THE ROLE OF R-RAS IN ARF6 ACTIVATION DOWNSTREAM OF THE HGF-C-MET SIGNAL TRANSDUCTION PATHWAY

Open Access
Author:
Pursel, Heather
Graduate Program:
Biochemistry, Microbiology, and Molecular Biology
Degree:
Master of Science
Document Type:
Master Thesis
Date of Defense:
March 24, 2011
Committee Members:
  • Lorraine C Santy, Thesis Advisor
Keywords:
  • cell migration
  • GTPase
Abstract:
Epithelial cells grow as sheets that function as a selectively permeable barrier that is sealed together with an extensive network of junctions. Under normal circumstances, this network forces the cells to be non-motile. However, it is possible for these cells to become motile during embryonic morphogenesis and the metastasis of epithelial cancers. This movement is prompted by a variety of growth factors such as HGF that can cause epithelial cell line scattering and motility in vitro. The binding of HGF to its receptor c-Met has the ability to stimulate the activation of PI3-kinase and the small GTPase Ras. Ras and other subfamily members such as R-ras have been known to play a role in proliferation and cell survival as well as cell migration and morphology. The small GTPase Arf6, which regulates actin cytoskeletal organization and can be activated by HGF, has also been associated with, and is required for, epithelial cell movement. While it is known that R-Ras and Arf6 are both required for epithelial motility, the signaling pathway linking these components to cell shape changes remains unclear. Therefore, we hypothesize that R-ras and Arf6 are downstream of the HGF-cMet signal transduction pathway to regulate epithelial cell migration. The involvement of the Ras related protein, R-Ras, within this pathway was initially investigated via a morphological study. R-ras activity was altered using either dominant negative or constitutively active mutants and cell morphology and migration was monitored. A reduction in R-ras activity inhibited cell spreading and scattering after stimulation with HGF while constant R-ras activity led to cell scattering without further addition of HGF, as had been previously shown. The migration promoted by active R-ras was inhibited by the addition of SecinH3, a cytohesin inhibitor. These observations were further supported by western blot analysis of Arf6 activation in the mutant expressing cells. A decrease in R-ras activity reduced Arf6 activation in the presence of HGF. The constant activation of R-ras also caused a decrease in Arf6 activation in the presence of HGF. Together, this data shows that R-Ras is downstream of the HGF-c-Met signal transduction pathway and has the ability to alter cell shape and migration.