Absence Of Focal Adhesion Kinase Research Articles

The Chlamydia Effector TarP Mimics the Mammalian Leucine-Aspartic Acid Motif of Paxillin to Subvert the Focal Adhesion Kinase during Invasion

Host cell signal transduction pathways are often targets of bacterial pathogens, especially during the process of invasion when robust actin remodeling is required. We demonstrate that the host cell focal adhesion kinase (FAK) was necessary for the invasion by the obligate intracellular pathogen Chlamydia caviae. Bacterial adhesion triggered the transient recruitment of FAK to the plasma membrane to mediate a Cdc42- and Arp2/3-dependent actin assembly. FAK recruitment was via binding to a domain within the virulence factor TarP that mimicked the LD2 motif of the FAK binding partner paxillin. Importantly, bacterial two-hybrid and quantitative imaging assays revealed a similar level of interaction between paxillin-LD2 and TarP-LD. The conserved leucine residues within the L(D/E)XLLXXL motif were essential to the recruitment of FAK, Cdc42, p34(Arc), and actin to the plasma membrane. In the absence of FAK, TarP-LD-mediated F-actin assembly was reduced, highlighting the functional relevance of this interaction. Together, the data indicate that a prokaryotic version of the paxillin LD2 domain targets the FAK signaling pathway, with TarP representing the first example of an LD-containing Type III virulence effector.

Abstract 2879: Expression of Focal Adhesion Kinase (FAK) in myeloid lineage cells increases with maturation and regulates primary tumor growth in MMTV-PyVmT murine model of breast cancer.

Abstract Myeloid lineage cells are critical mediators of innate immunity that contribute to the inflammatory environment associated with tumor formation and progression. Our research explores the role of adhesion signaling through Focal Adhesion Kinase (FAK) in myeloid lineage cells as a potential regulator of breast tumor outgrowth. Previously, we have demonstrated that recruitment of cd11b-positive monocytes to sites of thioglycollate-induced inflammation was impaired in the absence of FAK. Additionally, we showed that bone marrow-derived macrophages exhibit reduced migration toward chemotactic factors commonly found in the breast tumor milieu. Given these results, the goal of the current study is to address the fundamental contribution of FAK toward myeloid cell functions that regulate breast tumor growth and progression. Mice with FAK conditionally deleted in myeloid lineage cells (FAKΔmyeloid) were crossed with mice that develop spontaneous breast carcinomas due to expression of the MMTV-PyMT transgene. FAKΔmyeloid MMTV-PyMT mice showed significantly enhanced primary tumor outgrowth compared to wildtype (WT) littermate MMTV-PyMT animals. Tumors from both genotypes contained equivalent numbers of mature, F4/80-positive tumor-associated macrophages (TAMs), demonstrating that these cells were not dependent upon FAK to infiltrate/survive in the tumor. This was reinforced by adoptive transfer studies showing equal numbers of WT and FAKΔmyeloid bone marrow monocytes at the tumor site 30 minutes post-injection. Further analysis revealed that FAK expression was developmentally regulated, such that bone marrow cells and the immature Ly6C+cd11b+ cells present in circulation expressed nearly undetectable amounts of FAK protein. However, a substantial number of the cd11b+ cells within the tumor were Lyc6C-negative and had gained expression of FAK. Interestingly, the Ly6C-negative cells were shown to be more migratory than the Lyc6C+ monocytes, and this increased migration was dependent upon FAK expression. Based on these data, we propose that monocytes mature and upregulate FAK once they have successfully extravasated out of the tumor vasculature. This, in turn, allows increased movement of these cells within the tumor microenvironment, which may help to control tumor size/outgrowth through more effective clearance of necrotic cells and/or more global distribution of factors that could ultimately mediate an anti-tumor effect. Experiments are currently underway to test these possibilities. Citation Format: Ryan Llewellyn, Michael F. Gutknecht, Amy H. Bouton. Expression of Focal Adhesion Kinase (FAK) in myeloid lineage cells increases with maturation and regulates primary tumor growth in MMTV-PyVmT murine model of breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2879. doi:10.1158/1538-7445.AM2013-2879

FAK deletion promotes p53-mediated induction of p21, DNA-damage responses and radio-resistance in advanced squamous cancer cells.

