Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.10.2 (focal adhesion kinase)
44,029 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Glycosyltransferase gene transfection into cell lines has been an approach used successfully to elucidate the functional role of cell surface glycoconjugates. We have transfected the rat CMP-NeuAc:Galbeta1,4GlcNAc alpha2,6-sialyltransferase (EC 2.4.99.1) gene into a human, tumorigenic, glioma cell line, U373 MG. This transfection led to a marked inhibition of invasivity, alterations in adhesivity to fibronectin and collagen matrices, and inappropriately sialylated alpha3beta1 integrin. Adhesion-mediated protein tyrosine phosphorylation was reduced in the transfectants despite increased expression of focal adhesion kinase, p125fak. Furthermore, the transfectants showed a distinct cell morphology, an increased number of focal adhesion sites, and different sensitivity to cytochalasin D treatment than control U373 MG cells. These results suggest that inappropriate sialylation of cell surface glycoconjugates, such as integrins, can change focal adhesion as well as adhesion-mediated signal transduction and block glioma cell invasivity in vitro.
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PMID:alpha2,6-Sialyltransferase gene transfection into a human glioma cell line (U373 MG) results in decreased invasivity. 916 54

Assembly of a fibronectin (FN) matrix is a multistep process which influences a number of cellular functions including intracellular cytoskeletal organization and signaling responses. We have previously reported on a recombinant FN (recFN), FN delta III1-7, which differs from native FN in its rate of fibril formation. To determine the intracellular consequences of a delay in assembly, we compared the distribution of cytoskeletal proteins during the formation of native and recFN matrices by immunofluorescence at various time points. CHO alpha 5 cell cytoskeleton was reorganized in response to both native and recFN matrix formation. Assembly of native FN induced a rapid reorganization of actin into stress fibers and colocalization of alpha 5 beta 1 integrin, focal adhesion kinase (FAK), vinculin, and paxillin to regions of cell-matrix contact. alpha 5 beta 1 integrins and FAK are also clustered upon binding of FN delta III1-7 to cells but actin reorganization and focal adhesion formation are delayed and appear to be dependent on the formation of FN delta III1-7 fibrils. These results suggest that the structural framework of the matrix plays an important role in the ability of FN to initiate intracellular responses.
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PMID:Coordinated regulation of fibronectin fibril assembly and actin stress fiber formation. 917 3

Cell adhesive interactions play important roles during many normal physiological processes such as embryonic development and wound repair, and also during the progression of diseases such as cancer. Cell adhesion is mediated by the specific interactions of cell surface receptors with extracellular glycoproteins. The best characterized cell adhesion receptors are the integrins. Integrins comprise a family of more than 23 noncovalent, heterodimeric complexes consisting of an alpha and a beta subunit. Each subunit is a glycoprotein with a large, globular extracellular domain and a transmembrane domain. Most integrins have relatively small cytoplasmic domains consisting of fewer than 60 amino acids. Although many integrins can bind fibronectin, the alpha 5, beta 1, integrin is the major fibronectin receptor on most cells. This integrin mediates such cellular responses to fibronectin substrates as adhesion, migration, assembly of extracellular matrix, and signal transduction. Integrin ligands, such as fibronectin, are not passive adhesive molecules but are active participants in the cell adhesive process that leads to signal transduction. The best characterized integrin ligand is fibronectin. Fibronectin is a multifunctional glycoprotein comprised of three different types of homologous repeating units (termed type I, type II, and type III). Fibronectin has at least two independent cell adhesive regions: one located near the center of the polypeptide chain in the ninth and tenth type III modules binds to the alpha 5 beta 1 integrin. The biological function of the central cell adhesive region requires two critical amino acid sequences--an Arg-Gly-Asp (RGD) sequence and a Pro-His-Ser-Arg-Asn (PHSRN) sequence, which function in synergy--for optimal binding to the alpha 5 beta 1 integrin. Furthermore, the spacing between the crucial RGD and PHSRN sequences is also important for activity, suggesting the sequences themselves are necessary, but not sufficient, to account for the cell adhesive activity of fibronectin. One of the manifestations of integrin-mediated signal transduction including protein tyrosine phosphorylation. One cytoplasmic protein that is phosphorylated in response to cell adhesion is the focal adhesion kinase known as pp125FAK or FAK. The beta 1, beta 3, and beta 5 integrin intracellular domains are sufficient to initiate signal transduction pathways. Furthermore, alternative splicing can regulate the ability of beta integrin intracellular domains to participate in signal transduction. Other intracellular responses to cell adhesion include stimulation of migration, the assembly of an F-actin cytoskeleton and specialized structures called focal contacts, changes of cytoplasmic pH and calcium ion concentration, and modulation of proliferation and gene expression. Such varied modes of signal transduction are probably differentially controlled by a mechanism that requires either integrin receptor clustering alone, ligand occupancy in addition to clustering, or clustering and/or ligand occupancy plus tyrosine kinase activity for different responses. The examination of the fundamental mechanisms important for adhesion of cultured human cells and the resultant signaling processes has the potential of providing an understanding of molecular mechanisms involved in complex physiological processes and serving the basis for the development of novel therapeutic agents for the treatment of human disease.
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PMID:Integrins in cell adhesion and signaling. 918 47

