SEARCH · PubMed Health

Results for “Focal Adhesion”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 145 records · Page 8Linked to original sources

PubMed1999

The interaction of activated integrin lymphocyte function-associated antigen 1 with ligand intercellular adhesion molecule 1 induces activation and redistribution of focal adhesion kinase and proline-rich tyrosine kinase 2 in T lymphocytes.

Integrin receptors play a central role in the biology of lymphocytes, mediating crucial functional aspects of these cells, including adhesion, activation, polarization, migration, and signaling. Here we report that induction of activation of the beta2-integrin lymphocyte function-associated antigen 1 (LFA-1) in T lymphocytes with divalent cations, phorbol esters, or stimulatory antibodies is followed by a dramatic polarization, resulting in a characteristic elongated morphology of the cells and the arrest of migrating lymphoblasts. This cellular polarization was prevented by treatment of cells with the specific tyrosine kinase inhibitor genistein. Furthermore, the interaction of the activated integrin LFA-1 with its ligand intercellular adhesion molecule 1 induced the activation of the cytoplasmic tyrosine kinases focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2 (PYK-2). FAK activation reached a maximum after 45 min of stimulation; in contrast, PYK-2 activation peaked at 30 min, declining after 60 min. Upon polarization of lymphoblasts, FAK and PYK-2 redistributed from a diffuse localization in the cytoplasm to a region close to the microtubule-organizing center in these cells. FAK and PYK-2 activation was blocked when lymphoblasts were pretreated with actin and tubulin cytoskeleton-interfering agents, indicating its cytoskeletal dependence. Our results demonstrate that interaction of the beta2-integrin LFA-1 with its ligand intercellular adhesion molecule 1 induces remodeling of T lymphocyte morphology and activation and redistribution of the cytoplasmic tyrosine kinases FAK and PYK-2.

Actins↗

PubMed1999

Beta3-integrin-mediated focal adhesion complex formation: adult cardiocytes embedded in three-dimensional polymer matrices.

In vivo studies show that beta3-integrin-mediated focal adhesion formation (FAF) causes recruitment of nonreceptor tyrosine kinases to the cytoskeleton in pressure-overloaded myocardium. To define the mechanism of beta3-integrin-mediated signaling, we developed a cell culture model (adult feline cardiocytes embedded in a 3-dimensional matrix of native type 1 collagen, fibronectin, and vitronectin) wherein beta3-integrin-mediated focal adhesion kinase occurs. Focal adhesion kinase was analyzed immunocytochemically using confocal microscopy. Initial studies suggested that cardiocytes cultured in a 3-dimensional matrix formed focal adhesions consisting of both beta3-integrin and the muscle-specific isoform, beta1-integrin (beta1D). The focal adhesions were associated with focal adhesion kinase on both costameres and intercalated disks. To determine the cause of beta1D-integrin-mediated focal adhesion kinase in this model, time course studies were done. Beta3-integrin-mediated focal adhesion kinase occurred within 30 minutes after embedding cardiocytes and persisted for >24 hours, whereas beta1D-integrin-mediated focal adhesion kinase was present from the outset. Because confocal microscopy showed that laminin was present on the surface of freshly isolated cardiocytes, we hypothesized that this was causative of beta1D-integrin-mediated focal adhesion kinase. Freshly isolated cardiocytes washed with acidic medium (2 minutes, pH 3.0) to remove laminin and then embedded in a 3-dimensional matrix showed complete absence of beta1D-integrin-mediated focal adhesion kinase, but beta3-integrin-mediated focal adhesion kinase occurred with a time course similar to that seen in cultured, unwashed cardiocytes. Acid washing did not alter the binding ability of beta1D-integrin, because acid-washed cardiocytes in the presence of laminin showed beta1D-integrin-mediated focal adhesion kinase. Thus, cardiocytes embedded in a 3-dimensional matrix show beta3-integrin-mediated focal adhesion kinase and provide an in vitro model to study beta3-integrin-mediated signaling in response to hemodynamic cardiac loading.

