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Mol Cell Biol. Feb 2003; 23(3): 933–949.
PMCID: PMC140691

R-Ras Promotes Focal Adhesion Formation through Focal Adhesion Kinase and p130Cas 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 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 p130Cas 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 p130Cas 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 p130Cas 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 p130Cas. 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 p130Cas through a novel mechanism that differs from but synergizes with the α2β1 integrin.

Integrins regulate several aspects of cell growth, migration, differentiation, and transformation. Integrin binding to components of the extracellular matrix activates several signaling pathways, leading to the phosphorylation of multiple proteins, a process termed “outside-in” signaling. In addition, integrin ligand binding is regulated by the activation of signaling pathways within cells, a process termed “inside-out” signaling. 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, 27). 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, 29).

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 (16). Only a few effectors for R-Ras have been identified: phosphatidylinositol 3-kinase (PI3K), Raf, Ral-GDS/Rlf/Rgl, and Nore1 (10, 20, 37, 41, 42), all of which are shared with H-, N-, and K-Ras. Of these, PI3K has emerged as the predominant effector of R-Ras (20), while activation of Raf and Ral-GDS is variable in different cell types and biological assays (10, 20, 24) (37). R-Ras enhancement of integrin function is in opposition to H-Ras, which down-regulates integrin affinity (38, 46). R-Ras also differs from K-Ras, in that R-Ras-induced cell migration is insensitive to inhibition of the Raf-MEK-mitogen-activated protein kinase pathway (16). 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, 44). One of these cytosolic proteins is focal adhesion kinase (FAK). Upon integrin binding to ligand, FAK becomes phosphorylated at tyrosine 397 (Y397), the autophosphorylation site, which creates a high-affinity binding site for Src (34). Src, in turn, phosphorylates additional sites in FAK, including Y576/Y577 in the catalytic loop, enhancing FAK function (31). Activation of FAK by Src is likely a key event in integrin signaling, because FAK phosphorylation, even at Y397, is minimal in the absence of Src activity (25, 33). This is in contrast to FAK activation downstream of G-protein-coupled receptors (GPCRs), where FAK phosphorylation at Y397 is Src independent (33). The activity of Src and FAK is important for integrin-stimulated phosphorylation of p130Cas (2, 35, 43), which creates multiple docking sites for additional downstream signaling molecules and focal adhesion components. FAK and p130Cas appear to be necessary regulators of focal adhesion dynamics and the promotion of cell migration (2, 11, 14, 18, 40). 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 (36). 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. R-Ras effects differ from K-Ras, in that R-Ras promotes migration through the α2β1 integrin across collagen but not through the α5β1 integrin across fibronectin. Additionally, R-Ras-induced migration is not sensitive to inhibition of MEK, unlike K-Ras (16). These results suggest that R-Ras regulates α2β1 integrin signaling pathways and that this regulation may differ from K-Ras. In this study, we further investigated the molecular basis for R-Ras effects on integrin signaling pathways. R-Ras dramatically enhanced focal adhesion formation and FAK and p130Cas phosphorylation. 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 p130Cas phosphorylation even in the absence of increased ligand binding. Moreover, signaling to FAK and p130Cas differed from integrins in dependence on Src and an intact actin cytoskeleton. Our results demonstrate a role for R-Ras in the regulation of focal adhesions and suggest that R-Ras promotes FAK and p130Cas phosphorylation by a unique mechanism that differs from, yet synergizes with, α2β1 integrin signaling.



Rat tail collagen I, paxillin, and p130Cas antibodies were obtained from BD Biosciences. FAK and 4G10 monoclonal antibodies were from Upstate Biotechnology. R-Ras, FAK, and pY99 antibodies were from Santa Cruz Biotechnology. 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. The inhibitors PP2 and cytochalasin D were obtained from Calbiochem, and LY294002 was obtained from Alexis Biochemicals. GammaBind G Sepharose beads and ECL Plus was from Amersham Pharmacia Biotech. The antibody to the α2 cytoplasmic tail was a generous gift from Eugene Marcantonio. pKH-Cas-SH3 was a generous gift of Jun-Lin Guan. pcDNA3-FRNK was a generous gift of Andrew Aplin. pBABE-Ral(S31N) was a generous gift of Channing Der. R-Ras effector loop constructs, i.e., pRK5myc-V38R-Ras/S61,/G63,/C66, were a generous gift of Alan Hall (37). Six-domain VCAM-1 (lacking the fourth domain and containing only one α4β1 integrin binding site) was prepared in pACBPG7.COCO in the laboratory of Deane Mosher by methods previously described for the seven-domain form (18a).

Cell culture.

T47D cells were stably transfected with pZIP-R-Ras(38V), pZIP-K-Ras(12V), or the vector alone, pZIP, and have been previously characterized (16). Cells were maintained in RPMI medium (Cellgro) supplemented with 10% fetal bovine serum (Life Technologies, Inc.) and 8 μg of insulin (Sigma)/ml at 37°C and 5% CO2. Cells expressing chimeric α4/α2 integrins (X4C2) have been previously described and characterized (16). 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 0.5 mM EDTA in phosphate-buffered saline (PBS), resuspended in RPMI medium with 5 mg of fatty-acid-free bovine serum albumin (BSA)/ml, and counted. Cells were subsequently stimulated with rat tail collagen type 1 (Collaborative Biosciences) either in suspension (0.5 mg/ml) or plated onto collagen-coated petri dishes (30 μg/ml) as indicated. For pharmacological inhibition, the cells were treated for 15 min with PP2 (10 μM) or LY294002 (25 μM) or for 1 h with cytochalasin D (1 μM) prior to collagen stimulation. Cells were then lysed with ice-cold lysis buffer (25 mM HEPES, 75 mM NaCl, 1% NP-40, 0.25% deoxycholate, 2 mM EDTA, 2 mM NaF, protease cocktail inhibitor, and 1 mM Na3VO4). Cell lysates were centrifuged at full speed in a tabletop centrifuge for 12 min at 4°C. For the detection of FAK and p130Cas phosphorylation, cell lysates were immunoprecipitated with anti-FAK and anti-p130Cas antibodies by using Gamma Bind G Sepharose beads. This was allowed to complex while rotating for 3 h at 4°C. The immunocomplexes were then washed three times with ice-cold lysis buffer after which Laemmli sample buffer containing β-mercaptoethanol was added and samples were heated to 100°C for 5 min. 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). The blots were blocked with 5% BSA in Tris-buffered saline with 0.3% Tween overnight. 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. For the immunoblots of FAK specific phosphorylation sites, we used anti-FAK phospho-specific antibodies at 0.3 μg/ml. Blots were stripped and reprobed with anti-FAK or anti-p130Cas.

