Focal adhesion kinase is not required for integrin function or viability in Drosophila

The mammalian focal adhesion kinase (FAK) family of non-receptor protein-tyrosine kinases has been implicated in controlling a multitude of cellular responses to the engagement of cell-surface integrins and G-protein-coupled receptors. The high level of sequence conservation between the mammalian proteins and the Drosophila homologue of FAK, Fak56, suggested that it would have similar functions. However, we show here that Drosophila Fak56 is not essential for integrin functions in adhesion, migration or signaling in vivo. Furthermore, animals lacking Fak56 are viable and fertile, demonstrating that Fak56 is not essential for other developmental or physiological functions. Despite this, overexpressed Fak56 is a potent inhibitor of integrins binding to the extracellular matrix, suggesting that Fak56 may play a subtle role in the negative regulation of integrin adhesion.     

DFak56 is a novel Drosophila melanogaster focal adhesion kinase

The mammalian focal adhesion kinase (FAK) family of nonreceptor protein-tyrosine kinases have been implicated in controlling a multitude of cellular responses to the engagement of cell surface integrins and G protein-coupled receptors. We describe here a Drosophila melanogaster FAK homologue, DFak56, which maps to band 56D on the right arm of the second chromosome. Full-length DFak56 cDNA encodes a phosphoprotein of 140 kDa, which shares strong sequence similarity not only with mammalian p125(FAK) but also with the more recently described mammalian Pyk2 (also known as CAKbeta, RAFTK, FAK2, and CADTK) FAK family member. DFak56 has intrinsic tyrosine kinase activity and is phosphorylated on tyrosine in vivo. As is the case for FAK, tyrosine phosphorylation of DFak56 is increased upon plating Drosophila embryo cells on extracellular matrix proteins. In situ hybridization and immunofluorescence staining analysis showed that DFak56 is ubiquitously expressed with particularly high levels within the developing central nervous system. We utilized the UAS-GAL4 expression system to express DFak56 and analyze its function in vivo. Overexpression of DFak56 in the wing imaginal disc results in wing blistering in adults, a phenotype also observed with both position-specific integrin loss of function and position-specific integrin overexpression. Our results imply a role for DFak56 in adhesion-dependent signaling pathways in vivo during D. melanogaster development.     

Cloning and characterization of Dfak56, a homolog of focal adhesion kinase, in Drosophila melanogaster.

The focal adhesion kinase (FAK) protein-tyrosine kinase plays important roles in cell adhesion in vertebrates. Using polymerase chain reaction-based cloning strategy, we cloned a Drosophila gene that is homologous to the vertebrate FAK family of protein-tyrosine kinases. We designated this gene Dfak56 and characterized its gene product. The overall protein structure and deduced amino acid sequence of Dfak56 show significant similarity to those of FAK and PYK2. Dfak56 has in vitro autophosphorylation activity at tyrosine residues. Expression of the Dfak56 mRNA and the protein was observed in the central nervous system and the muscle-epidermis attachment site in the embryo, where Drosophila position-specific integrins are localized. The results suggest that like FAK in vertebrates, Dfak56 functions downstream of integrins. Dfak56 was tyrosine-phosphorylated upon integrin-dependent attachment of the cell to the extracellular matrix. We conclude that the Dfak56 tyrosine kinase is involved in integrin-mediated cell adhesion signaling and thus is a functional homolog of vertebrate FAK.     

Structural insight into the mechanisms of targeting and signaling of focal adhesion kinase

Focal adhesion kinase (FAK) is a nonreceptor tyrosine kinase whose focal adhesion targeting (FAT) domain interacts with other focal adhesion molecules in integrin-mediated signaling. Localization of activated FAK to focal adhesions is indispensable for its function. Here we describe a solution structure of the FAT domain bound to a peptide derived from paxillin, a FAK-binding partner. The FAT domain is composed of four helices that form a "right-turn" elongated bundle; the globular fold is mainly maintained by hydrophobic interactions. The bound peptide further stabilizes the structure. Certain signaling events such as phosphorylation and molecule interplay may induce opening of the helix bundle. Such conformational change is proposed to precede departure of FAK from focal adhesions, which starts focal adhesion turnover.     

