Co-localization of cortactin and phosphotyrosine identifies active invadopodia in human breast cancer cells

Invadopodia are filopodia-like projections possessing protease activity that participate in tumor cell invasion. We demonstrate that co-localization of cortactin and phosphotyrosine identifies a subset of cortactin puncta termed "invadopodial complexes" that we find to be closely associated with the plasma membrane at active sites of focal degradation of the extracellular matrix in MDA-MB-231 breast cancer cells. Manipulation of c-Src activity in cells by transfection with kinase activated c-Src(527) or kinase inactive c-Src(295) results in a dramatic increase or decrease, respectively, in the number of these structures associated with changes in the number of sites of active matrix degradation. Overexpression of kinase-inactive c-Src(295) does not prevent localization of cortactin at the membrane; however, co-localized phosphotyrosine staining is decreased. Thus, elevated phosphotyrosine at invadopodial complexes is specifically associated with the proteolytic activity of invadopodia. Further, invadopodial complexes are spatially, morphologically and compositionally distinct from focal adhesions as determined by localization of focal adhesion kinase (FAK), which is not present in invadopodial complexes. Expression of kinase-inactive c-Src(295) blocks invadopodia activity, but does not block filopodia formation. Thus, invadopodia, but not filopodia, are highly correlated with matrix invasion, and sites of invadopodial activity can be identified by the formation of invadopodial complexes.     

Ezrin regulates focal adhesion and invadopodia dynamics by altering calpain activity to promote breast cancer cell invasion

Up-regulation of the cytoskeleton linker protein ezrin frequently occurs in aggressive cancer types and is closely linked with metastatic progression. However, the underlying molecular mechanisms detailing how ezrin is involved in the invasive and metastatic phenotype remain unclear. Here we report a novel function of ezrin in regulating focal adhesion (FA) and invadopodia dynamics, two key processes required for efficient invasion to occur. We show that depletion of ezrin expression in invasive breast cancer cells impairs both FA and invadopodia turnover. We also demonstrate that ezrin-depleted cells display reduced calpain-mediated cleavage of the FA and invadopodia-associated proteins talin, focal adhesion kinase (FAK), and cortactin and reduced calpain-1–specific membrane localization, suggesting a requirement for ezrin in maintaining proper localization and activity of calpain-1. Furthermore, we show that ezrin is required for cell directionality, early lung seeding, and distant organ colonization but not primary tumor growth. Collectively our results unveil a novel mechanism by which ezrin regulates breast cancer cell invasion and metastasis.     

Calpain 2 and PTP1B function in a novel pathway with Src to regulate invadopodia dynamics and breast cancer cell invasion

Invasive cancer cells form dynamic adhesive structures associated with matrix degradation called invadopodia. Calpain 2 is a calcium-dependent intracellular protease that regulates adhesion turnover and disassembly through the targeting of specific substrates such as talin. Here, we describe a novel function for calpain 2 in the formation of invadopodia and in the invasive abilities of breast cancer cells through the modulation of endogenous c-Src activity. Calpain-deficient breast cancer cells show impaired invadopodia formation that is rescued by expression of a truncated fragment of protein tyrosine phosphatase 1B (PTP1B) corresponding to the calpain proteolytic fragment, which indicates that calpain modulates invadopodia through PTP1B. Moreover, PTP1B activity is required for efficient invadopodia formation and breast cancer invasion, which suggests that PTP1B may modulate breast cancer progression through its effects on invadopodia. Collectively, our experiments implicate a novel signaling pathway involving calpain 2, PTP1B, and Src in the regulation of invadopodia and breast cancer invasion.     

