Indomethacin delays gastric restitution: association with the inhibition of focal adhesion kinase and tensin phosphorylation and reduced actin stress fibers

Repair of superficial gastric mucosal injury is accomplished by the process of restitution-migration of epithelial cells to restore continuity of the mucosal surface. Actin filaments, focal adhesions, and focal adhesion kinase (FAK) play crucial roles in cell motility essential for restitution. We studied whether epidermal growth factor (EGF) and/or indomethacin (IND) affect cell migration, actin stress fiber formation, and/or phosphorylation of FAK and tensin in wounded gastric monolayers. Human gastric epithelial monolayers (MKN 28 cells) were wounded and treated with either vehicle or 0.5 mM IND for 16 hr followed by EGF. EGF treatment significantly stimulated cell migration and actin stress fiber formation, and increased FAK localization to focal adhesions, and phosphorylation of FAK and tensin, whereas IND inhibited all these at the baseline and EGF-stimulated conditions. IND-induced inhibition of FAK phosphorylation preceded changes in actin polymerization, indicating that actin depolymerization might be the consequence of decreased FAK activity. In in vivo experiments, rats received either vehicle or IND (5 mg/kg i.g.), and 3 min later, they received water or 5% hypertonic NaCl; gastric mucosa was obtained at 1, 4, and 8 hr after injury. Four and 8 hr after hypertonic injury, FAK phosphorylation was induced in gastric mucosa compared with controls. IND pretreatment significantly delayed epithelial restitution in vivo, and reduced FAK phosphorylation and recruitment to adhesion points, as well as actin stress fiber formation in migrating surface epithelial cells. Our study indicates that FAK, tensin, and actin stress fibers are likely mediators of EGF-stimulated cell migration in wounded human gastric monolayers and potential targets for IND-induced inhibition of restitution.     

Helicobacter pylori vacuolating cytotoxin (VacA) alters cytoskeleton-associated proteins and interferes with re-epithelialization of wounded gastric epithelial monolayers

In previous studies, we demonstrated that Helicobacter pylori vacuolating cytotoxin (VacA) inhibits gastric epithelial cell proliferation and inhibits epidermal growth factor (EGF)-activated signal transduction. Cell proliferation and migration, both essential for mucosal healing are dependent on the cell cytoskeleton. Other investigators demonstrated that VacA induces vacuolation of eukaryotic cells. Since in some cells, control of actin cytoskeleton involves GTP-binding proteins of Rho family, in this study we examined whether VacA affects wound re-epithelialization, cell cytoskeleton-associated proteins Rho, Rac1 in a gastric epithelial (RGM1) cell monolayer wound model, and whether these changes correlate with vacuolation. VacA treatment significantly inhibited wound re-epithelialization, cell proliferation vs control. VacA-induced cell vacuolation strongly correlated with inhibition of wound re-epithelialization. Furthermore, VacA reduced Rac-1 protein expression and distribution, and C3-mediated ADP-ribosylation of Rho. These findings suggest that VacA may interfere with repair of gastric mucosal injury and ulcer re-epithelialization by altering cytoskeleton-dependent cell functions and signaling.     

FAK integrates growth-factor and integrin signals to promote cell migration

Here we show that cells lacking focal adhesion kinase (FAK) are refractory to motility signals from platelet-derived and epidermal growth factors (PDGF and EGF respectively), and that stable re-expression of FAK rescues these defects. FAK associates with activated PDGF- and EGF-receptor (PDGFR and EGFR) signalling complexes, and expression of the band-4.1-like domain at the FAK amino terminus is sufficient to mediate an interaction with activated EGFR. However, efficient EGF-stimulated cell migration also requires FAK to be targeted, by its carboxy-terminal domain, to sites of integrin-receptor clustering. Although the kinase activity of FAK is not needed to promote PDGF- or EGF-stimulated cell motility, kinase-inactive FAK is transphosphorylated at the indispensable Src-kinase-binding site, FAK Y397, after EGF stimulation of cells. Our results establish that FAK is an important receptor-proximal link between growth-factor-receptor and integrin signalling pathways.     

