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)

It is well appreciated that clustering of receptors for the extracellular matrix, most notably the integrins, elicits intracellular signal cascades. One of the first indications that integrin-dependent signaling has occurred is by the activation of focal adhesion kinase (FAK). Another, although less well understood, receptor for the extracellular matrix is (beta)1, 4-galactosyltransferase I (GalT). GalT participates during lamellipodia formation and cell migration by recognizing terminal N-acetylglucosamine residues on basal lamina glycosides. In this study, we investigated whether GalT is also capable of eliciting intracellular signal cascades, specifically FAK activation, in response to ligand binding and/or aggregation. 3T3 fibroblasts were treated with two different reagents capable of aggregating GalT, either antibodies raised against recombinant GalT or multivalent polymers of N-acetylglucosamine, and the effects on tyrosine phosphorylation were analyzed. Both reagents induced an initial tyrosine phosphorylation (1-2 minutes) and subsequent dephosphorylation (5-10 minutes) of proteins with molecular mass 67 and 125 kDa. These proteins were identified as paxillin and FAK, respectively, by immunoprecipitation with anti-paxillin and anti-FAK antibodies. Preimmune IgG, anti-GalT Fab fragments, irrelevant polymers and monomeric N-acetylglucosamine had no effect. The ability of GalT aggregation to induce transient tyrosine phosphorylation was dependent upon cell density. In addition, FAK dephosphorylation was found to be sensitive to the phosphatase inhibitor, sodium pervanadate. Similar to the integrins, GalT requires association with the cytoskeleton in order to function as a matrix receptor. To determine if the transient tyrosine phosphorylation of FAK was dependent upon GalT binding to the cytoskeleton, stably transfected fibroblasts expressing different amounts of GalT were treated with polymeric N-acetylglucosamine. Cells expressing increased levels of GalT associated with the cytoskeleton showed increased levels of FAK tyrosine phosphorylation and prolonged dephosphorylation, relative to control cells. In contrast, cells in which a dominant negative form of GalT prevents association with the cytoskeleton showed no or weak response to polymeric N-acetylglucosamine. Concomitant with the GalT-stimulated dephosphorylation of FAK, cells treated with anti-GalT antibodies or polymeric N-acetylglucosamine showed a loss of actin stress fibers and focal adhesions. Pervanadate treatment inhibited the GalT-dependent loss of actin stress fibers. To confirm the requirement of GalT in transient FAK phosphorylation and stress fiber reorganization in this system, we created cells homozygous null for the GalT isoform that functions as a matrix receptor. These cells were incapable of phosphorylating FAK in response to GalT agonists and, interestingly, showed a lack of lamellar stress fibers when cultured on basal lamina matrices. These data suggest that GalT function as a basal lamina receptor involves transient activation of FAK and an associated reorganization of stress fibers.
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PMID:Clustering of cell surface (beta)1,4-galactosyltransferase I induces transient tyrosine phosphorylation of focal adhesion kinase and loss of stress fibers. 1063 75

TEC-01 monoclonal antibody recognizes the oligosaccharide structure Galbeta1-->4[Fucalpha1-->3] GlcNAc (Le(X) hapten). To determine its fine specificity, reactivity of TEC-01 antibody with Le(X) glycosphingolipids, isolated from human liver metastasis of adenocarcinoma, and Le(X) neoglycolipid were analysed. Immunostaining of Le(X) glycosphingolipids, fractionated by thin-layer chromatography, showed that TEC-01 reacted with difucosyl and trifucosyl Le(X) glycosphingolipids but not with Le(X) pentasaccharide ceramide. Interestingly, TEC-01 also reacted with Le(X) neoglycolipid prepared by reductive amination from Le(X) pentasaccharide, lacto-N-fucopentaose III, and L-1,2-dipalmitoyl-sn-glycerophosphoethanolamine. The combined data suggest that TEC-01 recognizes Le(X) hapten in glycolipids with longer oligosaccharide chain moiety.
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PMID:Fine specificity of anti-Le(X) monoclonal antibody TEC-01. 1073 Aug 72