Laminin‐332–β1 integrin interactions negatively regulate invadopodia

Adhesion of epithelial cells to basement membranes (BM) occurs through two major structures: actin‐associated focal contacts and keratin‐associated hemidesmosomes, both of which form on laminin‐332 (Ln‐332). In epithelial‐derived cancer cells, additional actin‐linked structures with putative adhesive properties, invadopodia, are frequently present and mediate BM degradation. A recent study proposed that BM invasion requires a proper combination of focal contacts and invadopodia for invading cells to gain traction through degraded BM, and suggested that these structures may compete for common molecular components such as Src kinase. In this study, we tested the role of the Ln‐332 in regulating invadopodia in 804G rat bladder carcinoma cells, a cell line that secretes Ln‐332 and forms all three types of adhesions. Expression of shRNA to Ln‐332 γ2 chain (γ2‐kd) led to increased numbers of invadopodia and enhanced extracellular matrix degradation. Replating γ2‐kd cells on Ln‐332 or collagen‐I fully recovered cell spreading and inhibition of invadopodia. Inhibition of α3 or β1, but not α6 or β4, phenocopied the effect of γ2‐kd, suggesting that α3β1‐mediated focal contacts, rather than α6β4‐mediated hemidesmosome pathways, intersect with invadopodia regulation. γ2‐kd cells exhibited alterations in focal contact‐type structures and in activation of focal adhesion kinase (FAK) and Src kinase. Inhibition of FAK also increased invadopodia number, which was reversible with Src inhibition. These data are consistent with a model whereby actin‐based adhesions can limit the availability of active Src that is capable of invadopodia initiation and identifies Ln‐332‐β1 interactions as a potent upstream regulator that limits cell invasion. J. Cell. Physiol. 223: 134–142, 2010. © 2009 Wiley‐Liss, Inc.     

Disruption of Ca2+-dependent cell-matrix adhesion enhances c-Src kinase activity, but causes dissociation of the c-Src/FAK complex and dephosphorylation of tyrosine-577 of FAK in carcinoma cells

The nonreceptor tyrosine kinase c-Src is activated in most invasive cancers. Activated c-Src binds to FAK in the focal adhesion complex, resulting in the activation of the c-Src/FAK signaling cascade, which regulates cytoskeletal functions. However, the mechanisms by which c-Src/FAK signaling is regulated during conditions of anchorage-independent growth, a hallmark of tumor progression, are not clearly known. Here, an in vivo approach to measure c-Src activity was studied using phospho-specific antibodies against phosphorylated Y418 of c-Src (Src[pY418]), an autophosphorylation site of c-Src, and phosphorylated Y577 of FAK (FAK[pY577]), a known substrate of c-Src. Using genetic and pharmacological approaches to modulate c-Src activity, we showed that the levels of Src[pY418] and FAK[pY577], and the formation of a c-Src/FAK[pY577] complex correlated with the activation state of c-Src in adherent cells. Interestingly, both the in vivo level of Src[pY418] and in vitro c-Src kinase activity were increased in carcinoma cells following disruption of Ca(2+)-dependent cell-matrix adhesion. In contrast, the level of FAK[pY577] and its association with c-Src were reduced in suspended cells. The amount of FAK[pY577] in suspended cells was recovered following attachment of rounded cells to fibronectin-coated polystyrene beads, indicating that cell spreading was not required for phosphorylation of FAK. Moreover, cells expressing activated c-Src showed sustained Src[Y418] phosphorylation, but required Ca(2+)-dependent cell adhesion for phosphorylation of FAK[Y577] and association of c-Src with FAK[pY577]. These findings indicate an important role of integrin-based cell-matrix adhesion in regulating c-Src/FAK signaling under decreased anchorage conditions.     

v-Src SH3-enhanced interaction with focal adhesion kinase at beta 1 integrin-containing invadopodia promotes cell invasion

In viral Src (v-Src)-transformed cells, focal adhesion kinase (FAK) associates with v-Src by combined v-Src SH2 and gain-of-function v-Src SH3 domain binding to FAK. Here we assess the significance of the Arg-95 to Trp gain-of-function mutation in the v-Src SH3 domain through comparisons of Src-/- fibroblasts transformed with either Prague C v-Src or a point mutant (v-Src-RT) containing a normal (Arg-95) SH3 domain. Both v-Src isoforms exhibited equivalent kinase activity, enhanced Src-/- cell motility, and stimulated cell growth in both low serum and soft agar. The stability of a v-Src-RT.FAK signaling complex and FAK phosphorylation at Tyr-861 and Tyr-925 were reduced in v-Src-RT- compared with v-Src-transformed cells. v-Src but not v-Src-RT promoted Src-/- cell invasion through a reconstituted Matrigel basement membrane barrier and v-Src co-localized with FAK and beta(1) integrin at invadopodia. In contrast, v-Src-RT exhibited a partial perinuclear and focal contact distribution in Src-/- cells. Adenovirus-mediated FAK overexpression promoted v-Src-RT recruitment to invadopodia, the formation of a v-Src-RT.FAK signaling complex, and reversed the v-Src-RT invasion deficit. Adenovirus-mediated inhibition of FAK blocked v-Src-stimulated cell invasion. These studies establish that gain-of-function v-Src SH3 targeting interactions with FAK at beta(1) integrin-containing invadopodia act to stabilize a v-Src.FAK signaling complex promoting cell invasion.     