Focal adhesion regulation of cell behavior

Focal adhesions lie at the convergence of integrin adhesion, signaling and the actin cytoskeleton. Cells modify focal adhesions in response to changes in the molecular composition, two-dimensional (2D) vs. three-dimensional (3D) structure, and physical forces present in their extracellular matrix environment. We consider here how cells use focal adhesions to regulate signaling complexes and integrin function. Furthermore, we examine how this regulation controls complex cellular behaviors in response to matrices of diverse physical and biochemical properties. One event regulated by the physical structure of the ECM is phosphorylation of focal adhesion kinase (FAK) at Y397, which couples FAK to several signaling pathways that regulate cell proliferation, survival, migration, and invasion.     

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.     

Direct interaction of focal adhesion kinase with p190RhoGEF

Focal adhesion kinase (FAK) is a protein-tyrosine kinase that associates with multiple cell surface receptors and signaling proteins through which it can modulate the activity of several intracellular signaling pathways. FAK activity can influence the formation of distinct actin cytoskeletal structures such as lamellipodia and stress fibers in part through effects on small Rho GTPases, although the molecular interconnections of these events are not well defined. Here, we report that FAK interacts with p190RhoGEF, a RhoA-specific GDP/GTP exchange factor, in neuronal cells and in brain tissue extracts by co-immunoprecipitation and co-localization analyses. Using a two-hybrid assay and deletion mutagenesis, the binding site of the FAK C-terminal focal adhesion targeting (FAT) domain was identified within the C-terminal coiled-coil domain of p190RhoGEF. Binding was independent of a LD-like binding motif within p190RhoGEF, yet FAK association was disrupted by a mutation (Leu-1034 to Ser) that weakens the helical bundle structure of the FAK FAT domain. Neuro-2a cell binding to laminin increased endogenous FAK and p190RhoGEF tyrosine phosphorylation, and co-transfection of a dominant-negative inhibitor of FAK activity, termed FRNK, inhibited lamininstimulated p190RhoGEF tyrosine phosphorylation and p21 RhoA GTP binding. Overexpression of FAK in Neuro-2a cells increased both endogenous p190RhoGEF tyrosine phosphorylation and RhoA activity, whereas these events were inhibited by FRNK co-expression. Because insulin-like growth factor 1 treatment of Neuro-2a cells increased FAK tyrosine phosphorylation and enhanced p190RhoGEF-mediated activation of RhoA, our results support the conclusion that FAK association with p190RhoGEF functions as a signaling pathway downstream of integrins and growth factor receptors to stimulate Rho activity.     

Focal adhesion kinase is a component of antiviral RIG-I-like receptor signaling

Viruses modulate the actin cytoskeleton at almost every step of their cellular journey from entry to?egress. Cellular sensing of these cytoskeletal changes may function in the recognition of viral infection. Here we show that focal adhesion kinase (FAK), a focal adhesion localized tyrosine kinase that transmits signals between the extracellular matrix and the cytoplasm, serves as a RIG-I-like receptor antiviral signaling component by directing mitochondrial antiviral signaling adaptor (MAVS) activation. Cells deficient in FAK are highly susceptible to RNA virus?infection and attenuated in antiviral signaling. We show that FAK interacts with MAVS at the mitochondrial membrane in a virus infection-dependent manner and potentiates MAVS-mediated signaling via a kinase-independent mechanism. A cysteine protease encoded by enteroviruses cleaves FAK to suppress its role in innate immune signaling. These findings suggest that FAK serves as a link between cytoskeletal perturbations that occur during virus infection and activation of innate immune signaling.     

Giant lens, a gene involved in cell determination and axon guidance in the visual system of Drosophila melanogaster.

