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 FER locus in the mouse encodes two tyrosine kinases, p94fer and p51ferT. While p94fer accumulates in the cytoplasm and nucleus of most mammalian cells the expression of p51ferT is restricted to the nucleus of meiotic primary spermatocytes. The cellular function of the FER kinases is not understood, nor has a substrate for these enzymes been characterized. To identify putative substrates of p94fer and p51ferT, the two enzymes were used as 'baits' in the yeast two-hybrid screening system. cDNAs encoding the mouse TATA element modulatory factor (TMF) were repeatedly isolated in this assay. TMF was previously shown to bind the TATA element in RNA polymerase II promoters and impaired their functioning in a cell free transcription system. Both p94fer and p51ferT phosphorylated the TMF protein in in vitro and in vivo kinase assays. Sequential deletions showed that the carboxy-terminal region of TMF was essential for phosphorylation. In situ hybridization analysis revealed the preferential accumulation of TMF transcripts in meiotic spermatogenic and oogenic cells. p94fer and p51ferT may thus modulate the suppressive activity of TMF during cellular growth and in defined differentiation processes.
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PMID:Tyrosine phosphorylation of the TATA element modulatory factor by the FER nuclear tyrosine kinases. 974 51

IMP dehydrogenase is a rate-limiting enzyme involved in the synthesis of GTP. In mammalian cells it is regulated with respect to growth rate and is the target of numerous therapeutic agents. Mutations in the RNA polymerase II elongation machinery render yeast sensitive to inhibitors of IMP dehydrogenase and defective in inducing transcription of one of the IMP dehydrogenase-encoding genes, IMD2. Here we show that loss of IMD2, but not IMD1, IMD3, or IMD4, conferred upon yeast the same drug sensitivity found in elongation mutants. We tested whether the drug sensitivity of elongation mutants is due to their inability to induce IMD2 by providing them with exogenous copies of the gene. In some elongation mutants, overexpression reversed drug sensitivity and a transcriptional defect. Overexpression in mutants with a more severe phenotype partially suppressed drug sensitivity but was inconsequential in reversing a defect in transcription. These findings suggest that the drug sensitivity of elongation mutants is largely but not solely attributable to defects in the ability to induce IMD2, because transcription is compromised even when IMD2 mRNA levels are adequate. We describe two DNA sequence elements in the promoter of the gene that regulate it. We also found that IMD2 mRNA abundance is coupled to cell growth rate. These findings show that yeast possess a conserved system that gauges nucleotide pools and cell growth rate and responds through a uniquely regulated member of the IMD gene family.
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PMID:Regulation of an IMP dehydrogenase gene and its overexpression in drug-sensitive transcription elongation mutants of yeast. 1144 Oct 18

Transcriptional regulation by estrogen receptor alpha (ERalpha) involves protein-protein interactions among the receptor, its associated coactivators and the RNA polymerase II transcriptional machinery. We have used an in vitro chromatin assembly and transcription system to examine the biochemistry of interactions among ERalpha, the SRC proteins and p300/CBP. Using polypeptides designed to block specific receptor- cofactor or cofactor-cofactor interactions, we show that interactions among ERalpha, its coactivators and the RNA pol II machinery are all required for ERalpha- mediated transcription. Furthermore, we show that ERalpha-SRC-p300/CBP interactions are necessary and sufficient for the targeted acetylation of nucleosomal histones on estrogen-responsive promoters in the absence of transcription. The protein-protein interactions required for histone acetylation constitute a subset of the interactions required for transcriptional activation. Finally, we show that the major role of SRC-p300/CBP interactions is to enhance ERalpha- mediated transcription initiation, and they have little or no role in stimulating subsequent rounds of transcription. Together, our results indicate a specific role for the SRC and p300/CBP coactivators, as well as targeted histone acetylation, in ERalpha-mediated transcription.
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PMID:A role for coactivators and histone acetylation in estrogen receptor alpha-mediated transcription initiation. 1168 48