Focal adhesion kinase is not required for Src-induced formation of invadopodia in KM12C colon cancer cells and can interfere with their assembly

Overexpression of active Src induces invadopodia formation and associated matrix degradation in KM12C colon cancer cells. FAK is present with active Src at sites of matrix-degrading activity (invadopodia), specifically residing in rings surrounding the cortactin-containing invadopodia cores. Since FAK is a key effector protein in many aspects of Src function, we addressed whether FAK is necessary for Src-induced invadopodia formation and matrix degradation in KM12C colon cancer cells. We found that efficient knockdown of FAK expression by siRNA had no effect on invadopodia formation or matrix degradation. However, overexpression of FAK could actually suppress invadopodia formation and matrix degradation. FAK phosphorylation on the putative auto-phosphorylation tyrosine 397 and the Src-specific sites are all required for overexpressed FAK to inhibit invadopodia formation, while the kinase activity of exogenous FAK is apparently not required. These data imply that kinase activities other than FAK auto-phosphorylation may contribute to the phosphorylation of FAK tyrosine 397 in some contexts to promote an activity of FAK that can counteract invadopodia formation. Further work is required to determine how the strength of signalling through FAK suppresses invadopodia, but we propose that FAK controls the balance of adhesion types in cells, and that this is one of the determinants of whether a cancer cell can make stable matrix-degrading invadopodia.     

