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)

Interstitial cells of Cajal (ICC) are pacemaker cells for the spontaneous muscular contractions and neuromodulators that mediate neurotransmission from enteric neurons to smooth muscle cells in the gastrointestinal (GI) tract. They express c-Kit, and the antibody for c-Kit (especially ACK2) has been a useful tool for functional and morphological studies. ACK2, however, does not work on tissues fixed with paraformaldehyde, and not all ICC express c-Kit in human. Therefore, in order to find a new marker of ICC and/or new antibody resisting aldehyde fixation, we produced a new monoclonal antibody that identifies ICC and then investigated the properties of its antigen. Isolated ICC were used for immunization. Hybridomas fused with myeloma SP2 were screened by immunohistochemistry. ACK2 and each antibody were applied on serial sections, and the clone producing anti-ICC antibody (AIC) that stains ICC was established. The distribution of AIC immunopositive cells was examined in other organs and also GI muscles of W/Wv mice. The biochemical properties were studied using dot blot analysis. AIC recognized ICC; however, distribution of immunopositive cells in W/Wv mice and other organs was different from that of c-Kit. The immunoreactivity was stable for paraformaldehyde but was blocked by either Triton X-100 or SDS. In conclusion, new antibody AIC recognized ICC but the antigen was not c-Kit, which confirms the existence of good markers of ICC besides c-Kit. Although the antigen has not been isolated, AIC is suitable for morphological study and is useful for investigation of ICC in c-Kit mutants.
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PMID:New monoclonal antibody (AIC) identifies interstitial cells of Cajal in the musculature of the mouse gastrointestinal tract. 1529 91

We found a gamma-resorcylic acid (gamma-RA, 2,6-dihydroxybenzoic acid) decarboxylase, as a novel enzyme applicable to carboxylation of resorcinol (RE, 1,3-dihydroxybenzene) to form gamma-RA, in a bacterial strain Rhizobium radiobacter WU-0108 isolated through the screening of gamma-RA degrading microorganisms. The activities for carboxylation of RE and decarboxylation of gamma-RA were detected in the cell-free extracts of R. radiobacter WU-0108 grown aerobically with gamma-RA. The enzyme, gamma-RA decarboxylase, was purified to homogeneity on SDS-PAGE through the steps of one ion-exchange chromatography and two kinds of hydrophobic chromatography. The molecular weight of the enzyme was estimated to be 130 kDa by gel-filtration, and that of the subunit was determined to be 34 kDa by SDS-PAGE, suggesting that the enzyme is a homotetrameric structure. The enzyme catalyzed the decarboxylation of gamma-RA, but not alpha-RA or beta-RA. Without addition of any cofactors, the enzyme catalyzed the regio-selective carboxylation of RE to form gamma-RA, without formation of alpha-RA and beta-RA, and of catechol to 2,3-dihydroxybenzoic acid. In the presence of oxygen, this gamma-RA decarboxylase showed no decrease in both of the activities as for decarboxylation of gamma-RA and carboxylation of RE, different from other decarboxylases reported so far. The gene, rdc, encoding the gamma-RA decarboxylase was cloned into Escherichia coli, sequenced, and subjected to over-expression. The deduced amino acid sequence of the rdc gene consists of 327 amino acid residues corresponding to 34 kDa protein, and shows 42% and 30% identity to those of a 2,3-dihydroxybenzoic acid decarboxylase from Aspergillus niger and a 5- carboxyvanillate decarboxylase from Sphingomonas paucimobilis SYK-6. A site-directed mutagenesis study revealed the two histidine residues at positions of 164 and 218 in Rdc to be essential for the catalytic activities of decarboxylation of gamma-RA and carboxylation of RE.
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PMID:Reversible and nonoxidative gamma-resorcylic acid decarboxylase: characterization and gene cloning of a novel enzyme catalyzing carboxylation of resorcinol, 1,3-dihydroxybenzene, from Rhizobium radiobacter. 1547 71