Steroid receptor coactivator-3 (SRC-3/AIB1) is a coactivator for nuclear receptors and other transcription factors and an oncogene that contributes to growth regulation and development of mammary and other tumor types. Because of its biological functions, it is important to identify genes regulated by SRC-3. However, because coactivators do not bind DNA directly, extensive work is required to determine whether genes identified by RNA profiling approaches are direct or indirect targets. Here, we report the use of chromatin immunoprecipitation (ChIP)-based assays that involve genomic mapping and computational analyses of immunoprecipitated DNA to identify SRC-3-binding target genes in estradiol (E2)-treated MCF-7 breast cancer cells. We identified 18 SRC-3 genomic binding sites and demonstrated estrogen receptor-alpha (ERalpha) binding to all of them. Both E2-dependent and -independent SRC-3/ERalpha-binding sites were identified. RNA polymerase II ChIP assays were used to determine the correlation between SRC-3 and ERalpha binding and recruitment of the transcriptional machinery. These assays, in conjunction with analyses of RNA obtained from E2-treated cells, lead to the identification of SRC-3/ERalpha-associated genes. The ability of SRC family coactivators to regulate the expression of one of these genes, PARD6B/Par6, was confirmed by using cells individually depleted of SRC-1, SRC-2, or SRC-3 by small interfering RNA. The method described herein can be used to identify genes regulated by non-DNA-binding factors, such as other coactivators or corepressors, as well as DNA-binding transcription factors, and provides information on their binding location that can accelerate further gene characterization.
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PMID:Identification of target genes in breast cancer cells directly regulated by the SRC-3/AIB1 coactivator. 1567 24

Using chromatin immunoprecipitation assays, we studied the 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-mediated recruitment of the aryl hydrocarbon receptor (AhR) and several co-regulators to the CYP1A1 promoter. AhR displayed a time-dependent recruitment, reaching a peak at 75 min and maintaining promoter occupancy for the remainder of the time course. Recruitment of AhR was followed by TIF2/SRC2, which preceded CBP, histone H3 acetylation, and RNA polymerase II (RNAPII). Simultaneous recruitment to the enhancer and the TATA box region suggests the formation of a large multiprotein complex bridging the two promoter regions. Interestingly, estrogen receptor alpha (ERalpha) displayed a TCDD- and time-dependent recruitment to the CYP1A1 promoter, which was increased by co-treatment with estradiol. Transfection in HuH7 human liver cells confirmed previously reported ERalpha enhancement of AhR activity. In contrast, TCDD did not induce the recruitment of ERalpha to the estrogen-responsive pS2 promoter, and after 120 min of co-treatment with estradiol, ERalpha is still present on the CYP1A1 promoter but no longer at pS2. RNA interference studies with T47D cells support a role for ERalpha in TCDD-dependent CYP1A1 expression. Our data suggest that ERalpha acts as a coregulator of AhR-mediated transcriptional activation and that the recruitment of ERalpha by AhR represents a novel mechanism AhR-ERalpha cross talk.
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PMID:Aryl hydrocarbon receptor-mediated transcription: ligand-dependent recruitment of estrogen receptor alpha to 2,3,7,8-tetrachlorodibenzo-p-dioxin-responsive promoters. 1596 90

