Enterolobium contortisiliquum trypsin inhibitor (EcTI), a plant proteinase inhibitor, decreases in vitro cell adhesion and invasion by inhibition of Src protein-focal adhesion kinase (FAK) signaling pathways

Tumor cell invasion is vital for cancer progression and metastasis. Adhesion, migration, and degradation of the extracellular matrix are important events involved in the establishment of cancer cells at a new site, and therefore molecular targets are sought to inhibit such processes. The effect of a plant proteinase inhibitor, Enterolobium contortisiliquum trypsin inhibitor (EcTI), on the adhesion, migration, and invasion of gastric cancer cells was the focus of this study. EcTI showed no effect on the proliferation of gastric cancer cells or fibroblasts but inhibited the adhesion, migration, and cell invasion of gastric cancer cells; however, EcTI had no effect upon the adhesion of fibroblasts. EcTI was shown to decrease the expression and disrupt the cellular organization of molecules involved in the formation and maturation of invadopodia, such as integrin β1, cortactin, neuronal Wiskott-Aldrich syndrome protein, membrane type 1 metalloprotease, and metalloproteinase-2. Moreover, gastric cancer cells treated with EcTI presented a significant decrease in intracellular phosphorylated Src and focal adhesion kinase, integrin-dependent cell signaling components. Together, these results indicate that EcTI inhibits the invasion of gastric cancer cells through alterations in integrin-dependent cell signaling pathways.     

Focal adhesion kinase (FAK) activates and stabilizes IGF-1 receptor

Recent studies have shown a direct association between IGF-1R and FAK, two important mediators of cell growth, survival and migration. However, the mechanism by which FAK affects IGF-1R function remains unknown. This study investigates the potential role of FAK in mediating activation and stability of IGF-1R. Autophosphorylation and phosphorylation capacities of wild type and mutant IGF-1R were studied. Surprisingly, we found that the mutant IGF-1R lacking the three core tyrosine residues in the activation-loop can be phosphorylated although it is unable to undergo autophosphorylation, suggesting that another kinase possesses the ability to phosphorylate IGF-1R. By using wild type MEFs and FAK−/− MEFs we could demonstrate that FAK mediates activation-loop independent phosphorylation, as well as Akt and ERK activation. Furthermore, the stability of IGF-1R was decreased upon FAK siRNA or inactivation. Taken together, our data suggest a role for FAK in phosphorylation, signaling and stability of the IGF-1R.     

PTEN improve renal fibrosis in vitro and in vivo through inhibiting FAK/AKT signaling pathway

Renal fibrosis, the ultimate common pathway of progressive nephropathy, is characterized by excess accumulation and deposition of extracellular matrix (ECM) within the renal interstitium and glomeruli, finally resulting in end-stage kidney failure. TGFβ1 is not only abnormally increased during fibrosis but also involved in ECM induction and accumulation. Based on the bioinformative analyses, phosphatase and tensin homolog deleted on chromosome ten (PTEN) and focal adhesion kinase (FAK) signaling pathway might be involved in TGFβ1 functions on renal fibrosis development. In the present study, fibrosis was induced in HK-2 cells using TGFβ1 and PTEN expression was significantly suppressed by 24 or 48 hours TGFβ1 treatment. PTEN overexpression in HK-2 cells improved TGFβ1-induced fibrosis within α-SMA and E-cadherin. According to the KEGG signaling pathway annotation analyses on microarray profiles (GSE23338 and GSE20247) and immunoblotting validation, FAK signaling might be involved in PTEN functions in TGFβ1-induced fibrosis. PTEN overexpression significantly inhibited TGFβ1- or unilateral ureteral obstruction (UUO)-induced FAK signaling pathway activation both in vitro and in vivo; more importantly, PTEN silence enhanced TGFβ1- or UUO-induced fibrosis, while FAK inhibitor PF567721 significantly reversed the effects of PTEN silence, indicating that PTEN exerted its effects on TGFβ1- and UUO-induced fibrotic development in vitro and in vivo via inhibiting FAK signaling pathway. In summary, these findings indicate that PTEN could improve cellular fibrotic changes and renal fibrosis via inhibiting FAK/AKT signaling pathway. Restoring PTEN expression to target FAK/AKT signaling pathway might be a potent strategy for renal fibrosis treatment.     