Epidermal growth factor stimulates serine/threonine phosphorylation of the focal adhesion protein paxillin in a MEK-dependent manner in normal rat kidney cells

Epidermal growth factor (EGF)-stimulated proliferation of renal epithelial cells plays an important role in the recovery of kidney tubule epithelia following exposure to insult. Numerous studies have demonstrated that tyrosine phosphorylation of the focal adhesion protein paxillin mediates in part the effects of growth factors on cell growth, migration, and organization of the actin-based cytoskeleton. The experiments in this report were designed to determine the effect of EGF on paxillin phosphorylation in normal rat kidney (NRK) epithelial cells. Interestingly, treatment of NRK cells with EGF stimulated paxillin serine/threonine phosphorylation, which caused a reduction in the mobility of paxillin on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The EGF-stimulated mobility shift of paxillin was independent of an intact cytoskeleton, phosphatidylinositol 3-kinase (PI 3-kinase) activation, protein kinase C (PKC) activation, and cellular adhesion. However, inhibitors of the mitogen-activated protein kinase/extracellular signal-regulated kinase kinase abrogated the EGF-stimulated change in paxillin mobility. In addition, the EGF-stimulated change in paxillin serine/threonine phosphorylation was not accompanied by a profound reorganization of the actin cytoskeleton. These results identify paxillin as a component EGF signaling in renal epithelial cells and implicate members of the MAP kinase pathway as critical regulators of paxillin serine/threonine phosphorylation.     

Epidermal growth factor modulates tyrosine phosphorylation of a novel tensin family member, tensin3

Here, we report the identification of a new tensin family member, tensin3, and its role in epidermal growth factor (EGF) signaling pathway. Human tensin3 cDNA encodes a 1445 amino acid sequence that shares extensive homology with tensin1, tensin2, and COOH-terminal tensin-like protein. Tensin3 is expressed in various tissues and in different cell types such as endothelia, epithelia, and fibroblasts. The potential role of tensin3 in EGF-induced signaling pathway is explored. EGF induces tyrosine phosphorylation of tensin3 in MDA-MB-468 cells in a time- and dose-dependent manner, but it is independent of an intact actin cytoskeleton or phosphatidylinositol 3-kinase. Activation of EGF receptor is necessary but not sufficient for tyrosine phosphorylation of tensin3. It also requires Src family kinase activities. Furthermore, tensin3 forms a complex with focal adhesion kinase and p130Cas in MDA-MB-468 cells. Addition of EGF to the cells induces dephosphorylation of these two molecules, leads to disassociation of the tensin3-focal adhesion kinase-p130Cas complex, and enhances the interaction between tensin3 and EGF receptor. Our results demonstrate that tensin3 may function as a platform for the disassembly of EGF-related signaling complexes at focal adhesions.     

Focal adhesion kinase modulates tension signaling to control actin and focal adhesion dynamics

In response to alphabeta1 integrin signaling, transducers such as focal adhesion kinase (FAK) become activated, relaying to specific machineries and triggering distinct cellular responses. By conditionally ablating Fak in skin epidermis and culturing Fak-null keratinocytes, we show that FAK is dispensable for epidermal adhesion and basement membrane assembly, both of which require alphabeta1 integrins. FAK is also dispensible for proliferation/survival in enriched medium. In contrast, FAK functions downstream of alphabeta1 integrin in regulating cytoskeletal dynamics and orchestrating polarized keratinocyte migration out of epidermal explants. Fak-null keratinocytes display an aberrant actin cytoskeleton, which is tightly associated with robust, peripheral focal adhesions and microtubules. We find that without FAK, Src, p190RhoGAP, and PKL-PIX-PAK, localization and/or activation at focal adhesions are impaired, leading to elevated Rho activity, phosphorylation of myosin light chain kinase, and enhanced tensile stress fibers. We show that, together, these FAK-dependent activities are critical to control the turnover of focal adhesions, which is perturbed in the absence of FAK.     

Progesterone enhances vascular endothelial cell migration via activation of focal adhesion kinase

