ARF-GEF cytohesin-2/ARNO regulates R-Ras and α5-integrin recycling through an EHD1-positive compartment.

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 the 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), colocalizes with active R-Ras in transiently expressed HeLa cells. In addition, we show that EHD1-positive recycling endosomes are a novel compartment for cytohesin-2/ARNO. Knockdown or expression of GEF-inactive (E156K) cytohesin-2/ARNO causes R-Ras to accumulate on recycling endosomes containing EHD1 and inhibits cell spreading. E156K-ARNO also causes a reduction in focal adhesion size and number. 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.

CNK3 and IPCEF1 produce a single protein that is required for HGF dependent Arf6 activation and migration.

Epithelial cells are largely immotile under normal circumstances, but become motile during development, repair of tissue damage and during cancer metastasis. Numerous growth factors act to initiate epithelial cell movements. Hepatocyte growth factor (HGF) induces many epithelial cell lines to begin crawling. A number of small GTPases act downstream of HGF to alter cell shape and promote movement. Arf6 is one of these GTPases that can alter the cortical actin cytoskeleton and promote epithelial movement. Activation of Arf6 in MDCK cells by its guanine nucleotide exchange factor cytohesin 2/ARNO produces a scattering response strikingly reminiscent of the action of HGF. We have previously shown that IPCEF1, a scaffold that binds to cytohesin 2, is required for cytohesin-induced scattering. We report here that IPCEF1 is actually the C-terminal half of CNK3. CNKs are scaffolds involved in signal transduction downstream of Ras. We have found that both MDCK and CaCo-2 cells express a fused CNK3/IPCEF1 protein. Knockdown of this protein impairs HGF-induced Arf6 activation and migration in response to HGF treatment.