Animals↗

PubMed2004

Reactive oxygen species increased focal adhesion kinase production in pulmonary microvascular endothelial cells.

Focal adhesion kinase (FAK) is a 125 kDa protein tyrosine kinase (PTK) associated with focal adhesion in many cells, which plays a major role in the integrity of cytoskeletal structure. Reactive oxygen species produced during ischemia and reperfusion injury has been found to be an important mediator of signal transduction process. We found that low dose H2O2 induced increased FAK production in pulmonary microvascular endothelial cells, which could be blocked by cycloheximide (CHX), a protein synthesis inhibitor. Pulmonary endothelial cells were cultured on DMEM medium till 100% confluent. H2O2 was added at 100 uM for 30 min. The cells were collected and lysed, then immuno-blotted with anti-FAK antibody. After 30 min treatment, we found a 30%+/-6% (N=5) increase of FAK in H2O2 treated endothelial cells. This increase could be blocked by pretreatment of cells with CHX at 5 ug/ml for 60 min. In both groups, increased phosphorylation of ERK was observed. Immuno-fluorescence revealed increased staining of FAK in the peri-nuclear region of the H2O2 treated endothelial cells. These findings suggest that H2O2 activated MAP kinase pathway leading to increased FAK production at the protein level. FAK is a 125 kDa PTK associated with focal adhesion in many cells, and it plays a major role in the integrity of cytoskeletal structure. FAK is discretely localized to focal adhesions via its C-terminal focal adhesion-targeting (FAT) sequence. FAK is regulated by integrin-dependent cell adhesion and can control tyrosine phosphorylation of downstream substrates, like paxillin. The reactive oxygen species produced during ischemia and reperfusion injury has been found to be an important mediator of the signal transduction process. Although the signaling pathways leading to hydrogen peroxide induced endothelial monolayer permeability remain ambiguous, cytoskeletal proteins are known to be essential for maintaining endothelial integrity and regulating solute flux through the monolayer. Recent evidence has shown that H2O2 stimulates cytoskeleton reorganization, cell growth/proliferation, and DNA synthesis in various cells. In our previous study, we found a significantly increased amount of FAK in endothelial cells treated with low doses of H2O2. Mitogen-activated protein (MAP) kinases are a group of 30- to 110-kDa serine/threonine kinases. MAPKs belong to the group of kinases that are rapidly activated in response to growth factor stimulation. This family of MAPKs includes ERK, and ERK2. The activated MAPK can translocate to the nucleus where it can regulate transcription factors. Activation of p44 and p42 extracellular signal-regulated protein kinases (ERK1 and ERK2) is an important step in the cascade leading to cell growth and proliferation. In order to determine the mechanism of increased FAK production, we investigated the relationship of FAK production and ERK activation.

Animals↗

PubMed1995

SPARC mediates focal adhesion disassembly in endothelial cells through a follistatin-like region and the Ca(2+)-binding EF-hand.

SPARC is a one of a group of extracellular matrix proteins that regulate cell adhesion through a loss of focal adhesion plaques from spread cells. We previously reported that SPARC reduced the number of bovine aortic endothelial (BAE) cells positive for focal adhesions [Murphy-Ullrich et al. (1991): J Cell Biol 115:1127-1136]. We have now characterized the effect of SPARC on the cytoskeleton of BAE cells. Addition of SPARC to spread BAE cells caused a dose-dependent loss of focal adhesion-positive cells, that was maximal at approximately 1 microgram/ml (0.03 microM). Consistent with the loss of adhesion plaques as detected by interference reflection microscopy, vinculin appeared diffuse and F-actin was redistributed to the periphery of cells incubated with SPARC. However, the distribution of the integrin alpha v beta 3 remained clustered in a plaque-like distribution. These data, and the observation that SPARC binds to BAE cells but not to the extracellular matrix, indicate that SPARC acts via interactions with cell surface molecules and not by steric/physical disruption of integrin-extracellular matrix ligands. To determine the region(s) of SPARC that mediate a loss of focal adhesions, we tested peptides from the four distinct regions of SPARC. The cationic, cysteine-rich peptide 2.1 (amino acids 54-73) and the Ca(2+)-binding EF-hand-containing peptide 4.2 (amino acids 254-273) were active in focal adhesion disassembly. Furthermore, antibodies specific for these regions neutralized the focal adhesion-labilizing activity of SPARC. These results are consistent with previous data showing that peptide 2.1 and 4.2 interact with BAE cell surface proteins and indicate that the loss of focal adhesions from endothelial cells exposed to SPARC is a receptor-mediated event.