The mechanisms of progesterone on endothelial cell motility are poorly investigated. Previously we showed that progesterone stimulated endothelial cell migration via the activation of actin-binding protein moesin, leading to actin cytoskeleton remodelling and the formation of cell membrane structures required for cell movement. In this study, we investigated the effects of progesterone on the formation of focal adhesion complexes, which provide anchoring sites for cell movement. In cultured human umbilical endothelial cells, progesterone enhanced focal adhesion kinase (FAK) phosphorylation at Tyr(397) in a dose- and time-dependent manner. Several signalling inhibitors interfered with progesterone-induced FAK activation, including progesterone receptor (PR) antagonist ORG 31710, specific c-Src kinase inhibitor PP2, phosphatidylinosital-3 kinase (PI3K) inhibitor wortmannin as well as ρ-associated kinase (ROCK-2) inhibitor Y27632. It suggested that PR, c-Src, PI3K and ROCK-2 are implicated in this action. In line with this, we found that progesterone rapidly promoted c-Src/PI3K/Akt activity, which activated the small GTPase RhoA/ρ-associated kinase (ROCK-2) complex, resulting in FAK phosphorylation. In the presence of progesterone, endothelial cells displayed enhanced horizontal migration, which was reversed by small interfering RNAs abrogating FAK expression. In conclusion, progesterone promotes endothelial cell movement via the rapid regulation of FAK. These findings provide new information on the biological actions of progesterone on human endothelial cells that are relevant for vascular function.     

Adhesion, actin cytoskeleton organisation and the spreading of colon adenocarcinoma cells induced by EGF are mediated by α2β1 integrin low clustering through focal adhesion kinase

Both epidermal growth factor (EGF) and the extracellular matrix components have been implicated in the pathobiology of adenocarcinomas by somewhat poorly understood mechanisms. We have addressed this problem using an in vitro model comprising the colon adenocarcinoma cell line HT29-D4, wherein the role of EGF and type IV collagen on cell adhesion was examined. We demonstrated that the effect of EGF on HT29-D4 cell adhesion was regulated by type IV collagen in a time- and dose-dependent manner. The incorporation of a panel of monoclonal antibodies to integrins alpha1beta1, alpha2beta1 and alpha3beta1 in adhesion medium revealed that EGF-mediated increase in the cell adhesion was mediated essentially by alpha2beta1, and the use of flow cytometry led us to conclude that this EGF effect was mediated by an increase in alpha2beta1 activation and not by an increase in cell surface expression of integrin. An indirect immunofluorescence technique was employed to demonstrate that focal adhesion kinase (FAK) and alpha2beta1 integrin were present in focal complexes in large EGF-induced lamellipodia whereas actin cytoskeleton was organised in small tips that colocalised with FAK. This pattern was observed at early time points (15 min) with a strong FAK tyrosine phosphorylation and with an increase in mitogen-activated protein kinase activity (5-15 min) as measured by immunoprecipitation and immunoblotting. We conclude that at early time points of cell adhesion and spreading, EGF exerted an inside-out regulation of alpha2beta1 integrin in HT29-D4 cells. This regulation seemed to be mediated by EGF-dependent FAK phosphorylation entailing an increase in integrin activation and their recruitment in numerous focal complexes. Furthermore after activation, FAK induced aggregation of actin-associated proteins (paxillin, vinculin and other tyrosine phosphorylated proteins) in focal complexes, leading to organisation of actin cytoskeleton that is involved in lamellipodia formation. Finally, activated alpha2beta1 integrins intervened in all these processes clustered in small focal complexes but not in focal adhesions.     

Protein-tyrosine phosphatase PTPD1 regulates focal adhesion kinase autophosphorylation and cell migration

PTPD1 is a cytosolic nonreceptor tyrosine phosphatase and a positive regulator of the Src-epidermal growth factor transduction pathway. We show that PTPD1 localizes along actin filaments and at adhesion plaques. PTPD1 forms a stable complex via distinct molecular modules with actin, Src tyrosine kinase, and focal adhesion kinase (FAK), a scaffold protein kinase enriched at adhesion plaques. Overexpression of PTPD1 promoted cell scattering and migration, short hairpin RNA-mediated silencing of endogenous PTPD1, or expression of PTPD1 mutants lacking either catalytic activity (PTPD1(C1108S)) or the FERM domain (PTPD1(Delta1-325)) significantly reduced cell motility. PTPD1 and Src catalytic activities were both required for epidermal growth factor-induced FAK autophosphorylation at its active site and for downstream propagation of ERK1/2 signaling. Our findings demonstrate that PTPD1 is a component of a multivalent scaffold complex nucleated by FAK at specific intracellular sites. By modulating Src-FAK signaling at adhesion sites, PTPD1 promotes the cytoskeleton events that induce cell adhesion and migration.     