Tyrosine phosphorylation of paxillin by the focal adhesion kinase (FAK) has been implicated as a signal transduction mechanism associated with cell adhesion and cytoskeletal reorganization. The potential role of serine phosphorylation of paxillin in these events has not been well characterized. In this study we have examined the phosphorylation profile of paxillin both in vitro and in vivo. By using glutathione S-transferase-paxillin fusion proteins in precipitation-kinase assays in vitro we observed that a fusion protein spanning amino acid residues 54-313 of paxillin, and containing a FAK-binding site, precipitated substantial serine kinase activity as well as FAK activity from a smooth-muscle lysate. Together these kinases phosphorylated paxillin on tyrosine residue 118, a site that has been identified previously as a target for FAK phosphorylation, and on serine residues 188 and/or 190. The binding site for the serine kinase, the identity of which is currently unknown, was further mapped to residues 168-191 of paxillin. To assess the physiological relevance of these sites phosphorylated in vitro, the profile of paxillin phosphorylation in vivo stimulated by seeding fibroblasts on fibronectin was characterized. As expected, plating cells on fibronectin enhanced the tyrosine phosphorylation of paxillin. However, 96% of the phosphorylation of paxillin occurred on serine residues. Comparison by two-dimensional phosphopeptide analyses indicated that the major sites of tyrosine and serine phosphorylation detected in the assays in vitro co-migrate with phosphopeptides derived from paxillin phosphorylated in vivo in response to plating cells on fibronectin. These findings support a role for both tyrosine and serine kinases in the signal transduction pathway linking integrin activation to paxillin phosphorylation.
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PMID:Adhesion of fibroblasts to fibronectin stimulates both serine and tyrosine phosphorylation of paxillin. 923 Jan 16

The inappropriate activation of protein-tyrosine kinases (PTKs) has been associated with initiation and progression of several types of human cancers. We therefore postulated that immortalization by DNA tumor viruses results in the induction of PTKs fundamental to these processes. An RT-PCR-based screen was thus used to identify PTKs that were abundantly expressed in HPV-18-immortalized epithelial cells and HPV-containing carcinoma cell lines. One of the genes isolated in this screen was the focal adhesion kinase (FAK; pp125FAK), a cytoplasmic protein kinase that is activated in v-src transformed cells or by stimulation with mitogenic polypeptides. FAK also becomes catalytically active upon integrin engagement with extracellular matrix proteins, such as fibronectin. We found that FAK expression and activity were significantly elevated in HPV-18 E6/E7-immortalized human genital epithelial cells relative to their primary cell counterparts. Protein expression and tyrosine phosphorylation of the putative FAK substrate, paxillin, were also notably increased upon HPV-18 immortalization of genital epithelial cells and in HPV-containing cervical carcinoma cell lines. Most significantly, these cells expressed markedly higher levels of both intracellular and extracellular fibronectin, thus providing a mechanism for activation of FAK and increased tyrosine phosphorylation of paxillin. These findings suggest a role for the integrin/FAK-mediated signaling pathway in cervical carcinogenesis and represent one of the first demonstrations of a tyrosine kinase whose activity is elevated following viral immortalization.
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PMID:Activation of the focal adhesion kinase signal transduction pathway in cervical carcinoma cell lines and human genital epithelial cells immortalized with human papillomavirus type 18. 923 61

