Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.11.1 (protein kinase)
 81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Transcription of the phosphoenolpyruvate carboxykinase (PEPCK) gene is induced upon activation of protein kinase A by cAMP and phosphorylation of Ser-133 in the transcription factor, cAMP-response element binding protein (CREB), and this induction is inhibited by insulin. We show here that insulin does not act by dephosphorylating CREB or by affecting heterologous kinases that phosphorylate Ser-129 or Ser-142 in CREB. In addition, insulin inhibition of minimal PEPCK promoter activity induced by CREB-GAL4 + protein kinase A was equivalent to inhibition of basal transcription, and thus cAMP-independent. On the other hand, nearly complete insulin inhibition is observed with the full PEPCK promoter (-600/+69), indicating that other factors are involved. The additional promoter elements required for induction by protein kinase A lie within -271 nucleotides of the start site and correspond to putative binding sites for activator protein-1 and CAAT/enhancer-binding protein (C/EBP), first identified by Roesler et al. (Roesler, W. J., McFie, P. J., and Puttick, D. M., (1993) J. Biol. Chem. 268, 3791-3796). This tripartite array of binding sites for CREB, C/EBP, and activator protein-1 (AP-1) factors forms a cAMP response unit that, together with the minimal promoter, can mediate both induction by cAMP and inhibition by insulin. Thus, for the PEPCK gene with a single CREB site, the CREB.CBP.RNA polymerase II complex cannot mediate either induction by cAMP or inhibition by insulin.
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PMID:A tripartite array of transcription factor binding sites mediates cAMP induction of phosphoenolpyruvate carboxykinase gene transcription and its inhibition by insulin. 966 47

A single far-upstream enhancer is sufficient to confer hepatocyte-specific, glucocorticoid- and cyclic AMP-inducible periportal expression to the carbamoylphosphate synthetase I (CPS) gene. To identify the mechanism of hormone-dependent activation, the composition and function of the enhancer have been analyzed. DNase I protection and gel mobility shift assays revealed the presence of a cyclic AMP response element, a glucocorticoid response element (GRE), and several sites for the liver-enriched transcription factor families HNF3 and C/EBP. The in vivo relevance of the transcription factors interacting with the enhancer in the regulation of CPS expression in the liver was assessed by the analysis of knockout mice. A strong reduction of CPS mRNA levels was observed in glucocorticoid receptor- and C/EBPalpha-deficient mice, whereas the CPS mRNA was normally expressed in C/EBPbeta knockout mice and in HNF3alpha and -gamma double-knockout mice. (The role of HNFbeta could not be assessed, because the corresponding knockout mice die at embryonic day 10). In hepatoma cells, most of the activity of the enhancer is contained within a 103-bp fragment, which depends for its activity on the simultaneous occupation of the GRE, HNF3, and C/EBP sites, thus meeting the requirement of a glucocorticoid response unit. In fibroblast-like CHO cells, on the other hand, the GRE in the CPS enhancer does not cooperate with the C/EBP and HNF3 elements in transactivation of the CPS promoter. In both hepatoma and CHO cells, stimulation of expression by cyclic AMP depends mainly on the integrity of the glucocorticoid pathway, demonstrating cross talk between this pathway and the cyclic AMP (protein kinase A) pathway.
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PMID:Glucocorticoid receptor, C/EBP, HNF3, and protein kinase A coordinately activate the glucocorticoid response unit of the carbamoylphosphate synthetase I gene. 977 47

We report here the cloning, characterisation and developmental expression profile of the Xenopus laevis CCAAT-enhancer binding protein beta (xC/EBPbeta) gene. The protein synthesised from the xC/EBPbeta gene interacts specifically with a C/EBP-recognition sequence and acts as a transcriptional activator. Several conserved regions are present in the xC/EBPbeta sequence, including the basic region, leucine zipper, activation domains, three in-frame AUG codons, and a consensus site for mitogen activated protein kinase. The corresponding mRNA is present at high levels in the kidney, liver, lung, muscle and adipose tissue, and at low levels in the ovary, brain and heart. Although the xC/EBPbeta mRNA and protein are present throughout embryogenesis, there is a biphasic increase in their expression levels during development. Whole-mount in situ hybridisation shows a restricted spatial expression profile of the xC/EBPbeta gene during early embryogenesis, with transcripts present around the blastopore lip and in the endodermal cells at the mid-gastrula stage, and, the whole dorsal side at the neurula and early tailbud stage. The expression domain becomes almost ubiquitous during later embryonic development, and includes the brain, spinal cord, somites and regions that give rise to the liver and the heart.
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PMID:Characterisation and developmental regulation of the Xenopus laevis CCAAT-enhancer binding protein beta gene. 983 41

