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)

To clarify the function of osteopontin in osteoblast differentiation, we have examined the signal transduction pathway in an osteoblastic cell line (UMR106-6) bound to osteopontin, fibronectin, vitronectin and collagen type I surfaces. This was done by investigating the production and autophosphorylation of focal adhesion kinase (FAK) and the expression of alkaline phosphatase (ALP) at the transcription level. Results suggest that osteopontin was not only responsible for the autophosphorylation of FAK but regulated the expression of ALP, which was strongly correlated with FAK activity. These results suggest that osteopontin might act as a trigger in the early differentiation of osteoblasts.
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PMID:Osteopontin involvement in integrin-mediated cell signaling and regulation of expression of alkaline phosphatase during early differentiation of UMR cells. 945 May 60

The vinculin gene codes for a cytoskeletal protein, found in focal adhesion plaques and in cell-cell adherens junctions. Vinculin was inactivated by homologous recombination using a targeting vector in embryonic stem (ES) cells. The heterozygous ES cells were introduced into mice by established procedures to produce heterozygous animals that were normal and fertile. No homozygous vinculin-/- embryos were born and analyses during the gestational period showed that the vinculin null embryos were small and abnormal from day E8 but some survived until E10. The most prominent defect was lack of midline fusion of the rostral neural tube, producing a cranial bilobular appearance and attenuation of cranial and spinal nerve development. Heart development was curtailed at E9.5, with severely reduced and akinetic myocardial and endocardial structures. Mutant embryos were 30-40% smaller, somites and limbs were retarded and ectodermal tissues were sparse and fragile. Fibroblasts (MEF) isolated from mutant embryos were shown to have reduced adhesion to fibronectin, vitronectin, laminin and collagen compared to wild-type levels. In addition, migration rates over these substrata were two-fold higher and the level of focal adhesion kinase (FAK) activity was three-fold higher. We conclude that vinculin is necessary for normal embryonic development, probably because of its role in the regulation of cell adhesion and locomotion, cell behaviors essential for normal embryonic morphogenesis, although specific roles in neural and cardiac development cannot be ruled out.
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PMID:Vinculin knockout results in heart and brain defects during embryonic development. 948 5

Cell adhesion to the extracellular matrix appears to trigger a cascade of intracellular signalings. We have shown previously that treatment of ovarian cancer cells with peritoneal conditioned medium or purified fibronectin (FN) activated matrix metalloproteinase 9 secretion and, thereby, cancer cell invasion. By use of antisense oligonucleotides to focal adhesion kinase (FAK) and a dominant-negative mutant of ras (S17Nras), we found that both FAK and c-Ras were required for the activation of matrix metalloproteinase 9 secretion by FN. In addition, both antisense oligonucleotides to FAK and S17Nras inhibited mitogen-activated protein kinase activation by FN treatment, suggesting the involvement of mitogen-activated protein kinase in the FN-dependent signaling.
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PMID:Both focal adhesion kinase and c-Ras are required for the enhanced matrix metalloproteinase 9 secretion by fibronectin in ovarian cancer cells. 950 Apr 47

Balb/c 3T3 cell adhesion on substrata coated with fibronectin's (FN) alternatively-spliced EDb, implicated in some tumor cell systems, and its neighboring type III repeats (III7 and III8) induced intracellular signaling coincident with morphological responses. These events were analysed using minigene-expressed proteins containing various permutations of type III repeats of FN. Cells adherent to the tri-repeat protein 7-EDb-8 were compared to those attached to the tri-repeat 8-9-10 which can interact with integrins through RGD and its synergistic sequences. Cell adhesion to 7-EDb-8 generated rapid tyrosine phosphorylation of several intracellular proteins (particularly the complex at 120-130 kD), with the overall phosphorylation level and its sensitivity to tyrosine kinase inhibitors similar to responses on the 8-9-10 tri-repeat. This similarity contrasted with the differential morphological responses of cells mediated by these proteins. Further analysis of the kinetics of phosphorylation through immunoblotting of two focal adhesion proteins, p125FAK and pl30cas, revealed a profile for Balb/c 3T3 adhesion to 7-EDb-8 distinct from that on 8-9-10. EDb mono-repeat was also more potent for inducing both limited cell spreading and FAK tyrosine phosphorylation than its neighboring repeats III7 or III8. Examination of cellular localization of FAK and vinculin showed that cells spread on the 7-EDb-8 substratum displayed vinculin-positive focal complex-like structures at the cell periphery, in contrast to the classical focal adhesions seen in 8-9-10-adherent cells. These results suggest that EDb induces cell signaling events, leading to tyrosine phosphorylation of focal adhesion proteins, through a mechanism different from that mediated by integrins recognizing sequences in III8-9-10. EDb-dependent signaling may have special significance in some tumor systems.
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PMID:Adhesion to fibronectin's EDb domain induces tyrosine phosphorylation of focal adhesion proteins in Balb/c 3T3 cells. 950 75