Keratin 8/18 Modulation of Protein Kinase C-mediated Integrin-dependent Adhesion and Migration of Liver Epithelial Cells

Keratins are intermediate filament (IF) proteins of epithelial cells, expressed as pairs in a lineage/differentiation manner. Hepatocyte and hepatoma cell IFs are made solely of keratins 8/18 (K8/K18), the hallmark of all simple epithelia. Cell attachment/spreading (adhesion) and migration involve the formation of focal adhesions at sites of integrin interactions with extracellular matrix, actin adaptors such as talin and vinculin, and signaling molecules such as focal adhesion kinase (FAK) and member(s) of the protein kinase C (PKC) family. Here, we identify the novel PKCδ as mediator of the K8/K18 modulation of hepatoma cell adhesion and migration. We also demonstrate a K8/K18-dependent relationship between PKCδ and FAK activation through an integrin/FAK-positive feedback loop, in correlation with a reduced FAK time residency at focal adhesions. Notably, a K8/K18 loss results to a time course modulation of the receptor of activated C-kinase-1, β1-integrin, plectin, PKC, and c-Src complex formation. Although the K8/K18 modulation of hepatocyte adhesion also occurs through a PKC mediation, these differentiated epithelial cells exhibit minimal migrating ability, in link with marked differences in protein partner content and distribution. Together, these results uncover a key regulatory function for K8/K18 IFs in the PKC-mediated integrin/FAK-dependent adhesion and migration of simple epithelial cells.     

RAFTK/Pyk2 regulates EGF-induced PC12 cell spreading and movement

The protein tyrosine kinase RAFTK, also termed Pyk2, is a member of the focal adhesion kinase (FAK) subfamily. In this report, we show the role of RAFTK in neuroendocrine PC12 cells upon epidermal growth factor (EGF) stimulation. Following EGF treatment, we observed that RAFTK was tyrosine-phosphorylated in a time- and dose-dependent manner, while FAK was constitutively phosphorylated and primarily regulated by cell adhesion. Moreover, we found that RAFTK associated with the phosphorylated EGF receptor (EGFR) upon EGF stimulation. RAFTK phosphorylation was mediated primarily through PLCgamma-IP3-Ca(2+) signaling and partially through PI3-Kinase. Furthermore, overexpression of PRNK, a specific dominant-negative construct of RAFTK, was sufficient to block EGF-induced cell spreading and movement. Paxillin, a key modulator of the actin cytoskeleton and an RAFTK substrate, was also phosphorylated following EGF treatment. EGF induced a dynamic reorganization of RAFTK and paxillin at neuronal adhesion sites, with the specific localization of paxillin at the inner juxtaposition of RAFTK. Additionally, we observed that RAFTK associated with the scaffold protein c-Cbl and mediated its phosphorylation. Our data demonstrate that while FAK mediated cell adhesion, RAFTK was localized at the cytoplasm where it mediated inside-out signaling through intracellular Ca(2+), thus leading to cell spreading and movement upon EGF stimulation.     

Growth factors stimulate kidney proximal tubule cell migration independent of augmented tyrosine phosphorylation of focal adhesion kinase

Migration of human proximal tubule cells (HKC-5) was stimulated by epidermal growth factor (EGF), hepatocyte growth factor (HGF), and insulin-like growth factor-1 (IGF-1). Integrin signaling via phosphorylation of focal adhesion kinase (FAK) appears to play a central role in cell migration. Once stimulated, FAK undergoes autophosphorylation at tyrosine (Y) 397, followed by phosphorylation of several sites including Y576/Y577 which increases FAK's kinase activity, as well as at Y407, Y861, and Y925. EGF, HGF, and IGF-1 stimulate FAK phosphorylation in various cells. We showed that endothelin stimulated phosphorylation of Y397 in fibroblasts but not HKC-5 cells. After EGF stimulation, HKC-5 cells showed no change in tyrosine phosphorylation at FAK Y397, 407, 576, 861, or 925. Similarly, HGF and IGF-1 did not stimulate the phosphorylation of FAK Y397 in HKC-5 cells. Further, after inhibition of FAK expression by siRNA, cell migration was similar to cells treated with non-target siRNA and responded to EGF with increased migration. Thus, in proximal tubule cells, stimulation of cell migration by growth factors was independent of augmented FAK tyrosine phosphorylation.     