Kinase assays.

To determine PI3K activity, cells were treated with dimethyl sulfoxide (DMSO) or 25 μM LY294002 for 15 min prior to 10 min of incubation with collagen. Cells were lysed in buffer (20 mM Tris-HCl, pH 7.4, 137 mM NaCl, 2 mM EDTA, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, and a protease inhibitor cocktail) and cell lysates were immunoprecipitated with anti-PI3K antibody as described above. Immunoprecipitates were washed once with buffer A (20 mM Tris-HCl, pH 7.4, 137 mM NaCl, 1 mM MgCl2, 1 mM CaCl2, and 100 μM Na3VO4) and were then washed twice with 100 mM Tris-HCl pH 7.5, 500 mM LiCl, and 100 μM Na3VO4 and finally washed twice with Tris-NaCl-EDTA (10 mM Tris-HCl, pH 7.4, 100 mM NaCl, 1 mM EDTA, and 100 μM Na3VO4). The kinase reaction was performed at room temperature for 15 min in the presence of Tris-NaCl-EDTA, 100 mM MgCl2, 1 mM ATP, 2 μg of phosphatidyl inositol (Avanti)/ml, and 15 μCi of [γ-32P]ATP and was terminated by addition of 8 N HCl. Samples were extracted with CHCl3-MeOH. The organic phase was spotted onto oxalate-treated thin-layer chromatography plates and was developed in CHCl3-MeOH-H2O-NH4OH. The plate was air dried, and the amount of 32P incorporated into PI phosphate was determined by autoradiography.

To determine Src kinase activity, cells were treated with DMSO or 10 μM PP2 for 15 min prior to a 10-min incubation with collagen and were lysed with buffer containing 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% NP-40, 1 mM Na3VO4, and protease inhibitor cocktail. 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 7.5, 100 mM NaCl, 10 mM MnCl2, 500 mM LiCl, and 100 μM 1 mM Na3VO4 and once with kinase buffer (100 mM NaCl, 5 mM MnCl2, 50 mM Tris-HCl, pH 7.5, 10 mM MgCl2, and 1 mM Na3VO4). Beads were resuspended in a total of 30 μl with the addition of 2 μg of enolase (Sigma)/ml, 1 mM ATP, and 15 μCi of [γ-32P]ATP. Samples were run on an SDS-12% PAGE gel, dried, and incorporation of 32P was determined by autoradiography.

Flow cytometry.

Cells were detached from tissue culture dishes by using 0.5 mM EDTA in PBS and were resuspended in PBS supplemented with 5 mg of BSA/ml. Cells held in suspension at 4°C were treated with 2 mM Mn2+ or left untreated for 5 min and were then incubated with 12G10 monoclonal antibody, which recognizes the conformationally active form of the β1 integrin subunit. As a control, cells were stained with isotype-matched immunoglobulin G (IgG). Excess antibody was removed by washing once in ice-cold PBS, and cells were stained with fluorescein isothiocynate (FITC)-conjugated anti-mouse IgG (Jackson Labs). Mean channel fluorescence was determined on a Beckman FACScan as previously described (16).

For ligand binding studies, previously characterized T47D cells expressing X4C2 chimeric integrin (16) were detached as described and 500,000 cells were resuspended in RPMI medium with 0.2% fatty-acid-free BSA. 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 (1:100) was incubated with cells for 20 min on ice and analyzed by flow cytometry.

Immunofluorescence microscopy.

Cells harvested as described above were allowed to adhere and were spread on collagen-coated coverslips (30 μg/ml) for 45 min. If treated with inhibitors, cells were treated for 15 min at 37°C in suspension prior to being cultured on collagen. Cells were rinsed with PBS and fixed for 15 min in 4% paraformaldehyde. After being rinsed again in PBS, paraformaldehyde was quenched with 0.15 M glycine in PBS. Cells were then permeabilized in 0.02% Triton X-100 in PBS for 10 min. Coverslips with adherent cells were blocked with donkey serum and BSA in PBS for 30 min, followed by primary antibody for 15 min at 37°C. Primary antibodies used were as follows: 1:200 antiphosphotyrosine pY99, 1:400 antivinculin, 1:100 anti-FAK 397, and 1:100 antipaxillin. The coverslips were rinsed in PBS, and their contents were then incubated with secondary antibody for 15 min at 37°C. Secondary antibodies used were anti-mouse tetramethyl rhodamine isocyanate and anti-rabbit FITC, both used at a 1:100 dilution. After a final rinse with PBS, coverslips were mounted using ProLong Antifade mounting medium (Molecular Probes, Inc.). Slides were viewed with a Nikon Eclipse TE300 inverted microscope. Images were collected, and three-dimensional deconvolution was performed using Inovision software.

Cell migration assays.