The hexosamine pathway provides UDP-N:-acetylhexosamine donor substrates used in cytosolic and Golgi-mediated glycosylation of proteins and for formation of glycosylphosphatidylinositol (GPI) anchors, which tether proteins to the outer plasma membrane. We have recently identified the murine glucosamine-6-phosphate (GlcN6P) acetyltransferase, EMeg32, as a developmentally regulated enzyme on the route to UDP-N:-acetylglucosamine (UDP-GlcNAc). Here we describe embryos and cells that have the EMeg32 gene inactivated by homologous recombination. Homozygous mutant embryos die at around embryonic day (E) 7.5 with a general proliferative delay of development. In vitro differentiated EMeg32(-/-) ES cells show reduced proliferation. Mouse embryonic fibroblasts (MEFs) deficient for EMeg32 exhibit defects in proliferation and adhesiveness, which could be complemented by stable re-expression of EMeg32 or by nutritional restoration of intracellular UDP-GlcNAc levels. Reduced UDP-GlcNAc levels predominantly translated into decreased O-GlcNAc modifications of cytosolic and nuclear proteins. Interestingly, growth-impaired EMeg32(-/-) MEFs withstand a number of apoptotic stimuli and express activated PKB/AKT. Thus, EMeg32-dependent UDP-GlcNAc levels influence cell cycle progression and susceptibility to apoptotic stimuli.
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PMID:Decreased UDP-GlcNAc levels abrogate proliferation control in EMeg32-deficient cells. 1101 12

The interaction of sialyl Lewis(x), Lewis(x), and alpha-L-Fuc-(1-->3)-beta-D-GlcNAc with isolectin A from Lotus tetragonolobus (LTL-A), and with Aleuria aurantia agglutinin (AAA) was studied using NMR experiments and surface plasmon resonance. Both lectins are specific for fucose residues. From NMR experiments it was concluded that alpha-L-Fuc-(1-->3)-beta-D-GlcNAc and Lewis(x) bound to both lectins, whereas sialyl Lewis(x) only bound to AAA. Increased line broadening of 1H NMR signals of the carbohydrate ligands upon binding to AAA and LTL-A suggested that AAA bound to the ligands more tightly. Further comparison of line widths showed that for both lectins binding strengths decreased from alpha-L-Fuc-(1-->3)-beta-D-GlcNAc to Lewis(x) and were lowest for sialyl Lewis(x). Surface plasmon resonance measurements were then employed to yield accurate dissociation constants. TrNOESY, QUIET-trNOESY, and trROESY experiments delivered bioactive conformations of the carbohydrate ligands, and STD NMR experiments allowed a precise epitope mapping of the carbohydrates bound to the lectins. The bioactive conformation of Lewis(x) bound to LTL-A, or AAA revealed an unusual orientation of the fucose residue, with negative values for both dihedral angles, phi and psi, at the alpha(1-->3)-glycosidic linkage. A similar distortion of the fucose orientation was also observed for sialyl Lewis(x) bound to AAA. From STD NMR experiments it followed that only the L-fucose residues are in intimate contact with the protein. Presumably steric interactions are responsible for locking the sialic acid residue of sialyl Lewis(x) in one out of many orientations that are present in aqueous solution. The sialic acid residue of sialyl Lewis(x) bound to AAA adopts an orientation similar to that in the corresponding sialyl Lewis(x)/E-selectin complex.
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PMID:Molecular recognition of sialyl Lewis(x) and related saccharides by two lectins. 1167 3

Transferred nuclear Overhauser enhancement (TRNOE) experiments have been performed at 800 MHz to investigate the bound conformation of the hexapeptide DRPVPY, a functional molecular mimic of the group A Streptococcus cell-wall polysaccharide. The hexapeptide binds to the monoclonal antibody SA-3, mimicking the branched trisaccharide repeating unit, L-Rha-alpha-(1 --> 2)-(D-GlcNAc-beta-(1 --> 3))-alpha-L-Rha (Rha, rhamnose; GlcNAc, N-acetylglucosamine). The peptide adopts a tight turn conformation with close contacts between the side chains of valine and tyrosine. Relaxation network editing experiments (QUIET-NOESY) were used to confirm the validity of the observed contacts and to evaluate the presence of spin diffusion pathways. Saturation transfer difference (STD-NMR) experiments with selective saturation of protein resonances revealed enhancements of many of the peptide resonances due to close contacts between the peptide and the protein within the antibody combining site.
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PMID:NMR studies of the antibody-bound conformation of a carbohydrate-mimetic peptide. 1184 Dec 5