Actins↗

PubMed2005

The role of the cytoskeleton in differentially regulating pressure-mediated effects on malignant colonocyte focal adhesion signaling and cell adhesion.

Increased extracellular pressure stimulates colon cancer cell adhesion by activating focal adhesion kinase (FAK) and Src. We investigated the role of the cytoskeleton in pressure-induced inside-out FAK and Src phosphorylation and pressure-stimulated adhesion. We perturbed actin polymerization with phalloidin, cytochalasin D and latrunculin B, and microtubule organization with colchicine and paclitaxol. We compared the effects of these agents on pressure-induced SW620 and human primary colon cancer cell adhesion and inside-out FAK/Src activation with outside-in adhesion-dependent FAK/Src activation. Cells pretreated with cytoskeletal inhibitors were subjected to 15 mmHg increased pressure and allowed to adhere to collagen I coated plates or prevented from adhesion to pacificated plates for 30 min. Phalloidin, cytochalasin D, latrunculin B and colchicine pretreatment completely prevented pressure-stimulated and significantly inhibited basal SW620 cell adhesion. Taxol did not inhibit pressure-induced colon cancer cell adhesion, but significantly lowered basal adhesion. Cytochalasin D and colchicine had similar effects in pressure-stimulated primary human malignant colonocytes. Phalloidin, cytochalasin D, latrunculin B and colchicine prevented pressure-induced SW620 FAK phosphorylation but not Src phosphorylation. FAK phosphorylation in response to collagen I adhesion was significantly attenuated but not completely prevented by these inhibitors. Although Src phosphorylation was not increased on adhesion, the cytoskeleton disrupting agents significantly lowered basal Src phosphorylation in adherent cells. These results suggest that both cytoskeleton-dependent FAK activation and cytoskeleton-independent Src activation may be required for extracellular pressure to stimulate colon cancer cell adhesion. Furthermore, the cytoskeleton plays a different role in pressure-activated FAK and Src signaling than in FAK and Src activation in adherent cells. We, therefore, hypothesize that cytoskeletal interactions with focal adhesion signals mediate the effects of extracellular pressure on colon cancer cell adhesion.

Cell Adhesion↗

PubMed1999

Astrocytoma adhesion to extracellular matrix: functional significance of integrin and focal adhesion kinase expression.

