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.     

Cellular characterization of a novel focal adhesion kinase inhibitor

Focal adhesion kinase (FAK) is a member of a family of non-receptor protein-tyrosine kinases that regulates integrin and growth factor signaling pathways involved in cell migration, proliferation, and survival. FAK expression is increased in many cancers, including breast and prostate cancer. Here we describe perturbation of adhesion-mediated signaling with a FAK inhibitor, PF-573,228. In vitro, this compound inhibited purified recombinant catalytic fragment of FAK with an IC(50) of 4 nM. In cultured cells, PF-573,228 inhibited FAK phosphorylation on Tyr(397) with an IC(50) of 30-100 nM. Treatment of cells with concentrations of PF-573,228 that significantly decreased FAK Tyr(397) phosphorylation failed to inhibit cell growth or induce apoptosis. In contrast, treatment with PF-573,228 inhibited both chemotactic and haptotactic migration concomitant with the inhibition of focal adhesion turnover. These studies show that PF-573,228 serves as a useful tool to dissect the functions of FAK in integrin-dependent signaling pathways in normal and cancer cells and forms the basis for the generation of compounds amenable for preclinical and patient trials.     

Identification of a novel nuclear-localized adenylate kinase from Drosophila melanogaster

As a step to further understand the role of adenylate kinase (AK) in the energy metabolism network, we identified, purified, and characterized a previously undescribed adenylate kinase in Drosophila melanogaster. The cDNA encodes a 175-amino acid protein, which shows 47.85% identity in 163 amino acids to human AK6. The recombinant protein was successfully expressed in Escherichia coli BL21(DE3) strain. Characterization of this protein by enzyme activity assay showed adenylate kinase activity. AMP and CMP were the preferred substrates, and UMP can also be phosphorylated to some extent, with ATP as the best phosphate donor. Subcellular localization study showed a predominantly nuclear localization. Therefore, based on the substrate specificity, the specific nuclear localization in the cell, and the sequence similarity with human AK6, we named this novel adenylate kinase identified from the fly DAK6.     

Microtubule-induced focal adhesion disassembly is mediated by dynamin and focal adhesion kinase

Imaging studies implicate microtubule targeting of focal adhesions in focal adhesion disassembly, although the molecular mechanism is unknown. Here, we develop a model system of focal adhesion disassembly based on the finding that microtubule regrowth after nocodazole washout induces disassembly of focal adhesions, and that this disassembly occurs independently of Rho and Rac, but depends on focal adhesion kinase (FAK) and dynamin. During disassembly, dynamin interacts with FAK and colocalizes with focal adhesions. Inhibition of dynamin prevents migration of cells with a focal adhesion phenotype. Our results show that focal adhesion disassembly involves microtubules, dynamin and FAK, and is not simply the reversal of focal adhesion formation.     

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.     

Regulation of focal adhesion kinase by a novel protein inhibitor FIP200

Focal adhesion kinase (FAK) is a major mediator of integrin signaling pathways. The mechanisms of regulation of FAK activity and its associated cellular functions are not very well understood. Here, we present data suggesting that a novel protein FIP200 functions as an inhibitor for FAK. We show the association of endogenous FIP200 with FAK, which is decreased upon integrin-mediated cell adhesion concomitant with FAK activation. In vitro- and in vivo-binding studies indicate that FIP200 interacts with FAK through multiple domains directly. FIP200 bound to the kinase domain of FAK inhibited its kinase activity in vitro and its autophosphorylation in vivo. Overexpression of FIP200 or its segments inhibited cell spreading, cell migration, and cell cycle progression, which correlated with their inhibition of FAK activity in vivo. The inhibition of these cellular functions by FIP200 could be rescued by coexpression of FAK. Last, we show that disruption of the functional interaction between endogenous FIP200 with FAK leads to increased FAK phosphorylation and partial restoration of cell cycle progression in cells plated on poly-L-lysine, providing further support for FIP200 as a negative regulator of FAK. Together, these results identify FIP200 as a novel protein inhibitor for FAK.     

