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)

L6 myoblasts stably transfected with a GLUT4 cDNA harboring an exofacial myc epitope tag (L6-GLUT4myc myoblasts) were used to study the role of protein kinase B alpha (PKBalpha)/Akt1 in the insulin-induced translocation of GLUT4 to the cell surface. Surface GLUT4myc was detected by immunofluorescent labeling of the myc epitope in nonpermeabilized cells. Insulin induced a marked translocation of GLUT4myc to the plasma membrane within 20 min. This was prevented by transient transfection of a dominant inhibitory construct of phosphatidylinositol (PI) 3-kinase (Deltap85alpha). Transiently transfected cells were identified by cotransfection of green fluorescent protein. A constitutively active PKBalpha, created by fusion of a viral Gag protein at its N terminus (GagPKB), increased the cell surface density of GLUT4myc compared to that of neighboring nontransfected cells. A kinase-inactive, phosphorylation-deficient PKBalpha/Akt1 construct with the mutations K179A (substitution of alanine for the lysine at position 179), T308A, and S473A (AAA-PKB) behaved as a dominant-negative inhibitor of insulin-dependent activation of cotransfected wild-type hemagglutinin (HA)-tagged PKB. Furthermore, AAA-PKB markedly inhibited the insulin-induced phosphorylation of cotransfected BAD, demonstrating inhibition of the endogenous PKB/Akt. Under the same conditions, AAA-PKB almost entirely blocked the insulin-dependent increase in surface GLUT4myc. PKBalpha with alanine substitutions T308A and S473A (AA-PKB) or K179A (A-PKB) alone was a less potent inhibitor of insulin-dependent activation of wild-type HA-PKB or GLUT4myc translocation than was AAA-PKB. Cotransfection of AAA-PKB with a fourfold DNA excess of HA-PKB rescued insulin-stimulated GLUT4myc translocation. AAA-PKB did not prevent actin bundling (membrane ruffling), though this response was PI 3-kinase dependent. Therefore, it is unlikely that AAA-PKB acted by inhibiting PI 3-kinase signaling. These results outline an important role for PKBalpha/Akt1 in the stimulation of glucose transport by insulin in muscle cells in culture.
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PMID:Protein kinase B/Akt participates in GLUT4 translocation by insulin in L6 myoblasts. 1033 Jan 41

Urokinase-type plasminogen activator (uPA) and its receptor (uPAR) participate in matrix degradation and cell migration by focusing proteolysis and functioning as a signaling ligand/receptor complex. uPAR, anchored by a lipid moiety in the membrane, is thought to require a transmembrane adapter to transduce signals into the cytoplasm. To study uPAR signaling, we transfected the prostate carcinoma cell line LNCaP, which does not express endogenous uPA or uPAR, with a uPAR encoding cDNA, resulting in high-level surface expression. We studied migration of these cells on fibronectin, which is mediated by the integrin alpha5beta1. Ligation of uPAR with uPA or its amino-terminal fragment enhanced haptotactic migration to fibronectin. In cells on fibronectin, but not on poly-l-lysine, ligation of uPAR also resulted in tyrosine phosphorylation of several proteins, including two proteins involved in integrin signaling, focal adhesion kinase and the crk-associated substrate p130(Cas). Furthermore, after uPAR ligation, uPAR was co-immunoprecipitated with beta1 integrins from the detergent-insoluble fraction of cell lysates. Thus, our data suggest that uPAR occupancy results in an interaction between uPAR and integrins and a potentiation of integrin-mediated signaling, which leads to enhanced cell migration.
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PMID:Urokinase-type plasminogen activator binding to its receptor stimulates tumor cell migration by enhancing integrin-mediated signal transduction. 1038 37