Focal adhesion kinase (FAK) is a cytoplasmic tyrosine kinase that is elevated in a variety of human cancers. While FAK is implicated in many cellular processes that are perturbed in cancer, including proliferation, actin and adhesion dynamics, polarisation and invasion, there is only some limited information regarding the role of FAK in radiation survival. We have evaluated whether FAK is a general radio-sensitising target, as has been suggested by previous reports. We used a clean genetic system in which FAK was deleted from mouse squamous cell carcinoma (SCC) cells (FAK −/−), and reconstituted with exogenous FAK wild type (wt). Surprisingly, the absence of FAK was associated with increased radio-resistance in advanced SCC cells. FAK re-expression inhibited p53-mediated transcriptional up-regulation of p21, and a sub-set of other p53 target genes involved in DNA repair, after treatment with ionizing radiation. Moreover, p21 depletion promoted radio-sensitisation, implying that FAK-mediated inhibition of p21 induction is responsible for the relative radio-sensitivity of FAK-proficient SCC cells. Our work adds to a growing body of evidence that there is a close functional relationship between integrin/FAK signalling and the p53/p21 pathway, but demonstrates that FAK's role in survival after stress is context-dependent, at least in cancer cells. We suggest that there should be caution when considering inhibiting FAK in combination with radiation, as this may not always be clinically advantageous.

FAK Regulates Intestinal Epithelial Cell Survival and Proliferation during Mucosal Wound Healing

BackgroundFollowing damage to the intestinal epithelium, restoration of epithelial barrier integrity is triggered by a robust proliferative response. In other tissues, focal adhesion kinase (FAK) regulates many of the cellular processes that are critical for epithelial homeostasis and restitution, including cell migration, proliferation and survival. However, few studies to date have determined how FAK contributes to mucosal wound healing in vivo.Methodology and Principal FindingsTo examine the role of FAK in intestinal epithelial homeostasis and during injury, we generated intestinal epithelium (IE)-specific conditional FAK knockout mice. Colitis was induced with dextran-sulfate-sodium (DSS) and intestinal tissues were analyzed by immunohistochemistry and immunoblotting. While intestinal development occurred normally in mice lacking FAK, FAK-deficient animals were profoundly susceptible to colitis. The loss of epithelial FAK resulted in elevated p53 expression and an increased sensitivity to apoptosis, coincident with a failure to upregulate epithelial cell proliferation. FAK has been reported to function as a mechanosensor, inducing cyclin D1 expression and promoting cell cycle progression under conditions in which tissue/matrix stiffness is increased. Collagen deposition, a hallmark of inflammatory injury resulting in increased tissue rigidity, was observed in control and FAK knockout mice during colitis. Despite this fibrotic response, the colonic epithelium in FAK-deficient mice exhibited significantly reduced cyclin D1 expression, suggesting that proliferation is uncoupled from fibrosis in the absence of FAK. In support of this hypothesis, proliferation of Caco-2 cells increased proportionally with matrix stiffness in vitro only under conditions of normal FAK expression; FAK depleted cells exhibited reduced proliferation concomitant with attenuated cyclin D1 expression.ConclusionsIn the colon, FAK functions as a regulator of epithelial cell survival and proliferation under conditions of mucosal injury and a mechanosensor of tissue compliance, inducing repair-driven proliferation in the colonic epithelium through upregulation of cyclin D1.

Complete focal adhesion kinase deficiency in the mammary gland causes ductal dilation and aberrant branching morphogenesis through defects in Rho kinase‐dependent cell contractility

The adult, virgin mammary gland is a highly organized branched ductal network comprising two major cell types: myoepithelial and luminal epithelial cells. To study the role and mechanism of focal adhesion kinase (FAK)-mediated signaling in mammary gland development and differentiation, we used a conditional Fak-knockout mammary epithelial cell (MEC) transplantation model. Conditional Cre recombinase (Cre)-mediated Fak deletion in primary cultured MECs isolated from FAK(lox/lox)/Rosa26Cre-ERT2 donor mice caused loss of FAK in all mammary cells. Transplantation of Fak-knockout MECs in a cleared mammary fat pad of immune-deficient recipient mice resulted in development of new but dilated virgin ducts with a disrupted myoepithelial and luminal epithelial cell multilayer and aberrant ductal morphogenesis during pregnancy. In the absence of FAK, MECs spread poorly, showed enhanced Rho kinase (ROCK)-mediated cytoskeletal contractility, and failed to respond to receptor-mediated cytoskeletal remodeling. Likewise, FAK deficiency fully inhibited branching morphogenesis of mammary gland organoids in a ROCK-dependent manner. Altogether these data suggest a model in which FAK coordinates contractile forces in MECs to maintain the bilayered cellular organization of myoepithelial and luminal epithelial cells in ducts, thus allowing proper mammary gland development and function.