A cDNA encoding farnesyl diphosphate synthase (FPS; EC2.5.1.1/EC2.5.1.10) was isolated from Centella asiacita (L.) Urban, using degenerate primers based on two highly conserved domains. A full-length cDNA clone was subsequently isolated by rapid amplification of cDNA ends (RACE) PCR. The sequence of the CaFPS (C. asiatica farnesyl diphosphate synthase) cDNA contains an open reading frame of 1029 nucleotides encoding 343 amino acids with a molecular mass of 39.6 kDa. The deduced CaFPS amino acid sequence exhibits 84, 79, and 72%, identity to the FPSs of Artemisia annua, Arabidopsis thaliana, and Oryza sativa, respectively. Southern blot analysis suggested that the C. asiatica genome contains only one FPS gene. An artificially expressed soluble form of the CaFPS was identified by SDS-PAGE. It had high specific activity and produced farnesyl diphosphate as the major isoprenoid.
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PMID:Cloning and expression of a farnesyl diphosphate synthase in Centella asiatica (L.) Urban. 1587 17

The capability of the suppressed conductometric detection ion chromatography (IC) was investigated for the separation and determination of inorganic anions (F-, Cl-, NO3- and SO4(2-)) in standard reference materials SRM-1648 urban particulate matter following ultrasonic extraction. The effects of the cationic surfactant (SDS) and the anionic surfactant (CTAB) on ultrasonic extraction efficiency of inorganic anions from complex matrix of airborne particulate matter were investigated. The results showed that surfactant can enhance the extraction efficiency. Finally, the concentrations of inorganic anions in the atmosphere of the city of Isfahan were determined. The results showed a trend of SO4(2-) > NO3- > Cl- > F-.
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PMID:Determination of atmospheric concentrations of inorganic anions by ion chromatography following ultrasonic extraction. 1625 97

Ste20-like kinase, SLK, a germinal center kinase found in kidney epithelial cells, signals to promote apoptosis. Expression of SLK mRNA and protein and kinase activity are increased during kidney development and recovery from ischemic acute renal failure. The 3'-untranslated region (3'-UTR) of SLK mRNA contains multiple adenine and uridine-rich elements, suggesting that 3'-UTR may regulate mRNA stability. This was confirmed in COS cell transient transfection studies, which showed that expression of the SLK open-reading frame plus 3'-UTR mRNA was reduced by 35% relative to the open-reading frame alone. To further characterize the SLK-3'-UTR, this nucleotide sequence was subcloned downstream of enhanced green fluorescent protein (EGFP) cDNA. In COS, 293T, and glomerular epithelial cells, expression of EGFP mRNA and protein was markedly reduced in the presence of the SLK-3'-UTR. After transfection and subsequent addition of actinomycin D, EGFP mRNA remained stable in cells for at least 6 h, whereas EGFP-SLK-3'-UTR mRNA decayed with a half-life of approximately 4 h. A region containing five AUUUA motifs within the SLK-3'-UTR destabilized EGFP mRNA. Deletion of this region from the SLK-3'-UTR, in part, restored mRNA stability. By UV cross-linking and SDS-PAGE, the SLK-3'-UTR bound to protein(s) of approximately 30 kDa in extracts of COS cells, glomerular epithelial cells, and kidney. Cotransfection of HuR (a RNA binding protein of approximately 30 kDa) increased the steady-state mRNA level of EGFP-SLK-3'-UTR but not EGFP. Thus the SLK-3'-UTR may interact with kidney RNA-binding proteins to regulate expression of SLK mRNA during kidney development and after ischemic injury.
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PMID:The 3'-untranslated region of the Ste20-like kinase SLK regulates SLK expression. 1700 24

