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)

Many cell-surface and extracellular matrix proteins contain multiple modular domains known as fibronectin type III (FNIII) repeats. Cells adhere to the extracellular matrix proteins fibronectin and tenascin in part by the interaction of certain integrins with the Arg-Gly-Asp (RGD) sequence, displayed on specific FNIII repeats. We have found that, after experimental activation of beta1 integrins, a number of cell types adhere and spread on FNIII repeats lacking RGD, derived from extracellular matrix proteins and cytokine receptors. Interaction between individual FNIII domains and beta1 integrins mediates focal adhesion kinase phosphorylation and subsequent stress fiber and focal contact formation. These data suggest that many FNIII-containing proteins may bind and signal through activated beta1 integrins, dramatically expanding the potential for integrin-dependent intercellular and cell-matrix communication.
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PMID:Fibronectin type III repeats mediate RGD-independent adhesion and signaling through activated beta1 integrins. 939 78

The rat L6 skeletal muscle cell line was used to study expression of the dystrophin-containing glycoprotein complex and its interaction with the integrin system involved in the cell-matrix adhesion reaction. A complex of dystrophin and its associated proteins was fully expressed in L6 myotubes, from which anti-dystrophin or anti-alpha-sarcoglycan co-precipitated integrin alpha 5 beta 1 and other focal adhesion-associated proteins vinculin, talin, paxillin, and focal adhesion kinase. Immunostaining and confocal microscopy revealed that dystrophin, alpha-sarcoglycan, integrin alpha 5 beta 1, and vinculin exhibited overlapping distribution in the sarcolemma, especially at focal adhesion-like, spotty structures. Adhesion of cells to fibronectin- or collagen type I-coated dishes resulted in induction of tyrosine phosphorylation of alpha- and gamma-sarcoglycans but not beta-sarcoglycan. The same proteins were also tyrosine-phosphorylated when L6 cells in suspension were exposed to Arg-Gly-Asp-Ser peptide. All of these tyrosine phosphorylations were inhibited by herbimycin A. On the other hand, treatment of L6 myotubes with alpha- and gamma-sarcoglycan antisense oligodeoxynucleotides resulted in complete disappearance of alpha- and gamma-sarcoglycans and in significant reduction of levels of the associated focal adhesion proteins, which caused about 50% reduction of cell adhesion. These results indicate the existence of bidirectional communication between the dystrophin-containing complex and the integrin adhesion system in cultured L6 myocytes.
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PMID:Bidirectional signaling between sarcoglycans and the integrin adhesion system in cultured L6 myocytes. 943 Jun 99

The beta1 integrin adhesion receptors activate signal transduction pathways that induce tyrosine phosphorylation of a variety of substrates. Increased tyrosine phosphorylation is mediated by the beta1 subunit cytoplasmic domain, which consists of 46 amino acids and contains no intrinsic kinase activity. In the H9 T cell line, beta1 integrin engagement leads to the increased tyrosine phosphorylation of three 105 to 115-kDa substrates that are distinct from focal adhesion kinase (FAK): HEF1 (human enhancer of filamentation 1), a protein with structural homology to p130Cas, and two novel substrates, pp105 and pp115. DNA-mediated gene transfer was used to explore the role of the beta1 cytoplasmic domain in integrin-mediated tyrosine phosphorylation of HEF1, pp105, and pp115 in human T cells. Using a chimeric receptor composed of the cytoplasmic domain of the beta1 integrin subunit and the extracellular and transmembrane domains of the CD2 Ag, we demonstrate that the beta1 cytoplasmic domain is necessary and sufficient for inducing tyrosine phosphorylation of each of these three substrates in H9 T cells. Analysis of a series of beta1 cytoplasmic domain truncations reveals that a truncation of only five amino acids from the carboxyl-terminal end of the beta1 cytoplasmic domain abrogates the ability of the CD2/beta1 chimera to activate tyrosine phosphorylation of HEF1, pp105, or pp115. Thus, the carboxyl-terminal five amino acids, Lys-Tyr-Glu-Gly-Lys (KYEGK), of the beta1 integrin cytoplasmic domain are critical for the coordinate tyrosine phosphorylation of three non-FAK substrates in human T cells.
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PMID:Structural requirements for beta1 integrin-mediated tyrosine phosphorylation in human T cells. 954 75

