R-Ras Promotes Focal Adhesion Formation through Focal Adhesion Kinase and p130^Cas by a Novel Mechanism That Differs from Integrins

Abstract

R-Ras regulates integrin function, but its effects on integrin signaling pathways have not been well described. We demonstrate that activation of R-Ras promoted focal adhesion formation and altered localization of the α2β1 integrin from cell-cell to cell-matrix adhesions in breast epithelial cells. Constitutively activated R-Ras(38V) dramatically enhanced focal adhesion kinase (FAK) and p130^Cas phosphorylation upon collagen stimulation or clustering of the α2β1 integrin, even in the absence of increased ligand binding. Signaling events downstream of R-Ras differed from integrins and K-Ras, since pharmacological inhibition of Src or disruption of actin inhibited integrin-mediated FAK and p130^Cas phosphorylation, focal adhesion formation, and migration in control and K-Ras(12V)-expressing cells but had minimal effect in cells expressing R-Ras(38V). Therefore, signaling from R-Ras to FAK and p130^Cas has a component that is Src independent and not through classic integrin signaling pathways and a component that is Src dependent. R-Ras effector domain mutants and pharmacological inhibition suggest a partial role for phosphatidylinositol 3-kinase (PI3K), but not Raf, in R-Ras signaling to FAK and p130^Cas. However, PI3K cannot account for the Src-independent pathway, since simultaneous inhibition of both PI3K and Src did not completely block effects of R-Ras on FAK phosphorylation. Our results suggest that R-Ras promotes focal adhesion formation by signaling to FAK and p130^Cas through a novel mechanism that differs from but synergizes with the α2β1 integrin.

ACKNOWLEDGMENTS

We thank Deane Mosher and Steve Barthel for VCAM and assistance with VCAM binding assays, Matt Bunce for assistance with PI3K assays, Andrew Aplin for helpful discussion and for pcDNA3-FRNK, Alan Hall for R-Ras effector loop constructs, Jun-Lin Guan for Cas-SH3, Channing Der for Ral(31N), and Eugene Marcantonio for anti-α2 integrin cytoplasmic tail antibody.

This work was supported by an AACR-Susan G. Komen career development award, NIH grant R29 CA76537-06, and an HHMI Medical School Start-up Award (P.J.K.).