TRAF6 is a novel regulator of Notch signaling in Drosophila melanogaster

Notch signaling pathway unravels a fundamental cellular communication system that plays an elemental role in development. It is evident from different studies that the outcome of Notch signaling depends on signal strength, timing, cell type, and cellular context. Since Notch signaling affects a spectrum of cellular activity at various developmental stages by reorganizing itself in more than one way to produce different intensities in the signaling output, it is important to understand the context dependent complexity of Notch signaling and different routes of its regulation. We identified, TRAF6 (Drosophila homolog of mammalian TRAF6) as an interacting partner of Notch intracellular domain (Notch-ICD). TRAF6 genetically interacts with Notch pathway components in trans-heterozygous combinations. Immunocytochemical analysis shows that TRAF6 co-localizes with Notch in Drosophila third instar larval tissues. Our genetic interaction data suggests that the loss-of-function of TRAF6 leads to the rescue of previously identified Kurtz–Deltex mediated wing notching phenotype and enhances Notch protein survival. Co-expression of TRAF6 and Deltex results in depletion of Notch in the larval wing discs and down-regulates Notch targets, Wingless and Cut. Taken together, our results suggest that TRAF6 may function as a negative regulator of Notch signaling.     

The focal adhesion targeting (FAT) region of focal adhesion kinase is a four-helix bundle that binds paxillin.

Focal adhesion kinase (FAK) is a tyrosine kinase found in focal adhesions, intracellular signaling complexes that are formed following engagement of the extracellular matrix by integrins. The C-terminal 'focal adhesion targeting' (FAT) region is necessary and sufficient for localizing FAK to focal adhesions. We have determined the crystal structure of FAT and show that it forms a four-helix bundle that resembles those found in two other proteins involved in cell adhesion, alpha-catenin and vinculin. The binding of FAT to the focal adhesion protein, paxillin, requires the integrity of the helical bundle, whereas binding to another focal adhesion protein, talin, does not. We show by mutagenesis that paxillin binding involves two hydrophobic patches on opposite faces of the bundle and propose a model in which two LD motifs of paxillin adopt amphipathic helices that augment the hydrophobic core of FAT, creating a six-helix bundle.     

Characterization of a novel Drosophila melanogaster acylphosphatase

Analysis of the Drosophila melanogaster EST database led to the characterization of a novel acylphosphatase (AcPDro2). This is coded by the CG18505 (Acyp2) gene and is clearly distinct from a previously described AcPDro coded by the CG16870 (Acyp) gene from D. melanogaster. The two proteins show a 60% homology with both vertebrate isoenzymes. All the residues involved in the catalytic mechanism are conserved. AcPDro2 is a stable enzyme with a correct globular folded structure. Its activity on benzoylphosphate shows higher K(cat) but lower K(m) with respect to AcPDro. It is possible that AcPDro and AcPDro2 genes are not the direct ancestor of MT and CT vertebrate isoenzymes.     

Signaling through focal adhesion kinase

Focal adhesion kinase (FAK) is a nonreceptor protein-tyrosine kinase implicated in controlling cellular responses to the engagement of cell-surface integrins, including cell spreading and migration, survival and proliferation. Aberrant FAK signaling may contribute to the process of cell transformation by certain oncoproteins, including v-Src. Progress toward elucidating the events leading to FAK activation following integrin-mediated cell adhesion, as well as events downstream of FAK, has come through the identification of FAK phosphorylation sites and interacting proteins. A signaling partnership is formed between FAK and Src-family kinases, leading to tyrosine phosphorylation of FAK and associated ‘docking’ proteins Cas and paxillin. Subsequent recruitment of proteins containing Src homology 2 domains, including Grb2 and c-Crk, to the complex is likely to trigger adhesion-induced cellular responses, including changes to the actin cytoskeleton and activation of the Ras-MAP kinase pathway.     

Paxillin binding is not the sole determinant of focal adhesion localization or dominant-negative activity of focal adhesion kinase/focal adhesion kinase-related nonkinase

The carboxy-terminal 150 residues of the focal adhesion kinase (FAK) comprise the focal adhesion-targeting sequence, which is responsible for its subcellular localization. The mechanism of focal adhesion targeting has not been fully elucidated. We describe a mutational analysis of the focal adhesion-targeting sequence of FAK to further examine the mechanism of focal adhesion targeting and explore additional functions encoded by the carboxy-terminus of FAK. The results demonstrate that paxillin binding is dispensable for focal adhesion targeting of FAK. Cell adhesion-dependent tyrosine phosphorylation strictly correlated with the ability of mutants to target to focal adhesions. Focal adhesion targeting was also a requirement for maximal FAK-dependent tyrosine phosphorylation of paxillin and FAK-related nonkinase (FRNK)-dependent inhibition of endogenous FAK function. However, there were additional requirements for these latter functions because we identified mutants that target to focal adhesions, yet are defective for the induction of paxillin phosphorylation or the dominant-negative function of FRNK. Furthermore, the paxillin-binding activity of FRNK mutants did not correlate with their ability to inhibit FAK, suggesting that FRNK has other targets in addition to paxillin.     