FAK competes for Src to promote migration against invasion in melanoma cells

Melanoma is one of the most deadly cancers because of its high propensity to metastasis, a process that requires migration and invasion of tumor cells driven by the regulated formation of adhesives structures like focal adhesions (FAs) and invasive structures like invadopodia. FAK, the major kinase of FAs, has been implicated in many cellular processes, including migration and invasion. In this study, we investigated the role of FAK in the regulation of invasion. We report that suppression of FAK in B16F10 melanoma cells led to increased invadopodia formation and invasion through Matrigel, but impaired migration. These effects are rescued by FAK WT but not by FAK^Y397F reexpression. Invadopodia formation requires local Src activation downstream of FAK and in a FAK phosphorylation-dependant manner. FAK deletion correlates with increased phosphorylation of Tks-5 (tyrosine kinase substrate with five SH3 domain) and reactive oxygen species production. In conclusion, our data show that FAK is able to mediate opposite effects on cell migration and invasion. Accordingly, beneficial effects of FAK inhibition are context dependent and may depend on the cell response to environmental cues and/or on the primary or secondary changes that melanoma experienced through the invasion cycle.Patients with spreading melanoma diseases have a very poor prognosis with a 5-year survival rate <5%. The metastatic spread of melanoma is a complex process involving several genetic alterations. In melanoma,¹ as in many highly invasive cancer cell types like head and neck squamous cell carcinoma² or breast carcinoma,³ specialized matrix-degrading organelles termed invadopodia have been identified. Invadopodia consist of dynamic actin-based protrusions of 0, 1 to 2 μm in diameter emanating from the ventral edge of tumor cells.⁴ Besides their actin scaffold, these structures are enriched in proteolytic enzymes such as matrix metalloproteinases (MMPs), which mediate extracellular matrix (ECM) degradation. Indeed, MMP are upregulated in invasive melanoma and there is extensive evidence that they have a role in promoting the dissemination of melanoma.^{5, 6, 7} Several proteins like integrins, Src and paxillin, found at sites of cell adhesion to the matrix, are also present in invadopodia.^{8, 9} On the other hand, other proteins like the Src substrate proteins cortactin¹⁰ and the tyrosine kinase substrate with five SH3 domain (Tks-5)¹¹ are specifically localized at invadopodia and not found at focal adhesion (FA). In addition, reactive oxygen species (ROS)¹² have been localized at invadopodia and are supposed to have a prominent role in inducing invadopodia function.^{13, 14} Although significant efforts have been made to characterize components of invadopodia, the precise mechanisms of their regulation, especially in a melanoma context, remain poorly understood.Tumor invasion is a multistep process that requires cell adhesion to the environing substratum, migration and invasion. In many cell types, migration requires fine control of FA turn-over. FAs are formed by the cluster of up to 200 proteins¹⁵ ensuring cell anchorage to the ECM. The cyclic process of FA formation and disruption is crucial for cell migration. Because both anchorage and migration involve cellular interactions with ECM components, FAs are endowed with transmembrane ECM receptor proteins such as integrins that interact with ECM molecules and intra-cellular proteins composed of scaffold proteins, as well as signal-transducing molecules. Among those, focal adhesion kinase (FAK) is a crucial signaling protein that integrates signals from integrins to the actin filaments during cell migration.¹⁶ Structurally, FAK is a 125-kDa protein that contains an N-terminal 4.1-ezrin–radixin–moesin domain, a central kinase domain and a C-terminal domain that contains the focal adhesion targeting site.¹⁷ The phosphorylation of FAK at Y397 creates a binding site for Src, which can phosphorylate other tyrosines on the FAK sequence, thus creating new binding sites for SH2 domain-containing proteins.FAK is involved in many aspects of the metastatic process and thus, overexpression, hyperphosphorylation and/or elevated activity of FAK have been reported in a variety of human cancers, including sarcomas and carcinomas of the breast, colon, thyroid, prostate, oral cavity, liver, stomach and ovary.¹⁸ In human melanoma cell lines, early studies reported high FAK expression and requirement of FAK for cell substrate adhesion.¹⁹ Later, it was reported that FAK promotes the aggressive melanoma phenotype.²⁰ Indeed, immunohistochemical analyses revealed high levels of FAK phosphorylation at Tyr397 and Tyr576, a marker of FAK kinase activity, in late-stage cutaneous and uveal melanoma, which correlated with their increased invasion and migration properties.²¹ Furthermore, melanoma differentiation-associated gene-9 (mda-9)/syntenin was also reported to mediate adhesion-dependant activation of protein kinase Cα (PKCα) and FAK in melanoma cells. Thus, inhibiting either mda-9/syntenin or PKCα suppressed fibronectin-induced formation of integrin-β1/FAK/c-Src signaling complexes and reduced migration and invasion toward fibronectin.²² Therefore, FAK appears to be a major player of melanoma invasion, but how this kinase controls the formation and proteolytic activity of invadopodia in melanoma cells was never investigated.In this study, we uncovered a surprising negative regulation of invadopodia activity in B16F10 cells by FAK. The depletion of FAK was associated with increased ROS production and Tks-5 phosphorylation. Using mutation of FAK at Tyr397, a binding site for Src, we found that these sites are implicated in FAK-mediated inhibition of invadopodia activity. In addition, we report that this mutation induced decreased migration speed but increased invasive properties. Taken together, our data suggest a competition between FA and invadopodia substrates for Src phosphorylation that might depend on environmental cues, thus leading to the engagement of either migration or degradation pathways.     

Beta2-Adaptin Binds Actopaxin and Regulates Cell Spreading,Migration and Matrix Degradation

Cell adhesion to the extracellular matrix is a key event in cell migration and invasion and endocytic trafficking of adhesion receptors and signaling proteins plays a major role in regulating these processes. Beta2-adaptin is a subunit of the AP-2 complex and is involved in clathrin-mediated endocytosis. Herein, β2-adaptin is shown to bind to the focal adhesion protein actopaxin and localize to focal adhesions during cells spreading in an actopaxin dependent manner. Furthermore, β2-adaptin is enriched in adhesions at the leading edge of migrating cells and depletion of β2-adaptin by RNAi increases cell spreading and inhibits directional cell migration via a loss of cellular polarity. Knockdown of β2-adaptin in both U2OS osteosarcoma cells and MCF10A normal breast epithelial cells promotes the formation of matrix degrading invadopodia, adhesion structures linked to invasive migration in cancer cells. These data therefore suggest that actopaxin-dependent recruitment of the AP-2 complex, via an interaction with β2-adaptin, to focal adhesions mediates cell polarity and migration and that β2-adaptin may control the balance between the formation of normal cell adhesions and invasive adhesion structures.     