The androgen receptor (AR) is a ligand-activated transcription factor that mediates the actions of the steroid hormones testosterone and dihydrotestosterone at the level of gene transcription. The main transactivation function is modular in structure, maps to the N-terminal domain (NTD), and is termed AF1. This region of the AR is structurally flexible and functions in multiple protein-protein interactions with coregulatory proteins and components of the general transcription machinery. Using surface plasmon resonance, the binding kinetics for the interaction of AR-AF1 with the large subunit of the general transcription factor TFIIF, termed RAP74, and the coactivator SRC-1a were measured. AR-AF1 interacts with both the NTD and CTD of RAP74 and the CTD of SRC-1a. The dissociation constants ( Kd) for the binding of polypeptides derived from RAP74 are in the submicromolar range, while a peptide from SRC-1a bound with a Kd of 14 microM. Significantly, the individual NTD and CTD of RAP74 interacted with AR-AF1 with distinct binding kinetics, with the NTD exhibiting slower on and off rates. TFIIF is involved in transcription initiation and elongation, and the CTD of RAP74 binds to the RNA polymerase II enzyme, the general transcription factor TFIIB, and a CTD phosphatase, FCP1. We have mutated hydrophobic residues in the RAP74-CTD structure to disrupt secondary structure elements and show that binding of AR-AF1 depends upon helix 3 in the winged-helix domain of the RAP74-CTD polypeptide. Altogether, a model is suggested for AR-AF1-dependent transactivation of receptor-target genes.
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PMID:Functional characterization of the native NH2-terminal transactivation domain of the human androgen receptor: binding kinetics for interactions with TFIIF and SRC-1a. 1828 9

After interaction with its receptor, GM-CSF induces phosphorylation of the beta-chain in two distinct domains in macrophages. One induces activation of mitogen-activated protein kinases and the PI3K/Akt pathway, and the other induces JAK2-STAT5. In this study we describe how trichostatin A (TSA), which inhibits deacetylase activity, blocks JAK2-STAT5-dependent gene expression but not the expression of genes that depend on the signal transduction induced by the other domain of the receptor. TSA treatment inhibited the GM-CSF-dependent proliferation of macrophages by interfering with c-myc and cyclin D1 expression. However, M-CSF-dependent proliferation, which requires ERK1/2, was unaffected. Protection from apoptosis, which involves Akt phosphorylation and p21(waf-1) expression, was not modified by TSA. GM-CSF-dependent expression of MHC class II molecules was inhibited because CIITA was not induced. The generation of dendritic cells was also impaired by TSA treatment because of the inhibition of IRF4, IRF2, and RelB expression. TSA mediates its effects by preventing the recruitment of RNA polymerase II to the promoter of STAT5 target genes and by inhibiting their expression. However, this drug did not affect STAT5A or STAT5B phosphorylation or DNA binding. These results in GM-CSF-treated macrophages reveal a relationship between histone deacetylase complexes and STAT5 in the regulation of gene expression.
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PMID:Deacetylase activity is required for STAT5-dependent GM-CSF functional activity in macrophages and differentiation to dendritic cells. 1842 9

The expression of carbonic anhydrase XII (CA12), a gene that encodes a zinc metalloenzyme responsible for acidification of the microenvironment of cancer cells, is highly correlated with estrogen receptor alpha (ER alpha) in human breast tumors. Here, we show that CA12 is robustly regulated by estrogen via ER alpha in breast cancer cells, and that this regulation involves a distal estrogen-responsive enhancer region. Upon the addition of estradiol, ER alpha binds directly to this distal enhancer in vivo, resulting in the recruitment of RNA polymerase II and steroid receptor coactivators SRC-2 and SRC-3, and changes in histone acetylation. Mutagenesis of an imperfect estrogen-responsive element within this enhancer region abolishes estrogen-dependent activity, and chromosome conformation capture and chromatin immunoprecipitation assays show that this distal enhancer communicates with the transcriptional start site of the CA12 gene via intrachromosomal looping upon hormone treatment. This distal enhancer element is observed in the homologous mouse genomic sequence, and the expression of the mouse homologue, Car12, is rapidly and robustly stimulated by estradiol in the mouse uterus in vivo, suggesting that the ER regulation of CA12 is mechanistically and evolutionarily conserved. Our findings highlight the crucial role of ER in the regulation of the CA12 gene, and provide insight into the transcriptional regulatory mechanism that accounts for the strong association of CA12 and ER in human breast cancers.
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PMID:Estrogen receptor regulation of carbonic anhydrase XII through a distal enhancer in breast cancer. 1845 Nov 79

