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)

The tumor-promoting phorbol ester, phorbol 12-myristate 13-acetate (PMA), acutely stimulates the tyrosine phosphorylation of proteins of approximately 190, 120, and 70 kDa in the well differentiated Fao rat hepatoma cell line. This phosphorylation is dependent on protein kinase C (PKC) and is abolished by down-regulation of PKC or pretreatment with a PKC inhibitor. Purification of the 190-kDa tyrosine-phosphorylated protein revealed that it consists of both ErbB2 and ErbB3. Following PMA-induced tyrosine phosphorylation, ErbB2 and ErbB3 were able to associate with the SH2 domains of several signaling proteins including the p85alpha subunit of phosphatidylinositol 3-kinase, Syp, and Grb2. The 120-kDa protein phosphorylated in response to PMA consists of at least two proteins: focal adhesion kinase that exhibits a minor increase in tyrosine phosphorylation following treatment with PMA, and a major 120-kDa tyrosine-phosphorylated species in PMA-stimulated Fao cells which as yet is unidentified. Similarly, the 70-kDa tyrosine-phosphorylated protein also appears to represent more than one protein, including paxillin and a second protein of similar mobility which appears to be the major tyrosine phosphorylation in response to PMA. Both ErbB2 and paxillin also exhibit reduced migration on SDS-polyacrylamide gel electrophoresis following PMA treatment, suggesting that they are also phosphorylated on serine/threonine residues. The mobility shift of both of these proteins is abolished by treatment with inhibitors of PKC or mitogen-activated protein kinase/extracellular signal-related kinase kinase. These results suggest a novel mechanism of cross-talk between the serine/threonine kinase PKC and tyrosine phosphorylation pathways. The activation of ErbB2 and ErbB3 that is initiated by PMA may contribute to the tumor promoting activity of these compounds.
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PMID:Cross-talk between phorbol ester-mediated signaling and tyrosine kinase proto-oncogenes. I. Activation of protein kinase C stimulates tyrosine phosphorylation and activation of ErbB2 and ErbB3. 938 71

The tyrosine phosphatase SHP-1 functions as a negative regulator in hematopoietic cell development, proliferation, and receptor-mediated cellular activation. In Jurkat T cells, a major 68-kDa band and a minor 70-kDa band were immunoprecipitated by a monoclonal antibody against the SHP-1 protein-tyrosine phosphatase domain, while an antibody against the SHP-1 C-terminal 19 amino acids recognized only the 68-kDa SHP-1. The SDS-gel-purified 70-kDa protein was subjected to tryptic mapping and microsequencing, which was followed by molecular cloning. It revealed that the 70-kDa protein, termed SHP-1L, is a C-terminal alternatively spliced form of SHP-1. SHP-1L is 29 amino acids longer than SHP-1, and its 66 C-terminal amino acids are different from SHP-1. The C terminus of SHP-1L contains a proline-rich motif PVPGPPVLSP, a potential Src homology 3 domain-binding site. In contrast to SHP-1, tyrosine phosphorylation of SHP-1L is not detected upon stimulation in Jurkat T cells. This is apparently due to the lack of a single in vivo tyrosine phosphorylation site, which only exists in the C terminus of SHP-1 (Y564). COS cell-expressed glutathione S-transferase-SHP-1L can dephosphorylate tyrosine-phosphorylated ZAP70. At pH 7.4, SHP-1L was shown to be more active than SHP-1 in the dephosphorylation of ZAP70. At pH 5.4, SHP-1L and SHP-1 exhibited similar catalytic activity. It is likely that these two isoforms play different roles in the regulation of hematopoietic cell signal transduction.
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PMID:Human 70-kDa SHP-1L differs from 68-kDa SHP-1 in its C-terminal structure and catalytic activity. 1049 87