Evidence is accumulating implicating a role for integrins in the pathogenesis of cancer, a disease in which alterations in cellular growth, differentiation, and adhesive characteristics are defining features. In the present report we studied a panel of 8 human astrocytoma cell lines for their expression of integrin subunits by RT-PCR, and of integrin heterodimers by immunoprecipitation analyses. The functionality of integrin heterodimers was assessed using cell attachment assays to plastic or single matrix substrates. Downstream effects of integrin activation were studied by western blot analyses of FAK expression in human astrocytoma cell lines growing on plastic and on a fibronectin matrix, and in 13 primary human brain tumor specimens of varying histopathological grade. Furthermore, we studied tyrosine phosphorylation of FAK in astrocytoma cells growing on plastic versus fibronectin. Finally, we analyzed the effects of intermediate filament gene transfer on FAK phosphorylation in SF-126 astrocytoma cells. Our data show that astrocytoma cell lines express various integrin subunits by RT-PCR, and heterodimers by immunoprecipitation analyses. The beta1 and alphav integrin subunits were expressed by all astrocytoma cell lines. The alpha3 subunit was expressed by all cell lines except SF-188. By immunoprecipitation, the fibronectin receptor (alpha5beta1 integrin heterodimer) and the vitronectin receptor (alphavbeta3) were identified in several cell lines. Astrocytoma cell attachment studies to human matrix proteins suggested that these integrin heterodimers were functional. Using confocal laser microscopy, we showed that FAK was colocalized to actin stress fibers at sites of focal adhesion complexes. By western blot, FAK was variably but quite ubiquitously expressed in human astrocytoma cell lines, and in several primary human astrocytoma specimens. When U373 and U87 MG astrocytoma cells bind to a fibronectin matrix, FAK is phosphorylated. GFAP-transfected SF-126 human astrocytoma cells were shown to overexpress the phosphorylated form of FAK only when these cells were placed on a fibronectin matrix. This result is of interest because it suggests that manipulations of the astrocytoma cytoskeleton per se can bring about potential signaling changes that channel through integrins and focal adhesion sites leading to activation of key kinases such as FAK.

Adult↗

PubMed1999

Syndecan-4 signals cooperatively with integrins in a Rho-dependent manner in the assembly of focal adhesions and actin stress fibers.

The assembly of focal adhesions and actin stress fibers by cells plated on fibronectin depends on adhesion-mediated signals involving both integrins and cell-surface heparan sulfate proteoglycans. These two cell-surface receptors interact with different domains of fibronectin. To attempt to identify the heparan sulfate proteoglycans involved, we used fibronectin-null (FN-/-) mouse fibroblasts to eliminate the contribution of endogenous fibronectin during the analysis. FN-/- fibroblasts plated on the cell-binding domain of fibronectin or on antibodies directed against mouse beta1 integrin chains attach but fail to spread and do not form focal adhesions or actin stress fibers. When such cells are treated with antibodies directed against the ectodomain of mouse syndecan-4, they spread fully and assemble focal adhesions and actin stress fibers indistinguishable from those seen in cells plated on intact fibronectin. These results identify syndecan-4 as a heparan sulfate proteoglycan involved in the assembly process. The antibody-stimulated assembly of focal adhesions and actin stress fibers in cells plated on the cell-binding domain of fibronectin can be blocked with C3 exotransferase, an inhibitor of the small GTP-binding protein Rho. Treatment of cells with lysophosphatidic acid, which activates Rho, results in full spreading and assembly of focal adhesions and actin stress fibers in fibroblasts plated on the cell-binding domain of fibronectin. We conclude that syndecan-4 and integrins can act cooperatively in generating signals for cell spreading and for the assembly of focal adhesions and actin stress fibers. We conclude further that these joint signals are regulated in a Rho-dependent manner.

Actin Cytoskeleton↗

PubMed1995

Identification and characterization of a novel related adhesion focal tyrosine kinase (RAFTK) from megakaryocytes and brain.