Mutations in the Drosophila gene giant lens (gil) affect ommatidial development, photoreceptor axon guidance and optic lobe development. We have cloned the gene using an enhancer trap line. Molecular analysis of gil suggests that it encodes a secreted protein with an epidermal-growth-factor-like motif. We have generated mutations at the gil locus by imprecise excision of the enhancer trap P-element. In the absence of gil, additional photoreceptors develop at the expense of pigment cells, suggesting an involvement of gil in cell determination during eye development. In addition, gil mutants show drastic effects on photoreceptor axon guidance and optic lobe development. In wildtype flies, photoreceptor axons grow from the eye disc through the optic stalk into the larval brain hemisphere, where retinal innervation is required for the normal development of the lamina and distal medulla. The projection pattern of these axons in the developing lamina and medulla is highly regular and reproducible. In gil, photoreceptor axons enter the larval brain but fail to establish proper connections in the lamina or medulla. We propose that gil encodes a new type of signalling molecule involved in the process of axon pathfinding and cell determination in the visual system of Drosophila.     

Regulation of stress granule dynamics by Grb7 and FAK signalling pathway

Cells form stress granules (SGs) in response to environmental stresses, which constitute cytoplasmic domains where mRNAs are stored and translation is halted. Although several components are found in SGs, it is poorly understood as to how SGs are formed and dissolved. We identified growth factor receptor-bound protein 7 (Grb7), an RNA-binding, translational regulator, as an integral component of SGs, which directly interacts with Hu antigen R (HuR) and is required for cells to form SGs. When stress is terminated, Grb7 is hyperphosphorylated by focal adhesion kinase (FAK), loses its ability to directly interact with HuR and is dissociated from SG components, thereby disrupting SGs in recovering cells. Consistently, dominant-negative hypophospho mutants of FAK and Grb7 significantly attenuate SG disassembly during recovery. FAK activation followed by its phosphorylating Grb7 constitutes a cell-autonomous signalling pathway that regulates the disassembly of SGs and translational stimulation during recovery. This is the first reported pathway actively regulating the dynamics of SGs.     

Signal-transducing adaptor protein-2 regulates integrin-mediated T cell adhesion through protein degradation of focal adhesion kinase

Signal-transducing adaptor protein-2 (STAP-2) is a recently identified adaptor protein that contains pleckstrin homology- and Src homology 2-like domains as well as a YXXQ motif in its C-terminal region. Our previous studies demonstrated that STAP-2 binds to STAT3 and STAT5, and regulates their signaling pathways. In the present study, we find that STAP-2-deficient splenocytes or T cells exhibit enhanced cell adhesion to fibronectin after PMA treatment, and that STAP-2-deficient T cells contain the increased protein contents of focal adhesion kinase (FAK). Furthermore, overexpression of STAP-2 induces a dramatic decrease in the protein contents of FAK and integrin-mediated T cell adhesion to fibronectin in Jurkat T cells via the degradation of FAK. Regarding the mechanism for this effect, we found that STAP-2 associates with FAK and enhances its degradation, proteasome inhibitors block FAK degradation, and STAP-2 recruits an endogenous E3 ubiquitin ligase, Cbl, to FAK. These results reveal a novel regulation mechanism for integrin-mediated signaling in T cells via STAP-2, which directly interacts with and degrades FAK.     

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.     

Knock-in Mutation Reveals an Essential Role for Focal Adhesion Kinase Activity in Blood Vessel Morphogenesis and Cell Motility-Polarity but Not Cell Proliferation

Focal adhesion kinase (FAK) associates with both integrins and growth factor receptors in the control of cell motility and survival. Loss of FAK during mouse development results in lethality at embryonic day 8.5 (E8.5) and a block in cell proliferation. Because FAK serves as both a scaffold and signaling protein, gene knock-outs do not provide mechanistic insights in distinguishing between these modes of FAK function. To determine the role of FAK activity during development, a knock-in point mutation (lysine 454 to arginine (R454)) within the catalytic domain was introduced by homologous recombination. Homozygous FAK^R454/R454 mutation was lethal at E9.5 with defects in blood vessel formation as determined by lack of yolk sac primary capillary plexus formation and disorganized endothelial cell patterning in FAK^R454/R454 embryos. In contrast to the inability of embryonic FAK^−/− cells to proliferate ex vivo, primary FAK^R454/R454 mouse embryo fibroblasts (MEFs) were established from E8.5 embryos. R454 MEFs exhibited no difference in cell growth compared with normal MEFs, and R454 FAK localized to focal adhesions but was not phosphorylated at Tyr-397. In E8.5 embryos and primary MEFs, FAK R454 mutation resulted in decreased c-Src Tyr-416 phosphorylation. R454 MEFs exhibited enhanced focal adhesion formation, decreased migration, and defects in cell polarity. Within immortalized MEFs, FAK activity was required for fibronectin-stimulated FAK-p190RhoGAP association and p190RhoGAP tyrosine phosphorylation linked to decreased RhoA GTPase activity, focal adhesion turnover, and directional motility. Our results establish that intrinsic FAK activity is essential for developmental processes controlling blood vessel formation and cell motility-polarity but not cell proliferation. This work supports the use of FAK inhibitors to disrupt neovascularization.     