Stimulation of quiescent Swiss 3T3 cells with bombesin induces a rapid increase in the formation of complexes between focal adhesion kinase (FAK) and Src family members, which can be extracted with a buffer containing Triton, deoxycholate, and SDS but not with a buffer containing Triton alone. An increase in complex formation between FAK and Src in response to bombesin could be detected within 1 min, reached a maximum after 10 min, and declined toward base-line levels after 60 min of bombesin treatment. Bradykinin, endothelin, and lysophosphatidic acid also stimulated FAK-Src complex formation. Bombesin stimulated FAK/Src association through a Ca(2+) and phosphatidylinositol 3'-kinase-independent pathway that requires the integrity of the actin filament network and is partly dependent on functional protein kinase C. Treatment with the selective Src kinase inhibitor PP-2 inhibited both FAK activation and phosphorylation of FAK at Tyr(577) induced by bombesin in intact cells. Platelet-derived growth factor at low concentrations (1-10 ng/ml) also induced FAK-Src complex formation via a pathway that depended on the integrity of the actin cytoskeleton and phosphatidylinositol 3'-kinase. Thus, G protein-coupled receptor agonists and platelet-derived growth factor promote complex formation between endogenous FAK and Src in attached cells through different signal transduction pathways.
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PMID:Bombesin and platelet-derived growth factor induce association of endogenous focal adhesion kinase with Src in intact Swiss 3T3 cells. 1049 97

The growth hormone receptor (GHR), a cytokine receptor superfamily member, requires the JAK2 tyrosine kinase for signaling. We now examine functional interactions between growth hormone (GH) and epidermal growth factor (EGF) in 3T3-F442A fibroblasts. Although EGF enhanced ErbB-2 tyrosine phosphorylation, GH, while causing retardation of its migration on SDS-polyacrylamide gel electrophoresis, decreased ErbB-2's tyrosine phosphorylation. GH-induced retardation was reversed by treatment of anti-ErbB-2 precipitates with both alkaline phosphatase and protein phosphatase 2A, suggesting that GH induced serine/threonine phosphorylation of ErbB-2. Both GH-induced shift in ErbB-2 migration and GH-induced MAP kinase activation were unaffected by a protein kinase C inhibitor but were blocked by the mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1 (MEK1) inhibitor, PD98059. Notably, leukemia inhibitory factor, but not interferon-gamma, also promoted ErbB-2 shift and mitogen-activated protein kinase activation. Cotreatment with EGF and GH versus EGF alone resulted in a 35% decline in acute ErbB-2 tyrosine 1248 autophosphorylation, a marked decline (approximately 50%) in DNA synthesis, and substantially decreased cyclin D1 expression. We conclude that in 3T3-F442A cells, 1) the GH-induced decrease in ErbB-2 tyrosine phosphorylation correlates with MEK1/mitogen-activated protein kinase activity and 2) GH antagonizes EGF-induced DNA synthesis and cyclin D1 expression in a pattern consistent with its alteration in ErbB-2 phosphorylation status.
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PMID:Growth hormone-induced alteration in ErbB-2 phosphorylation status in 3T3-F442A fibroblasts. 1058 92

Our previous studies have shown that the Galbeta1-3GalNAcalpha- (Thomsen-Friedenreich antigen)-binding lectin from the common edible mushroom Agaricus bisporus (ABL) reversibly inhibits cell proliferation, and this effect is a consequence of inhibition of nuclear localization sequence-dependent nuclear protein import after ABL internalization [Yu, L.G., Fernig, D.G., White, M.R.H., Spiller, D.G., Appleton, P., Evans, R.C., Grierson, I., Smith, J.A., Davies, H., Gerasimenko, O.V., Petersen, O.H., Milton, J.D. & Rhodes, J.M. (1999) J. Biol. Chem. 274, 4890-4899]. Here, we have investigated further the intracellular trafficking and fate of ABL after internalization in HT29 human colon cancer cells. Internalization of 125I-ABL occurred within 30 min of the lectin being bound to the cell surface. Subcellular fractionation after pulse labelling of the cells with 125I-ABL for 2 h at 4 degrees C followed by culture of the cells at 37 degrees C demonstrated a steady increase in radioactivity in a crude nuclear extract. The radioactivity in this extract reached a maximum after 10 h and declined after 20 h. Release of ABL from the cell, after pulse labelling, was assessed using both fluorescein isothiocyanate-labelled ABL and 125I-ABL and was slow, with a t1/2 of 48 h. Most of the 125I-ABL both inside cells and in the medium remained intact, as determined by trichloroacetic acid precipitation and SDS/PAGE, and after 48 h only 22 +/- 2% of ABL in the medium and 14 +/- 2% inside the cells was degraded. This study suggests that the reversibility of the antiproliferative effect of ABL is associated with its release from cells after internalization. The internalization and subsequent slow release, with little degradation of ABL, reflects the tendency of lectins to resist biodegradation and implies that other endogenous or exogenous lectins may be processed in this way by intestinal epithelial cells.
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PMID:Intracellular trafficking and release of intact edible mushroom lectin from HT29 human colon cancer cells. 1072 53