We previously isolated and characterized the novel human gene MOST-1 (C8orf17) that is ubiquitously expressed in all cancer cell lines tested but differentially expressed in normal adult tissues. MOST-1 maps to chromosome region 8q24.2 whose amplification is frequently associated with breast and prostate cancers. RT-PCR analyses of breast and prostatic biopsies revealed MOST-1 overexpression and/or amplification in high-grade carcinomas. We raised and characterized a polyclonal antibody against a MOST-1-specific synthetic peptide. in vitro expression of MOST-1 protein revealed a tendency to exist as high molecular mass isoforms which are SDS-insoluble upon thermal stress. MOST-1 displayed cytoplasmic localization in four human cell lines (hTERT-HME1 normal mammary epithelial, MCF7 breast adenocarcinoma, PrEC normal prostate epithelial and DU145 prostate carcinoma), with polar expression during cell division. Knockdown of MOST-1 expression in DU145 cells resulted in reduced cell proliferation but enhanced apoptosis implying a putative mitogenic role of MOST-1. Yeast two-hybrid analyses demonstrated interaction with seven human proteins, most of which are overexpressed in tumors or involved in metabolic pathways. The interacting proteins were creatine kinase, Gardner feline sarcoma v-FGR oncogene product, telethonin, SNC73 protein, ferritin light chain, peripheral benzodiazepine receptor, and immunoglobulin C (mu) and C (delta) heavy chain. Co-immunoprecipitation assays validated the interactions of MOST-1 with the latter three proteins. Our results suggest that MOST-1 is associated with cell survival, proliferation and progression of cancer cells.
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PMID:Cellular expression, localization and interactions of the product of the human MOST-1 gene associated with breast and prostate cancers. 1714 15

A key factor governing cellular sensitivity to GH is cell surface GH receptor (GHR) abundance, which is affected transcriptionally and posttranscriptionally. Mature cell surface GHR abundance is regulated by constitutive and inducible metalloproteolysis and constitutive endosomal/lysosomal degradation. We previously found that Janus kinase 2 (JAK2)-deficient GHR-expressing cells have a greater precursor/mature GHR ratio, exhibit diminished inducible metalloproteolysis, and have a cytoplasmic domain-containing GHR fragment called the basal remnant (by virtue of comigration on SDS-PAGE with the inducible, metalloprotease-generated remnant). Herein we examined the mechanism of generation of basal remnant in JAK2-deficient cells, asking whether it originates from precursor vs. mature receptor and which protease(s) catalyzes its appearance. Prolonged metalloprotease inhibitor treatment or small interfering RNA knockdown of TNF-alpha converting enzyme (TACE) and a disintegrin and metalloprotease-10 (ADAM10) (both implicated in inducible GHR proteolysis) did not reduce basal remnant, indicating its generation is not metalloprotease dependent. However, a mutant GHR resistant to metalloprotease cleavage did not yield basal remnant when expressed in JAK2-deficient cells, suggesting common structural determinants for generation of the inducible remnant and the non-metalloprotease-generated basal remnant seen in JAK2-deficient cells. Treatment of JAK2-deficient cells with a proteasome inhibitor, but not two separate lysosome inhibitors, dramatically decreased basal remnant, accompanied by decreased precursor GHR and increased mature GHR abundance. Disruption of endoplasmic reticulum-to-Golgi transport with brefeldin A (BFA) also reduced basal remnant, and washout of BFA allowed regeneration of basal remnant along with GHR precursor. Notably, BFA washout in the presence of cycloheximide blocked both basal remnant and precursor GHR reappearance, but BFA washout in the presence of lactacystin blocked only basal remnant reappearance, suggesting that basal remnant is generated proteasome dependently from precursor GHR. Collectively, our data suggest that JAK2, by association with GHR in the secretory pathway, blunts proteasome activity-dependent discrete GHR cleavage and endoplasmic reticulum-dependent degradation of the precursor receptor. In so doing, JAK2 enables efficient processing of precursor receptor to mature GHR.
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PMID:Endoplasmic reticulum-associated degradation of growth hormone receptor in Janus kinase 2-deficient cells. 1776 66

Tristetraprolin/zinc finger protein 36 (TTP/ ZFP36) binds and destabilizes some proinflammatory cytokine mRNAs. TTP-deficient mice develop a profound inflammatory syndrome due to excessive production of proinflammatory cytokines. TTP gene expression is induced by various factors including insulin, cinnamon, and green tea extracts. Previous studies have shown that TTP is highly phosphorylated in vivo and multiple phosphorylation sites are identified in human TTP. This study evaluated the potential protein kinases that could phosphorylate recombinant TTP in vitro. Motif scanning suggested that TTP was a potential substrate for various kinases. SDS-PAGE showed that in vitro phosphorylation of TTP with p42 and p38 MAP kinases resulted in visible electrophoretic mobility shift of TTP to higher molecular masses. Autoradiography showed that TTP was phosphorylated in vitro by GSK3b, PKA, PKB, PKC, but not Cdc2, in addition to p42, p38, and JNK. These results demonstrate that TTP is a substrate for a number of protein kinases in vitro.
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PMID:Phosphorylation of recombinant tristetraprolin in vitro. 1807 86