Interactions of mesangial cells (MCs) with components of the extracellular matrix (ECM) profoundly influence the MC phenotype, such as attachment, contraction, migration, survival and proliferation. Here, we investigated the effects of exogenous nitric oxide (NO) on the process of MC adhesion to ECM molecules. Incubation of rat MCs with the NO donor S-nitroso-N-acetylpenicillamine (SNAP) dose- and time-dependently inhibited MC adhesion and spreading on various ECM substrata, being more pronounced on collagen type I than on collagen type IV, laminin or fibronectin. In contrast, SNAP did not inhibit MC adhesion to L-polylysine-coated plates. The inhibitory effects of SNAP were reduced by hemoglobin and enhanced by superoxide dismutase. The anti-adhesive action of SNAP was mimicked not only by other NO donors but also by 8-bromo-cGMP, and significantly reversed by the soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3,-alpha]quinoxalin-1-one (ODQ). Moreover, SNAP and 8-bromo-cGMP decreased the adhesion-induced phosphorylation of focal adhesion kinase (pp125FAK). In the presence of SNAP or 8-bromo-cGMP, adherent MCs exhibited disturbed organization of alpha-actin filaments and reduced numbers of focal adhesions, as shown by immunocytochemistry. In additional experiments with adherent MCs, it was found that exposure to SNAP or 8-bromo-cGMP for 12 and 24 hours induced detachment of MCs. The results indicate that exogenous NO interferes with the establishment and maintenance of MC adhesion to ECM components. This inhibitory NO effect is mediated predominantly by cGMP-signaling. Disturbance of MC attachment to ECM molecules could represent an important mechanism by which NO affects MC behavior in vitro and in vivo.
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PMID:Exogenous nitric oxide inhibits mesangial cell adhesion to extracellular matrix components. 950 4

To investigate the role of the potential phosphorylation sites in the cytoplasmic domain of integrin beta1A, point mutated variants of the protein were stably expressed in the beta1-deficient cell line GD25. Mutants T777A, Y783F, S785A, and Y795F were fully active in promoting cell adhesion, de novo formation of focal contacts, formation of fibronectin fibrils, and activation of focal adhesion kinase. Thus, phosphorylation of these residues is not required for several basic functions of integrin beta1A. On the other hand, the TT788-9AA mutant, was defective in mediating cell attachment and did not contribute to fibronectin fibril formation. The conformation of the extracellular domain was shifted towards an inactive state as measured by binding of the monoclonal antibody 9EG7. Antibody induced clustering of beta1ATT788-9AA demonstrated that the mutant cytoplasmic part was functional in mediating activation of focal adhesion kinase. Therefore, we conclude that threonines 788-789, which are conserved among most integrin beta subunits, are of critical importance for integrin function due to effects on the extracellular conformation of the receptor.
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PMID:Mutational analysis of the potential phosphorylation sites in the cytoplasmic domain of integrin beta1A. Requirement for threonines 788-789 in receptor activation. 951 7

The beta1-integrin cytoplasmic domain consists of a membrane proximal subdomain common to the four known isoforms ("common" region) and a distal subdomain specific for each isoform ("variable" region). To investigate in detail the role of these subdomains in integrin-dependent cellular functions, we used beta1A and beta1B isoforms as well as four mutants lacking the entire cytoplasmic domain (beta1TR), the variable region (beta1COM), or the common region (beta1 deltaCOM-B and beta1 deltaCOM-A). By expressing these constructs in Chinese hamster ovary and beta1 integrin-deficient GD25 cells (Wennerberg et al., J Cell Biol 132, 227-238, 1996), we show that beta1B, beta1COM, beta1 deltaCOM-B, and beta1 deltaCOM-A molecules are unable to support efficient cell adhesion to matrix proteins. On exposure to Mn++ ions, however, beta1B, but none of the mutants, can mediate cell adhesion, indicating specific functional properties of this isoform. Analysis of adhesive functions of transfected cells shows that beta1B interferes in a dominant negative manner with beta1A and beta3/beta5 integrins in cell spreading, focal adhesion formation, focal adhesion kinase tyrosine phosphorylation, and fibronectin matrix assembly. None of the beta1 mutants tested shows this property, indicating that the dominant negative effect depends on the specific combination of common and B subdomains, rather than from the absence of the A subdomain in the beta1B isoform.
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PMID:beta1-integrin cytoplasmic subdomains involved in dominant negative function. 952 73