Enterotoxigenic Escherichia coli (ETEC) expresses a broad spectrum of O:H antigens. Serogroup O20 is one of the most prevalent among the ETEC strains lacking any of the defined colonization factors (CFs), in Argentina. An O20:H- strain, ARG-3, adhered to Caco-2 cells and exhibited a thermoregulated 15.7-kDa protein band upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). An antiserum against this protein inhibited ARG-3 adhesion to Caco-2 cells and bound to very thin fibrilla-like structures on the bacterial surface. A 15.7-kDa protein-defective mutant failed to adhere to Caco-2 cells and lacked immunogold-labeled surface structures. The N-terminal amino acid sequence of the structural subunit showed 95% homology to that of CS15 of ETEC (former antigen 8786) and 65% homology with fimbria SEF14 of Salmonella enterica serovar Enteritidis. Nevertheless, the molecular size of ARG-3 adhesin was different from that of CS15, as revealed by SDS-PAGE and mass spectrometry. Both proteins are immunologically related, yet not identical, since an antiserum against the 15.7-kDa protein reacted solely with ARG-3 after absorption with bacteria bearing CS15. Moreover, only under low stringency conditions could DNA from strain ARG-3 be amplified by PCR using primers derived from the nfaA sequence of CS15. Thus, from the DNA sequence obtained from the ARG-3 PCR product, it could be deduced that the subunit protein differed in 30 residues from that of CS15. ARG-3 adhesin was found in 60% of the O20:H- CF-negative ETEC strains from Argentina; however, it appeared restricted to this serotype. We propose the designation CS22 for the herein identified nonfimbrial adhesin of human ETEC.
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PMID:CS22, a novel human enterotoxigenic Escherichia coli adhesin, is related to CS15. 1081 74

RAK antigens p120, p42, and p25 exhibit molecular and immunological similarity to the proteins encoded by HIV-1 and are expressed by 95% of breast and gynecological cancer cases in women and prostate cancer cases in men. Binding of the monoclonal antibody (MAb) RAK-BrI to cancer RAK antigens has been found to be inhibited by a peptide derived from the variable loop V3 of HIV-1. Since MAb RAK-BrI has been developed against denatured froms of breast cancer proteins, and it binds to a short epitope, GRAF, this MAb does not recognize the native, three-dimensional structure of proteins. Subsequently Western blot, after electrophoretic separation in gels with SDS, has been used to detect these unique cancer markers. The current studies were focused on the immunohistochemical evaluation of the novel marker RAK. Serial sections, 5 microm thick, were cut from frozen or Formalin-fixed, paraffin-embedded tissue blocks and immunostained with MAb RAK-BrI. All of the 53 cases of breast cancer tested RAK positive and no differences were observed in the immunohistochemical staining of lobular and ductal carcinoma cases. In contrast, MAb RAK-BrI antigens were detected in only 3 of 15 cases of macroscopically normal breast removed during mastectomy for breast cancer. It is noteworthy that Western blots of breast samples from the same series demonstrated a high expression of three RAK antigens in 20/20 of invasive breast carcinomas, while there was only a very weak expression of RAK antigens in 2/7 of the macroscopically "normal" breast samples. Due to the suspected viral origin of RAK markers, immunohistochemical staining with MAb RAK-BrI might be a useful tool in the early detection of malignant changes occurring in breast tissues.
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PMID:Immunohistochemical versus molecular detection of RAK antigens in breast cancer. 1089 Dec 90