Focal Adhesion Kinase (FAK)-related Non-kinase Inhibits Myofibroblast Differentiation through Differential MAPK Activation in a FAK-dependent Manner

Transforming growth factor (TGF)-beta1 induces fibroblast transdifferentiation to myofibroblasts, a process that requires the involvement of integrin-mediated signaling and focal adhesion kinase (FAK). FAK-related non-kinase (FRNK) is known for its role in inhibiting integrin-mediated cell migration; however, its role in myofibroblast differentiation has not been defined. Here, we report that FRNK abrogates TGF-beta1-induced myofibroblast differentiation in vitro and in vivo. TGF-beta1 can induce alpha-smooth muscle actin (alpha-SMA) expression in the presence or absence of FAK; however, TGF-beta1-induced alpha-SMA expression is reduced (approximately 73%) in FAK-deficient fibroblasts. Although both ERK and p38 MAPK activation is required for maximal TGF-beta1-induced alpha-SMA expression, ERK is the major signaling intermediate in cells that express FAK. In contrast, p38 MAPK is the dominant mediator of TGF-beta1-induced alpha-SMA expression in FAK-deficient cells. FRNK overexpression blocks TGF-beta1-induced ERK or p38 MAPK activation in the presence, and surprisingly, in the absence of FAK. The loss of FRNK was tested in vivo during experimentally induced pulmonary fibrosis in mice. FRNK knock-out mice have a greater increase in alpha-SMA-expressing cells in response to a pulmonary fibrotic stimulus in vivo, as compared with congenic wild type mice. This is the first time that FRNK loss has been shown to modify the pathobiology in any animal disease model. Together, the data demonstrate that FRNK negatively regulates myofibroblast differentiation in vitro and in vivo. These data further suggest that modulation FRNK expression may be a novel avenue for therapeutic intervention in tissue fibrosis.

Regulation of lamellipodial persistence, adhesion turnover, and motility in macrophages by focal adhesion kinase

Macrophages are a key component of the innate immune system. In this study, we investigate how focal adhesion kinase (FAK) and the related kinase Pyk2 integrate adhesion signaling and growth factor receptor signaling to regulate diverse macrophage functions. Primary bone marrow macrophages isolated from mice in which FAK is conditionally deleted from cells of the myeloid lineage exhibited elevated protrusive activity, altered adhesion dynamics, impaired chemotaxis, elevated basal Rac1 activity, and a marked inability to form stable lamellipodia necessary for directional locomotion. The contribution of FAK to macrophage function in vitro was substantiated in vivo by the finding that recruitment of monocytes to sites of inflammation was impaired in the absence of FAK. Decreased Pyk2 expression in primary macrophages also resulted in a diminution of invasive capacity. However, the combined loss of FAK and Pyk2 had no greater effect than the loss of either molecule alone, indicating that both kinases function within the same pathway to promote invasion.

Tumor Necrosis Factor-α Stimulates Focal Adhesion Kinase Activity Required for Mitogen-activated Kinase-associated Interleukin 6 Expression

Tumor Necrosis Factor-α Stimulates Focal Adhesion Kinase Activity Required for Mitogen-activated Kinase-associated Interleukin 6 Expression

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

In response to αβ1 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 αβ1 integrins. FAK is also dispensible for proliferation/survival in enriched medium. In contrast, FAK functions downstream of αβ1 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.

Focal Adhesion Kinase Is Critical for Entry of Kaposi's Sarcoma-Associated Herpesvirus into Target Cells

Kaposi's sarcoma-associated herpesvirus/human herpesvirus 8 (KSHV/HHV-8) interacts with cell surface alpha3beta1 integrin early during in vitro infection of human endothelial cells and fibroblasts and activates the focal adhesion kinase (FAK) that is immediately downstream in the outside-in signaling pathway by integrins, leading to the activation of several downstream signaling molecules. In this study, using real-time DNA and reverse transcription-PCR assays to measure total internalized viral DNA, viral DNA associated with infected nuclei, and viral gene expression, we examined the stage of infection at which FAK plays the most significant role. Early during KSHV infection, FAK was phosphorylated in FAK-positive Du17 mouse embryonic fibroblasts. The absence of FAK in Du3 (FAK(-/-)) cells resulted in about 70% reduction in the internalization of viral DNA, suggesting that FAK plays a role in KSHV entry. Expression of FAK in Du3 (FAK(-/-)) cells via an adenovirus vector augmented the internalization of viral DNA. Expression of the FAK dominant-negative mutant FAK-related nonkinase (FRNK) in Du17 cells significantly reduced the entry of virus. Virus entry in Du3 cells, albeit in reduced quantity, delivery of viral DNA to the infected cell nuclei, and expression of KSHV genes suggested that in the absence of FAK, another molecule(s) may be partially compensating for FAK function. Infection of Du3 cells induced the phosphorylation of the FAK-related proline-rich tyrosine kinase (Pyk2) molecule, which has been shown to complement some of the functions of FAK. Expression of an autophosphorylation site mutant of Pyk2 in which Y402 is mutated to F (F402 Pyk2) reduced viral entry in Du3 cells, suggesting that Pyk2 facilitates viral entry moderately in the absence of FAK. These results suggest a critical role for KSHV infection-induced FAK in the internalization of viral DNA into target cells.