We previously showed that the rate of hepatocyte proliferation in livers from newborn C/EBPalpha knockout mice was increased. An examination of cell cycle-related proteins showed that the cyclin-dependent kinase (CDK) inhibitor p21 level was reduced in the knockout animals compared to that in wild-type littermates. Here we show additional cell cycle-associated proteins that are affected by C/EBPalpha. We have observed that C/EBPalpha controls the composition of E2F complexes through interaction with the retinoblastoma (Rb)-like protein, p107, during prenatal liver development. S-phase-specific E2F complexes containing E2F, DP, cdk2, cyclin A, and p107 are observed in the developing liver. In wild-type animals these complexes disappear by day 18 of gestation and are no longer present in the newborn animals. In the C/EBPalpha mutant, the S-phase-specific complexes do not diminish and persist to birth. The elevation of levels of the S-phase-specific E2F-p107 complexes in C/EBPalpha knockout mice correlates with the increased expression of several E2F-dependent genes such as those that encode cyclin A, proliferating cell nuclear antigen, and p107. The C/EBPalpha-mediated regulation of E2F binding is specific, since the deletion of another C/EBP family member, C/EBPbeta, does not change the pattern of E2F binding during prenatal liver development. The addition of bacterially expressed, purified His-C/EBPalpha to the E2F binding reaction resulted in the disruption of E2F complexes containing p107 in nuclear extracts from C/EBPalpha knockout mouse livers. Ectopic expression of C/EBPalpha in cultured cells also leads to a reduction of E2F complexes containing Rb family proteins. Coimmunoprecipitation analyses revealed an interaction of C/EBPalpha with p107 but none with cdk2, E2F1, or cyclin A. A region of C/EBPalpha that has sequence similarity to E2F is sufficient for the disruption of the E2F-p107 complexes. Despite its role as a DNA binding protein, C/EBPalpha brings about a change in E2F complex composition through a protein-protein interaction. The disruption of E2F-p107 complexes correlates with C/EBPalpha-mediated growth arrest of hepatocytes in newborn animals.
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PMID:C/EBPalpha regulates formation of S-phase-specific E2F-p107 complexes in livers of newborn mice. 1008 61

In the course of decidualization, human endometrial stromal cells (ESC) activate the alternative upstream promoter of the decidual prolactin (dPRL) gene. The dPRL promoter is induced by the protein kinase A pathway in a delayed fashion via the region -332/-270 which contains two overlapping consensus binding sequences, B and D, for CCAAT/enhancer-binding proteins (C/EBP). Here we show that sites B and D both bind C/EBPbeta and -delta from ESC nuclear extracts. When decidualization of cultured ESC was induced by treatment with 8-Br-cAMP, complex formation on sites B and D was enhanced. Western blot analysis revealed an elevation of both C/EBPbeta isoforms, liver-enriched activator protein and liver-enriched inhibitory protein, with a delayed onset between 8 and 24 h of cAMP treatment, while C/EBPdelta expression remained unaffected. Cyclic AMP-mediated activation of dPRL promoter construct dPRL-332/luc3 was abrogated by mutation of sites B and D at -310/-285. An expression vector for liver-enriched activator protein potently induced transcription of dPRL-332/luc3 and further enhanced cAMP-mediated induction, while liver-enriched inhibitory protein expression vector abolished the cAMP response, implying that C/EBPs serve as mediators in the delayed cAMP signal transduction to the dPRL promoter. The ratio between activating and repressing isoforms is likely to dictate the transcriptional output.
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PMID:CCAAT/enhancer-binding proteins are mediators in the protein kinase A-dependent activation of the decidual prolactin promoter. 1045 53