To clarify the mechanisms of purvalanol A in the induction of apoptosis, we investigated whether purvalanol A influenced the RNA synthesis and expression of RNA polymerase II and signal transducer and activator of transcription 3 (STAT3). When MKN45 cells were treated with 30 micromol/l purvalanol A, mitochondrial dysfunction occurred before the induction of the apoptosis and the expression of antiapoptotic proteins survivin, Bcl-XL, and Bcl-2 was reduced. The treatment with parvalanol A was also shown to reduce not only mRNA for these proteins but also global RNA synthesis. The phosphorylation of the carboxy-terminal domain of RNA polymerase II, which was involved in transcriptional regulation, was strongly inhibited by purvalanol A, followed by the partial inhibition of the expression of RNA polymerase II. Furthermore, the phosphorylation at Tyr705 of STAT3, which is known to be a phosphorylation site for Janus kinase 2 (JAK2), was completely inhibited by purvalanol A early (3 h) after drug treatment, although the phosphorylation of STAT3 at Ser727, which is a phosphorylation site for Ras/Raf/MEK and extracellular signal-regulated protein kinase 1/2, was still detectable until late (12 h) after treatment. In addition, the tyrosine phosphorylation of JAK2 was efficiently inhibited by purvalanol A. These results suggest that the inhibition of JAK2/STAT3 and RNA polymerase II is crucial in the downregulation of antiapoptotic proteins leading to the apoptotic cell death induced by parvalanol A.
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PMID:Purvalanol A induces apoptosis and downregulation of antiapoptotic proteins through abrogation of phosphorylation of JAK2/STAT3 and RNA polymerase II. 1852 15

Cytochrome P450 (CYP) 1A1 and CYP1B1 are inducible by 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin) in the human breast cancer cell line, MCF-7. Since CYP1A1 was inducible to a much greater degree than CYP1B1, we hypothesized that there may be differences in coactivator recruitment to the promoter and/or enhancer regions of these genes. Dioxin treatment leads to recruitment of the aryl hydrocarbon receptor to the enhancer regions but not to the proximal promoter regions of both the CYP1A1 and CYP1B1 genes. On the other hand, dioxin treatment facilitated recruitment of RNA polymerase II to the promoters but not the enhancer regions. Dioxin treatment also elicited recruitment of the transcriptional coactivators, steroid receptor coactivator 1 (SRC-1) and steroid receptor coactivator 2 (SRC-2) and p300, which possess intrinsic histone acetyltranferase activities, to both genes, whereas Brahma (BRM)/Switch 2-related gene 1 (BRG-1), a subunit of nucleosomal remodeling factors, was recruited more robustly to CYP1A1 relative to CYP1B1. Small inhibitory RNA-mediated knockdown of p300 and SRC-2 adversely affected dioxin induction of both genes, whereas knockdown of BRM/BRG-1 reduced CYP1A1 induction but had little, if any, effect on CYP1B1 induction. These results suggest that nucleosomal remodeling is less significant for dioxin-mediated induction of CYP1B1 than that of CYP1A1 and may be related to the more modest inducibility of the former. Interestingly, simultaneous knockdown of SRC-2 and BRM/BRG-1 had no greater effect on CYP1A1 induction than knockdown of each coactivator individually, while simultaneous knockdown of p300 and BRM/BRG-1 had a much greater effect than knockdown of each individual gene, suggesting that the recruitment of SRC-2 to CYP1A1 depends upon BRM/BRG-1, while the recruitments of p300 and BRM/BRG-1 are independent of each other. These observations provide novel insights into the functional roles of the endogenous coactivators in dioxin induction of the human CYP1A1 and CYP1B1 genes in their natural chromosomal configurations.
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PMID:Roles of coactivator proteins in dioxin induction of CYP1A1 and CYP1B1 in human breast cancer cells. 1884 20


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