The human colon adenocarcinoma cell lines Colo 201 and Colo 205 lose adhevise capacity to the extracellular matrix (ECM) and take on a round and floating cell shape. Treatment of these cells with all-trans-retinoic acid (RA) results in inhibition of growth and in a marked increase in the production of carcinoembryonic antigen, thereby indicating that the cells undergo differentiation. This RA-induced differentiation was accompanied by a large increase in the degree of cell adhesion with localization of E-cadherin molecules at cell-cell contact sites. We examined several adhesion molecules involved in cell-cell and cell-ECM interaction by immunoblotting, but no change in E-cadherin, intercellular adhesion molecule-1, or CD44 was observed in RA-treated Colo 201 cells. Although the adhesion of Colo 201 cells to ECM depends on the Arg-Gly-Asp sequence, levels of integrins, alpha 2, alpha 3, alpha 5, alpha V, and beta 1 in differentiated adherent cells were similar to those in untreated cells. In contrast to equivalent amounts of cell surface adhesion molecules before and after differentiation, intracellular focal adhesion kinase (FAK) was markedly induced during RA treatment, and the increase in FAK resulted in elevation of tyrosine-phosphorylated FAK. These findings suggest a role for FAK in activation of cell adhesion of RA-induced differentiation of these colon cancer cells. This may serve as an appropriate model to examine the mode of activation of the adhesive capacity of cancer cells.
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PMID:Acquisition of cell adhesion and induction of focal adhesion kinase of human colon cancer Colo 201 cells by retinoic acid-induced differentiation. 956 10

HOE 901 is a new biosynthetic long-acting human insulin analog (GLY[A21]ARG[B31]ARG[B32]). We compared HOE 901 with normal human insulin and the insulin analog Asp(B10), which is known to have increased mitogenic activity at least partially mediated through the insulin receptor. We have analyzed receptor binding, insulin-induced receptor autophosphorylation and phosphorylation of receptor substrates in rat-1 fibroblasts overexpressing human insulin receptor isoform A (HIR A) or B (HIR B). In HIR A expressing cells, insulin and its analogs showed no significant differences in receptor association while clearly different dissociation kinetics were observed. In HIR B expressing cells, no significant differences in association and dissociation kinetics were observed. All insulins induced rapid autophosphorylation of the insulin receptor reaching a maximum after 10 min of stimulation. Asp(B10)insulin induced a prolonged phosphorylation state (over 60 minutes) of the 95 kDa receptor beta-subunit and of the substrates IRS-1/IRS-2 and Shc in contrast to normal human insulin and to HOE 901. In addition, we observed an increased and prolonged tyrosine phosphorylation of an unidentified protein with Asp(B10)insulin at about 60 kDa. Insulin-dependent dephosphorylation of the focal adhesion kinase (p125FAK) was equally induced by all these ligands. With respect to [3H]thymidine incorporation into DNA, HOE 901 had similar effects as normal human insulin, while Asp(B10)insulin showed increased [3H]thymidine incorporation. In summary, the data show that the increased mitogenic activity of Asp(B10)insulin may be explained with a prolonged kinetics of tyrosine phosphorylation of the insulin receptor and of insulin signalling elements together with the preferential phosphorylation of an yet unidentified 60 kDa protein. HOE 901 behaves with respect to insulin receptor binding, receptor autophosphorylation, phosphorylation of signalling elements and promotion of mitogenesis like regular human insulin.
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PMID:The long acting human insulin analog HOE 901: characteristics of insulin signalling in comparison to Asp(B10) and regular insulin. 956 52

Interaction of epithelial cells with the extracellular matrix is mediated through integrin receptors, which transmit signals regulating cell growth, differentiation and death. Occupation of these receptors, via Arg-Gly-Asp (RGD) recognition sequences, leads to activation of focal adhesion kinase (FAK). We treated human breast cancer cell lines with RGD-containing peptides, which can disrupt integrin attachment, and investigated alterations in FAK phosphorylation, cell detachment and death. Cells grown in vitro were treated with insulin-like growth factor-binding protein-1 (IGFBP-1) and a small, synthetic RGD-containing peptide (Gly-Arg-Gly-Asp-Thr-Pro) and its negative control peptide RGE (Arg-Gly-Glu-Ser) for either 30 min followed by immunoprecipitation of cell lysates with anti-phosphotyrosine and Western immunoblotting with anti-FAK or for 24 h followed by cell counting, immunocytochemistry and flow cytometry. Both IGFBP-1 (0-800 ng/ml) and the synthetic RGD-containing peptide (1-100 microg/ml) caused significant dephosphorylation of FAK. Furthermore, after 24 h both peptides caused detachment from the matrix and the induction of apoptosis. We conclude from these data that IGFBP-1 can interact with integrin receptors to induce FAK dephosphorylation and subsequently influence attachment and cell death.
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PMID:Effect of insulin-like growth factor binding protein-1 on integrin signalling and the induction of apoptosis in human breast cancer cells. 1019 17