Heregulin and HER2 signaling selectively activates c-Src phosphorylation at tyrosine 215

To elucidate the molecular mechanisms by which human epidermal growth factor receptor/heregulin (HER2/HRG) influence the migratory potential of breast cancer cells, we have used phospho-specific antibodies against c-Src kinase and focal adhesion kinase (FAK). This study establishes that HER2/HRG signaling selectively upregulates Tyr phosphorylation of c-Src at Tyr-215 located within the SH2 domain, increases c-Src kinase activity and selectively upregulates Tyr phosphorylation of FAK at Tyr-861. HER2-overexpressing tumors showed increased levels of c-Src phosphorylation at Tyr-215. These findings suggest that HER2/HRG influence metastasis of breast cancer cells through a novel signaling pathway involving phosphorylation of FAK tyrosine 861 via activation of c-Src tyrosine 215.     

Estrogen receptor-alpha promotes breast cancer cell motility and invasion via focal adhesion kinase and N-WASP

The ability of cancer cells to move and invade the surrounding environment is the basis of local and distant metastasis. Cancer cell movement requires dynamic remodeling of the cytoskeleton and cell membrane and is controlled by multiprotein complexes including focal adhesion kinase (FAK) or the Neural Wiskott-Aldrich Syndrome Protein (N-WASP). We show that 17β-estradiol induces phosphorylation of FAK and its translocation toward membrane sites where focal adhesion complexes are assembled. This process is triggered via a Gα/Gβ protein-dependent, rapid extranuclear signaling of estrogen receptor α interacts in a multiprotein complex with c-Src, phosphatidylinositol 3-OH kinase, and FAK. Within this complex FAK autophosphorylation ensues, and activated FAK recruits the small GTPase cdc42, which, in turn, triggers N-WASP phosphorylation. This results in the translocation of Arp2/3 complexes at sites where membrane structures related to cell movement are formed. Recruitment of FAK and N-WASP is necessary for cell migration and invasion induced by 17β-estradiol in breast cancer cells. Our findings identify an original mechanism through which estrogen promotes breast cancer cell motility and invasion. This information helps to understand the effects of estrogen on breast cancer metastasis and may provide new targets for therapeutic interventions.     

Proximal, selective, and dynamic interactions between integrin alphaIIbbeta3 and protein tyrosine kinases in living cells

Stable platelet aggregation, adhesion, and spreading during hemostasis are promoted by outside-in alphaIIbbeta3 signals that feature rapid activation of c-Src and Syk, delayed activation of FAK, and cytoskeletal reorganization. To evaluate these alphaIIbbeta3-tyrosine kinase interactions at nanometer proximity in living cells, we monitored bioluminescence resonance energy transfer between GFP and Renilla luciferase chimeras and bimolecular fluorescence complementation between YFP half-molecule chimeras. These techniques revealed that alphaIIbbeta3 interacts with c-Src at the periphery of nonadherent CHO cells. After plating cells on fibrinogen, complexes of alphaIIbbeta3-c-Src, alphaIIbbeta3-Syk, and c-Src-Syk are observed in membrane ruffles and focal complexes, and the interactions involving Syk require Src activity. In contrast, FAK interacts with alphaIIbbeta3 and c-Src, but not with Syk, in focal complexes and adhesions. All of these interactions require the integrin beta3 cytoplasmic tail. Thus, alphaIIbbeta3 interacts proximally, if not directly, with tyrosine kinases in a coordinated, selective, and dynamic manner during sequential phases of alphaIIbbeta3 signaling to the actin cytoskeleton.     

Src-dependent autophagic degradation of Ret in FAK-signalling-defective cancer cells

We have recently described that autophagic targeting of Src maintains cancer cell viability when FAK signalling is defective. Here, we show that the Ret tyrosine kinase is also degraded by autophagy in cancer cells with altered/reduced FAK signalling, preventing its binding to FAK at integrin adhesions. Inhibition of autophagy restores Ret localization to focal adhesions. Importantly, Src kinase activity is required to target Ret to autophagosomes and enhance Ret degradation. Src is thus a general mediator of selective autophagic targeting of adhesion-linked kinases, and Ret a second FAK-binding tyrosine kinase degraded through autophagy in cancer cells under adhesion stress. Src-by controlling not only its own degradation but also that of other FAK-binding partners-allows cancer cell survival, suggesting a new therapeutic strategy.     