Cell attachment to fibronectin stimulates the integrin-dependent interaction of p85-associated phosphatidylinositol (PI) 3-kinase with integrin-dependent focal adhesion kinase (FAK) as well as activation of the Ras/mitogen-activated protein (MAP) kinase pathway. However, it is not known if this PI 3-kinase-FAK interaction increases the synthesis of the 3-phosphorylated phosphoinositides (3-PPIs) or what role, if any, is played by activated PI 3-kinase in integrin signaling. We demonstrate here the integrin-dependent accumulation of the PI 3-kinase products, PI 3,4-bisphosphate [PI(3,4)P2] and PI(3,4,5)P3, as well as activation of AKT kinase, a serine/threonine kinase that can be stimulated by binding of PI(3,4)P2. The PI 3-kinase inhibitors wortmannin and LY294002 significantly decreased the integrin-induced accumulation of the 3-PPIs and activation of AKT kinase, without having significant effects on the levels of PI(4,5)P2 or tyrosine phosphorylation of paxillin. These inhibitors also reduced cell adhesion/spreading onto fibronectin but had no effect on attachment to polylysine. Interestingly, integrin-mediated Erk-2, Mek-1, and Raf-1 activation, but not Ras-GTP loading, was inhibited at least 80% by wortmannin and LY294002. In support of the pharmacologic results, fibronectin activation of Erk-2 and AKT kinases was completely inhibited by overexpression of a dominant interfering p85 subunit of PI 3-kinase. We conclude that integrin-mediated adhesion to fibronectin results in the accumulation of the PI 3-kinase products PI(3,4)P2 and PI(3,4,5)P3 as well as the PI 3-kinase-dependent activation of the kinases Raf-1, Mek-1, Erk-2, and AKT and that PI 3-kinase may function upstream of Raf-1 but downstream of Ras in integrin activation of Erk-2 MAP and AKT kinases.
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PMID:Phosphatidylinositol 3-kinase is required for integrin-stimulated AKT and Raf-1/mitogen-activated protein kinase pathway activation. 923 99

Leukocyte integrins are fundamentally important in modulating adhesion to extracellular matrix components and to other cells. This integrin-mediated adhesion controls leukocyte arrest and extravasation during the onset of inflammatory responses. Moreover, integrin-ligand interactions trigger signaling pathways that may influence leukocyte phenotype and function at sites of inflammation. In the current studies, we evaluated the combinatorial effects of monocyte adhesion and IFN-gamma on intracellular signaling pathways. IFN-gamma triggers a well-defined signal transduction pathway, which although not directly stimulated by monocyte adherence to fibronectin or arginine-glycine-aspartate (RGD)-coated substrata, was enhanced significantly in these matrix-adherent cells. Compared with monocytes in suspension or adherent on plastic surfaces, monocytes adherent to fibronectin or RGD exhibited a greater than threefold increase in steady state levels of IFN-gamma-induced mRNA for the high affinity Fc gammaRI receptor. By electrophoretic mobility shift assays, this increase in mRNA was associated with a 5- to 10-fold increase in the STAT1-containing DNA-binding complex that binds to Fc gammaRI promoter elements. Furthermore, the tyrosine phosphorylation of STAT1 and the tyrosine kinases JAK1 and JAK2 was enhanced significantly in RGD-adherent monocytes compared with control cells. These results suggest a novel mechanism by which integrin-mediated cell adhesion can modulate the magnitude of cytokine-induced signal transduction pathways, thereby amplifying cellular events leading to monocyte activation and inflammation.
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PMID:Human monocyte binding to fibronectin enhances IFN-gamma-induced early signaling events. 927 34