For the construction of macromolecule-drug conjugates, it is important to provide rational basis to the selection of proper carrier. With respect to the importance of the side-chain structure and charge of the branched polypeptides in biological properties, we have prepared a new class of branched polypeptides with single or multiple hydroxyl groups and studied their solution conformation, in vitro cytotoxicity, biodistribution, and immunoreactivity. For comparative studies, polypeptides were designed to contain serine at various positions of the side chains, varying also the number. Ser was attached to the end of oligo(DL-Ala) side chains grafted to polylysine resulting polypeptides with the general formula poly[Lys(Ser(i)-DL-Ala(m))], (SAK). Ser was also coupled directly to the polylysine backbone poly[Lys(Ser(i))] (S(i)K) and then elongated by polymerization of N-carboxy-DL-Ala anhydride resulting poly[Lys(DL-Ala(m)-Ser(i))] (ASK). An additional polymer was also prepared, but instead of the oligo(DL-Ala) branches, oligo(DL-Ser) side chains were introduced (poly[Lys(DL-Ser(m))], SK). The presence of hydroxyl groups resulted in compounds with improved of water solubility. CD spectra of polypeptides showed significant differences correlating with the position and numbers of Ser residues in the side chains. Under physiological conditions, polycationic polypeptides assumed ordered secondary structure (S(i)K and LSK) or partially unordered conformation (SK, SAK, and ASK). Data of selected polymers demonstrate that these polycationic compounds are essentially nontoxic in vitro on normal rat liver or mouse spleen cells and have no cytostatic effect on mouse colorectal carcinoma C26 cells. The blood clearance and biodistribution of these derivatives were greatly dependent on the position and number of Ser residues in the branches and possess a rather extended blood survival in mice. Polypeptides were taken up predominantly by the liver and kidney (S(i)K, LSK, and ASK) or kidney and lung (SK and SAK). The best survival in the blood was found with SAK, representing the first polycationic branched polypeptide, which show extended blood clearance. The relative position of Ser residue had also a marked influence on the immunogenicity of polypeptides. The characteristics of the antibody response to polypeptide containing Ser at the end of the branches (SAK) or adjacent to the polylysine backbone (ASK) was also dependent on the genetic background of the mouse strains. We also found that these compounds have no effect on to the SRBC-specific humoral immune response, indicating the lack of nonspecific immunostimulatory potential. In conclusion, these studies suggest that synthetic branched polypeptides with Ser can be considered as candidates for constructing suitable conjugates for drug/epitope delivery. It is not only due to the presence of hydroxyl group to be used for oxime chemistry but also to their beneficial biological features.
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PMID:Carrier design: new generation of polycationic branched polypeptides containing OH groups with prolonged blood survival and diminished in vitro cytotoxicity. 1050 43

The preparation of antibodies against the liver toxin microcystin, as described here, is of major importance for its detection and purification in food and water, and for a therapeutic approach to neutralize the toxin by passive immunization. Microcystin-LR (MLR) and microcystin-RR (MRR) were purified from cyanobacterial cell materials by extraction, Sephadex LH-20-, ODS silica gel-, ionic exchange and RP-HPLC-chromatography. In order to reduce the toxicity for parenteral administration, microcystins were coupled by the carbodiimide method to poly-L-lysine (PLL(50.000)). Mice and rabbits were immunized with the conjugates in the presence of two lipopeptide immunoadjuvants (P(3)CSK(4) and P(3)CS-T(h)). High MLR-specific antibody levels were observed after parenteral coadministration of antigen and lipopeptides, whereas no anti-MLR antibodies were obtained with free microcystin or the microcystin-PLL(50.000)-conjugate in the absence of lipopeptide. In oral immunization, coadministration of antigen and adjuvants resulted in an accelerated development of anti MLR-specific antibodies and high antibody levels. Using the antisera, we could detect different microcystins and nodularin down to a concentration range of 10-50 ng/ml by a competitive inhibition ELISA; detection of microcystins in crude cell preparations was also possible. Furthermore, microcystins from different sources could be detected and discriminated from cyclic cyanopeptolines.
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PMID:Generation of antibodies directed against the low-immunogenic peptide-toxins microcystin-LR/RR and nodularin. 1070 82

Src-homology 3 (SH3) domains recognize PXXP core motif preceded or followed by positively charged residue(s). Whether SH3 domains recognize motifs other than proline-based sequences is unclear. In this study, we report SH3 domain binding to a novel proline-independent motif in immune cell adaptor SKAP55, which is comprised of two N-terminal lysine and arginine residues followed by two tyrosines (i.e. RKxxYxxY). Domains capable of binding to class I proline motifs bound to the motif, while the class II domains failed to bind. Peptide precipitation, alanine scanning and in vivo co-expression studies demonstrated a requirement for the arginine, lysine and tandem tyrosines of the motif. Two-dimensional NMR analysis of the peptide bound FYN-SH3 domain showed overlap with the binding site of a proline-rich peptide on the charged surface of the SH3 domain, while resonance signals for other residues (W119, W120, Y137) were not perturbed by the RKGDYASY based peptide. Expression of the RKGDYASY peptide potently inhibited TcRzeta/CD3-mediated NF-AT transcription in T cells. Our findings extend the repertoire of SH3 domain binding motifs to include a tyrosine-based motif and demonstrate a regulatory role for this motif in receptor signaling.
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PMID:SH3 domain recognition of a proline-independent tyrosine-based RKxxYxxY motif in immune cell adaptor SKAP55. 1085 34