For migration assays, cells were transiently transfected with 10 μg of pcDNA3-FRNK, pKH-Cas-SH3, dominant-negative pBABE-Ral(S31N), or vector alone, together with 5 μg of green fluorescent protein (GFP) using Lipofectamine Plus and following the manufacturer's instructions (Life Technologies Inc.). The migration assay was performed using transwells (Corning Costar Corp.) as described earlier (15). The underside of the transwell was coated with 3 μg of collagen/ml. At 36 h after transfection, cells were trypsinized and were transferred to the upper chamber of the transwell. After incubation at 37°C in 5% CO2 overnight, the migration assay was terminated by 4% paraformaldehyde fixation. 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.


R-Ras enhances focal adhesion formation.

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. (Fig.1A1A and E). 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). R-Ras also altered the localization of the α2β1 integrin, the major collagen receptor on T47D breast epithelial cells (16). In control cells, the α2β1 integrin was localized to both cell-cell contacts and focal adhesions (Fig. (Fig.1B),1B), whereas, in cells expressing constitutively active R-Ras(38V), α2β1 was lost from cell-cell contacts and relocalized to focal adhesions (Fig. (Fig.1F).1F). 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. (Fig.1G).1G). R-Ras was not obvious in focal adhesions, although occasionally it was found colocalized with the α2β1 integrin (Fig. (Fig.1H1H).

FIG. 1.
R-Ras enhances focal adhesion formation. Control cells (A to D) and R-Ras(38V)-expressing cells (E to H) cells were cultured on collagen-coated coverslips for 45 min and were immunostained with antiphosphotyrosine antibody (A and E) or were coimmunostained ...

R-Ras enhances the phosphorylation of FAK and p130Cas in a collagen-dependent manner.

Integrin-mediated cell migration and adhesion to the ECM involve signaling to FAK and p130Cas, both of which are found in focal adhesions. Because R-Ras enhances integrin-mediated adhesion and migration (16, 23, 38, 46), we examined whether R-Ras could enhance integrin signaling events. The effects of R-Ras on tyrosine phosphorylation of FAK and p130Cas were determined by immunoprecipitating these molecules from lysates of T47D cells stimulated in suspension with collagen. Expression of constitutively active R-Ras(38V) enhanced phosphorylation of FAK by two- to fourfold and p130Cas by ~10-fold in a collagen-dependent manner relative to control cells (Fig. (Fig.2A2A).

FIG. 2.FIG. 2.
R-Ras enhances FAK and p130Cas phosphorylation, leading to an enhancement of cell migration. (A) Expression of activated R-Ras(38V) enhances FAK and p130Cas phosphorylation. Cells were stimulated with collagen in suspension for 10 min; FAK and p130Cas ...

To determine whether FAK and p130Cas play a role in R-Ras-induced migration across collagen, haptotactic migration assays were performed following transfection of dominant-negative forms of FAK (FRNK) and p130Cas (Cas-SH3). Expression of FRNK (Fig. (Fig.2B)2B) or Cas-SH3 (Fig. (Fig.2C)2C) significantly decreased migration in cells expressing R-Ras(38V). In addition, FAK and p130Cas were also necessary for focal adhesion formation since dominant-negative FAK (FRNK) and p130Cas (Cas-SH3) decreased the number of cells able to form focal adhesions in both control and R-Ras(38V)-expressing cells (Fig. (Fig.2D).2D). Taken together, these results suggest that FAK and p130Cas play important roles in R-Ras-induced migration and focal adhesion formation via the α2β1 integrin. Interestingly, the role of FAK appears to be more important for effects of R-Ras on focal adhesion formation, while the role of p130Cas appears to be more important for effects of R-Ras on cell migration.

R-Ras effects on α2β1 integrin signaling do not require increased ligand binding and differ from the conformational changes induced by Mn2+.

In certain cell types, constitutively active R-Ras(38V) enhances cell adhesion by increasing ligand binding activity of the integrin receptor (46). Such affinity changes in integrin binding can be reflected by conformational changes of the integrin. The enhancement of FAK and p130Cas phosphorylation by R-Ras(38V) expression could be due to effects of R-Ras on α2β1 integrin affinity. To determine whether this was a possibility, cells were treated with Mn2+, which can conformationally activate integrins, enhancing their affinity and exposing ligand binding sites (reviewed in reference 21). Treatment of control cells with Mn2+ dramatically increased the phosphorylation of FAK (an average of 2.7-fold for three separate experiments, compared to control cells plus collagen in the absence of Mn2+) and p130Cas (an average of sevenfold) following collagen stimulation (Fig. (Fig.3A,3A, lane 2 versus lane 4). R-Ras synergized with Mn2+, since its activation resulted in a further increase in the phosphorylation of both FAK (an average of 4.8-fold above control cells, lane 8 versus lane 4) and p130Cas (an average of eightfold above control cells) (Fig. (Fig.3).3). The ability of Mn2+ and R-Ras to synergize with one another suggests that R-Ras does not mimic the effects of Mn2+ on integrin conformation and ligand affinity. To more directly determine if R-Ras induced conformational changes in the α2β1 integrin on T47D cells, we made use of an anti-β1 antibody, 12G10, whose epitope is conformationally sensitive. Flow cytometry demonstrated that expression of R-Ras(38V) did not enhance the binding of 12G10 relative to control cells in the absence of Mn2+ (Fig. (Fig.3B).3B). Therefore, R-Ras does not induce the conformational change detected by the 12G10 antibody.

FIG. 3.FIG. 3.
R-Ras effects on integrin signaling are not due to increased ligand binding. (A) Expression of activated R-Ras(38V) synergizes with Mn2+ to enhance phosphorylation of FAK and p130Cas. Cells were treated with 1 mM Mn2+, stimulated with ...