GH binds dimerized GH receptors (GHRs) to form a trimolecular complex and induces downstream signaling events. The mechanism by which GH binding converts the inactive predimerized GHR to its active signaling conformation is uncertain. GH has no axis of symmetry. Its interaction with GHR is mediated by two asymmetric binding sites on GH, each with distinct affinity. Site 1 is of high affinity and is thought to mediate the first binding step. Mutation of binding site 2 (as in the human GH mutant, G120R) disrupts the second binding but leaves site 1 binding intact. G120R is a GH antagonist; it binds only one GHR and thus fails to signal, and it prevents productive GHR binding by normal GH. We previously demonstrated that prolactin receptor signaling was achieved by a dimeric version of a prolactin antagonist. We now employ assays of cellular signaling and receptor conformational changes to examine whether GH molecules harboring two site 1 regions can trigger GHR activation. We used recombinantly produced GH-GH and G120R-G120R dimers in which monomers in tandem are connected by a short linker peptide. Rabbit GHR-expressing human fibrosarcoma cells (C14) were treated with GH, G120R, GH-GH, or G120R-G120R. As expected, GH and GH-GH, but not G120R, induced GHR disulfide linkage, as assessed by anti-GHR blotting of cell extracts resolved by SDS-PAGE under nonreducing conditions. Disulfide linkage of GHRs reflects attainment of the active signaling conformation. Likewise, GH and GH-GH, but not G120R, caused Janus kinase 2 (JAK2) and signal transducer and activator of transcription 5 (STAT5) activation. Notably, G120R-G120R, despite its lack of an intact site 2 in either dimer partner, also promoted GHR disulfide linkage and JAK2 and STAT5 activation, albeit less potently than either GH or GH-GH. Time-course responses of the three agonists were similar in terms of JAK2 and STAT5 activation. Pretreatment of cells with our conformation-sensitive inhibitory monoclonal antibody, anti-GHR ext-mAb, prevented ligand-induced receptor activation for all three agonists. GHR was also rendered less immunoprecipitable by anti-GHR ext-mAb after treatment with these agonists. These results are important in that they indicate that a ligand with two intact binding sites 1 causes GHR to adopt similar conformational changes as does GH and thus triggers activation of JAK2 and downstream signaling. Furthermore, we infer that there is substantial flexibility in the GHR extracellular domain, such that it productively accommodates GH dimers that are much larger than GH.
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PMID:Activation of growth hormone receptors by growth hormone and growth hormone antagonist dimers: insights into receptor triggering. 1809 90

Chronic exposure to arsenic has been linked to tumorigenesis, cardiovascular disease, hypertension, atherosclerosis, and peripheral vascular disease; however, the molecular mechanisms underlying its pathological effects remain elusive. In this study, we investigated arsenic-induced alteration of focal adhesion protein complexes in normal, primary vascular smooth muscle cells. We demonstrate that exposure to environmentally relevant concentrations of arsenic (50 ppb As(3+)) can alter focal adhesion protein co-association leading to activation of downstream pathways. Co-associated proteins were identified and quantitated via co-immunoprecipitation, SDS-PAGE, and Western blot analysis followed by scanning densitometry. Activation of MAPK pathways in total cell lysates was evaluated using phosphor-specific antibodies. In our model, arsenic treatment caused a sustained increase in FAK-src association and activation, and induced the formation of unique signaling complexes (beginning after 3-hour As(3+) exposure and continuing throughout the 12-hour time course studied). The effects of these alterations were manifested as chronic stimulation of downstream PAK, ERK and JNK pathways. Past studies have demonstrated that these pathways are involved in cellular survival, growth, proliferation, and migration in VSMCs.
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PMID:Arsenic alters vascular smooth muscle cell focal adhesion complexes leading to activation of FAK-src mediated pathways. 1848 77


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