Enhanced v-Src-induced oncogenic transformation in the absence of focal adhesion kinase is mediated by phosphatidylinositol 3-kinase

We showed previously [K. Moissoglu, I.H. Gelman, J. Biol. Chem. 278 (2003) 47946–47959] that oncogenic v-Src could induce 7- to 10-fold greater anchorage-independent growth (AIG) in FAK-null mouse embryo fibroblasts (MEF) compared to those expressing FAK. Here, we demonstrate that the enhanced AIG (eAIG) correlates with increased activation levels of phosphatidylinositol 3-kinase (PI3K) and not with changes in the protein levels of the p85 regulatory subunit of PI3K, PDK1 or PTEN- modulators, and/or mediators of PI3K activity. eAIG could be blunted selectively by treatment with the PI3K inhibitor, LY294002, or by overexpression of either the PI3K antagonist, PTEN, dominant-interfering alleles of PI3K or a downstream PI3K mediator, AKT, but not by the MEK inhibitor, PD98059, dominant-interfering alleles of MEK or the signal transducer and activator of transcription (STAT)-3. In contrast, RNAi-mediated knockdown of FAK resulted in increased v-Src-induced AIG. Expression of a constitutively active PI3K allele was sufficient to induce higher levels of AIG, whereas overexpression of v-Src produced only larger-sized colonies in soft agar. Interestingly, FAK was required for full activation of PI3K by PDGF whereas the activation of PI3K by insulin was significantly increased in FAK−/− cells. Thus, although FAK is dispensable for v-Src-induced oncogenic transformation in vitro, it may exert either positive or negative effects on signaling or motility depending on which pathways are activated in cancer cells.

V-Src Rescues Actin-based Cytoskeletal Architecture and Cell Motility and Induces Enhanced Anchorage Independence during Oncogenic Transformation of Focal Adhesion Kinase-null Fibroblasts

The ability of the focal adhesion kinase (FAK) to integrate signals from extracellular matrix and growth factor receptors requires the integrity of Tyr397, a major autophosphorylation site that mediates the Src homology 2-dependent binding of Src family kinases. However, the precise roles played by FAK in specific Src-induced pathways, especially as they relate to oncogenic transformation, remain unclear. Here, we investigate the role of FAK in v-Src-induced oncogenic transformation by transducing temperature-sensitive v-Src (ts72v-Src) into p53-null FAK+/+ or FAK-/- mouse embryo fibroblasts (MEF). At the permissive temperature (PT), ts72v-Src induced abundant tyrosine phosphorylation, morphological transformation and cytoskeletal rearrangement in FAK-/- MEF, including the restoration of cell polarity, typical focal adhesion complexes, and longitudinal F-actin stress fibers. v-Src rescued the haptotactic, linear directional, and invasive motility defects of FAK-/- cells to levels found in FAK+/+ or FAK+/+-[ts72v-Src] cells, and, in the case of monolayer wound healing motility, there was an enhancement. Src activation failed to increase the high basal tyrosine phosphorylation of the Crk-associated substrate, CAS, found in FAK-/- MEF, indicating that CAS phosphorylation alone is insufficient to induce motility in the absence of FAK- or v-Src-induced cytoskeletal remodeling. Compared with FAK+/+[ts72v-Src] controls, FAK-/-[ts72v-Src] clones exhibited 7-10-fold higher anchorage-independent proliferation that could not be attributed to variations in either v-Src protein level or stability. Re-expression of FAK diminished the colony-forming activities of FAK-/-[ts72v-Src] without altering ts72v-Src expression levels, suggesting that FAK attenuates Src-induced anchorage independence. Our data also indicate that the enhanced Pyk2 level found in FAK-/- MEF plays no role in v-Src-induced anchorage independence. Overall, our data indicate that FAK, although dispensable, attenuates v-Src-induced oncogenic transformation by modulating distinct signaling and cytoskeletal pathways.