Multiple mechanisms are involved in the resistance of cancer cells to cisplatin, including the expression of multidrug resistance-associated protein (MRP) and enhanced DNA repair. Here, we report findings to show that oligosaccharide changes in alpha5beta1 integrin are associated with cisplatin resistance in a head and neck squamous cell carcinoma cell line, HSC-2. Cisplatin-resistant HSC-2 (HSC-2/CR) cells were established by stepwise treatment with various concentrations of cisplatin. The oligosaccharides containing beta1, 6-N-acetylglucosamine (beta1-6GlcNAc) branching, detected by leukoagglutinating phytohemagglutinin (L(4)-PHA) lectin blot, were found to be dramatically decreased in alpha5beta1 integrin immunoprecipitated from HSC-2/CR cells. To better understand the mechanisms underlying cisplatin resistance and oligosaccharide alteration, we analyzed the downstream signaling of alpha5beta1 integrin, one of the target glycoproteins of beta1-6GlcNAc transferase [UDP-GlcNAc:alpha-D-mannoside beta1, 6-N-acetylglucosaminyltransferase (GnT-V)]. Cell adhesion to fibronectin and phosphorylation of focal adhesion kinase (FAK), which are associated with alpha5beta1 integrin and involved in a cell survival signaling, were found to be increased in the cisplatin-resistant cells. Enhancement of the inhibition of cell adhesion and FAK phosphorylation also support the above data in GnT-V transfectants of HSC-2 cells. Interestingly, the differences in sensitivity to cisplatin and FAK phosphorylation between cisplatin-sensitive and -resistant cells were completely abolished by treatment with a neutral antibody of alpha5beta1 integrin. These results suggest that modification of oligosaccharides of alpha5beta1 integrin represents one of the possible mechanisms of drug resistance in head and neck cancer cells.
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PMID:Involvement of oligosaccharide changes in alpha5beta1 integrin in a cisplatin-resistant human squamous cell carcinoma cell line. 1461 94

The enzyme beta1,4-N-acetylglucosaminyltransferase III (GnT-III) catalyzes the addition of a bisecting GlcNAc residue to glycoproteins, resulting in a modulation in biological function. Our previous studies showed that the transfection of the GnT-III gene into B16 melanoma cells results in a suppression of invasive ability and lung colonization. The suppression has been postulated to be due to an increased level of E-cadherin expression on the cell surface, which in turn leads to the up-regulation of cell-cell adhesion. In this study, we report on the effects of overexpression of GnT-III on cell-matrix adhesion. The overexpression of GnT-III, but not that of an enzymatic inactive GnT-III (D323A), inhibits cell spreading and migration on fibronectin, a specific ligand for integrin alpha(5)beta(1), and the focal adhesion kinase phosphorylation. E(4)-PHA lectin blot analyses showed that the levels of bisecting GlcNAc structures on the integrin alpha(5) subunit as well as alpha(2) and alpha(3) subunits immunoprecipitated from GnT-III transfectants were substantially increased. In addition, the affinity of the binding of integrin alpha(5)beta(1) to fibronectin was significantly reduced by the introduction of the bisecting GlcNAc, to the alpha(5) subunit. These findings suggest that the modification of N-glycan of integrin by GnT-III inhibits its ligand binding ability, subsequently leading to the down-regulation of integrin-mediated signaling.
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PMID:Introduction of bisecting GlcNAc into integrin alpha5beta1 reduces ligand binding and down-regulates cell adhesion and cell migration. 1499 99

