R-Ras promotes focal adhesion formation through focal adhesion kinase and p130(Cas) by a novel mechanism that differs from integrins.
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 alpha2beta1 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 alpha2beta1 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 alpha2beta1 integrin.
Kwong
Lina
L
Department of Pharmacology, University of Wisconsin Medical School, Madison, Wisconsin 53706, USA.
Wozniak
Michele A
MA
Collins
Asha S
AS
Wilson
Siobhan D
SD
Keely
Patricia J
PJ
D000199:Actins / Q000378:metabolism; D002460:Cell Line; D003094:Collagen / Q000378:metabolism; D050719:Crk-Associated Substrate Protein; D005260:Female; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D022001:Focal Adhesions / Q000502:physiology*; D020558:GTP Phosphohydrolases / Q000737:chemistry / Q000502:physiology*; D006801:Humans; D038982:Integrin alpha2beta1 / Q000502:physiology; D016023:Integrins / Q000502:physiology*; D008954:Models, Biological; D019869:Phosphatidylinositol 3-Kinases / Q000502:physiology; D010750:Phosphoproteins / Q000502:physiology*; D010766:Phosphorylation; D011487:Protein Conformation; D011505:Protein-Tyrosine Kinases / Q000502:physiology*; D011506:Proteins; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000502:physiology; D050717:Retinoblastoma-Like Protein p130; D015398:Signal Transduction; D018631:ras Proteins / Q000737:chemistry / Q000502:physiology*
D000199:Actins; C493841:BCAR1 protein, human; D050719:Crk-Associated Substrate Protein; D038982:Integrin alpha2beta1; D016023:Integrins; D010750:Phosphoproteins; D011506:Proteins; D050717:Retinoblastoma-Like Protein p130; D003094:Collagen; D019869:Phosphatidylinositol 3-Kinases; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496050:PTK2 protein, human; D016392:Proto-Oncogene Proteins pp60(c-src); D020558:GTP Phosphohydrolases; C085929:RRAS protein, human; D018631:ras Proteins