The change in amino acid enrichment, an indicator of a change in protein synthesis and/or degradation, is usually measured using gas chromatography-mass spectrometry and/or (GC-combustion) isotope ratio mass spectrometry. Unfortunately, often a complex and sensitive derivatization procedure and/or a large amount of sample is required. Also, these techniques are less suited to study intermediary metabolism, in which the simultaneous application (and thus measurement) of multiple amino acid tracers is preferred. Alternatively, in this study the possibilities of the coupling of liquid chromatography and mass spectrometry were explored, resulting in the measurement of both the concentration and isotope enrichment of o-phthaldialdehyde (OPA)-derivatizated plasma amino acids in one run. This was achieved by the injection of OPA-derivatizated amino acids into an automated HPLC system. After the elution of buffer salts and reagent excess to drain using column switching, the column effluent was directed via a fluorescence detector into a Thermoquest Model LCQ benchtop LC-MS. Mass spectrometric measurements were performed in "zoom-scan" mode, employing multiple scan events if the target components were not baseline separated. Best signal-to-noise ratio's were obtained using the LCQ's electrospray probe in the negative mode. Still, when working under standard conditions the total ion current of OPA-amino acid derivatives eluting at the beginning of the chromatogram (e.g., citrulline, arginine and glycine) was by a factor of 5 lower, compared to components eluting in the last part of the chromatogram (leucine, valine, and ornithine). These differences could be minimized by increasing the temperature of the heated capillary to 260 degrees C and by applying 5% collision energy (between the skimmer and the first octapole) to the first eluting components. A further improvement could not be obtained by the addition of makeup liquids like ammonia, acetic acid, methanol, or acetonitrile (up to 25% of column effluent flow). Considering these results and the fact that the first eluting amino acid derivatives are the most polar ones, we hypothesized that hydration of these components interferes with the ionization process. A linear calibration curve was obtained for both fluorescent response and total ion current (TIC) for all amino acids in the range from 5 to 1000 pmol per injection. The coefficient of variation of the fluorescent response was typically on the order of 1-4%, for the TIC this was between 4 and 9%. However, measurement of isotope ratios requires not only the determination of the area of the base peak, but also of the area of the (enriched) isotopomeric peak(s), having a much lower abundance. Therefore, isotope ratio measurements require the injection of at least 25 pmol of the amino acid derivative of interest (except for ARG 50 pmol) to obtain true ratio's. The accuracy of the isotope enrichment measurement was determined by the injection of a standard containing all major physiological amino acids (400 pmol each) and a standard at physiological concentrations (ranging from 50 pmol (CIT) to 350 pmol (VAL). Standard deviation of the isotopic ratios ranged from 0.1 to 0. 5% for the high (400 pmol) standards and from 0.2 to 0.8% for the low (physiological) standard, which is comparable with GC-MS. A plot of the results against the theoretical values gave a linear curve for all isotopes studied (R2 ranged from 0.9984 to 0.9997). However, the [1-13C]-enriched amino acids measured (LEU, GLY, and VAL) gave a closer agreement to the expected values as was found for [ureido-13C-5,5-2H2]-enriched citrulline and [guanidino-15N2]-enriched arginine. We could not determine whether this was due to the measurement procedure itself or resulting from an instability of the tracers in solution. Nevertheless, the results were reproducible and the theoretical value could be calculated using the tangent of the enrichment curves. (ABSTRACT TRUNCATED)
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PMID:Determination of amino acid isotope enrichment using liquid chromatography-mass spectrometry. 1036 Sep 99

