The role of a R-ras and cytohesin-2 signaling pathway in epithelial cell migration

Open Access
Salem, Joseph Charles
Graduate Program:
Biochemistry, Microbiology, and Molecular Biology
Doctor of Philosophy
Document Type:
Date of Defense:
October 08, 2015
Committee Members:
  • Lorraine C Santy, Dissertation Advisor
  • Lorraine C Santy, Committee Chair
  • Melissa Rolls, Committee Member
  • Andrea Marie Mastro, Committee Member
  • Graham Hugh Thomas, Committee Member
  • Pamela Hankey Giblin, Special Member
  • cytohesin-2
  • R-Ras
  • EHD1
  • cell migration
  • ARNO
When expressed in epithelial cells, cytohesin-2/ARNO, a guanine nucleotide exchange factor (GEF) for ARF small GTPases, causes a robust migration response. Recent evidence suggests that cytohesin-2/ARNO acts downstream of small GTPase R- Ras to promote spreading and migration. We hypothesized that cytohesin-2/ARNO could transmit R-Ras signals by regulating the recycling of R-Ras through ARF activation. We found that Eps15-homology domain 1 (EHD1), a protein that associates with the endocytic recycling compartment (ERC), co-localizes with active R-Ras in HeLa cells. Additionally, we show that EHD1-positive recycling endosomes are a novel compartment for cytohesin-2/ARNO. Expression of GEF-inactive (E156K) cytohesin-2/ARNO causes R-Ras to accumulate on recycling endosomes containing EHD1, and inhibits cell spreading. Knockdown of endogenous cytohesin-2/ARNO inhibits cell spreading and also leads to accumulation of R-Ras on EHD1-positive compartments. Finally, we demonstrate that R-Ras/ARNO signaling is required for recycling of α5-integrin and R- Ras to the plasma membrane. These data establish a role for cytohesin-2/ARNO as a regulator of R-Ras and integrin recycling, and suggest that ARF-regulated trafficking of R-Ras is required for R-Ras-dependent effects on spreading and adhesion formation.