Atorvastatin inhibited Rho-associated kinase 1 (ROCK1) and focal adhesion kinase (FAK) mediated adhesion and differentiation of CD133CD44 prostate cancer stem cells

Prostate cancer has become a global health concern and is one of the leading causes of cancer death of men after lung and gastric cancers. It has been suggested that the 3-hydroxy-3-methyl-glutarylcoenzyme-CoA (HMG-CoA) reductase inhibitor atorvastatin shows anticancer activity in prostate cancer cell lines. To this end, we analyzed the influence of atorvastatin on the cell adhesion and differentiation of CD133⁺CD44⁺ cells derived from prostate cancer biopsies and peripheral blood. CD133⁺CD44⁺ cells were treated with atorvastatin (16–64 μM) for different time periods. Cell adhesion to endothelial cell monolayers and differentiation into prostate cancer cells were evaluated. α1, β1 and α2β1 integrins adhesion receptors and the downstream target of atorvastatin Rho-dependent kinase (ROCK) and focal adhesion kinase (FAK) were analyzed by Western blot. Further blocking studies with the ROCK inhibitor H1152, anti-FAK antibody and anti-integrin α1 and β1 antibodies were carried out. Atorvastatin treatment inhibited dose-dependently cell attachment to endothelium and differentiation. The inhibitory effect of atorvastatin on cell adhesion was associated with decreased expression of integrins α1 and β1 and phosphorylated MYPT1 and FAK. Furthermore, atorvastatin strongly reduced ROCK1 and FAK mediated differentiation of CD133⁺CD44⁺ cells, which was confirmed by antibody treatment. Atorvastatin modified the expression of cell adhesion molecules and differentiation markers. These beneficial effects of atorvastatin may be mediated by ROCK and FAK signaling pathway. The data presented may point to novel treatment options for prostate cancer.     

Inhibition of focal adhesion kinase (FAK) signaling in focal adhesions decreases cell motility and proliferation.

It has been proposed that the focal adhesion kinase (FAK) mediates focal adhesion formation through tyrosine phosphorylation during cell adhesion. We investigated the role of FAK in focal adhesion structure and function. Loading cells with a glutathione-S-transferase fusion protein (GST-Cterm) containing the FAK focal adhesion targeting sequence, but not the kinase domain, decreased the association of endogenous FAK with focal adhesions. This displacement of endogenous FAK in both BALB/c 3T3 cells and human umbilical vein endothelial cells loaded with GST-Cterm decreased focal adhesion phosphotyrosine content. Neither cell type, however, exhibited a reduction in focal adhesions after GST-Cterm loading. These results indicate that FAK mediates adhesion-associated tyrosine phosphorylation, but not the formation of focal adhesions. We then examined the effect of inhibiting FAK function on other adhesion-dependent cell behavior. Cells microinjected with GST-Cterm exhibited decreased migration. In addition, cells injected with GST-Cterm had decreased DNA synthesis compared with control-injected or noninjected cells. These findings suggest that FAK functions in the regulation of cell migration and cell proliferation.     

Tac-beta1 inhibits FAK activation and Src signaling

The binding of integrins to extracellular matrix triggers signals that promote cell spreading. We previously demonstrated that expression of the integrin β1 cytoplasmic domain in the context of a chimeric transmembrane receptor with the Tac subunit of the interleukin-2 receptor (Tac-β1) inhibits cell spreading. To study the mechanism whereby Tac-β1 inhibits cell spreading, we examined the effect of Tac-β1 on early signaling events following integrin engagement namely FAK and Src signaling. We infected primary fibroblasts with adenoviruses expressing Tac or Tac-β1 and found that Tac-β1 prevented FAK activation by inhibiting the phosphorylation of FAK at Tyr-397. In contrast, Src activation was maintained, as phosphorylation of Src at Tyr-419 and Tyr-530 were not responsive to expression of Tac-β1. Importantly, adhesion-induced tyrosine phosphorylation of the Src substrates p130Cas and paxillin was inhibited, indicating that Src signaling was blocked by Tac-β1. These Src-dependent signaling events were found to require FAK signaling. Our results suggest that Tac-β1 inhibits cell spreading, at least in part, by preventing the phosphorylation of FAK at Tyr-397 and the assembly of signaling complexes necessary for phosphorylation of p130Cas and other downstream effectors.     

p56(lck) Controls phosphorylation of filamin (ABP-280) and regulates focal adhesion kinase (pp125(FAK))

Transformation of cells by src -like kinases leads to altered cell morphology associated with the disassembly of focal contacts and concomitant increase in tyrosine phosphorylation of pp125(FAK) x p56(lck) is a lymphocyte-specific member of the src family of protein tyrosine kinases that associates with cell surface glycoproteins such as CD4 and CD8. It phosphorylates and activates pp125(FAK) and increases its autokinase activity, thus pretreatment of pp125(FAK) with protein kinase C (PKC) markedly attenuates its phosphorylation and activation, suggesting a potential regulatory pathway of pp125(FAK) activation in focal contacts. p56(lck) further phosphorylates and activates actin binding protein (ABP-280; filamin) and controls its association with cell surface receptors such as beta-2 integrins, actin filament cross-linking, and possibly lipid membrane insertion.     

