound the cell polarity protein Par6 in a GTP-dependent manner. Activated Wrch-1 negatively regulated the kinetics of tight junction assembly during epithelial cell polarization, without a detectable effect on overall cell polarity in confluent monolayers. Disruption of tight junction dynamics driven by activated Wrch-1 was accompanied by dramatic cytoskeletal reorganization and multilayering of MDCK cells grown in two-dimensional (2D) culture. Additionally, expression of constitutively active Wrch-1 disrupted cystogenesis of cells grown in three-dimensional (3D) culture, resulting in aberrant multilumenal structures. The loss of epithelial morphogenesis mediated by activated Wrch-1 correlated with the ability of Wrch-1 to promote cellular transformation. Finally, an effector domain mutation in activated Wrch-1 that prevents Par6 binding repressed the transforming activity and abrogated the ability of Wrch-1 to disrupt tight junction formation, actin organization, and epithelial morphogenesis. Thus, I potentially uncovered a mechanism by which activated Wrch-1 induces morphological and growth transformation that involves Par6-mediated disruption of tight junctions and actin organization.
|Advisor:||Cox, Adrienne D.|
|Commitee:||Anderson, James M., Der, Channing J., Nicholas, Robert A., Peifer, Mark|
|School:||The University of North Carolina at Chapel Hill|
|School Location:||United States -- North Carolina|
|Source:||DAI-B 69/04, Dissertation Abstracts International|
|Subjects:||Molecular biology, Cellular biology|
|Keywords:||Actin cytoskeleton, Cell junctions, Cytoskeleton, Epithelial cells, Epithelial morphogenesis, Rho GTPases, Small GTPase, Transformation, Wrch-1|
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