To determine whether increased ligand binding might contribute to the effects of R-Ras, we investigated the binding of soluble VCAM to cells that express a chimeric α4/α2 integrin, having an extracellular α4 domain and an intracellular α2 domain (X4C2). We previously demonstrated that R-Ras enhances cell migration via X4C2 and that cell surface expression of X4C2 is similar in control and R-Ras-expressing cells (16). Soluble VCAM was bound to cells in suspension, and then binding was determined with anti-VCAM antibody by flow cytometry analogous to the approach of Rose et al. (30). Expression of R-Ras(38V) did not enhance binding of VCAM (Fig. (Fig.3C).3C). As a positive control, activation of the integrin by Mn2+ increased VCAM binding to both control and R-Ras-expressing cells (Fig. (Fig.3C3C).

As an additional means to determine whether increased ligand binding is necessary for R-Ras effects on FAK and p130Cas phosphorylation, the α2β1 integrin was stimulated with P1E6, an anti-α2 antibody that binds within the ligand binding region and inhibits collagen binding to α2β1. Importantly, we previously demonstrated by flow cytometry that expression of activated R-Ras does not enhance binding of P1E6 to the α2β1 integrin (16). Expression of R-Ras(38V) enhanced p130Cas phosphorylation following clustering of the α2β1 integrin with P1E6, whereas signaling in control cells was minimal (Fig. (Fig.3D).3D). Similar results were obtained for FAK (data not shown) and when cells adhered to plates coated with P1E6 antibody (not shown). Therefore, in the absence of changes in ligand binding, R-Ras increases signaling to FAK and p130Cas in an integrin-dependent manner. The effects of R-Ras were also not due to changes in the surface expression of α2β1 integrin, since we previously demonstrated that R-Ras does not alter the expression of several integrin subunits, including α2 and β1, on the surface of T47D cells (16).

R-Ras enhances FAK and p130Cas phosphorylation independent of the actin cytoskeleton.

To determine whether the effects of R-Ras on FAK and p130Cas phosphorylation are secondary to enhanced focal adhesion formation, we treated cells with an inhibitor of actin polymerization, cytochalasin D. Cytochalasin D (1 μM) caused complete disruption of focal adhesions in both control and R-Ras-expressing cells (Fig. (Fig.4A).4A). Treatment with cytochalasin D inhibited both FAK and p130Cas phosphorylation in control cells (Fig. (Fig.4B).4B). However, in cells expressing R-Ras(38V), FAK phosphorylation was only partially inhibited (by 48% as an average of three experiments) and p130Cas phosphorylation was inhibited only by 10% overall when treated with cytochalasin D in the presence of collagen (Fig. (Fig.4B4B).

FIG. 4.
R-Ras enhancement of FAK and p130Cas phosphorylation is not dependent on an intact actin cytoskeleton and focal adhesions. (A) Cytochalasin D disrupts focal adhesion formation in control, R-Ras(38V)-, and K-Ras(12V)-expressing cells. Cells were pretreated ...

Since we previously noted that cell migration induced by R-Ras differed from that induced by K-Ras (16), a comparison to K-Ras was made. Expression of K-Ras(12V) enhanced FAK and p130Cas phosphorylation, although usually to a lesser extent than R-Ras. Interestingly, cytochalasin D completely inhibited FAK and p130Cas phosphorylation in K-Ras(12V)-expressing cells, unlike its effects in R-Ras(38V)-expressing cells (Fig. (Fig.4B).4B). These data suggest that R-Ras can signal to FAK and p130Cas independent of the integrity of the actin cytoskeleton, whereas, in normal cells and cells expressing activated K-Ras, FAK and p130Cas phosphorylation is dependent on the actin cytoskeleton.

The role of focal adhesion formation and polymerization of the actin cytoskeleton was further investigated by comparing cells stimulated by collagen in suspension to those stimulated by adhesion to collagen. FAK and p130Cas phosphorylation was similar whether cells were stimulated with collagen in suspension or by adhesion to collagen-coated dishes, demonstrating that the enhancement of FAK and p130Cas phosphorylation is independent of cell shape and does not rely on focal adhesion formation (Fig. (Fig.4C).4C). Because we wanted to investigate proximal integrin signaling events in the absence of focal adhesion formation and cell spreading, subsequent experiments were performed by stimulating cells with collagen in suspension.

R-Ras enhances FAK Y397 phosphorylation.

To further define the effects of R-Ras on FAK phosphorylation, phospho-specific antibodies that recognize the different phosphorylation sites of FAK were used. Expression of constitutively active R-Ras(38V) enhanced phosphorylation at Y397 (≥3-fold) and at Y576 (2.2-fold) relative to control cells in response to collagen as analyzed by densitometry when normalized to total FAK levels for three separate experiments (Fig. (Fig.5A).5A). Collagen did not appreciably enhance phosphorylation at Y925 in either control or R-Ras-expressing cells (Fig. (Fig.5A).5A). The function of the anti-Y925 antibody was verified with positive control CEF cell lysates (not shown). Immunostaining of FAK Y397 was also enhanced at focal adhesions in R-Ras-expressing cells compared to that found in control cells. Phospho-specific FAK 397 was found at both large peripheral and more central focal adhesions (Fig. (Fig.5B).5B). These results indicate that R-Ras enhances FAK phosphorylation at the autophosphorylation site, Y397, which may help recruit other signaling molecules.

FIG. 5.
R-Ras enhances FAK phosphorylation at specific sites. (A) R-Ras enhances FAK phosphorylation at Y397 (threefold) and Y576 (twofold) but not at Y925. Lysates from control cells and cells expressing activated R-Ras(38V) stimulated with collagen were subjected ...

R-Ras signals to FAK and p130Cas, focal adhesion formation, and migration through Src-dependent and -independent pathways.