Autophagic targeting of Src promotes cancer cell survival following reduced FAK signalling

Here we describe a mechanism that cancer cells use to survive when flux through the Src/FAK pathway is severely perturbed. Depletion of FAK, detachment of FAK-proficient cells or expression of non-phosphorylatable FAK proteins causes sequestration of active Src away from focal adhesions into intracellular puncta that co-stain with several autophagy regulators. Inhibition of autophagy results in restoration of active Src at peripheral adhesions, and this leads to cancer cell death. Autophagic targeting of active Src is associated with a Src-LC3B complex, and is mediated by c-Cbl. However, this is independent of c-Cbl E3 ligase activity, but is mediated by an LC3-interacting region. Thus, c-Cbl-mediated autophagic targeting of active Src can occur in cancer cells to maintain viability when flux through the integrin/Src/FAK pathway is disrupted. This exposes a previously unrecognized cancer cell vulnerability that may provide a new therapeutic opportunity.     

Coordination of cell polarization and migration by the Rho family GTPases requires Src tyrosine kinase activity.

BACKGROUND: The ability of a cell to polarize and move is governed by remodeling of the cellular adhesion/cytoskeletal network that is in turn controlled by the Rho family of small GTPases. However, it is not known what signals lie downstream of Rac1 and Cdc42 during peripheral actin and adhesion remodeling that is required for directional migration. RESULTS: We show here that individual members of the Rho family, RhoA, Rac1, and Cdc42, direct the specific intracellular targeting of c-Src tyrosine kinase to focal adhesions, lamellipodia, or filopodia, respectively, and that the adaptor function of c-Src (the combined SH3/SH2 domains coupled to green fluorescent protein) is sufficient for targeting. Furthermore, Src's catalytic activity is absolutely required at these peripheral cell-matrix attachment sites for remodeling that converts RhoA-dependent focal adhesions into smaller focal complexes along Rac1-induced lamellipodia (or Cdc42-induced filopodia). Consequently, cells in which kinase-deficient c-Src occupies peripheral adhesion sites exhibit impaired polarization toward migratory stimuli and reduced motility. Furthermore, phosphorylation of FAK, an Src adhesion substrate, is suppressed under these conditions. CONCLUSIONS: Our findings demonstrate that individual Rho GTPases specify Src's exact peripheral localization and that Rac1- and Cdc42-induced adhesion remodeling and directed cell migration require Src activity at peripheral adhesion sites.     

Endothelin-1 promotes migration of endothelial cells through the activation of ARF6 and the regulation of FAK activity

Several proteins act in concert to promote remodeling of the actin cytoskeleton during migration. This process is highly regulated by small GTP-binding proteins of the ADP-ribosylation factor (ARF) family of proteins. Here, we show that endothelin-1 (ET-1) can promote the activation of ARF6 and migration of endothelial cells through the activation of ET_B receptors. Inhibition of ARF6 expression using RNA interference markedly impairs basal and ET-1 stimulated cell migration. In contrast, depletion of ARF1 has no significant effect. In order to delineate the underlying mechanism, we examined the signaling events activated in endothelial cells following ET-1 stimulation. Here, we show that this hormone promotes the phosphorylation of focal adhesion kinase (FAK), Erk1/2, and the association of FAK to Src, as well as of FAK to GIT1. These have been shown to be important for the formation and turnover of focal adhesions. In non-stimulated cells, depletion of ARF6 leads to increased FAK and Erk1/2 phosphorylation, similar to what is observed in ET-1 treated cells. In these conditions, FAK is found constitutively associated with the soluble tyrosine kinase, Src. In contrast, depletion of ARF6 impairs the ability of GIT1 to form an agonist promoted complex with FAK, thereby preventing disassembly of focal adhesions. As a consequence, ARF6 depleted endothelial cells are impaired in their ability to form capillary tubes. Taken together, our data suggest that ARF6 is central in regulating focal adhesion turnover in endothelial cells. Our study provides a molecular mechanism by which, this small GTPase regulates cell motility, and ultimately angiogenesis.     