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.     

DWnt4 regulates cell movement and focal adhesion kinase during Drosophila ovarian morphogenesis

Cell motility is regulated by extracellular cues and by intracellular factors that accumulate at sites of contact between cells and the extracellular matrix. One of these factors, focal adhesion kinase (FAK), regulates the cycle of focal adhesion formation and disassembly that is required for cell movement to occur. Recently, Wnt signaling has also been implicated in the control of cell movement in vertebrates, but the mechanism through which Wnt proteins influence motility is unclear. We demonstrate that Drosphila Wnt4 is required for cell movement and FAK regulation during ovarian morphogenesis. Dfrizzled2, Disheveled, and protein kinase C are also required. The DWnt4 cell motility pathway is distinct from both the canonical Wnt pathway and the planar polarity pathway. Our data suggest that DWnt4 facilitates motility through regulation of focal adhesions.     

Copper deficiency induced emphysema is associated with focal adhesion kinase inactivation

Background

Copper is an important regulator of hypoxia inducible factor 1 alpha (HIF-1α) dependent vascular endothelial growth factor (VEGF) expression, and is also required for the activity of lysyl oxidase (LOX) to effect matrix protein cross-linking. Cell detachment from the extracellular matrix can induce apoptosis (anoikis) via inactivation of focal adhesion kinase (FAK).

Methodology

To examine the molecular mechanisms whereby copper depletion causes the destruction of the normal alveolar architecture via anoikis, Male Sprague-Dawley rats were fed a copper deficient diet for 6 weeks while being treated with the copper chelator, tetrathiomolybdate. Other groups of rats were treated with the inhibitor of auto-phosphorylation of FAK, 1,2,4,5-benzenetetraamine tetrahydrochloride (1,2,4,5-BT) or FAK small interfering RNA (siRNA).

Principal Findings

Copper depletion caused emphysematous changes, decreased HIF-1α activity, and downregulated VEGF expression in the rat lungs. Cleaved caspase-3, caspase-8 and Bcl-2 interacting mediator of cell death (Bim) expression was increased, and the phosphorylation of FAK was decreased in copper depleted rat lungs. Administration of 1,2,4,5-BT and FAK siRNA caused emphysematous lung destruction associated with increased expression of cleaved capase-3, caspase-8 and Bim.

Conclusions

These data indicate that copper-dependent mechanisms contribute to the pathogenesis of emphysema, which may be associated with decreased HIF-1α and FAK activity in the lung.     

In vitro phosphorylation of the focal adhesion targeting domain of focal adhesion kinase by Src kinase

Focal adhesion kinase (FAK), a key regulator of cell adhesion and migration, is overexpressed in many types of cancer. The C-terminal focal adhesion targeting (FAT) domain of FAK is necessary for proper localization of FAK to focal adhesions and subsequent activation. Phosphorylation of Y926 in the FAT domain by the tyrosine kinase Src has been shown to promote metastasis and invasion in vivo by linking the FAT domain to the MAPK pathway via its interaction with growth factor receptor-bound protein 2. Several groups have reported that inherent conformational dynamics in the FAT domain likely regulate phosphorylation of Y926; however, what regulates these dynamics is unknown. In this paper, we demonstrate that there are two sites of in vitro Src-mediated phosphorylation in the FAT domain: Y926, which has been shown to affect FAK function in vivo, and Y1008, which has no known biological role. The phosphorylation of these two tyrosine residues is pH-dependent, but this does not reflect the pH dependence of Src kinase activity. Circular dichroism and nuclear magnetic resonance data indicate that the stability and conformational dynamics of the FAT domain are sensitive to changes in pH over a physiological pH range. In particular, regions of the FAT domain previously shown to regulate phosphorylation of Y926 as well as regions near Y1008 show pH-dependent dynamics on the microsecond to millisecond time scale.     