We have isolated a cDNA encoding a novel human intracytoplasmic tyrosine kinase, termed RAFTK (for a related adhesion focal tyrosine kinase). In addition, we have cloned and characterized the murine homolog of the human RAFTK cDNA. Comparison of the deduced amino acid sequences of human RAFTK and murine Raftk cDNAs revealed 95% homology, indicating that RAFTK is highly conserved between these species. The RAFTK cDNA clone, encoding a polypeptide of 1009 amino acids, has closest homology (48% identity, 65% similarity) to the focal adhesion kinase (pp125FAK). Comparison of the deduced amino acid sequences also indicates that RAFTK, like pp125FAK, lacks a transmembrane region, myristylation sites, and SH2 and SH3 domains. In addition, like pp125FAK, RAFTK contains a kinase domain flanked by large N-terminal (426 residues) and C-terminal (331 residues) domains, and the C-terminal region contains a predicted proline-rich stretch of residues. In fetal tissues, RAFTK expression was abundant in brain, and low levels were observed in lung and liver. In adult tissues, it was less restricted, indicating that RAFTK expression is developmentally up-regulated. Expression of RAFTK was also observed in human CD34+ marrow cells, primary bone marrow megakaryocytes, platelets, and various areas of brain. The human RAFTK gene was assigned to human chromosome 8 using genomic DNAs from human/rodent somatic cell hybrid lines. The mouse Raftk gene was mapped to chromosome 14, closely linked to gonadotropin-releasing hormone. Using specific antibodies for RAFTK, a approximately 123-kDa protein from the human megakaryocytic CMK cell line was immunoprecipitated. Treatment of the megakaryocytic CMK cells with thrombin caused a rapid induction of tyrosine phosphorylation of RAFTK protein. The structural features of RAFTK suggest that it is a member of the focal adhesion kinase gene family and may participate in signal transduction in human megakaryocytes and brain as well as in other cell types.

Amino Acid Sequence↗

PubMed2002

Class A scavenger receptors mediate cell adhesion via activation of G(i/o) and formation of focal adhesion complexes.

Class A macrophage scavenger receptors (SR-A) are multifunctional receptors with roles in modified lipoprotein uptake, innate immunity, and macrophage adhesion. Our previous studies conducted in mouse peritoneal macrophages demonstrated that pertussis toxin (PTX) mediated inhibition of G(i/o) attenuated SR-A-dependent uptake of modified lipoprotein. The finding that SR-A-mediated lipoprotein internalization was PTX-sensitive led us to hypothesize that SR-A-mediated cell adhesion might be similarly regulated by G(i/o)-dependent signaling pathways. To test this hypothesis, SR-A was expressed in HEK cells under inducible control. Relative to HEK cells that lack SR-A, SR-A expressing cells displayed enhanced adhesion to tissue culture dishes. SR-A-mediated adhesion was significantly reduced following PTX treatment and was insensitive to chelating divalent cations with EDTA. SR-A-expressing cells exhibited a distinct cell morphology characterized by fine filopodia-like projections. Both polymerized actin and vinculin were codistributed with SR-A in the filopodia-like projections indicating the formation of focal adhesion complexes. Overall, our results indicate that the ability of SR-A to enhance cell adhesion involves G(i/o) activation and formation of focal adhesion complexes.

Actins↗

PubMed1995

v-Src-induced degradation of focal adhesion kinase during morphological transformation of chicken embryo fibroblasts.

Morphological transformation of cells by the v-Src tyrosine kinase is incompletely understood. However, it is independent of nuclear functions and probably involves phosphorylation of targets associated with the cytoskeleton and focal adhesions, structures which tether the cytoskeleton to the points of cell attachment. v-Src activity both stimulates tyrosine phosphorylation of a tyrosine kinase present in focal adhesions (focal adhesion kinase or pp125FAK) and disrupts focal adhesions, leading to cell rounding and detachment. However, pp125FAK is also phosphorylated on tyrosine as a result of integrin stimulation which induces quite different biological consequences including the organisation of focal adhesions when cells spread on fibronectin (reviewed in Schaller and Parsons, 1993). To address this paradox, we examined changes in pp125FAK during activation and shut-off of temperature sensitive mutant v-Src proteins that induce varying degrees of transformation in chick embryo fibroblasts. An efficiently transforming v-Src mutant initially stimulated pp125FAK tyrosine phosphorylation, but induced subsequent pp125FAK degradation prior to the onset of cell rounding and detachment. v-Src mutants which are impaired in their ability to induce morphological transformation were much less efficient at inducing degradation of pp125FAK. Moreover, cell spreading during restitution of normal morphology did not require detectable tyrosine phosphorylation of pp125FAK, or its potential substrate paxillin, suggesting that pp125FAK may function more in the turnover of focal adhesions than in their assembly.

Animals↗

PubMed2004

Surface chemistry modulates focal adhesion composition and signaling through changes in integrin binding.