RACK1 regulates Src activity and modulates paxillin dynamics during cell migration

Receptor for Activated C Kinase, RACK1, is an adaptor protein that regulates signaling via Src and PKC-dependent pathways, and has been implicated in cell migration. In this study we demonstrate novel functions for RACK1 in regulating adhesion dynamics during cell migration. We report that cells lacking RACK1 are less motile and show reduced dynamics of paxillin and talin at focal complexes. To investigate the role of the RACK1/Src interactions in adhesion dynamics, we used RACK1 in which the putative Src binding site has been mutated (RACK Y246F). RACK1-deficient cells showed enhanced c-Src activity that was rescued by expression of wild type RACK1, but not by RACK Y246F. Expression of wild type RACK1, but not RACK Y246F, was also able to rescue the adhesion and migration defects observed in the RACK1-deficient cells. Furthermore, our findings indicate that RACK1 functions to regulate paxillin phosphorylation and that its effects on paxillin dynamics require the Src-mediated phosphorylation of tyrosine 31/118 on paxillin. Taken together, these findings support a novel role for RACK1 as a key regulator of cell migration and adhesion dynamics through the regulation of Src activity, and the modulation of paxillin phosphorylation at early adhesions.     

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.     

Src and FAK mediate cell–matrix adhesion‐dependent activation of met during transformation of breast epithelial cells

Cell–matrix adhesion has been shown to promote activation of the hepatocyte growth factor receptor, Met, in a ligand‐independent manner. This process has been linked to transformation and tumorigenesis in a variety of cancer types. In the present report, we describe a key role of integrin signaling via the Src/FAK axis in the activation of Met in breast epithelial and carcinoma cells. Expression of an activated Src mutant in non‐neoplastic breast epithelial cells or in carcinoma cells was found to increase phosphorylation of Met at regulatory tyrosines in the auto‐activation loop domain, correlating with increased cell spreading and filopodia extensions. Furthermore, phosphorylated Met is complexed with β1 integrins and is co‐localized with vinculin and FAK at focal adhesions in epithelial cells expressing activated Src. Conversely, genetic or pharmacological inhibition of Src abrogates constitutive Met phosphorylation in carcinoma cells or epithelial cells expressing activated Src, and inhibits filopodia formation. Interestingly, Src‐dependent phosphorylation of Met requires cell–matrix adhesion, as well as actin stress fiber assembly. Phosphorylation of FAK by Src is also required for Src‐induced Met phosphorylation, emphasizing the importance of the Src/FAK signaling pathway. However, stimulation of Met phosphorylation by addition of exogenous HGF in epithelial cells is refractory to inhibition of Src family kinases, indicating that HGF‐dependent and Src/integrin‐dependent Met activation occur via distinct mechanisms. Together these findings demonstrate a novel mechanism by which the Src/FAK axis links signals from the integrin adhesion complex to promote Met activation in breast epithelial cells. J. Cell. Biochem. 107: 1168–1181, 2009. © 2009 Wiley‐Liss, Inc.     

FAK signaling is critical for ErbB-2/ErbB-3 receptor cooperation for oncogenic transformation and invasion

The overexpression of members of the ErbB tyrosine kinase receptor family has been associated with cancer progression. We demonstrate that focal adhesion kinase (FAK) is essential for oncogenic transformation and cell invasion that is induced by ErbB-2 and -3 receptor signaling. ErbB-2/3 overexpression in FAK-deficient cells fails to promote cell transformation and rescue chemotaxis deficiency. Restoration of FAK rescues both oncogenic transformation and invasion that is induced by ErbB-2/3 in vitro and in vivo. In contrast, the inhibition of FAK in FAK-proficient invasive cancer cells prevented cell invasion and metastasis formation. The activation of ErbB-2/3 regulates FAK phosphorylation at Tyr-397, -861, and -925. ErbB-induced oncogenic transformation correlates with the ability of FAK to restore ErbB-2/3-induced mitogen-activated protein kinase (MAPK) activation; the inhibition of MAPK prevented oncogenic transformation. In contrast, the inhibition of Src but not MAPK prevented ErbB-FAK-induced chemotaxis. In migratory cells, activated ErbB-2/3 receptors colocalize with activated FAK at cell protrusions. This colocalization requires intact FAK. In summary, distinct FAK signaling has an essential function in ErbB-induced oncogenesis and invasiveness.