We have previously shown that in a HEK-293 cell line that overexpresses the C1a isoform of the calcitonin receptor (C1a-HEK), calcitonin induces the tyrosine phosphorylation of the focal adhesion-associated proteins HEF1 (a p130(Cas)-like docking protein), paxillin, and focal adhesion kinase and that it also stimulates the phosphorylation and activation of Erk1 and Erk2. We report here that cell attachment to the extracellular matrix, an intact actin cytoskeleton, and c-Src are absolutely required for the calcitonin-induced phosphorylation of focal adhesion-associated proteins. In contrast to the phosphorylation of paxillin and HEF1 in cells attached to fibronectin-coated dishes, calcitonin failed to stimulate the phosphorylation of paxillin and HEF1 in suspended cells, in cells attached to poly-d-lysine-coated dishes, and in attached cells pretreated with the RGD-containing peptide GRGDS. Overexpression of wild-type c-Src increased calcitonin-induced paxillin and HEF1 phosphorylation, whereas overexpression of kinase-dead Src or Src lacking a functional SH2 domain inhibited the calcitonin-stimulated tyrosine phosphorylation of these proteins. Overexpression of Src lacking the SH3 domain did not affect the calcitonin-induced phosphorylation of paxillin and HEF1. In contrast to the regulation of paxillin and HEF1 phosphorylation, the calcitonin-induced phosphorylation of Erk1 and Erk2 did not appear to involve c-Src and was only partially dependent on cell adhesion to the extracellular matrix and an intact actin cytoskeleton. Furthermore, inhibition of Erk1 and Erk2 phosphorylation had no effect on the calcitonin-induced phosphorylation of paxillin and HEF1. Thus, in C1a-HEK cells, the calcitonin receptor is coupled to the tyrosine phosphorylation of focal adhesion-associated proteins and to Erk1/2 phosphorylation by mechanisms that are in large part independent.
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PMID:Integrin engagement, the actin cytoskeleton, and c-Src are required for the calcitonin-induced tyrosine phosphorylation of paxillin and HEF1, but not for calcitonin-induced Erk1/2 phosphorylation. 1095 2

Since osteoblast proliferation is critical for bone development, the effect of bone extracellular matrix (ECM) proteins on osteoblast signaling and proliferation in serum-free medium was investigated. Proliferation was highest in primary rat calvarial osteoblasts cells grown on fibronectin but less on type I collagen; osteonectin and poly-L-lysine did not support early proliferation. Fibronectin and type I collagen binding requires integrins, whereas cell adhesion to osteonectin or poly-L-lysine does not involve integrins. Therefore, the role of integrins in osteoblast signaling, leading to the induction of AP-1 transcription factors (c-fos and c-jun) which are important in cell proliferation, was studied. c-fos and c-jun message levels were increased at 60 min in osteoblasts plated onto fibronectin or collagen, but not in cells on osteonectin or poly-L-lysine. Protein synthesis was not required for c-fos mRNA expression; however, kinase activity was necessary for c-fos induction. In cells plated onto fibronectin, c-fos mRNA levels were controlled by protein kinase C and phosphotyrosine kinase signaling pathways. In contrast, c-fos levels in collagen-adhering cells may involve protein kinase A. The signaling pathway involving the phosphorylation of focal adhesion kinase and mitogen-activated kinases was also shown to be transiently increased in osteoblasts on fibronectin and type I collagen, but not in cells on poly-L-lysine. These results demonstrate that osteoblast binding to the extracellular matrix through integrins induces c-fos and c-jun, and that both fibronectin and collagen affect these AP-1 transcription factors through protein kinase-sensitive pathways. Thus, osteoblast proliferation is modulated differentially by specific ECM components.
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PMID:Integrin-mediated signaling regulates AP-1 transcription factors and proliferation in osteoblasts. 1103 56