Biomaterial surface properties influence protein adsorption and elicit diverse cellular responses in biomedical and biotechnological applications. However, the molecular mechanisms directing cellular activities remain poorly understood. Using a model system with well-defined chemistries (CH3, OH, COOH, NH2) and a fixed density of the single adhesive ligand fibronectin, we investigated the effects of surface chemistry on focal adhesion assembly and signaling. Surface chemistry strongly modulated integrin binding and specificity--alpha5beta1 integrin binding affinity followed the pattern OH>NH2=COOH>CH3, while integrin alphaVbeta3 displayed the relationship COOH>NH2>>OH=CH3. Immunostaining and biochemical analyses revealed that surface chemistry modulates the structure and molecular composition of cell-matrix adhesions as well as focal adhesion kinase (FAK) signaling. The neutral hydrophilic OH functionality supported the highest levels of recruitment of talin, alpha-actinin, paxillin, and tyrosine-phosphorylated proteins to adhesive structures. The positively charged NH2 and negatively charged COOH surfaces exhibited intermediate levels of recruitment of focal adhesion components, while the hydrophobic CH3 substrate displayed the lowest levels. These patterns in focal adhesion assembly correlated well with integrin alpha5beta1 binding. Phosphorylation of specific tyrosine residues in FAK also showed differential sensitivity to surface chemistry. Finally, surface chemistry-dependent differences in adhesive interactions modulated osteoblastic differentiation. These differences in focal adhesion assembly and signaling provide a potential mechanism for the diverse cellular responses elicited by different material properties.

3T3 Cells↗

PubMed1998

Thrombospondin signaling of focal adhesion disassembly requires activation of phosphoinositide 3-kinase.

Thrombospondin is an extracellular matrix protein involved in modulating cell adhesion. Thrombospondin stimulates a rapid loss of focal adhesion plaques and reorganization of the actin cytoskeleton in cultured bovine aortic endothelial cells. The focal adhesion labilizing activity of thrombospondin is localized to the amino-terminal domain, specifically amino acids 17-35. Use of a synthetic peptide (hep I), containing amino acids 17-35 of thrombospondin, enables us to examine the signaling mechanisms specifically involved in thrombospondin-induced disassembly of focal adhesions. We tested the hypothesis that activation of phosphoinositide 3-kinase is a necessary step in the thrombospondin-induced signaling pathway regulating focal adhesion disassembly. Both wortmannin and LY294002, membrane permeable inhibitors of phosphoinositide 3-kinase activity, blocked hep I-induced disassembly of focal adhesions. Similarly, wortmannin inhibited hep I-mediated actin microfilament reorganization and the hep I-induced translocation of alpha-actinin from focal adhesion plaques. Hep I also stimulated phosphoinositide 3-kinase activity approximately 2-3-fold as measured in anti-phosphoinositide 3-kinase and anti-phosphotyrosine immunoprecipitates. Increased immunoreactivity for the 85-kDa regulatory subunit in anti-phosphotyrosine immunoprecipitates suggests that the p85/p110 form of phosphoinositide 3-kinase is involved in this pathway. In 32Pi-labeled cells, hep I increased levels of phosphatidylinositol (3,4,5)-trisphosphate, the major product of phosphoinositide 3-kinase phosphorylation. These results suggest that thrombospondin signals the disassembly of focal adhesions and reorganization of the actin cytoskeleton by a pathway involving stimulation of phosphoinositide 3-kinase activity.

Amino Acid Sequence↗

PubMed2005

Focal adhesion kinase: in command and control of cell motility.

A central question in cell biology is how membrane-spanning receptors transmit extracellular signals inside cells to modulate cell adhesion and motility. Focal adhesion kinase (FAK) is a crucial signalling component that is activated by numerous stimuli and functions as a biosensor or integrator to control cell motility. Through multifaceted and diverse molecular connections, FAK can influence the cytoskeleton, structures of cell adhesion sites and membrane protrusions to regulate cell movement.