Platelet activation has been a focus of numerous studies in normal and abnormal states. Morphological changes and calcium signals found with activated platelets in vitro have been well characterized. However, the rate of cell spreading on substrates and the frequency of calcium oscillation within individual platelets upon activation have not yet been reported. In this study, we first examined the ability of a recombinant fusion protein of rhodostomin (GST-rhodostomin), a snake disintegrin containing an Arg-Gly-Asp (RGD) motif, to activate platelets when GST-rhodostomin served as a substrate. Four aspects of platelet activities induced by immobilized GST-rhodostomin and fibrinogen were analyzed in parallel. Examinations of (1) translocation of P-selectin from intracellular compartments to the plasma membrane, (2) platelet adhesion to and spreading on substrates, (3) platelet contact pattern on substrates, and (4) the degree of phosphorylation of focal adhesion kinase in platelets indicated that GST-rhodostomin was a better substrate for platelet activation than fibrinogen. Analysis of the rate of platelet spreading on GST-rhodostomin was examined by time-lapsed video microscopy. The spreading rate averaged 0.43 micrometer/minute, while cell spreading averaged 0.22 micrometer/minute when platelets were plated on fibrinogen and treated with thrombin. A newly developed method, using time-lapsed microscopy and the Metamorph program, was used to analyze calcium signals within platelets. We found that platelets on GST-rhodostomin evoked calcium oscillation at a frequency of 4.77 spike/cell/minute vs 2.76 spike/cell/minute on fibrinogen. The results of cell spreading and calcium oscillation were consistent with the results of microscopic and biochemical assays. We therefore conclude that the determination of the rate of platelet spreading and the frequency of calcium oscillation within platelets performed in this study provides more quantitative parameters for measuring platelet activities. Our results also suggest that GST-rhodostomin might potentially be used as a probe to dissect the molecular mechanisms underlying the kinetic processes of platelet activation.
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PMID:Recombinant rhodostomin substrates induce transformation and active calcium oscillation in human platelets. 1041 93

CD47-binding sequences from the carboxyl-terminal domain of thrombospondin-1 (TSP1) are known to regulate activity of the alpha(v)beta(3) integrin (Gao, G., Lindberg, F. P., Dimitry, J. M., Brown, E. J., and Frazier, W. A. (1996) J. Cell Biol. 135, 533-544). Here we show that peptides from the type 1 repeats of TSP1 also stimulate alpha(v)beta(3) integrin function in melanoma cells. Addition of soluble peptide 246 (KRFKQDGGWSHWSPWSS) enhances spreading of A2058 melanoma cells on several alpha(v)beta(3) integrin ligands, including vitronectin, recombinant TSP1 fragments containing the Arg-Gly-Asp sequence, and native TSP1. This activity requires the Trp residues and is independent of CD36-binding sequences in the type 1 repeats. Recombinant type 1 repeats expressed as a glutathione S-transferase fusion protein also enhance spreading on vitronectin and TSP1. Activation of alpha(v)beta(3) integrin by the soluble peptide 246 stimulates organization of F-actin and increases tyrosine phosphorylation of focal adhesion kinase. In contrast, direct adhesion of melanoma cells on immobilized peptide 246 inhibits tyrosine phosphorylation of focal adhesion kinase. Stimulation of alpha(v)beta(3) integrin function by the type 1 repeat peptide differs from that induced by CD47-binding TSP1 peptides in that heparan sulfate proteoglycans are required and pertussis toxin does not inhibit the former activity. Thus, the type 1 repeats contain a second sequence of TSP1 that can enhance alpha(v)beta(3) integrin signaling, and these two sequences stimulate recognition of both vitronectin and TSP1 by the alpha(v)beta(3) integrin.
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PMID:Cooperation between thrombospondin-1 type 1 repeat peptides and alpha(v)beta(3) integrin ligands to promote melanoma cell spreading and focal adhesion kinase phosphorylation. 1042 59

In order to obtain a mutant of Sindbis virus (SV) with a low methionine-resistant (LMR) phenotype, i.e., able to replicate in methionine-deprived Aedes albopictus mosquito cells, standard SV (SV(STD)) was passaged 17 times in mosquito cells maintained in a low methionine medium and then plaque-purified, also in mosquito cells. Although the virus obtained by this procedure, SV(LM17), did have the desired LMR phenotype, it also appeared to have acquired a host-range phenotype. We have now characterized the host-range phenotype of SV(LM17) in greater detail. In yield assays, the titer of SV(LM17) produced by chick embryo fibroblasts (CEF) was 100- to 1000-fold lower than that from mosquito cells. SV(STD), in contrast, produced a similar titer of virus from the two cell types. On the other hand, when SV(LM17) was assayed directly by plaque formation on CEF and on mosquito cell monolayers, no host restriction in CEF was observed. When CEF were infected with SV(LM17), viral proteins were synthesized normally, pE2 was processed to E2, and E2 was demonstrated by the fluorescent antibody method to reach the cell surface. However, electron microscopy of SV(LM17)-infected cells revealed an absence of extracellular virions and of budding particles; also, nucleocapsids were not aligned beneath the plasma membrane. By sequence determination and by site-directed mutagenesis, it was determined that the host restriction of SV(LM17) was due to a change from Ala to Val at position 251 of the E2 protein. Substitution of Gly or Leu at this position also resulted in the same host range phenotype.
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PMID:An amino acid change in the exodomain of the E2 protein of Sindbis virus, which impairs the release of virus from chicken cells but not from mosquito cells. 1054 44


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