Signaling through focal adhesion kinase

Integrin receptor binding to extracellular matrix proteins generates intracellular signals via enhanced tyrosine phosphorylation events that are important for cell growth, survival, and migration. This review will focus on the functions of the focal adhesion kinase (FAK) protein-tyrosine kinase (PTK) and its role in linking integrin receptors to intracellular signaling pathways. FAK associates with several different signaling proteins such as Src-family PTKs, p130^Cas, Shc, Grb2, PI 3-kinase, and paxillin. This enables FAK to function within a network of integrin-stimulated signaling pathways leading to the activation of targets such as the ERK and JNK/mitogen-activated protein kinase pathways. Focus will be placed on the structural domains and sites of FAK tyrosine phosphorylation important for FAK-mediated signaling events and how these sites are conserved in the FAK-related PTK, Pyk2. We will review what is known about FAK activation by integrin receptor-mediated events and also non-integrin stimuli. In addition, we discuss the emergence of a consensus FAK substrate phosphorylation sequence. Emphasis will also be placed on the role of FAK in generating cell survival signals and the cleavage of FAK during caspase-mediated apoptosis. An in-depth discussion will be presented of integrin-stimulated signaling events occurring in the FAK knockout fibroblasts (FAK⁻) and how these cells exhibit deficits in cell migration. FAK re-expression in the FAK⁻ cells confirms the role of this PTK in the regulation of cell morphology and in promoting cell migration events. In addition, these results reinforce the potential role for FAK in promoting an invasive phenotype in human tumors.     

Cellular settings mediating Src Substrate switching between focal adhesion kinase tyrosine 861 and CUB-domain-containing protein 1 (CDCP1) tyrosine 734

Reciprocal interactions between Src family kinases (SFKs) and focal adhesion kinase (FAK) are critical during changes in cell attachment. Recently it has been recognized that another SFK substrate, CUB-domain-containing protein 1 (CDCP1), is differentially phosphorylated during these events. However, the molecular processes underlying SFK-mediated phosphorylation of CDCP1 are poorly understood. Here we identify a novel mechanism in which FAK tyrosine 861 and CDCP1-Tyr-734 compete as SFK substrates and demonstrate cellular settings in which SFKs switch between these sites. Our results show that stable CDCP1 expression induces robust SFK-mediated phosphorylation of CDCP1-Tyr-734 with concomitant loss of p-FAK-Tyr-861 in adherent HeLa cells. SFK substrate switching in these cells is dependent on the level of expression of CDCP1 and is also dependent on CDCP1-Tyr-734 but is independent of CDCP1-Tyr-743 and -Tyr-762. In HeLa CDCP1 cells, engagement of SFKs with CDCP1 is accompanied by an increase in phosphorylation of Src-Tyr-416 and a change in cell morphology to a fibroblastic appearance dependent on CDCP1-Tyr-734. SFK switching between FAK-Tyr-861 and CDCP1-Tyr-734 also occurs during changes in adhesion of colorectal cancer cell lines endogenously expressing these two proteins. Consistently, increased p-FAK-Tyr-861 levels and a more epithelial morphology are seen in colon cancer SW480 cells silenced for CDCP1. Unlike protein kinase Cδ, FAK does not appear to form a trimeric complex with Src and CDCP1. These data demonstrate novel aspects of the dynamics of SFK-mediated cell signaling that may be relevant during cancer progression.     

