Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.3.1.28 (chloramphenicol acetyltransferase)
 5,100 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Two novel cAMP response element binding protein (CREB) splice variants were found by reverse transcription-polymerase chain reaction cloning by using mouse brain RNA as a template. One splice variant, named Delta-14, lacks 14 nucleotides at the beginning of exon 9 of the CREBDelta isoform. The other, named Delta-35, lacks 35 nucleotides at the beginning of exon 8 of CREBDelta. These nucleotide deletions cause frame shifts for codon usage, producing proteins which conserve the major phosphorylation site (Ser(133)) but lack the basic/leucine zipper domain, which is essential for binding to DNA and to other transcription factors. Both variants are widely expressed in peripheral tissues, but are enriched in brain, thymus, and testis. CREBDelta-14 and Delta-35 variant proteins were expressed by using an in vitro translation system and by transfecting into human embryonic kidney 293 cells. Both variants were detected by a CREB antibody that recognizes the CREBDelta amino terminus, but not by an antibody which recognizes the CREBDelta carboxy terminus, as would be predicted based on the frame shift. Activation of the cAMP pathway increased phospho-CREB immunoreactivity, indicating that these variants are substrates of cAMP-dependent protein kinase. In addition, immunocytochemical analysis demonstrated that CREBDelta-14 and Delta-35 are primarily cytosolic, whereas CREBalpha is predominantly in the nucleus. Finally, expression of CREBDelta-14 or Delta-35 decreased cAMP responsive element-chloramphenicol acetyltransferase reporter activity, demonstrating that both can function as repressors of endogenous CREB.
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PMID:Identification and functional analysis of novel cAMP response element binding protein splice variants lacking the basic/leucine zipper domain. 1053 95

In liver and kidney, the terminal step in gluconeogenesis is catalyzed by glucose-6-phosphatase. To examine the effect of the cAMP signal transduction pathway on transcription of the gene encoding the catalytic subunit of glucose-6-phosphatase (G6Pase), G6Pase-chloramphenicol acetyltransferase (CAT) fusion genes were transiently transfected into either the liver-derived HepG2 or kidney-derived LLC-PK cell line. Co-transfection of an expression vector encoding the catalytic subunit of cAMP-dependent protein kinase (PKA) markedly stimulated G6Pase-CAT fusion gene expression, and mutational analysis of the G6Pase promoter revealed that multiple regions are required for this PKA response in both the HepG2 and LLC-PK cell lines. A sequence in the G6Pase promoter that resembles a cAMP response element is required for the full PKA response in both HepG2 and LLC-PK cells. However, in LLC-PK cells, but not in HepG2 cells, a hepatocyte nuclear factor-1 (HNF-1) binding site was critical for the full induction of G6Pase-CAT expression by PKA. Changing this HNF-1 motif to that for the yeast transcription factor GAL4 reduces the PKA response in LLC-PK cells to the same degree as deleting the HNF-1 site. However, co-transfection of this mutated construct with chimeric proteins comprising the GAL4-DNA binding domain ligated to the coding sequence for HNF-1alpha, HNF-1beta, HNF-3, or HNF-4 completely restored the PKA response. Thus, we hypothesize that, in LLC-PK cells, HNF-1 is acting as an accessory factor to enhance PKA signaling through the cAMP response element by altering G6Pase promoter conformation or accessibility rather than specifically affecting some component of the PKA signal transduction pathway.
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PMID:Differential role of hepatocyte nuclear factor-1 in the regulation of glucose-6-phosphatase catalytic subunit gene transcription by cAMP in liver- and kidney-derived cell lines. 1076 45

The first and rate-controlling step of the haem biosynthetic pathway in mammals and fungi is catalysed by the mitochondrial-matrix enzyme 5-aminolaevulinate synthase (ALAS). The purpose of this work was to explore the molecular mechanisms involved in the cAMP regulation of rat housekeeping ALAS gene expression. Thus we have examined the ALAS promoter for putative transcription-factor-binding sites that may regulate transcription in a cAMP-dependent protein kinase (PKA)-induced context. Applying both transient transfection assays with a chloramphenicol acetyltransferase reporter gene driven by progressive ALAS promoter deletions in HepG2, and electrophoresis mobility-shift assays we have identified two putative cAMP-response elements (CREs) at positions -38 and -142. Functional analysis showed that both CRE-like sites were necessary for complete PKA induction, but only one for basal expression. Co-transfection with a CRE-binding protein (CREB) expression vector increased PKA-mediated induction of ALAS promoter transcriptional activity. However, in the absence of co-transfected PKA, CREB worked as a specific repressor for ALAS promoter activity. A CREB mutant deficient in a PKA phosphorylation site was unable to induce expression of the ALAS gene but could inhibit non-stimulated promoter activity. Furthermore, a DNA-binding mutant of CREB did not interfere with ALAS promoter basal activity. Site-directed-mutagenesis studies showed that only the nearest element to the transcription start site was able to inhibit the activity of the promoter. Therefore, we conclude that CREB, through its binding to CRE-like sites, mediates the effect of cAMP on ALAS gene expression. Moreover, we propose that CREB could also act as a repressor of ALAS transcription, but is able to reverse its role after PKA activation. Dephosphorylated CREB would interfere in a spatial-disposition-dependent manner with the transcriptional machinery driving inhibition of gene expression.
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PMID:5-Aminolaevulinate synthase gene promoter contains two cAMP-response element (CRE)-like sites that confer positive and negative responsiveness to CRE-binding protein (CREB). 1113 95


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