Elevated levels of fibronectin (Fn) in articular cartilage have been linked to the progression of both rheumatoid and osteoarthritis. In this study, we examined intracellular events which follow ligation of Fn to its receptor, the integrin alpha5beta1. In addition, we examined the regulatory influence of nitric oxide on these events, since this free radical has been implicated in cartilage degradation. Exposure of chondrocytes to Fn-coated beads resulted in the circumferential clustering of the alpha5beta1 integrin receptor, which was accompanied by the subplasmalemmal assembly of a focal activation complex comprised of F-actin, the tyrosine kinase, focal adhesion kinase (FAK), the ras related G protein rho A, as well as tyrosine-phosphorylated proteins. Treatment with exogenous nitric oxide (NO) or catabolic cytokines which induce nitric oxide synthase blocked the assembly of F-actin, FAK, rho A and tyrosine-phosphorylated proteins while not affecting the total number of beads bound per cell nor the clustering of alpha5beta1 integrin. Use of a cGMP antagonist (Rp-8-Br cGMPS) or cGMP agonist (Sp-cGMPS) either abolished or mimicked the NO effect, respectively. Adherence of chondrocytes to fibronectin enhanced proteoglycan synthesis by twofold (vs. albumin). In addition, basic fibroblast growth factor (FGF) and insulin growth factor (IGF-1) induced proteoglycan synthesis in chondrocytes adherent to Fn but not albumin suggesting a costimulatory signal transduced by alpha5betal and the FGF receptor. Both constitutive and FGF stimulated proteoglycan synthesis were completely inhibited by nitric oxide. These data indicate that the ligation of alpha5beta1 in the chondrocyte induced the intracellular assembly of an activation complex comprised of the cytoplasmic tail of alpha5beta1 integrin, actin, and the signaling molecules rho A and FAK. We show that NO inhibits the assembly of the intracellular activation complex and the synthesis of proteoglycans, but has no effect on the extracellular aggregation of alpha5beta1 integrin. These observations provide a basis by which nitric oxide can interfere with chondrocyte functions by affecting chondrocyte-matrix interactions.
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PMID:Outside-in signaling in the chondrocyte. Nitric oxide disrupts fibronectin-induced assembly of a subplasmalemmal actin/rho A/focal adhesion kinase signaling complex. 931 79

Cell-extracellular matrix interactions support the ability of cells to migrate into areas of inflammation and injury. The present study evaluated the ability of different matrix proteins to support bronchial epithelial cell attachment and survival. Collagens were able to support attachment and survival of normal cultured human bronchial epithelial cells but only in the presence of added soluble growth factors such as insulin, epidermal growth factor, platelet-derived growth factor, and bovine pituitary extract. In contrast, fibronectin was able to support attachment and survival of normal human bronchial epithelial cells in growth factor-deficient medium. In addition, fibronectin, in the absence of added growth factors, was able to induce integrin clustering, focal adhesion formation, and phosphorylation of focal adhesion kinase. A 120-kDa chymotryptic fragment of fibronectin containing the Arg-Gly-Asp peptide sequence was able to reproduce the effects of the whole fibronectin molecule. This study supports the concept that fibronectin has specialized roles in injury and repair.
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PMID:Fibronectin supports bronchial epithelial cell adhesion and survival in the absence of growth factors. 931 5

Fibronectin (FN) promotes the modulation of freshly isolated arterial smooth muscle cells (SMCs) from a contractile to a synthetic phenotype by interacting with integrins on the cell surface. This process is characterized by a structural and functional transformation of the cells, including a reorganization of the cytoskeleton, the formation of a large secretory apparatus, and the acquisition of proliferative capacity. In this study we have investigated the role of integrin signaling through tyrosine kinases in the structural changes that occur in SMCs during primary culture on FN. A gradual increase in phosphotyrosine staining in focal adhesions and a concomitant increase in tyrosine phosphorylation of proteins including focal adhesion kinase were observed. In contrast, cells seeded on laminin formed few focal adhesions, and tyrosine phosphorylation of proteins was less than in cells cultured on FN. Treatment of cells cultured on FN with the tyrosine kinase inhibitor genistein strongly suppressed focal adhesion formation, cell spreading, and cytoskeletal reorganization. In addition, electron microscopic analysis demonstrated that the phenotypic modulation was slowed down. These results indicate that the ability of extracellular matrix components to promote a change in the phenotypic properties of SMCs depends on the assembly of focal adhesions with associated tyrosine kinase activity.
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PMID:Role of tyrosine kinases in extracellular matrix-mediated modulation of arterial smooth muscle cell phenotype. 935 62


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