Epidermal growth factor-induced activation and translocation of c-Src to the cytoskeleton depends on the actin binding domain of the EGF-receptor

In the epidermal growth factor (EGF)-receptor signal transduction cascade, the non-receptor tyrosine kinase c-Src has been demonstrated to become activated upon EGF stimulation. In this paper we show that c-Src associates with the cytoskeleton and co-isolates with actin filaments upon EGF treatment of NIH-3T3 cells transfected with the EGF receptor. Immunofluorescence studies using CLSM show colocalization of F-actin and endogenous c-Src predominantly around endosomes and not on stress fibers and cell–cell contacts. Stimulation of EGF receptor-transfected NIH-3T3 cells with EGF induces an activation and translocation of c-Src to the cytoskeleton. These processes depend upon the presence of the actin binding domain of the EGF-receptor since in cells that express EGF-receptors lacking this domain, EGF fails to induce an activation and translocation to the cytoskeleton of c-Src. These data suggest a role for the actin binding domain of the EGF-receptor in the translocation of c-Src.     

PTP alpha regulates integrin-stimulated FAK autophosphorylation and cytoskeletal rearrangement in cell spreading and migration

We investigated the molecular and cellular actions of receptor protein tyrosine phosphatase (PTP) alpha in integrin signaling using immortalized fibroblasts derived from wild-type and PTP alpha-deficient mouse embryos. Defects in PTP alpha-/- migration in a wound healing assay were associated with altered cell shape and focal adhesion kinase (FAK) phosphorylation. The reduced haptotaxis to fibronectin (FN) of PTP alpha-/- cells was increased by expression of active (but not inactive) PTP alpha. Integrin-mediated formation of src-FAK and fyn-FAK complexes was reduced or abolished in PTP alpha-/- cells on FN, concomitant with markedly reduced phosphorylation of FAK at Tyr397. Reintroduction of active (but not inactive) PTP alpha restored FAK Tyr-397 phosphorylation. FN-induced cytoskeletal rearrangement was retarded in PTP alpha-/- cells, with delayed filamentous actin stress fiber assembly and focal adhesion formation. This mimicked the effects of treating wild-type fibroblasts with the src family protein tyrosine kinase (Src-PTK) inhibitor PP2. These results, together with the reduced src/fyn tyrosine kinase activity in PTP alpha-/- fibroblasts (Ponniah et al., 1999; Su et al., 1999), suggest that PTP alpha functions in integrin signaling and cell migration as an Src-PTK activator. Our paper establishes that PTP alpha is required for early integrin-proximal events, acting upstream of FAK to affect the timely and efficient phosphorylation of FAK Tyr-397.     

Tensins: a new switch in cell migration

Epidermal Growth Factor (EGF) is an important regulator of normal epithelial and carcinoma cell migration. The mechanism by which EGF induces cell migration is not fully understood. A recent report in Nature Cell Biology (Katz et al., 2007) demonstrates that EGF regulates migration through a switch in the expression of two tensin isoforms, weakening the association of beta1 integrin with the actin cytoskeleton in focal adhesions.     

RNA interference reveals a differential role of FAK and Pyk2 in cell migration, leading edge formation and increase in focal adhesions induced by LPA in intestinal epithelial cells

In the gastrointestinal mucosa, cell migration plays a crucial role in the organization and maintenance of tissue integrity but the mechanisms involved remain incompletely understood. Here, we used small-interfering RNA (siRNA)-mediated depletion of focal adhesion kinase (FAK) protein to determine the role of FAK in wound-induced migration and cytoskeletal organization in the non-transformed intestinal epithelial cells IEC-6 and IEC-18 stimulated with the G protein-coupled receptors (GPCR) agonist lysophosphatidic acid (LPA). Treatment of these cells with FAK siRNA substantially reduced FAK expression, but did not affect the expression of proline-rich tyrosine kinase 2 (Pyk2). Knockdown of FAK protein significantly inhibited LPA-induced migration of both IEC-18 and IEC-6 cells. LPA induced reorganization of actin and microtubule cytoskeleton in the leading edge was largely inhibited in FAK siRNA-transfected IEC-18 cells. Interestingly, in contrast to the FAK-/- cells, which exhibit an increased number of prominent focal adhesions when plated on fibronectin, FAK knockdown IEC-18 cells exhibited dramatically decreased number of focal adhesions in response to both LPA and fibronectin as compared with the control cells. We also used siRNAs to knockdown Pyk2 expression without reducing FAK expression. Depletion of Pyk2 did not prevent LPA-induced migration or cytoskeletal reorganization in IEC-18 cells. In conclusion, our study shows that FAK plays a critical role in LPA-induced migration, cytoskeletal reorganization, and assembly of focal adhesions in intestinal epithelial cells whereas depletion of Pyk2 did not interfere with any of these responses elicited by LPA.     