The Src inhibitor, PP2, was used to determine whether Src plays a role in the effects of R-Ras on phosphorylation of FAK and p130Cas. In control cells stimulated with collagen, both FAK and p130Cas phosphorylation was completely inhibited by PP2 to unstimulated levels (Fig. (Fig.6A).6A). In contrast, in cells expressing constitutively active R-Ras(38V), FAK and p130Cas phosphorylation was only partially inhibited by PP2 (Fig. (Fig.6A).6A). PP2 completely inhibited FAK and p130Cas phosphorylation in K-Ras(12V)-expressing cells, suggesting that K-Ras effects on FAK and p130Cas are Src dependent, in marked contrast to results with R-Ras cells. The failure of PP2 to block signaling in R-Ras cells was not due to resistance of these cells to PP2, as PP2 was as effective at inhibiting Src in an in vitro kinase assay in R-Ras(38V)-expressing cells as in control cells (Fig. (Fig.6A).6A). Interestingly, Src kinase activity was less in R-Ras(38V)-expressing cells (Fig. (Fig.6A),6A), although total levels of Src were the same in lysates of control and R-Ras(38V) cells (not shown).

FIG. 6.FIG. 6.
R-Ras signals to FAK and p130Cas through Src-dependent and Src-independent pathways. (A) Analysis of the effects of the Src inhibitor, PP2, on FAK and p130Cas phosphorylation. Cells were preincubated with 10 μM PP2 (+) or DMSO (−) ...

Src is required for integrin-dependent stimulation of FAK phosphorylation at Y397, whereas G-protein coupled receptor stimulation of FAK phosphorylation at Y397 is Src independent (33). We next examined the effects of PP2 on phosphorylation of FAK at specific sites. In control cells stimulated with collagen, PP2 inhibited phosphorylation at both Y397 and Y576 (Fig. (Fig.6B).6B). In R-Ras(38V)-expressing cells, PP2 diminished phosphorylation at Y576 but had minimal effect on phosphorylation at Y397 (Fig. (Fig.6B).6B). Therefore, R-Ras induces FAK Y397 phosphorylation by a Src-independent mechanism, unlike integrin signaling, which is Src dependent.

Like the phosphorylation events, the formation of focal adhesions in cells expressing R-Ras(38V) was much less sensitive to the effects of PP2 than in control cells or cells expressing K-Ras(12V). In control cells, treatment with PP2 resulted in a complete loss of focal adhesions in more than 50% of the cells and diminished cell spreading (Fig. (Fig.6C6C and D). K-Ras(12V)-expressing cells were even more sensitive to PP2, with greater than 75% of cells demonstrating complete loss of focal adhesions. In contrast, R-Ras-expressing cells treated with PP2 were still able to spread and retained peripheral focal adhesions, although there was a decrease in the apparent size and number of focal adhesions and a loss of large, central adhesions (Fig. (Fig.6C6C and D). The results obtained were similar when cells were stained for phosphotyrosine (not shown) or FAK Y397 (Fig. (Fig.6C6C).

Cell migration demonstrated a similar trend, since PP2 partially inhibited R-Ras-induced cell migration (Fig. (Fig.6E),6E), but had more striking effects on control cell migration. Collectively, these results suggest that there are Src-dependent and Src-independent pathways in R-Ras signaling to FAK, p130Cas, focal adhesion formation, and cell migration. In contrast, collagen-induced signaling to FAK and p130Cas in the absence of constitutively activated R-Ras is Src dependent. Therefore, R-Ras effects on FAK and p130Cas are not completely due to enhanced integrin binding to collagen, but R-Ras instead signals to FAK and p130Cas by a mechanism that differs from classic integrin pathways.

R-Ras signaling to FAK and p130Cas requires interactions through the effector loop.

The involvement of specific effectors was investigated with effector loop mutations (37), which have a second point mutation in the effector-binding loop of R-Ras, in addition to the activating 38V mutation (Fig. (Fig.7A).7A). R-Ras has at least three effectors, Raf, Ral-GDS, and PI3K. R-Ras(38V)/61S, which binds to Raf but does not bind Ral-GDS or PI3K (17, 23, 24, 37), decreased FAK phosphorylation by 47% compared to R-Ras(38V)-expressing cells (Fig. (Fig.7B).7B). R-Ras(38V)/63G, which binds Ral-GDS and PI3K, had only minimal effect on FAK phosphorylation (a 20% decrease compared to that observed with R-Ras[38V]). R-Ras(38V)/66C, which does not bind Ral-GDS and has diminished PI3K binding (17, 23, 24, 37), decreased FAK phosphorylation by 44%. These effector mutations had less of an impact on p130Cas phosphorylation: Ras(38V)/61S, 63G, and 66C decreased p130Cas phosphorylation by 28, 12, and 28%, respectively. Cell migration for these effector mutants somewhat mimicked the phosphorylation events, since R-Ras(38V)/63G did not have a significant effect on cell migration compared to R-Ras(38V)-expressing cells (Fig. (Fig.7C).7C). Since R-Ras(38V)/63G retains binding to both Ral-GDS and PI3K, we determined whether Ral plays a role in R-Ras-induced migration. Dominant-negative Ral(S31N) did not significantly inhibit R-Ras-induced migration across collagen (Fig. (Fig.7C)7C) or focal adhesion formation (data not shown). Consistent with the results for cell migration, cells expressing R-Ras(38V)/61S and R-Ras(38V)/66C demonstrated loss of focal adhesions, but R-Ras(38V)/63G cells did not (not shown). Therefore, R-Ras effects are independent of Raf and partially PI3K dependent. All of the mutations in the R-Ras effector loop diminished the formation of large, central focal adhesions promoted by R-Ras(38V) (Fig. (Fig.7D),7D), suggesting the contribution of either a novel effector or multiple effector pathways to these large focal adhesions.

FIG. 7.
Analysis of effector loop mutations on R-Ras-induced phosphorylation, migration, and focal adhesion formation. (A) R-Ras effector loop mutants used in this study and their reported binding activities based on data compiled from several labs (17, 23, ...

R-Ras effects on p130Cas and focal adhesions are partially PI3K dependent.