Changes in integrin expression during malignant transformation have been observed in many tumors. Colon-carcinoma cells show reduced expression or even loss of the alpha5beta1 integrin compared to normal or adenoma cells. To determine the significance of absent alpha5beta1 integrin signaling, we transfected the cDNA coding for the alpha5 integrin sub-unit into the human colon-carcinoma cell line HT29, which constitutively lacks this subunit but does express the beta1 subunit. We show here that the newly expressed fibronectin receptor alpha5beta1 generates multiple signals, causing marked changes in cytoskeletal arrangements within a few minutes of adhesion to fibronectin. Cells expressing the alpha5beta1 integrin exhibit the formation of actin stress fibers and focal adhesions, as well as the induction of tyrosine phosphorylation of several proteins, within 10 min. We identified the focal adhesion kinase pp125FAK and the cytoskeletal protein paxillin as major phosphorylation substrates in these cells. These proteins remained hypophosphorylated when alpha5-negative control cells were plated on fibronectin. The tyrosine kinase pp60c-src, regarded as central in the regulation of cellular proliferation and constitutively over-expressed in HT29 and in colon-carcinoma cells, showed reduced intrinsic kinase activity in unstimulated HT29alpha5 cells. In contrast, fibronectin-induced signaling through alpha5beta1 increased pp60c-src activity. Moreover, immunoprecipitation of pp60c-src from extracts of HT29alpha5 cells cultivated on fibronectin for 20 min revealed complex formation of pp60c-src and tyrosine-phosphorylated pp125FAK. Our data suggest that de novo expression of the alpha5beta1 integrin in HT29 colon-cancer cells restores signaling via pp125FAK and pp60c-src. Thus, loss of this receptor during malignant transformation may contribute to tumor-cell autonomy, while reduced activity of pp60c-src in HT29alpha5-cells may participate directly in growth control.
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PMID:De novo expression of the alpha5beta1-fibronectin receptor in HT29 colon-cancer cells reduces activity of C-SRC. Increase of C-SRC activity by attachment on fibronectin. 953 67

Freshly isolated human monocytes do not express p125(FAK) but upon adherence to substrata activate the highly related calcium-dependent tyrosine kinase (CADTK), also known as Pyk2, CAKbeta, RAFTK, and FAK2. The monocyte CADTK was 5 kDa smaller than protein from epithelial cells; isolation and sequencing of the monocyte CADTK cDNA revealed a predicted 42-amino acid deletion between the two proline-rich domains of the enzyme. The nucleic acid sequence suggests that the deletion is caused by alternative RNA splicing. This species was also found in T and B lymphocytes and appears to be the predominant form of cytoskeletal associated tyrosine kinase in non-neoplastic, circulating, hematopoietic cells. CADTK was not activated when monocytes maintained in suspension were treated with agents that produce an intracellular calcium (thapsigargin) or protein kinase C (phorbol 12-myristate 13-acetate) signal including a chemokine, RANTES, that binds to the HIV co-receptor, CCK5. In contrast, monocyte adherence to tissue culture plastic-stimulated CADTK tyrosine phosphorylation, a process that was enhanced by thapsigargin, phorbol 12-myristate 13-acetate, and RANTES but that was completely blocked by preincubation with cytochalasin D. When compared with plastic, adherence to fibronectin- or collagen-coated surfaces produced only minimal CADTK activation but permitted significant stimulation by added thapsigargin. These data suggest that in a cell type that lacks p125(FAK), CADTK plays an early role in post-adherence signaling. Its activation involves two stages, cytoskeletal engagement, which is permissive, and co-stimulatory signals (calcium or protein kinase C) generated by extensive cell surface engagement, agonists, or inflammatory chemokines.
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PMID:A calcium-dependent tyrosine kinase splice variant in human monocytes. Activation by a two-stage process involving adherence and a subsequent intracellular signal. 954 57

Chronic myelogenous leukemia (CML) is characterized by the continuous proliferation and abnormal circulation of malignant hematopoietic progenitors. This may be related to the unresponsiveness of CML progenitors to beta1 integrin adhesion receptor-mediated inhibition of progenitor proliferation by the marrow microenvironment. In hematopoietic cell lines, the BCR-ABL oncogene product, p210(BCR-ABL), interacts with a variety of cytoskeletal elements important for normal integrin signaling. We studied the role of p210(BCR-ABL) in abnormal integrin function in CML by evaluating the effect of inhibition of BCR-ABL expression with antisense oligodeoxynucleotides (AS-ODNs) on integrin-mediated adhesion and proliferation inhibition of malignant primary progenitors from CML marrow. Preincubation of CML CD34(+)HLA-DR+ (DR+) cells with breakpoint-specific AS-ODNs significantly increased adhesion of CML progenitors to stroma and fibronectin (FN). Pretreatment with breakpoint-specific ODNs also resulted in significant inhibition of CML progenitor proliferation after ligand or antibody-mediated beta1 integrin engagement. Breakpoint-specific ODNs were significantly more effective in restoring CML progenitor adhesion and proliferation inhibition than control ODNs. BCR-ABL mRNA and p210(BCR-ABL) levels in CML CD34(+) cells were significantly reduced after incubation with breakpoint-specific AS-ODN. These studies indicate a role for BCR-ABL in abnormal circulation and defective integrin-dependent microenvironmental regulation of proliferation of CML hematopoietic progenitors.
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PMID:Inhibition of BCR-ABL expression with antisense oligodeoxynucleotides restores beta1 integrin-mediated adhesion and proliferation inhibition in chronic myelogenous leukemia hematopoietic progenitors. 955


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