Src-mediated coupling of focal adhesion kinase to integrin alpha(v)beta5 in vascular endothelial growth factor signaling

The p38 mitogen-activated protein kinase (p38) is activated in response to environmental stress and inflammatory cytokines. Although several growth factors, including fibroblast growth factor (FGF)-2, mediate activation of p38, the consequences for growth factor-dependent cellular functions have not been well defined. We investigated the role of p38 activation in FGF-2-induced angiogenesis. In collagen gel cultures, bovine capillary endothelial cells formed tubular growth-arrested structures in response to FGF-2. In these collagen gel cultures, p38 activation was induced more potently by FGF-2 treatment compared with that in proliferating cultures. Treatment with the p38 inhibitor SB202190 enhanced FGF-2-induced tubular morphogenesis by decreasing apoptosis, increasing DNA synthesis and cell proliferation, and enhancing the kinetics of cell differentiation including increased expression of the Notch ligand Jagged1. Overexpression of dominant negative mutants of the p38-activating kinases MKK3 and MKK6 also supported FGF-2-induced tubular morphogenesis. Sustained activation of p38 by FGF-2 was identified in vascular endothelial cells in vivo in the chick chorioallantoic membrane (CAM). SB202190 treatment enhanced FGF-2-induced neovascularization in the CAM, but the vessels displayed abnormal features indicative of hyperplasia of endothelial cells. These results implicate p38 in organization of new vessels and suggest that p38 is an essential regulator of FGF-2-driven angiogenesis.     

Characterization of a novel focal adhesion kinase inhibitor in human platelets

Focal adhesion kinase (FAK) is activated in human platelets downstream of integrins, e.g. α_IIbβ₃, and other adhesion receptors e.g. GPVI. Mice in which platelets lack FAK have been shown to exhibit extended bleeding times and their platelets have been shown to display decreased spreading on fibrinogen-coated surfaces. Recently, a novel FAK inhibitor (PF-573,228) has become available, its selectivity for FAK shown in vitro and in cell lines. We determined the effect of this inhibitor on platelet function and signaling pathways. Like murine platelets lacking FAK, we found that PF-573,228 was effective at blocking human platelet spreading on fibrinogen-coated surfaces but did not affect the initial adhesion. We also found a reduced spreading on CRP-coated surfaces. Further analysis of the morphology of platelets adhered to these surfaces showed the defect in spreading occurred at the transition from filopodia to lamellipodia. Similar to that seen with murine neutrophils lacking FAK, we also observed an unexpected defect in intracellular calcium release in human platelets pre-treated with PF-573,228 which correlated with impaired dense granule secretion and aggregation. The aggregation defect could be partially rescued by addition of ADP, normally secreted from dense granules, suggesting that PF-573,228 has effects on FAK downstream of α_IIbβ₃ and elsewhere. Our data show that PF-573,228 is a useful tool for analysis of FAK function in cells and reveal that in human platelets FAK may regulate a rise in cell calcium and platelet spreading.     

SNARE-mediated membrane traffic is required for focal adhesion kinase signaling and Src-regulated focal adhesion turnover

Integrin signaling is central to cell growth and differentiation, and critical for the processes of apoptosis, cell migration and wound repair. Previous research has demonstrated a requirement for SNARE-dependent membrane traffic in integrin trafficking, as well as cell adhesion and migration. The goal of the present research was to ascertain whether SNARE-dependent membrane trafficking is required specifically for integrin-mediated signaling. Membrane traffic was inhibited in Chinese hamster ovary cells by expression of dominant-negative (E329Q) N-ethylmaleimide-sensitive fusion protein (NSF) or a truncated form of the SNARE SNAP23. Integrin signaling was monitored as cells were plated on fibronectin under serum-free conditions. E329Q-NSF expression inhibited phosphorylation of focal adhesion kinase (FAK) on Tyr397 at early time points of adhesion. Phosphorylation of FAK on Tyr576, Tyr861 and Tyr925 was also impaired by expression of E329Q-NSF or truncated SNAP23, as was trafficking, localization and activation of Src and its interaction with FAK. Decreased FAK-Src interaction coincided with reduced Rac activation, decreased focal adhesion turnover, reduced Akt phosphorylation and lower phosphatidylinositol 3,4,5-trisphosphate levels in the cell periphery. Over-expression of plasma membrane-targeted Src or phosphatidylinositol 3-kinase (PI3K) rescued cell spreading and focal adhesion turnover. The results suggest that SNARE-dependent trafficking is required for integrin signaling through a FAK/Src/PI3K-dependent pathway.     