Our results with effector mutants suggest that PI3K may be an important effector for R-Ras signaling to FAK and p130Cas consistent with its known role in R-Ras function (20). In addition, R-Ras-induced cell migration is partially dependent on PI3K (16). Moreover, PI3K binding to FAK Y397 is required for FAK-promoted cell migration in CHO cells (26). To further determine whether PI3K plays a role in the enhancement of FAK and p130Cas phosphorylation and focal adhesion formation by R-Ras activation, cells were treated with an inhibitor of PI3K, LY294002 (25 μM). In cells expressing R-Ras(38V), LY294002 partially inhibited p130Cas phosphorylation (~75% inhibition) but had no effect on FAK phosphorylation (Fig. (Fig.8A).8A). In control cells, LY294002 partially inhibited FAK phosphorylation (30% inhibition) (Fig. (Fig.8A).8A). These results indicate that R-Ras signals to FAK and p130Cas by different mechanisms. Moreover, R-Ras signals to FAK and p130Cas via a pathway that is at least partially independent of its effector, PI3K. In vitro PI3K assays confirmed that LY294002 is as effective in R-Ras(38V)-expressing cells as in control cells (Fig. (Fig.8A,8A, right panel). Total levels of PI3K were the same in lysates of control and R-Ras(38V)-expressing cells (not shown). In striking contrast to R-Ras, in K-Ras(12V)-expressing cells LY294002 caused a 90% inhibition of FAK phosphorylation. Therefore, the signaling pathway by which R-Ras enhances FAK phosphorylation differs from the integrin pathway and from that of K-Ras.

FIG. 8.FIG. 8.
R-Ras enhances FAK and p130Cas phosphorylation via PI3K-dependent and -independent pathways. (A) Phosphorylation of FAK and p130Cas following PI3K inhibition. Cells were preincubated with LY294002 (25 μM), and phosphorylation of FAK and p130Cas ...

To determine whether PI3K is involved in the enhancement of focal adhesion formation by R-Ras(38V), the effects of PI3K inhibition were determined. Treatment of cells expressing R-Ras(38V) with LY294002 decreased the number of cells containing large, central focal adhesions and decreased the overall size of focal adhesions, although most cells still maintained focal adhesions even in the presence of LY294002 (Fig. (Fig.8B8B and C). Focal adhesions in control cells and cells expressing K-Ras(12V) were also diminished by treatment with LY294002, with greater than 50% of K-Ras(12V) cells having lost all focal adhesions (Fig. (Fig.8B8B and C). These results suggest that PI3K plays a role in R-Ras signaling to focal adhesions, especially in the enhancement of large, central focal adhesions, but that PI3K does not account for all effects of R-Ras on focal adhesion formation.

Src and PI3K represent separate pathways linking R-Ras to focal adhesions.

Since R-Ras promotes FAK and p130Cas phosphorylation by a mechanism that could be only partially blocked by inhibitors of either Src or PI3K, we determined whether Src and PI3K represent separate pathways downstream of R-Ras. Cells were treated with the PI3K and Src inhibitors simultaneously, with the expectation that an additive effect would indicate that two pathways exist. Treatment with both LY294002 and PP2 completely abolished FAK and p130Cas phosphorylation in control cells and cells expressing K-Ras(12V) (Fig. (Fig.9A).9A). In cells expressing R-Ras(38V), treatment with both LY294002 and PP2 completely inhibited p130Cas phosphorylation and dramatically inhibited FAK phosphorylation (80% inhibition) (Fig. (Fig.9A,9A, lane 8 versus lane 6). This result suggests that R-Ras signaling to FAK and especially p130Cas occurs through two pathways: one Src dependent and one PI3K dependent. Interestingly, FAK phosphorylation in R-Ras(38V) expressing cells was not completely inhibited by LY294002 and PP2, since levels of FAK phosphorylation that equal those observed in untreated control cells resisted inhibition (Fig. (Fig.9A,9A, lane 8 versus lane 2). Therefore, R-Ras can enhance FAK phosphorylation by a novel mechanism independent of both Src and PI3K activation.

FIG. 9.
R-Ras-enhanced phosphorylation of FAK and p130Cas involve separate Src- and PI3K-dependent pathways. (A) Simultaneous treatment with LY294002 (25 μM) and PP2 (10 μM) completely inhibited p130Cas phosphorylation in all three cell types. ...

Treatment with both inhibitors completely blocked focal adhesion formation in control cells and K-Ras(12V)-expressing cells (Fig. (Fig.9B).9B). These cells were unable to spread and appeared round. However, ~6% of R-Ras(38V)-expressing cells retained the ability to make a small number of peripheral focal adhesions, although focal adhesion formation was greatly diminished by the combined effects of LY294002 and PP2 even in R-Ras(38V) cells. Therefore, although R-Ras can signal to FAK via a yet-unidentified pathway, downstream effects on focal adhesion formation require PI3K and Src.


R-Ras has emerged as an important regulator of integrin function, but the mechanisms by which it affects integrin signaling pathways have not been well elucidated. We present evidence that activation of R-Ras enhances phosphorylation of FAK and p130Cas, focal adhesion formation, and cell migration. Importantly, R-Ras signals to FAK and p130Cas by a mechanism that is not due merely to enhanced integrin ligand binding, although it collaborates with integrin signaling. R-Ras signaling to FAK and p130Cas is distinct from K-Ras and integrins, in that it is in part Src independent and is independent of an intact actin cytoskeleton, focal adhesion formation, and cell shape changes. These results suggest that R-Ras can regulate integrin signaling pathways by a novel mechanism separate from enhanced integrin avidity or affinity.