Essential role of SRC suppressed C kinase substrates in Schwann cells adhesion, spreading and migration

Integrin-mediated substrate adhesion of endothelial cells leads to dynamic rearrangement of the actin cytoskeleton. Protein kinase C (PKC) stimulates reorganization of microfilaments and adhesion, while the responses of Schwann cells during adhesion and migration are unknown, so we examined the expression changes of SSeCKS and F-actin in Schwann cells after exposure to fibronectin. Src (sarcoma) suppressed C kinase substrate (SSeCKS) is a PKC substrate that may play an important role in regulating actin cytoskeleton. We found that SSeCKS was localized to focal adhesion sites soon after Schwann cells adhesion and that SSeCKS increased during the process of cell spreading. Using small interfering RNAs specific to SSeCKS, we showed that Schwann cells in which SSeCKS expression was inhibited reduced cellular adhesion, spreading and promoted cellular migration on fibronectin through reorganization of actin stress fibers and blocking formation of focal adhesions. These results demonstrated SSeCKS modulate Schwann cells adhesion, spreading and migration by reorganization of the actin cytoskeleton.     

Invited review: focal adhesion and small heat shock proteins in the regulation of actin remodeling and contractility in smooth muscle.

Smooth muscle cells are able to adapt rapidly to chemical and mechanical signals impinging on the cell surface. It has been suggested that dynamic changes in the actin cytoskeleton contribute to the processes of contractile activation and mechanical adaptation in smooth muscle. In this review, evidence for functionally important changes in actin polymerization during smooth muscle contraction is summarized. The functions and regulation of proteins associated with "focal adhesion complexes" (membrane-associated dense plaques) in differentiated smooth muscle, including integrins, focal adhesion kinase (FAK), c-Src, paxillin, and the 27-kDa small heat shock protein (HSP27) are described. Integrins in smooth muscles are key elements of mechanotransduction pathways that communicate with and are regulated by focal adhesion proteins that include FAK, c-Src, and paxillin as well as proteins known to mediate cytoskeletal remodeling. Evidence that functions of FAK and c-Src protein kinases are closely intertwined is discussed as well as evidence that focal adhesion proteins mediate key signal transduction events that regulate actin remodeling and contraction. HSP27 is reviewed as a potentially significant effector protein that may regulate actin dynamics and cross-bridge function in response to activation of p21-activated kinase and the p38 mitogen-activated protein kinase signaling pathway by signaling pathways linked to integrin proteins. These signaling pathways are only part of a large number of yet to be defined pathways that mediate acute adaptive responses of the cytoskeleton in smooth muscle to environmental stimuli.     

Focal adhesion kinase‐dependent regulation of adhesive forces involves vinculin recruitment to focal adhesions

Background information. FAK (focal adhesion kinase), an essential non‐receptor tyrosine kinase, plays pivotal roles in migratory responses, adhesive signalling and mechanotransduction. FAK‐dependent regulation of cell migration involves focal adhesion turnover dynamics as well as actin cytoskeleton polymerization and lamellipodia protrusion. Whereas roles for FAK in migratory and mechanosensing responses have been established, the contribution of FAK to the generation of adhesive forces is not well understood. Results. Using FAK‐null cells expressing wild‐type and mutant FAK under an inducible tetracycline promoter, we analysed the role of FAK in the generation of steady‐state adhesive forces using micropatterned substrates and a hydrodynamic adhesion assay. FAK expression reduced steady‐state strength by 30% compared with FAK‐null cells. FAK expression reduced VCL (vinculin) localization to focal adhesions by 35% independently of changes in integrin binding and localization of talin and paxillin. RNAi (RNA interference) knock‐down of VCL abrogated the FAK‐dependent differences in adhesive forces. FAK‐dependent changes in VCL localization and adhesive forces were confirmed in human primary fibroblasts with FAK knocked down by RNAi. The autophosphorylation Tyr‐397 and kinase domain Tyr‐576/Tyr‐577 sites were differentially required for FAK‐mediated adhesive responses. Conclusions. We demonstrate that FAK reduces steady‐state adhesion strength by modulating VCL recruitment to focal adhesions. These findings provide insights into the role of FAK in mechanical interactions between a cell and the extracellular matrix.     

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.