Perturbing Nuclear Transport in Drosophila Eye Imaginal Discs Causes Specific Cell Adhesion and Axon Guidance Defects

To study nucleocytoplasmic transport during multicellular development, we developed a sensitive nuclear protein import assay in living blastoderm embryos. We show that dominant negative truncations of the human nuclear transport receptor karyopherinbeta/Importinbeta (DNImpbeta) disrupt mRNA export and protein import in Drosophila. To test the sensitivity of different developmental processes to nuclear trafficking perturbations, we expressed DNImpbeta behind the morphogenetic furrow of the eye disc, at a time when photoreceptors are patterned and project their axons to the brain. DNImpbeta expression does not disrupt the correct specification of different photoreceptors, but causes a defect in cell adhesion that leads to some photoreceptors descending below the layer of ommatidia. The photoreceptors initially project their axons correctly to the posterior, but later their axons are unable to enter the optic stalk en route to the brain and continue to project an extensive network of misguided axons. The axon guidance and cell adhesion defects are both due to a disruption in the function of Ketel, the Drosophila ortholog of Importinbeta. We conclude that cell adhesion and axon guidance in the eye have specific requirements for nucleocytoplasmic transport, despite involving processes that occur primarily at the cell surface.     

Cytoskeletal reorganization dependence of signaling by the gonadotropin-releasing hormone receptor

Activation of classical G protein-coupled receptors (GPCRs) like the mammalian gonadotropin-releasing hormone receptor (GnRHR) typically stimulates heterotrimeric G protein molecules that subsequently activate downstream effectors. Receptor activation of heterotrimeric G protein pathways primarily controls intermediary cell metabolism by elevation or diminution of soluble cytoplasmic second messenger molecules. We have demonstrated here that stimulation of the GnRHR also results in a dramatic change in both cell adhesion and superstructural morphology. Gonadotropin-releasing hormone (GnRH) receptor activation rapidly increases the capacity of HEK293 cells expressing the GnRHR to remain matrix-adherent in the face of fluid insults. Coinciding with this profound elevation in matrix adherence, we demonstrated a GnRH-induced alteration in both cell morphology and the de novo generation of polymerized actin structures. GnRH induction of cytoskeletal remodeling was correlated with significant increases in the tyrosine phosphorylation status of a series of cytoskeletal associated proteins, e.g. focal adhesion kinase (FAK), c-Src, and microtubule-associated protein kinase (MAPK or ERK1/2). The activation of the distal downstream effector ERK1/2 was demonstrated to be sensitive to the disrupters of cytoskeletal rearrangement, cytochalasin D and latrunculin B. In addition to the sensitivity of ERKs to cytoskeletal integrity, GnRH-induced FAK and c-Src kinase activation were sensitive to these agents and the fibronectin-integrin antagonistic RGDS peptide. Activation of ERK was dependent on its protein-protein assembly with FAK and c-Src at focal adhesion complexes. Induction of the cell remodeling event leading to this signaling complex assembly occurred primarily via GnRHR activation of the monomeric G protein Rac but not RhoA. These findings demonstrated a clear divergence of GnRHR signaling via the Rac monomeric G protein focal adhesion signaling complex assembly and cytoskeletal remodeling independent of the classical heterotrimeric G protein-controlled phospholipase C-beta pathway.     