The intracellular signaling pathway for osteoblast adhesion to the orthopedic implant material Ti6Al4V (TIV) was investigated and compared to integrin-mediated adhesion to extracellular matrix proteins. Primary osteoblasts from fetal rat calvaria were plated onto TIV, fibronectin (FN), and poly-L-lysine (PLL) and the levels of focal adhesion kinase (FAK), mitogen-activated protein kinase (MAPK), and AP-1 transcription factors, c-fos and c-jun, were compared by Western and Northern blots. Cells on all substrates showed maximum FAK phosphorylation within 60 min and then a decrease at 2 and 24 h. However, the subsequent signal transduction pathway differed on PLL compared to TIV and FN. MAPK was phosphorylated similarly in osteoblasts attached to FN and TIV, whereas cells on PLL demonstrated no MAPK phosphorylation. On TIV and FN, c-fos and c-jun mRNA levels were maximal within 1 h and then plateaued or declined by 2 h. On PLL, they increased at 2 h. Within 1 h, c-fos protein was stimulated in cells attached to TIV and FN and decreased in cells on PLL. c-jun protein increased on all substrates compared to unplated cells. Cytoskeletal changes visualized by phalloidin fluorescence microscopy at 4 h of culture were delayed on TIV compared to FN. In addition, approximately 50% fewer cells adhered to TIV compared to FN or PLL. By 24 h, a well-spread cytoskeleton with focal adhesion sites was apparent on TIV and FN, but cells on PLL were rounded with minimal cell spreading. During 6 days of culture, cells on FN and TIV proliferated, whereas the number of cells on PLL remained the same or decreased, depending on the initial plating density. We conclude that osteoblast adhesion to TIV implants is similar to osteoblast adhesion to FN and leads to osteoblast proliferation. These data provide evidence for the biocompatibility of TIV at a molecular level.
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PMID:Integrin-mediated signaling in osteoblasts on titanium implant materials. 1103 57

PSE-4 is a class A beta-lactamase produced by strains of Pseudomonas aeruginosa and is highly active for the penicillin derivative carbenicillin. The crystal structure of the wild-type PSE-4 carbenicillinase has been determined to 1.95 A resolution by molecular replacement and represents the first structure of a carbenicillinase published to date. A superposition of the PSE-4 structure with that of TEM-1 shows a rms deviation of 1.3 A for 263 Calpha atoms. Most carbenicillinases are unique among class A beta-lactamases in that residue 234 is an arginine (ABL standard numbering scheme), while in all other class A enzymes this residue is a lysine. Kinetic characterization of a R234K PSE-4 mutant reveals a 50-fold reduction in k(cat)/K(m) and confirms the importance of Arg 234 for carbenicillinase activity. A comparison of the structure of the R234K mutant refined to 1.75 A resolution with the wild-type structure shows that Arg 234 stabilizes an alternate conformation of the Ser 130 side chain, not seen in other class A beta-lactamase structures. Our molecular modeling studies suggest that the position of a bound carbenicillin would be shifted relative to that of a bound benzylpenicillin in order to avoid a steric clash between the carbenicillin alpha-carboxylate group and the conserved side chain of Asn 170. The alternate conformation of the catalytic Ser 130 in wild-type PSE-4 may be involved in accommodating this shift in the bound substrate position.
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PMID:Insights into the molecular basis for the carbenicillinase activity of PSE-4 beta-lactamase from crystallographic and kinetic studies. 1114 33

Integrins are cell surface receptors for extracellular matrix, which play important roles in a variety of biological processes. 14-3-3 proteins are a highly conserved family of cytoplasmic proteins that associate with several intracellular signaling molecules in regulation of various cellular functions. Here, we report identification of an interaction between the integrin beta1 cytoplasmic domain and 14-3-3beta by using the yeast two-hybrid screen. Like several other proteins, the integrin beta1 cytoplasmic domain associated with 14-3-3beta by a non-phosphoserine mechanism. The 14-3-3beta/integrin beta1 interaction was confirmed by in vitro binding assays as well as co-precipitation in vivo. Furthermore, we found that 14-3-3beta co-localized with integrin beta1 during the early stage of cell spreading on fibronectin, suggesting a potential role of the 14-3-3beta/integrin beta1 interaction in the regulation of cell adhesion. Using tetracycline-regulated expression system, we showed that overexpression of 14-3-3beta stimulated cell spreading and migration on fibronectin but not on poly-L-lysine. However, the induced expression of 14-3-3beta did not affect tyrosine phosphorylation of FAK or its substrates, p130(cas) and paxillin, suggesting that 14-3-3beta regulated integrin-mediated cell spreading and migration by FAK-independent mechanisms. Taken together, these results identify an interaction between integrin and 14-3-3 proteins and suggest a potentially novel cellular function for 14-3-3 proteins in the regulation of integrin-mediated cell adhesion and signaling events.
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PMID:Identification of a novel interaction between integrin beta1 and 14-3-3beta. 1131 64


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