The physiological actions of nitric oxide (NO) as a signaling molecule in endothelial and brain cells and as a toxic molecule used by activated immune cells have been the focus of a wide range of studies. Nevertheless, the downstream effector molecules of this important neuromodulator are not well understood. We have previously demonstrated that expression of the gene for the reproductive neuropeptide, GnRH, is repressed by the glutamate/NO/cyclic GMP (cGMP) signal transduction pathway through cGMP-dependent protein kinase in the hypothalamic GnRH-secreting neuronal cell line GT1-7. This repression localized within a previously characterized 300-bp neuron-specific enhancer. Here, we find that mutation of either of two adjacent elements within the enhancer eliminates repression by this pathway. An AT-rich sequence located at -1695 has homology to the octamer motif known to bind POU-homeodomain proteins, while the adjacent element at -1676 has homology to the C/EBP (CCAAT/enhancer-binding protein) protein family consensus sequence. Antibody supershift assays reveal that one of the proteins bound at the -1695 sequence is Oct-1, and one of the proteins bound to the element at -1676 is C/EBPbeta. These two proteins can bind simultaneously to the adjacent -1695 and -1676 binding sites in vitro. In nuclear extracts of GT1-7 cells treated with an NO donor, the intensity of the Oct-1 complex is increased. However, although Western blot analysis indicates that neither Oct-1 nor C/EBPbeta protein levels are increased, the relative binding affinity of Oct-1 is increased. Dephosphorylation of the nuclear extracts decreases binding of the Oct-1 complex to the -1695 site only in NO donor-treated extracts. Thus, we conclude that Oct-1 and C/EBPbeta are both downstream transcriptional regulators involved in the repression of GnRH gene expression by the glutamate/NO/ cGMP signal transduction pathway.
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PMID:Transcription factors Oct-1 and C/EBPbeta (CCAAT/enhancer-binding protein-beta) are involved in the glutamate/nitric oxide/cyclic-guanosine 5'-monophosphate-mediated repression of mediated repression of gonadotropin-releasing hormone gene expression. 1067 95

Cyclooxygenase-2 (COX-2), the enzyme primarily responsible for induced prostaglandin synthesis, is an immediate early gene induced by endotoxin in macrophages. We investigated the cis-acting elements of the COX-2 5'-flanking sequence, the transcription factors and signaling pathways responsible for transcriptional activation of the COX-2 gene in endotoxin-treated murine RAW 264.7 macrophages. Luciferase reporter constructs with alterations in presumptive cis-acting transcriptional regulatory elements demonstrate that the cyclic AMP-response element and two nuclear factor interleukin-6 (CCAAT/enhancer-binding protein (C/EBP)) sites of the COX-2 promoter are required for optimal endotoxin-dependent induction. In contrast, the E-box and NF-kappaB sites are not required for endotoxin-dependent induction. Inhibition of endotoxin-induced NF-kappaB activation by expression of an inhibitor-kappaB alpha mutant does not block endotoxin-dependent COX-2 reporter activity. Overexpression of c-Jun, C/EBPbeta, and C/EBPdelta enhances induction of the COX-2 reporter, while overexpression of cyclic AMP-response element-binding protein or "dominant negative" C/EBPbeta represses COX-2 induction. In addition, endotoxin rapidly and transiently elicits c-Jun phosphorylation in RAW 264.7 macrophages. Cotransfection of the COX-2 reporter with dominant negative expression vectors shows that endotoxin-induced COX-2 gene expression requires signaling through a Ras-independent pathway involving the adapter protein ECSIT and the signaling kinases MEKK1 and JNK. In contrast, endotoxin-induced COX-2 reporter activity is not blocked by overexpression of dominant-negative forms of Raf-1, ERK1, or ERK2.
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PMID:Transcriptional activation of the cyclooxygenase-2 gene in endotoxin-treated RAW 264.7 macrophages. 1069 22