De novo expression of pp125FAK in human macrophages regulates CSK distribution and MAP kinase activation but does not affect focal contact structure

The protein tyrosine kinase pp125FAK (focal adhesion kinase, or FAK) is expressed by a variety of cell types and has been implicated in integrin-mediated signaling events. We explored the potential functions of FAK by expressing it de novo in a cell type lacking FAK. We showed previously that cultured human macrophages lack FAK yet still have well-formed focal contacts. Adenovirus-mediated expression of FAK results in the appearance of FAK protein, which localizes to focal contacts and becomes tyrosine-phosphorylated without perturbing overall cell morphology or focal contacts. FAK associates with CSK 48 h after infection and recruits it to focal contacts. Tyrosine phosphorylation of p130cas but not of paxillin is stimulated after FAK expression. The phosphorylation of p130cas is lost at 48 h in parallel with CSK accumulation in focal contacts. The ERK2 form of MAP kinase is similarly activated at 12-24 h, but it also returns to low levels at 48 h. These findings demonstrate that FAK can be reconstituted to focal contacts in cells that lack it without affecting cell morphology or focal contact structure. FAK can regulate the distribution and activities of elements of the MAP kinase signaling pathway.     

Bcl-2 induces cyclin D1 promoter activity in human breast epithelial cells independent of cell anchorage

Cyclin D1 expression is co-regulated by growth factor and cell adhesion signaling. Cell adhesion to the extracellular matrix activates focal adhesion kinase (FAK), which is essential for cyclin D1 expression. Upon the loss of cell adhesion, cyclin D1 expression is downregulated, followed by apoptosis in normal epithelial cells. Since bcl-2 prevents apoptosis induced by the loss of cell adhesion, we hypothesized that bcl-2 induces survival signaling complementary to cell adhesion-mediated gene regulation. In the present study, we investigated the role of bcl-2 on FAK activity and cyclin D1 expression. We found that bcl-2 overexpression induces cyclin D1 expression in human breast epithelial cell line MCF10A independent of cell anchorage. Increased cyclin D1 expression in stable bcl-2 transfectants is not related to bcl-2-increased G1 duration, but results from cyclin D1 promoter activation. Transient transfection studies confirmed anchorage-independent bcl-2 induction of cyclin D1 promoter activity in human breast epithelial cell lines (MCF10A, BT549, and MCF-7). We provide evidence that bcl-2 induction of cyclin D1 expression involves constitutive activation of focal adhesion kinase, regardless of cell adhesion. The present study suggests a potential oncogenic activity for bcl-2 through cyclin D1 induction, and provides an insight into the distinct proliferation-independent pathway leading to increased cyclin D1 expression in breast cancer.     

Differential regulation of cell migration and cell cycle progression by FAK complexes with Src, PI3K, Grb7 and Grb2 in focal contacts.

Focal adhesion kinase (FAK) is a key mediator of integrin signaling, which has been implicated in the regulation of cell migration and cell cycle progression. Using chimeric molecules that fuse the focal adhesion targeting (FAT) sequence directly to several signaling molecules, we investigated the potential role of FAK recruitments of signaling molecules to focal contacts in the regulation of cell migration and cell cycle progression. We found that fusion of FAT to Src, the p85 subunit of phosphatidylinositol 3-kinase, Grb7 and Grb2 resulted in the efficient focal adhesion targeting of these signaling molecules. We showed that expression of Src-FAT, p85-FAT, or Grb7-FAT, but not Grb2-FAT, each stimulated cell migration. Interestingly, tyrosine phosphorylation of paxillin, but not p130cas, was induced by expression of Src-FAT, suggesting a potential role of paxillin in mediating stimulation of cell migration by the chimeric molecule. In contrast, targeting of Grb2, but not Src, p85, or Grb7, to focal contacts increased cell cycle progression. Biochemical analyses correlated Erk activation by Grb2-FAT with its stimulation of cell cycle progression. Together, these results suggest that at least part of the role of FAK interaction with these signaling molecules is to recruit them to focal contacts and that distinct FAK signaling complexes are involved in the regulation of cell migration vs. cell cycle progression.