Although R-Ras enhances integrin affinity and cell adhesion (16, 46), several pieces of evidence suggest that R-Ras enhances FAK and p130Cas phosphorylation by a mechanism that cannot be explained entirely through increased ligand binding. First, expression of activated R-Ras did not increase binding of a soluble integrin ligand to cells (Fig. (Fig.3C).3C). Second, R-Ras enhanced p130Cas phosphorylation following clustering of the α2β1 integrin by an anti-α2 antibody that did not exhibit increased binding to the integrin (Fig. (Fig.3D).3D). Moreover, if R-Ras effects were strictly due to enhanced integrin affinity or avidity, we would expect that FAK and p130Cas phosphorylation would be completely Src and actin dependent, as is the case for their phosphorylation downstream of integrins (25, 33, 36), but this was not the case (Fig. (Fig.44 and and6).6). Our results suggest that R-Ras activates unique downstream signaling pathways that lead to FAK and p130Cas phosphorylation.

PI3K plays a role in R-Ras signaling to p130Cas, focal adhesion formation (Fig. (Fig.6),6), and cell migration (16), as suggested by inhibition of PI3K with LY294002. The role of PI3K may be as an effector of R-Ras, as suggested by results in which effector loop mutations (61S and 66C) having diminished PI3K binding had diminished FAK and p130Cas phosphorylation and diminished cell migration and focal adhesion formation. However, we also find evidence for PI3K-independent signaling, as inhibition of all these events by LY294002 was only partial in cells expressing activated R-Ras. Moreover, cells expressing effector loop mutations 61S and 66C had only minimally diminished p130Cas phosphorylation, while p130Cas was more sensitive to effects of LY294002 than was FAK. This difference suggests that PI3K may also play a role elsewhere in the signaling pathway separate from its role as an R-Ras effector. The presence of PI3K-dependent and -independent pathways has also been suggested in the work of Kinashi et al. (17), in which a PI3K-independent pathway induces adhesion in cells expressing activated R-Ras but not H-Ras. Interestingly, these investigators found that changes in affinity of the α5β1 integrin were dependent on PI3K but that enhanced avidity was PI3K independent (17).

Our results with R-Ras effector loop mutations demonstrate that the R-Ras(38V)/63G mutation was almost as effective as R-Ras(38V) in signaling to FAK and p130Cas and in promoting cell migration and focal adhesion formation. This mutant has been shown to bind to PI3K, to activate Akt, and to bind to the Ral exchange factors, Ral-GDS, Rgl, and Rlf (24, 37). Effector loop mutations that have decreased binding to PI3K and Ral-GDS, R-Ras(38V)/61S, and R-Ras(38V)/66C were not as effective in promoting FAK phosphorylation, cell migration, or focal adhesion formation. However, it is unlikely that the effects of R-Ras on cell migration are through activation of Ral, as dominant-negative Ral did not significantly inhibit cell migration. Moreover, because R-Ras(38V)/61S maintains binding to Raf, our results suggest that activation of the Raf-MEK-mitogen-activated protein kinase pathway is not involved in R-Ras signaling to FAK or focal adhesions. Signaling to p130Cas was less sensitive to the effects of mutations in the R-Ras effector loop, suggesting that R-Ras signaling to FAK and p130Cas differs. Our results are consistent with those demonstrating that R-Ras(38V/63G), but not R-Ras(38V/61S) or R-Ras(38V/66C), promotes cell adhesion and integrin-mediated phagocytosis (24, 37). In contrast, Oertli et al. demonstrated that R-Ras(38V/66C) reduced activation of the PI3K pathway and disabled binding to Ral-GDS, Raf-1, and Nore1 but could still regulate integrin function, suggesting that these effectors are not essential for integrin regulation (23). Effector pathways downstream of R-Ras have been somewhat elusive. To date, the major identified effector is PI3K (20), but PI3K does not account for all of the effects of R-Ras in our study and those of others (24, 37). Future studies identifying additional effectors of R-Ras may provide insight into R-Ras signaling to focal adhesion formation.

Our results suggest a model where R-Ras signals to FAK phosphorylation by two pathways, i.e., one Src-dependent pathway and an unknown pathway that is Src and PI3K independent (Fig. (Fig.10).10). This latter pathway is also likely to be independent of the R-Ras effectors Raf and Ral-GDS (Fig. (Fig.7).7). R-Ras enhancement of p130Cas phosphorylation also requires two pathways, one Src dependent and the other PI3K dependent. We predict that the Src-dependent pathway may reflect enhancement of integrin function by R-Ras, since both FAK and p130Cas phosphorylation downstream of integrins is Src dependent (Fig. (Fig.6)6) (32, 33). The net result of this signaling pathway is enhancement of cell migration, focal adhesion formation, and integrin avidity. R-Ras activation induces greater phosphorylation of FAK at Y397 than at Y576 or Y925. Inhibition of Src completely inhibited integrin, but not R-Ras, signaling to FAK Y397. Because FAK Y397 is the autophosphorylation site, these results have important implications for how R-Ras signals to FAK. One possibility is that R-Ras enhances FAK autophosphorylation by inducing the clustering of FAK. This clustering could be through binding of FAK to the β1 integrin subunit or via another mechanism. FAK may also be responsible for the enhanced Src-independent phosphorylation of p130Cas in R-Ras-expressing cells. Alternatively, a yet unidentified tyrosine kinase might be activated by R-Ras signaling pathways leading to the enhanced phosphorylation of FAK or p130Cas that we observe.

FIG. 10.
Model of signaling pathways linking R-Ras to FAK and p130Cas. Arrows represent predicted functional linkages. Others have demonstrated that some of these linkages are also physical (R-Ras, PI3K; FAK, Src, p130Cas; FAK, PI3K; and p130Cas, PI3K). See Discussion ...

Phosphorylation of Y576 and Y577 in the activation loop leads to increased FAK activity (31). We find that phosphorylation of Y576 was sensitive to Src inhibition in both control and R-Ras-expressing cells, consistent with the known role for Src in phosphorylating this site (31). These results are consistent with those of Salazar and Rozengurt, in which GPCR-induced phosphorylation of FAK Y397 was Src independent but in which phosphorylation of Y576/Y577 was Src dependent (33). An obvious possibility is that endogenous R-Ras is activated downstream of GPCRs and mediates the Src-independent signaling to FAK Y397. Presently, upstream activators of R-Ras have not been defined.