The in vivo pattern of induction of phosphoenolpyruvate carboxykinase (PEPCK) gene transcription by cAMP and its inhibition by insulin is reproduced in H4IIe cells and is mediated by a bipartite cAMP/insulin response unit (C/IRU) consisting of a cAMP response element (-95/-87) and an upstream enhancer, AC (-271/-225). Studies in HepG2 cells showed that binding of AP-1 and CAAT/enhancer-binding protein (C/EBP) to AC is required for induction by cAMP, but insulin did not inhibit cAMP-induced PEPCK expression in HepG2 cells. Binding of H4IIe nuclear proteins to an AC element probe was inhibited by antibodies or a consensus site for C/EBP, but not AP-1. Transfection with dominant negative bZIP factors, which prevent endogenous factors from binding to DNA, showed that elimination of cAMP regulatory element-binding protein CREB or C/EBP activity blocked induction by protein kinase A (PKA), whereas elimination of AP-1 activity had no effect. In addition, promoters with multiple CREB sites, or a single CREB site and multiple C/EBP sites, mediated PKA induction, but this was inhibited to no greater extent than basal activity was by insulin. These results indicate that an AC factor other than C/EBP must mediate insulin inhibition. An A-site probe (-265/-247) or a probe across the middle of the AC element (-256/-237) competed for complexes formed by factors other than AP-1 or C/EBP. However, analysis of competitor oligonucleotides and antibodies for candidate factors failed to identify other factors. Scanning mutations throughout the AC element interfered with induction but allowed us to define five overlapping sites for regulatory factors in AC and to design probes binding just one or two factors. Comparison of the protein-DNA complexes formed on these smaller probes revealed that a specific complex present in rat liver and H4IIe cell nuclear extracts differed from those formed by HepG2 cell nuclear extracts. Our results suggest that multiple factors binding the AC element of the C/IRU interact with each other and CREB to regulate PEPCK induction by cAMP and inhibition by insulin and that the unique factor expressed in H4IIe cells is a candidate for involvement in insulin regulation of PKA-induced PEPCK gene transcription.
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PMID:Characterization of elements mediating regulation of phosphoenolpyruvate carboxykinase gene transcription by protein kinase A and insulin. Identification of a distinct complex formed in cells that mediate insulin inhibition. 1074 64

Cyclic AMP-response element modulator alpha (CREMalpha) is a transcription factor that is highly related to cAMP-response element-binding protein (CREB) but represses cAMP-induced gene expression from simple artificial promoters containing a cAMP-response element (CRE). CREMalpha lacks two glutamine-rich Q regions that, in CREB, are thought to be necessary for transcriptional activation. Nevertheless, protein kinase A stimulation induces CREMalpha to activate the complex native promoter in the phosphoenolpyruvate carboxykinase (PEPCK) gene. To study this phenomenon in the absence of protein kinase A stimulation, we introduced a mutation into CREMalpha to allow constitutive binding to the coactivator CREB-binding protein. This mutant, CREMalpha(DIEDML), constitutively activated the PEPCK promoter. By engineering the leucine zipper regions of CREMalpha(DIEDML) and CREB(DIEDML) to direct their patterns of dimerization, we found that only CREMalpha(DIEDML) homodimers fully activated the PEPCK promoter. By using a series of deletion and block mutants of the PEPCK promoter, we found that activation by CREMalpha(DIEDML) depended on the CRE and two CCAAT/enhancer-binding protein (C/EBP) sites. A dominant negative inhibitor of C/EBP, A-C/EBP, suppressed activation by CREMalpha(DIEDML). Furthermore, a GAL4-C/EBPalpha fusion protein and CREMalpha(DIEDML) cooperatively activated a promoter containing three GAL4 sites and the PEPCK CRE. Thus, we propose that the C/EBP sites in the PEPCK promoter allow CREMalpha to activate transcription despite its lack of Q regions.
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PMID:Cooperative mechanism of transcriptional activation by a cyclic AMP-response element modulator alpha mutant containing a motif for constitutive binding to CREB-binding protein. 1109 86

The memory for sensitization of the gill withdrawal reflex in Aplysia is reflected in facilitation of the monosynaptic connection between the sensory and motor neurons of the reflex. The switch from short- to long-term facilitation requires activation of CREB1, derepression of ApCREB2, and induction of ApC/EBP. In search for genes that act downstream from CREB1, we have identified a transcription activator, ApAF, which is stimulated by protein kinase A and can dimerize with both ApC/EBP and ApCREB2. ApAF is necessary for long-term facilitation induced by five pulses of serotonin, by activation of CREB1, or by derepression of ApCREB2. Overexpression of ApAF enhances the long-term facilitation further. Thus, ApAF is a candidate memory enhancer gene downstream from both CREB1 and ApCREB2.
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PMID:Enhancement of memory-related long-term facilitation by ApAF, a novel transcription factor that acts downstream from both CREB1 and CREB2. 1110 30


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