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Phone: Fax: E-mail: ude. Abstract R-Ras regulates integrin function, but its effects on integrin signaling pathways have not been well described. 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 pCas 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 pCas has a component that is Src independent and not through classic integrin signaling pathways and a component that is Src dependent. Integrins regulate several aspects of cell growth, migration, differentiation, and transformation.

It is increasingly apparent that small GTPases of the Ras superfamily participate in both outside-in and inside-out integrin signaling pathways. Integrin function leads to activation of Ras, Rac, Rho, and Cdc42 6 , and cell adhesion is necessary for signaling through the Ras-Raf-mitogen-activated protein kinase pathway 4 , 5 , 12 , Conversely, Ras transformation results in anchorage independence and loss of actin stress fibers, possibly due to its ability to down-regulate integrin affinity 13 , while Rho family GTPases regulate the actin cytoskeleton and enhance focal adhesion formation 22 , R-Ras, a member of the Ras superfamily closely related to H-, N-, and K-Ras, directly regulates integrin affinity and avidity states, enhancing adhesion of several cell types 1 , 19 , 46 and migration of breast epithelial cells R-Ras enhancement of integrin function is in opposition to H-Ras, which down-regulates integrin affinity 38 , Although the biological functions of R-Ras are clearly distinct from those of H-, N-, and K-Ras, the molecular basis for this difference is poorly understood.

Because integrins have short cytoplasmic tails lacking enzymatic activity, integrin signaling events occur through associations with cytosolic proteins that initiate the activation of signaling pathways 8 , One of these cytosolic proteins is focal adhesion kinase FAK. Upon integrin binding to ligand, FAK becomes phosphorylated at tyrosine Y , the autophosphorylation site, which creates a high-affinity binding site for Src The activity of Src and FAK is important for integrin-stimulated phosphorylation of pCas 2 , 35 , 43 , which creates multiple docking sites for additional downstream signaling molecules and focal adhesion components.

FAK and pCas appear to be necessary regulators of focal adhesion dynamics and the promotion of cell migration 2 , 11 , 14 , 18 , Moreover, focal adhesion targeting is an important regulator of FAK function 39 , and integrin signaling to FAK is inhibited upon disruption of the actin cytoskeleton Despite the obvious importance of focal adhesion formation to integrin signaling events, regulation of focal adhesions and their components is not fully understood.

Previously, we found that expression of activated R-Ras alters breast epithelial phenotypes by disrupting differentiation and promoting cell migration. In this study, we further investigated the molecular basis for R-Ras effects on integrin signaling pathways. The effects of R-Ras were not due to conformational changes in the integrin leading to enhanced affinity, nor could they be explained entirely by increased avidity, since R-Ras enhanced FAK and pCas phosphorylation even in the absence of increased ligand binding.

Moreover, signaling to FAK and pCas differed from integrins in dependence on Src and an intact actin cytoskeleton. FAK phosphorylation site-specific antibodies were obtained from Biosource International, and vinculin antibody was obtained from Sigma. Secondary antibodies were obtained from Jackson ImmunoResearch Laboratories.

R-Ras effector loop constructs, i. COCO in the laboratory of Deane Mosher by methods previously described for the seven-domain form 18a. Cell culture. R-Ras constructs containing the 38V mutation and a second mutation in the effector loop a generous gift of Alan Hall [ 37 ] were transfected into T47D cells, and pools of stable transfectants were isolated. Clonal cell lines were also created, with similar phenotype. In parallel, vector-only, wild-type R-Ras, and R-Ras 38V were also transfected into cells and pools of stable transfectants, and individual clonal cell lines were created.

Immunoprecipitations and immunoblotting. Subconfluent cells were harvested by treating cells with 0. Cells were subsequently stimulated with rat tail collagen type 1 Collaborative Biosciences either in suspension 0. In experiments to determine differences in the FAK specific phosphorylation sites, lysates were analyzed directly by immunoblotting.

The immunoprecipitated proteins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis SDS-PAGE and were subsequently transferred to polyvinyl difluoride membranes Millipore.

Phosphorylation was analyzed by immunoblotting with antiphosphotyrosine antibody 4G10 followed by an anti-mouse secondary antibody conjugated to horseradish peroxidase and was visualized by using the ECL Plus detection reagent, exposing the blots to Fuji medical X-ray film, and developing the film using an automatic radiograph developer.

Blots were stripped and reprobed with anti-FAK or anti-pCas. Kinase assays. The plate was air dried, and the amount of 32P incorporated into PI phosphate was determined by autoradiography. Src was immunoprecipitated from the cell lysates using an anti-Src antibody and washed twice with the lysis buffer, once with 50 mM Tris-HCl, pH 7.

Flow cytometry. Cells were detached from tissue culture dishes by using 0. As a control, cells were stained with isotype-matched immunoglobulin G IgG. For ligand binding studies, previously characterized T47D cells expressing X4C2 chimeric integrin 16 were detached as described and , cells were resuspended in RPMI medium with 0.

Cells were incubated with soluble monomeric six-domain VCAM-1 at room temperature for 30 min and washed three times. Cells were then incubated with an anti-VCAM-1 antibody for 20 min on ice and washed three times. Secondary FITC-conjugated donkey antibody was incubated with cells for 20 min on ice and analyzed by flow cytometry.

Immunofluorescence microscopy. After being rinsed again in PBS, paraformaldehyde was quenched with 0. Cells were then permeabilized in 0. Primary antibodies used were as follows: antiphosphotyrosine pY99, antivinculin, anti-FAK , and antipaxillin. Secondary antibodies used were anti-mouse tetramethyl rhodamine isocyanate and anti-rabbit FITC, both used at a dilution. Slides were viewed with a Nikon Eclipse TE inverted microscope. Images were collected, and three-dimensional deconvolution was performed using Inovision software.

Cell migration assays. The migration assay was performed using transwells Corning Costar Corp. At 36 h after transfection, cells were trypsinized and were transferred to the upper chamber of the transwell. The transwell membranes were washed and mounted on slides with Vectashield mounting medium Vector Laboratories, Inc.

Cells expressing GFP were counted. For the migration assays using inhibitors, cells were treated with inhibitor for 15 min prior to being transferred to the transwell, allowed to migrate for 5 h, and then fixed and stained with the HEMA 3 stain set Fisher Scientific Co. All migration assays were performed in triplicate and repeated three times.

Focal adhesion formation is an essential process in cell migration and adhesion. In an effort to understand the role of R-Ras in integrin-mediated cell migration, we examined the effects of R-Ras on focal adhesions. Expression of constitutively active R-Ras 38V enhanced the size and number of focal adhesions in cells cultured on collagen-coated coverslips compared to control cells Fig.

R-Ras 38V expression not only promoted larger focal adhesions but also the appearance of focal adhesions localized to the center of the ventral surface of the cell. Phosphotyrosine staining colocalized with paxillin and vinculin not shown. These results demonstrate that activated R-Ras dramatically enhances focal adhesion formation and may regulate cell-cell contacts as well. Activated R-Ras 38V was found at the plasma membrane and at cell-cell contacts and localized to membrane ruffles Fig.


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