Animals↗

PubMed2002

Reduced focal adhesion kinase and paxillin phosphorylation in BCR-ABL-transfected cells.

BACKGROUND: BCR-ABL formation is critical to oncogenic transformation in chronic myelogenous leukemia and has been implicated as a key event leading to alterations in cytoskeletal structures and adhesion in the leukemic cells. The authors therefore investigated the effect of p210(BCR-ABL) on actin polymerization as well as on the expression and phosphorylation state of the adhesion proteins paxillin and focal adhesion kinase (FAK). METHODS: Transfection with BCR-ABL constructs abrogated the ability of NIH 3T3 fibroblasts to adhere and the cells underwent striking morphologic changes. RESULTS: Scanning electron microscopy revealed that the cells lost their elongated appearance and became rounded. This alteration was associated with significantly reduced actin polymerization. In addition, steady-state levels of paxillin and FAK protein were increased. However, while the overall level of phosphotyrosines was also increased, the amount of tyrosine phosphorylated paxillin and FAK was reduced in the BCR-ABL-transfected cells as compared to the parental cells. Culture on extracellular fibronectin matrix partially reversed the morphologic changes and resulted in a return, albeit incomplete, of filamentous actin in BCR-ABL-transfected 3T3 fibroblasts. In addition, phosphorylation of paxillin and FAK in the BCR-ABL-transfected NIH 3T3 cells was restored. CONCLUSIONS: The authors conclude that, in the current system, transfection of BCR-ABL attenuates FAK and paxillin phosphorylation and reduces actin polymerization, events accompanied by significant alterations in cellular morphology. The observation that exposure of the cells to fibronectin partially reverses all these changes suggests that the focal adhesion proteins and actin structures nevertheless remain responsive to signaling from the outside.

3T3 Cells↗

PubMed2002

GFP-FRNK disrupts focal adhesions and induces anoikis in neonatal rat ventricular myocytes.

Focal adhesion kinase (FAK) is a nonreceptor protein tyrosine kinase involved in adhesion-dependent signal transduction. FAK is highly expressed in cultured neonatal rat ventricular myocytes (NRVMs) and undergoes tyrosine autophosphorylation in response to cell adhesion, stretch, and growth factor stimulation. We previously showed that inhibition of FAK phosphorylation by adenovirally mediated overexpression of FRNK (the autonomously expressed C-terminal domain of FAK) prevented endothelin-1 (ET)-induced NRVM hypertrophy. One question raised by these studies was whether FRNK localized to focal adhesions and displaced FAK from sites required for downstream signaling. Therefore, we constructed a replication-defective adenovirus encoding a GFP-FRNK fusion protein (Adv-GFP-FRNK) and examined its effects on NRVM cytoarchitecture and signaling. Uninfected NRVMs contained small amounts of endogenous FRNK. NRVMs infected with Adv-GFP-FRNK expressed much larger amounts of a 66-/68-kDa protein that localized to costameres and focal adhesions. GFP-FRNK overexpression suppressed basal and ET-induced FAK phosphorylation and also inhibited ET-induced phosphorylation of PYK2, the other member of the FAK family of nonreceptor protein tyrosine kinases. In contrast, GFP-FRNK overexpression did not prevent ET-induced ERK, JNK, or p70S6K phosphorylation. Furthermore, GFP-FRNK resulted in the loss of detectable FAK and paxillin in focal adhesions, which was accompanied by reduced levels of total paxillin and, ultimately, cell detachment and apoptosis. We conclude that FRNK functions as a dominant-negative inhibitor of adhesion-dependent signaling by displacing FAK from focal adhesions and interfering with the anchorage of NRVMs that is necessary for cell survival, a process known as anoikis.

Adenoviridae↗

PubMed2000

Collagen IV-dependent ERK activation in human Caco-2 intestinal epithelial cells requires focal adhesion kinase.