R-Ras promotes focal adhesion formation through focal adhesion kinase and p130(Cas) by a novel mechanism that differs from integrins

R-Ras regulates integrin function, but its effects on integrin signaling pathways have not been well described. We demonstrate that activation of R-Ras promoted focal adhesion formation and altered localization of the alpha2beta1 integrin from cell-cell to cell-matrix adhesions in breast epithelial cells. Constitutively activated R-Ras(38V) dramatically enhanced focal adhesion kinase (FAK) and p130(Cas) phosphorylation upon collagen stimulation or clustering of the alpha2beta1 integrin, even in the absence of increased ligand binding. Signaling events downstream of R-Ras differed from integrins and K-Ras, since pharmacological inhibition of Src or disruption of actin inhibited integrin-mediated FAK and p130(Cas) phosphorylation, focal adhesion formation, and migration in control and K-Ras(12V)-expressing cells but had minimal effect in cells expressing R-Ras(38V). Therefore, signaling from R-Ras to FAK and p130(Cas) has a component that is Src independent and not through classic integrin signaling pathways and a component that is Src dependent. R-Ras effector domain mutants and pharmacological inhibition suggest a partial role for phosphatidylinositol 3-kinase (PI3K), but not Raf, in R-Ras signaling to FAK and p130(Cas). However, PI3K cannot account for the Src-independent pathway, since simultaneous inhibition of both PI3K and Src did not completely block effects of R-Ras on FAK phosphorylation. Our results suggest that R-Ras promotes focal adhesion formation by signaling to FAK and p130(Cas) through a novel mechanism that differs from but synergizes with the alpha2beta1 integrin.     

Activation of pyk2/related focal adhesion tyrosine kinase and focal adhesion kinase in cardiac remodeling

Cellular remodeling during progression of dilation involves focal adhesion contact reorganization. However, the signaling mechanisms and structural consequences leading to impaired cardiomyocyte adhesion are poorly defined. These events were studied in tropomodulin-overexpressing transgenic mice that develop dilated cardiomyopathy associated with chronic elevation of intracellular calcium. Analysis of tropomodulin-overexpressing transgenic hearts by immunoblot and confocal microscopy revealed activation and redistribution of signaling molecules known to regulate adhesion. Calcium-dependent pyk2/related focal adhesion tyrosine kinase (RAFTK) showed changes in expression and phosphorylation state, similar to changes observed for a related downstream target molecule of pyk2/RAFTK termed focal adhesion kinase. Paxillin, the target substrate molecule for focal adhesion kinase phosphorylation, was redistributed in tropomodulin-overexpressing transgenic hearts with enhanced paxillin phosphorylation and cleavage. Certain aspects of the in vivo signaling phenotype including increased paxillin phosphorylation could be recapitulated in vitro using neonatal rat cardiomyocytes infected with recombinant adenovirus to overexpress tropomodulin. In addition, increasing intracellular calcium levels with ionomycin induced pyk2/RAFTK phosphorylation, and adenovirally mediated expression of wild-type pyk2/RAFTK resulted in increased phospho-pyk2/RAFTK levels and concomitant paxillin phosphorylation. Collectively, these results delineate a cardiomyocyte signaling pathway associated with dilation that has potential relevance for cardiac remodeling, focal adhesion reorganization, and loss of contractility.     

VEGF-induced activation of phosphoinositide 3-kinase is dependent on focal adhesion kinase

Vascular endothelial growth factor (VEGF)-A stimulates formation of new blood vessels (angiogenesis). This process includes migration of endothelial cells from the preexisting vessel toward the source of the growth factor. We show that VEGF-A-induced migration of porcine aortic endothelial cells expressing VEGF receptor-2 (VEGFR-2) is dependent on activation of phosphoinositide 3-kinase (PI3-kinase). There is no direct interaction between VEGF receptor-2 and PI3-kinase; instead PI3-kinase is activated downstream of focal adhesion kinase (FAK) in VEGF-A-stimulated cells. Thus, VEGF-A stimulation leads to complex formation between FAK and PI3-kinase and overexpression of dominant-negative FAK decreases VEGF-A-induced PI3-kinase activation. FAK activation by VEGF-A increases with increasing concentration of growth factor, without apparent collapse of the cytoskeleton, in contrast to the effect of platelet-derived growth factor. FAK activation is mediated via the C-terminal tail of VEGFR-2 and loss of VEGF-A-induced FAK activation in cells expressing mutant VEGFR-2 correlates with loss of migration capacity. These data show that VEGF-A-induced FAK and PI3-kinase activation are required for migration of cells expressing VEGFR-2, via a pathway independent of direct interaction with the receptor.