In addition to Src, several other molecules can bind to FAK phosphorylated at Y397, including PI3K, Grb7, SHC, and phospholipase C-γ (3, 9, 36, 45). Because R-Ras enhances phosphorylation at Y397, R-Ras activation may recruit molecules other than Src to Y397. Phosphorylation of Y397 has been functionally linked to cell migration and cell spreading (25, 40), suggesting that this could be an important determinant in R-Ras induction of cell migration (16). R-Ras effects on FAK Y397 phosphorylation are likely to alter downstream signaling events.

R-Ras dramatically increases focal adhesion formation, a finding that has not been previously shown. There are two striking differences between focal adhesions in control cells and cells expressing activated R-Ras. First, R-Ras induces an increase in large, central focal adhesions. Second, peripheral focal adhesions in R-Ras-expressing cells are resistant to inhibition of Src by PP2, in striking contrast to focal adhesions in control cells and cells expressing activated K-Ras(12V). It is likely that the large, central focal adhesions induced by R-Ras are due to synergy of multiple pathways, since inhibition of either Src or PI3K caused a shift to smaller, peripheral focal adhesions. The contribution of multiple pathways is also suggested by the loss of these large central focal adhesions in cells expressing R-Ras effector loop mutations.

Effects of R-Ras on FAK and p130Cas likely contribute to the enhanced focal adhesion formation, since dominant-negative constructs of FAK and p130Cas could decrease focal adhesion formation. Interestingly, inhibition of FAK with FRNK was more effective at inhibiting focal adhesion formation, while inhibition of p130Cas with Cas-SH3 was more effective at inhibiting cell migration. This finding suggests a fundamental difference in the function of FAK and p130Cas, as well as differences in signaling from R-Ras to each. Results with FRNK, which diminished focal adhesion formation, differ somewhat from results with FAK−/− cells, which have increased focal adhesions due to decreased focal adhesion turnover. Our results are consistent with those of Richardson and Parsons (28), who demonstrated that FRNK delays the formation of focal adhesions at time points less than 60 min. This result is consistent with the results in FAK−/− cells, as it suggests a role for FAK in focal adhesion dynamics. For cells that have attached greater than 24 h, when dynamics are less likely to be a factor, others have reported that FRNK does not inhibit focal adhesion formation (7, 40). Others have also suggested a role for FAK signaling in not only focal adhesion turnover but also focal adhesion assembly (31). A role for p130Cas in focal adhesion formation has also been suggested by results with p130Cas−/− cells (11).

Despite evidence for a unique mechanism linking R-Ras to FAK and p130Cas, phosphorylation of these molecules was still strongly regulated by collagen stimulation in R-Ras-expressing cells. We believe R-Ras-enhanced FAK and p130Cas phosphorylation in response to collagen is not due to activation of another collagen receptor, since clustering of the α2β1 integrin with an anti-α2 antibody was sufficient to induce FAK and p130Cas phosphorylation in cells expressing activated R-Ras. Moreover, migration induced by R-Ras can be completely inhibited by function-blocking anti-α2 subunit antibodies (16). Therefore, it appears that R-Ras synergizes with α2β1 integrin signaling to enhance FAK and p130Cas phosphorylation. We do not yet know the exact molecular mechanism for this synergy. It is noteworthy that there is a small increase in the baseline level of FAK and p130Cas phosphorylation in R-Ras-expressing cells in the absence of collagen stimulation, suggesting that R-Ras may signal to these molecules independent of integrins.

Integrin ligand binding can be induced by inside-out signaling pathways leading to conformational changes reflected in alterations in affinity (21). In addition, changes in cell adhesion may be the result of enhanced integrin avidity due to clustering of integrins or their association with the actin cytoskeleton in focal adhesions. A clear role for R-Ras in inside-out integrin signaling events has been demonstrated (46). Notably, R-Ras induces conformational changes and increases integrin affinity in CHO and 32D cells (46). Although R-Ras increases breast epithelial cell adhesion to collagen, we think this is also through changes in avidity, not just conformational changes related to altered affinity (Fig. (Fig.3).3). Expression of activated R-Ras does not cause the same conformational changes that are observed when cells are treated with Mn2+, implying that R-Ras does not conformationally activate integrins in the same manner. Moreover, R-Ras and Mn2+ had a synergistic effect on FAK and p130Cas phosphorylation, again suggesting that the molecular mechanism differs. Enhanced focal adhesion formation in R-Ras-expressing cells could account for increased cell adhesion to collagen through enhanced integrin clustering and avidity. An attractive model is that R-Ras signals to FAK and p130Cas to enhance focal adhesion formation, resulting in increased integrin clustering and avidity. When combined with Mn2+, enhanced signaling to FAK and p130Cas may result from a synergistic effect of increasing both affinity and avidity.

Our results further define the molecular differences for R-Ras signaling when compared to K-Ras. While expression of activated K-Ras enhanced FAK and p130Cas phosphorylation, this enhancement differed from R-Ras in that it was sensitive to inhibitors of Src and the actin cytoskeleton (Fig. (Fig.44 and and6).6). Moreover, cells expressing activated K-Ras had focal adhesions that were similar to those in control cells, rather than the large, central focal adhesions noted in R-Ras-expressing cells. Like control cells, focal adhesions in K-Ras-expressing cells were sensitive to inhibition of Src and/or PI3K (Fig. (Fig.6,6, ,8,8, and and9).9). These differences may account for functional differences in R-Ras and K-Ras-induced cell migration. Whereas R-Ras induces epithelial cell migration on collagen through the α2β1 integrin, K-Ras induces migration on both collagen and fibronectin through α2β1 and α5β1 integrins, respectively (16).


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.).


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