Medullary thymic epithelial cells (mTEC) are able to present soluble antigens to CD4+ helper T cell lines, whereas cortical thymic epithelial cells (cTEC) are not (Mizuochi, T., et al., J. Exp. Med. 1992. 175: 1601-1605). In addition, class II heterodimers from mTEC migrated with apparently less relative molecular mass in SDS-PAGE than those from cTEC (Kasai, M., et al., Eur. J. Immunol. 1998. 28:1867-1876). To investigate the cause of the distinct migration profiles of class II heterodimers in both TEC types, class II heterodimer-associated peptides were analyzed by matrix-assisted laser desorption ionization mass spectrometry. Self peptides from cTEC were shown to vary moderately in length and to be highly diverse, including low amounts of CLIP (class II-associated invariant chain peptide) variants. On the other hand, self peptides from two mTEC consisted predominantly of two CLIP variants with exceptional C-terminal extensions. C-terminally overhanging residues of CLIP in mTEC may be responsible for the distinct migration of class II heterodimers in SDS-PAGE. Differences in migration of class II heterodimers on SDS gels was also observed in H2-DM+ vesicles isolated from both TEC. The possible contribution of self peptides bound to class II heterodimers in TEC to positive or negative selection of T cells in the thymus is discussed.
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PMID:CLIP-derived self peptides bound to MHC class II molecules of medullary thymic epithelial cells differ from those of cortical thymic epithelial cells in their diversity, length, and C-terminal processing. 1116 95

A pCb plasmid encoding a beta-lactamase from Haemophilus ducreyi was transferred to Escherichia coli, purified, and characterized. The beta-lactamase could be isolated from a culture filtrate and further purified by ammonium sulfate and chelating Sepharose fast flow loaded with Zn(2+). The purified enzyme resulted in a major band at approximately 30-kDa on SDS-PAGE and its pI was determined to be 5.4. The beta-lactamase could hydrolyze both penicillin antibiotics including ampicillin, benzylpenicillin, and carbenicillin as well as cephalosporin antibiotics including nitrocefin, cephalothin, cephaloridine, and cefoperazone. However, benzylpenicillin was the best substrate. The enzyme activity was inhibited by clavulanic acid but not by boric acid, cefotaxime, ethylenediaminetetraacetic acid, or phenylmethylsulfonyl fluoride. The sequence of the beta-lactamase gene was also determined. It confirmed that the enzyme belonged to a class A beta-lactamase which had 99% identity to the ampicillin resistance transposon Tn3 of pBR322. Two nucleotides were different between the E. coli (Tn3) and H. ducreyi (pCb) genes that affected the amino-acid sequence. The valine at position 82 (ABL 84) was changed to isoleucine and the alanine at position 182 (ABL 184) was changed to valine. Genetic homogeneity among beta-lactamases is remarkable. Amino acid sequencing of some beta-lactamases has shown that substitution of only a few amino acids in the bla gene leads to high-level resistance against specific cephalosporins.
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PMID:Purification and characterization of a beta-lactamase from Haemophilus ducreyi in Escherichia coli. 1157 Aug 57

Adipose tissue is a primary target of insulin, but knowledge about insulin signalling in human adipocytes is limited. We developed an electroporation technique for transfection of primary human adipocytes with a transfection efficiency of 15% +/- 5 (mean +/- S.D.). Human adipocytes were co-transfected with a mutant of IRS-3 (all four potential PI3-kinase binding motifs mutated: IRS-3F4) and HA-tagged protein kinase B (HA-PKB/Akt). HA-PKB/Akt was immunoprecipitated from cell lysates with anti-HA antibodies, resolved with SDS-PAGE, and immunoblotted with phospho-specific antibodies. We found that IRS-3F4 blocked insulin stimulation of HA-PKB/Akt phosphorylation and in further analyses also translocation of recombinant HA-tagged glucose transporter to the plasma membrane. IRS-3F4 also blocked insulin-induced activation of the transcription factor Elk-1. Our results demonstrate the critical importance of IRS for metabolic as well as mitogenic signalling by insulin. This method for transfection of primary human adipocytes will be useful for studying insulin signalling in human adipocytes with molecular biological techniques.
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PMID:Expression of a mutant IRS inhibits metabolic and mitogenic signalling of insulin in human adipocytes. 1522 27


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