An N-linked glycan often increased during oncogenic transformation contains beta(1,6)-linked GlcNAc, synthesized by the N-acetylglucosaminyltransferase V (GnT-V). The progression of polyoma middle T-antigen oncoprotein-induced mammary carcinomas in GnT-V null mice was significantly retarded compared with that observed in wild-type mice. The matrix adhesion of mouse embryonic fibroblasts (MEF) from GnT-V null and wild-type mice was investigated to understand the mechanism by which deletion of GnT-V could retard tumor progression. GnT-V null MEF displayed enhanced adhesion to and spreading on fibronectin-coated plates with concomitant inhibition of cell migration. GnT-V null MEF also showed increased focal adhesion kinase tyrosine phosphorylation, consistent with decreased cell motility on fibronectin-coated plates. Expression of GnT-V cDNA in the null MEF reversed these abnormal characteristics, indicating the direct involvement of N-glycosylation events in these phenotypic changes. The alpha5beta1 fibronectin receptors exhibited increased clustering on the null MEF cell surfaces, consistent with previous studies that observed less integrin clustering in cells overexpressing GnT-V. Most surprisingly, GnT-V null MEF displayed increased expression levels of both alpha5 and beta1 subunits in lysates and on the cell surface. Increased alpha5beta1 expression in the null MEF was because of increased alpha5beta1 transcript levels that declined after re-expression of GnT-V cDNA, confirming that increased alpha5beta1 expression in null MEF was because of changes in GnT-V expression. The increased null MEF transcripts were shown to be caused at least in part by increased integrin promoter activity. Moreover, increased alpha5beta1 integrin transcripts in GnT-V null MEF were not due to a differential response to fibronectin; rather, they appeared to be mediated by activation of a protein kinase C signaling pathway. These results demonstrate that deletion of MEF GnT-V resulted in enhanced integrin clustering and activation of alpha5beta1 transcription by protein kinase C signaling, which in turn up-regulated levels of cell surface alpha5beta1 fibronectin receptors that resulted in increased matrix adhesion and inhibition of migration.
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PMID:Deletion of mouse embryo fibroblast N-acetylglucosaminyltransferase V stimulates alpha5beta1 integrin expression mediated by the protein kinase C signaling pathway. 1561 21

Both tyrosine-phosphorylated caveolin-1 (pY14Cav1) and GlcNAc-transferase V (Mgat5) are linked with focal adhesions (FAs); however, their function in this context is unknown. Here, we show that galectin-3 binding to Mgat5-modified N-glycans functions together with pY14Cav1 to stabilize focal adhesion kinase (FAK) within FAs, and thereby promotes FA disassembly and turnover. Expression of the Mgat5/galectin lattice alone induces FAs and cell spreading. However, FAK stabilization in FAs also requires expression of pY14Cav1. In cells lacking the Mgat5/galectin lattice, pY14Cav1 is not sufficient to promote FAK stabilization, FA disassembly, and turnover. In human MDA-435 cancer cells, Cav1 expression, but not mutant Y14FCav1, stabilizes FAK exchange and stimulates de novo FA formation in protrusive cellular regions. Thus, transmembrane crosstalk between the galectin lattice and pY14Cav1 promotes FA turnover by stabilizing FAK within FAs defining previously unknown, interdependent roles for galectin-3 and pY14Cav1 in tumor cell migration.
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PMID:Concerted regulation of focal adhesion dynamics by galectin-3 and tyrosine-phosphorylated caveolin-1. 1834 68

Beta1,4-Galactosyltransferases (beta1,4-GalTase) exposed on the cell surface are involved in cell migration. Specifically, beta1,4-GalTase V is highly expressed in glioma and promotes invasion, growth, and survival of glioma cells. A glycocalix[8]arene exposing N-acetylglucosamine (GlcNAc) residues (compound 1) inhibited rat C6 glioma cell migration as assessed in a scratch wound model. This effect was related to inhibition of focal adhesion kinase phosphorylation, measured by western blot analysis, and specifically observed in the area bordering the scratch wound. Compound 1 inhibited also C6 cell proliferation, an effect unrelated to its ability to interact with GalTase as it was mimicked by different calix[8]arene derivatives, all characterized by multivalency and ureido groups. Compound 1 did not induce apoptotic death, but caused a different distribution of C6 cells within the cell cycle. The results here reported identify compound 1 as a molecule able to exert inhibitory effects on C6 cell migration and proliferation, independently, because of distinct components in its structure.
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PMID:Inhibition of rat glioma cell migration and proliferation by a calix[8]arene scaffold exposing multiple GlcNAc and ureido functionalities. 1877 7


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