Differential regulation of cell motility and invasion by FAK

Cell migration and invasion are fundamental components of tumor cell metastasis. Increased focal adhesion kinase (FAK) expression and tyrosine phosphorylation are connected with elevated tumorigenesis. Null mutation of FAK results in embryonic lethality, and FAK-/- fibroblasts exhibit cell migration defects in culture. Here we show that viral Src (v-Src) transformation of FAK-/- cells promotes integrin-stimulated motility equal to stable FAK reexpression. However, FAK-/- v-Src cells were not invasive, and FAK reexpression, Tyr-397 phosphorylation, and FAK kinase activity were required for the generation of an invasive cell phenotype. Cell invasion was linked to transient FAK accumulation at lamellipodia, formation of a FAK-Src-p130Cas-Dock180 signaling complex, elevated Rac and c-Jun NH2-terminal kinase activation, and increased matrix metalloproteinase expression and activity. Our studies support a dual role for FAK in promoting cell motility and invasion through the activation of distinct signaling pathways.     

Amplification and oscillations in the FAK/Src kinase system during integrin signaling

Integrin signaling is a major pathway of cell adhesion to extracellular matrices that regulates many physiological cell behaviors such as cell proliferation, migration or differentiation and is implied in pathologies such as tumor invasion. In this paper, we focused on the molecular system formed by the two kinases FAK (focal adhesion kinase) and Src, which undergo auto- and co-activation during early steps of integrin signaling. The system is modelled using classical kinetic equations and yields a set of three nonlinear ordinary differential equations describing the dynamics of the different phosphorylation forms of FAK. Analytical and numerical analysis of these equations show that this system may in certain cases amplify incoming signals from the integrins. A quantitative condition is obtained, which indicates that the total FAK charge in the system acts as a critical mass that must be exceeded for amplification to be effective. Furthermore, we show that when FAK activity is lower than Src activity, spontaneous oscillations of FAK phosphorylation forms may appear. The oscillatory behavior is studied using bifurcation and stability diagrams. We finally discuss the significance of this behavior with respect to recent experimental results evidencing FAK dynamics.     

Dephosphorylation of focal adhesion kinase (FAK) and loss of focal contacts precede caspase-mediated cleavage of FAK during apoptosis in renal epithelial cells

The relationship between focal adhesion protein (FAK) activity and loss of cell-matrix contact during apoptosis is not entirely clear nor has the role of FAK in chemically induced apoptosis been studied. We investigated the status of FAK phosphorylation and cleavage in renal epithelial cells during apoptosis caused by the nephrotoxicant dichlorovinylcysteine (DCVC). DCVC treatment caused a loss of cell-matrix contact which was preceded by a dissociation of FAK from the focal adhesions and tyrosine dephosphorylation of FAK. Paxillin was also dephosphorylated at tyrosine. DCVC treatment activated caspase-3 which was associated with cleavage of FAK. However, FAK cleavage occurred after cells had already lost focal adhesions indicating that cleavage of FAK by caspases is not responsible for loss of FAK from focal adhesions. Accordingly, although inhibition of caspase activity with zVAD-fmk blocked activation of caspase-3, FAK cleavage, and apoptosis, it neither affected dephosphorylation nor translocation of FAK or paxillin. However, zVAD-fmk completely blocked the cell detachment caused by DCVC treatment. Orthovanadate prevented DCVC-induced tyrosine dephosphorylation of both FAK and paxillin; however, it did not inhibit DCVC-induced apoptosis and actually potentiated focal adhesion disorganization and cell detachment. Thus, FAK dephosphorylation and loss of focal adhesions are not due to caspase activation; however, caspases are required for FAK proteolysis and cell detachment.     

Uniaxial cyclic stretch induces focal adhesion kinase (FAK) tyrosine phosphorylation followed by mitogen-activated protein kinase (MAPK) activation

We investigated the role of tyrosine phosphorylation of FAK in the stretch-induced MAPKs (extracellular signal-regulated kinase (ERK), p38MAPK) activation in mutant FAK-transfected fibroblasts. In response to uniaxial cyclic stretch (1 Hz, 120% in length), the levels of tyrosine phosphorylation of the Tyr-397 and Tyr-925 of FAK in control cells increased and peaked at 5 min (2.75 +/- 0.51, n = 3), and 20 min (2.98 +/- 0.58, n = 3), respectively, and the activities of MAPKs increased and peaked at approximately 10 min. On the other hand, in the mutant FAK-transfected cells, the stretch-induced MAPKs activation was significantly inhibited. The stretch-induced activation of MAPKs was also significantly abolished by either treatment with Gd(3+) or extracellular Ca(2+) removal which may inhibit intracellular Ca(2+) increase caused by the activation of cation selective (Ca(2+)-permeable) stretch activated (SACatC) channels. These results suggest that the stretch-induced tyrosine-phosphorylation of FAK via SACatC activation is critical for the stretch-induced MAPKs activation.     