Tenascin-C modulates matrix contraction via focal adhesion kinase- and Rho-mediated signaling pathways.

A provisional matrix consisting of fibrin and fibronectin (FN) is deposited at sites of tissue damage and repair. This matrix serves as a scaffold for fibroblast migration into the wound where these cells deposit new matrix to replace lost or damaged tissue and eventually contract the matrix to bring the margins of the wound together. Tenascin-C is expressed transiently during wound repair in tissue adjacent to areas of injury and contacts the provisional matrix in vivo. Using a synthetic model of the provisional matrix, we have found that tenascin-C regulates cell responses to a fibrin-FN matrix through modulation of focal adhesion kinase (FAK) and RhoA activation. Cells on fibrin-FN+tenascin-C redistribute their actin to the cell cortex, downregulate focal adhesion formation, and do not assemble a FN matrix. Cells surrounded by a fibrin-FN+tenascin-C matrix are unable to induce matrix contraction. The inhibitory effect of tenascin-C is circumvented by downstream activation of RhoA. FAK is also required for matrix contraction and the absence of FAK cannot be overcome by activation of RhoA. These observations show dual requirements for both FAK and RhoA activities during contraction of a fibrin-FN matrix. The effects of tenascin-C combined with its location around the wound bed suggest that this protein regulates fundamental processes of tissue repair by limiting the extent of matrix deposition and contraction to fibrin-FN-rich matrix in the primary wound area.

Focal adhesion kinase suppresses Rho activity to promote focal adhesion turnover.

Focal adhesion kinase (FAK) is activated and localized at focal adhesions upon cell adhesion to extracellular matrices. Cells lacking FAK show increased focal adhesion number and decreased cell migration, functions that are regulated by the small GTPase Rho. We now report that fibroblasts from FAK-/- mice failed to transiently inhibit Rho activity when plated on fibronectin. Re-expression of FAK restored normal Rho regulation. Turnover of focal adhesions correlated inversely with Rho activity. The presence or absence of FAK was mimicked by inhibiting or activating Rho, respectively. These data suggest that loss of FAK resulting in constitutive activation of Rho and inhibition of focal adhesion turnover can account for deficiencies in cell migration and embryonic lethality of the FAK knockout.

Pyk2 and Src-family protein-tyrosine kinases compensate for the loss of FAK in fibronectin-stimulated signaling events but Pyk2 does not fully function to enhance FAK- cell migration.

The focal adhesion kinase (FAK) protein-tyrosine kinase (PTK) links transmembrane integrin receptors to intracellular signaling pathways. We show that expression of the FAK-related PTK, Pyk2, is elevated in fibroblasts isolated from murine fak-/- embryos (FAK-) compared with cells from fak+/+ embryos (FAK+). Pyk2 was localized to perinuclear regions in both FAK+ and FAK- cells. Pyk2 tyrosine phosphorylation was enhanced by fibronectin (FN) stimulation of FAK- but not FAK+ cells. Increased Pyk2 tyrosine phosphorylation paralleled the time-course of Grb2 binding to Shc and activation of ERK2 in FAK- cells. Pyk2 in vitro autophosphorylation activity was not enhanced by FN plating of FAK- cells. However, Pyk2 associated with active Src-family PTKs after FN but not poly-L-lysine replating of the FAK- cells. Overexpression of both wild-type (WT) and kinase-inactive (Ala457), but not the autophosphorylation site mutant (Phe402) Pyk2, enhanced endogenous FN-stimulated c-Src in vitro kinase activity in FAK- cells, but only WT Pyk2 overexpression enhanced FN-stimulated activation of co-transfected ERK2. Interestingly, Pyk2 overexpression only weakly augmented FAK- cell migration to FN whereas transient FAK expression promoted FAK- cell migration to FN efficiently compared with FAK+ cells. Significantly, repression of endogenous Src-family PTK activity by p50(csk) overexpression inhibited FN-stimulated cell spreading, Pyk2 tyrosine phosphorylation, Grb2 binding to Shc, and ERK2 activation in the FAK- but not in FAK+ cells. These studies show that Pyk2 and Src-family PTKs combine to promote FN-stimulated signaling events to ERK2 in the absence of FAK, but that these signaling events are not sufficient to overcome the FAK- cell migration defects.