Integrin-initiated extracellular signal-regulated kinase (ERK) activation by matrix adhesion may require focal adhesion kinase (FAK) or be FAK-independent via caveolin and Shc. This remains controversial for fibroblast and endothelial cell adhesion to fibronectin and is less understood for other matrix proteins and cells. We investigated Caco-2 intestinal epithelial cell ERK activation by collagen I and IV, laminin, and fibronectin. Collagens or laminin, but not fibronectin, stimulated tyrosine phosphorylation of FAK, paxillin, and p130(cas) and activated ERK1/2. Shc, tyrosine-phosphorylated by matrix adhesion in many cells, was not phosphorylated in Caco-2 cells in response to any matrix. Caveolin expression did not affect Caco-2 Shc phosphorylation in response to fibronectin. FAK, ERK, and p130(cas) tyrosine phosphorylation were activated after 10-min adhesion to collagen IV. FAK activity increased for 45 min after collagen IV adhesion and persisted for 2 h, while p130(cas) phosphorylation increased only slightly after 10 min. ERK activity peaked at 10 min, declined after 30 min, and returned to base line after 1 h. Transfection with FAK-related nonkinase, but not substrate domain deleted p130(cas), strongly inhibited ERK2 activation in response to collagen IV, indicating Caco-2 ERK activation is at least partly regulated by FAK.

Caco-2 Cells↗

PubMed1994

Association of the amino-terminal half of c-Src with focal adhesions alters their properties and is regulated by phosphorylation of tyrosine 527.

We have characterized the mechanism by which the subcellular distribution of c-Src is controlled by the phosphorylation of tyrosine 527. Mutation of this tyrosine dramatically redistributes c-Src from endosomal membranes to focal adhesions. Redistribution to focal adhesions occurs independently of kinase activity and cellular transformation. In cells lacking the regulatory kinase (CSK) that phosphorylates tyrosine 527, c-Src is also found predominantly in focal adhesions, confirming that phosphorylation of tyrosine 527 affects the location of c-Src inside the cell. The first 251 amino acids of c-Src are sufficient to allow association with focal adhesions, indicating that at least one signal for positioning c-Src in focal adhesions resides in the amino-terminal half. Point mutations and deletions in the first 251 amino acids of c-Src reveal that association with focal adhesions requires the myristylation site needed for membrane attachment, as well as the SH3 domain. Expression of the amino-terminal region alters both the structural and biochemical properties of focal adhesions. Focal adhesions containing this non-catalytic portion of c-Src are larger and exhibit increased levels of phosphotyrosine staining. Our results suggest that c-Src may regulate focal adhesions and cellular adhesion by a kinase-independent mechanism.

Amino Acid Sequence↗

PubMed1997

Characterization of focal adhesion assembly in XR1 glial cells.

In the present communication, we have characterized focal adhesions in cultured glial cells derived from the Xenopus retina. Using antibodies directed against focal adhesion proteins we found that beta1 integrin immunoreactivity colocalized with talin, vinculin, and phosphotyrosine immunoreactivities in glial cells from primary cultures of Xenopus retina, as well as in the XR1 glial cell line, an immortal cell line derived from Xenopus retinal neuroepithelium. beta1 integrin immunoreactivity also colocalized with the termini of rhodamine phalloidin-labeled filamentous-actin at focal adhesions. The regulation of focal adhesion assembly was examined in XR1 glial cells using inhibitors against actin polymerization (cytochalasins) or tyrosine kinase activity (genistein). Compared to control cultures, those treated with the inhibitors exhibited a dose-dependent decrease in the proportion of cells displaying focal adhesions. Treatment with cytochalasin B also resulted in a dose-dependent decrease in cell area. Mature focal adhesions in XR1 cells with a flattened, spread morphology also were disrupted by the presence of these inhibitors. These results provide strong evidence that an intact actin cytoskeleton and tyrosine kinase activity regulate focal adhesion assembly and also play important roles in the maintenance of the integrity of focal adhesions in glial cells.

Actins↗