Optogenetic control of focal adhesion kinase signaling

Focal adhesion kinase (FAK) integrates signaling from integrins, growth factor receptors and mechanical stress to control cell adhesion, motility, survival and proliferation. Here, we developed a single-component, photo-activatable FAK, termed optoFAK, by using blue light-induced oligomerization of cryptochrome 2 (CRY2) to activate FAK-CRY2 fusion proteins. OptoFAK functions uncoupled from physiological stimuli and activates downstream signaling rapidly and reversibly upon blue light exposure. OptoFAK stimulates SRC creating a positive feedback loop on FAK activation, facilitating phosphorylation of paxillin and p130Cas in adherent cells. In detached cells or in mechanically stressed adherent cells, optoFAK is autophosphorylated upon exposure to blue light, however, downstream signaling is hampered indicating that the accessibility to these substrates is disturbed. OptoFAK may prove to be a useful tool to study the biological function of FAK in growth factor and integrin signaling, tension-mediated focal adhesion maturation or anoikis and could additionally serve as test system for kinase inhibitors.     

MG53 inhibits angiogenesis through regulating focal adhesion kinase signalling

Mitsugumin 53 (MG53), which is expressed predominantly in striated muscle, has been demonstrated to be a myokine/cardiokine secreted from striated muscle under specific conditions. The important roles of MG53 in non-striated muscle tissues have also been examined in multiple disease models. However, no previous study has implicated MG53 in the control of endothelial cell function. In order to explore the effects of MG53 on endothelial cells, human umbilical vein endothelial cells (HUVECs) were stimulated with recombinant human MG53 (rhMG53). Then, rhMG53 uptake, focal adhesion kinase (FAK)/Src/Akt/ERK1/2 signalling pathway activation, cell migration and tube formation were determined in vitro. The efficacy of rhMG53 in regulating angiogenesis was also detected in postnatal mouse retinas. The results demonstrated that rhMG53 directly entered into endothelial cells in a cholesterol-dependent manner. The uptake of rhMG53 directly bound to FAK in endothelial cells, which resulted in a significant decrease in FAK phosphorylation at Y397. Accompanied by the dephosphorylation of FAK, rhMG53 uncoupled FAK-Src interaction and reduced the phosphorylation of Src at Y416. Consequently, the activation of FAK/Src downstream signalling pathways, such as Akt and ERK1/2, was also significantly inhibited by rhMG53. Furthermore, rhMG53 remarkably decreased HUVEC migration and tube formation in vitro and postnatal mouse retinal angiogenesis in vivo. Taken together, these data indicate that rhMG53 inhibits angiogenesis through regulating FAK/Src/Akt/ERK1/2 signalling pathways. This may provide a novel molecular mechanism for the impaired angiogenesis in ischaemic diseases.     

Nanog increases focal adhesion kinase (FAK) promoter activity and expression and directly binds to FAK protein to be phosphorylated

Nanog and FAK were shown to be overexpressed in cancer cells. In this report, the Nanog overexpression increased FAK expression in 293, SW480, and SW620 cancer cells. Nanog binds the FAK promoter and up-regulates its activity, whereas Nanog siRNA decreases FAK promoter activity and FAK mRNA. The FAK promoter contains four Nanog-binding sites. The site-directed mutagenesis of these sites significantly decreased up-regulation of FAK promoter activity by Nanog. EMSA showed the specific binding of Nanog to each of the four sites, and binding was confirmed by ChIP assay. Nanog directly binds the FAK protein by pulldown and immunoprecipitation assays, and proteins co-localize by confocal microscopy. Nanog binds the N-terminal domain of FAK. In addition, FAK directly phosphorylates Nanog in a dose-dependent manner by in vitro kinase assay and in cancer cells in vivo. The site-directed mutagenesis of Nanog tyrosines, Y35F and Y174F, blocked phosphorylation and binding by FAK. Moreover, overexpression of wild type Nanog increased filopodia/lamellipodia formation, whereas mutant Y35F and Y174F Nanog did not. The wild type Nanog increased cell invasion that was inhibited by the FAK inhibitor and increased by FAK more significantly than with the mutants Y35F and Y174F Nanog. Down-regulation of Nanog with siRNA decreased cell growth reversed by FAK overexpression. Thus, these data demonstrate the regulation of the FAK promoter by Nanog, the direct binding of the proteins, the phosphorylation of Nanog by FAK, and the effect of FAK and Nanog cross-regulation on cancer cell morphology, invasion, and growth that plays a significant role in carcinogenesis.