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:4.1.1.32 (phosphoenolpyruvate carboxykinase)
4,204 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Recently, Kalvakolanu et al. (Kalvakolanu, D. V. R., Liu, J., Hanson, R. W., Harter, M. L., and Sen, G. C. (1992) J. Biol. Chem. 267, 2530-2536) showed that E1A inhibited the basal and cAMP-stimulated transcription of the gene for phosphoenolpyruvate carboxykinase (PEPCK). This inhibition was mediated by the conserved region 1 (CR1) domain of E1A, which has been shown by other laboratories to bind to the cellular transcriptional adaptor proteins, p300 and cAMP response element binding protein (CREB)-binding protein (CBP). The PEPCK gene promoter contains a functional cAMP-response element, through which CREB and, therefore, CBP modulate transcription, and a consensus p300 DNA binding sequence is also present in a distal protein binding site of the promoter. We hypothesized that E1A might inhibit PEPCK gene transcription by binding to p300 and/or CBP. Surprisingly, we found that E1A consistently stimulated basal transcription from the PEPCK promoter in transfection assays in adenovirus (Ad)-infected HepG2 hepatoma cells or E1A-expressing, stably transfected 3T3 fibroblasts and nuclear run-on assays in Ad-infected H4IIE hepatoma cells. E1A also enhanced the stimulation of PEPCK gene transcription by Bt2cAMP. In transfection assays, wild type Ad5 expressing both 243R and 289R forms of E1A or a mutant virus expressing the 289R form alone stimulated transcription from the PEPCK promoter by approximately 5-fold 20 h postinfection. However, no stimulation was observed in cells infected with a virus expressing either the 243R protein alone or a 289R protein from which conserved region 3 (CR3) was mutated. Mutation or deletion of CR1 of E1A had no significant effect on transcription from the PEPCK promoter. Mutations within conserved region 2 (CR2) of E1A that inhibit the binding of E1A to the retinoblastoma gene product (pRb) further enhanced the stimulation of transcription from the PEPCK promoter by 2 3-fold compared with wild type E1A. These findings suggested that the normal function of pRb is to stimulate PEPCK gene transcription, and that this process is inhibited by the binding of E1A to pRb. This hypothesis was confirmed by overexpressing pRb in HepG2 cells, which stimulated transcription from the PEPCK promoter. Our findings indicate that Ad E1A regulates PEPCK gene transcription through a stimulatory mechanism involving CR3, and by attenuating a stimulatory effect of pRb through CR2.
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PMID:Adenovirus E1A proteins regulate phosphoenolpyruvate carboxykinase gene transcription through multiple mechanisms. 862 93

The onset of metabolic acidosis causes an increased transcription of the renal phosphoenolpyruvate carboxykinase (PCK) gene. When transgenic mice carrying a bovine growth hormone (bGH) gene driven by the -460 to +73 segment of the PCK promoter were made chronically acidotic, the bGH mRNA was increased twofold after 4 days. Confluent and well-differentiated cultures of LLC-PK1-F+ cells exhibit a 2.5-fold increase in PCK mRNA when transferred to acidic media (pH 6.9, 10 mM HCO3-) for 16 h. Confluent cultures transfected with PCK-490 CAT exhibit an increase (3.5-fold) in chloramphenicol acetyltransferase (CAT) activity when shifted to acidic medium for 48 h. Mutation or deletion of the P2 element causes a four- to fivefold decrease in basal CAT activity but does not affect the pH response. In contrast, mutations of the P3(II) element or the CRE-1 cAMP-response element have little effect on basal activity but cause a 50% decrease in the pH response. Other deletions or mutations have little effect on either activity. Thus changes in the activity or levels of the protein(s) in the renal proximal tubule that binds to the P3(II) and CRE-1 elements may mediate increased transcription of the PCK gene during metabolic acidosis.
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PMID:Promoter elements that mediate the pH response of PCK mRNA in LLC-PK1-F+ cells. 877 Jan 65

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

The glucagon-stimulated transcription of the cytosolic phosphoenolpyruvate carboxykinase-1 (PCK1) gene is mediated by cAMP and positively modulated by oxygen in primary hepatocytes. Rat hepatocytes were transfected with constructs containing the first 2500, 493 or 281 bp of the PCK1 5'-flanking region in front of the chloramphenicol acetyltransferase (CAT) reporter gene. With all three constructs glucagon induced CAT activity with decreasing efficiency maximally under arterial pO2 and to about 65% under venous pO2. Rat hepatocytes were then transfected with constructs containing the first 493 bp of the PCK1 5'-flanking region in front of the luciferase (LUC) reporter gene, which were block-mutated at the CRE1 (cAMP-response element-1; -93/-86), putative CRE2 (-146/-139), promoter element (P) 1 (-118/-104), P2 (-193/-181) or P4 (-291/-273) sites. Glucagon induced LUC activity strongly when the P1 and P2 sites were mutated and weakly when the P4 site was mutated; induction of the P1, P2 and P4 mutants was positively modulated by the pO2. Glucagon also induced LUC activity strongly when the putative CRE2 site was altered; however, induction of the CRE2 mutant was not modulated by the pO2. Glucagon did not induce LUC activity when the CRE1 site was modified. These experiments suggested that the CRE1 but not the putative CRE2 was an essential site necessary for the cAMP-mediated PCK1 gene activation by glucagon and that the putative CRE2 site was involved in the oxygen-dependent modulation of PCK1 gene activation. To confirm these conclusions rat hepatocytes were transfected with simian virus 40 (SV40)-promoter-driven LUC-gene constructs containing three CRE1 sequences (-95/-84), three CRE2 sequences (-148/-137) or three CRE1 sequences plus two CRE2 sequences of the PCK1 gene in front of the SV40 promoter. Glucagon induced LUC activity markedly when the CRE1, but not when the CRE2, sites were in front of the SV40-LUC gene; however, induction of the (CRE1)3SV40-LUC constructs was not modulated by the pO2. Glucagon also induced LUC activity very strongly when the CRE1 and CRE2 sites were combined; induction of the (CRE1)3(CRE2)2SV40-LUC constructs was positively modulated by the pO2. These findings corroborated that sequences of the putative CRE2 site were responsible for the modulation by oxygen of the CRE1-dependent induction by glucagon of PCK1 gene transcription.
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PMID:Identification of an oxygen-responsive element in the 5'-flanking sequence of the rat cytosolic phosphoenolpyruvate carboxykinase-1 gene, modulating its glucagon-dependent activation. 1021 94

cAMP increases transcription of the mitochondrial (mit.) gene for 3-hydroxy-3-methylglutaryl (HMG)-CoA synthase, which encodes an enzyme that has been proposed as a control site of ketogenesis. The incubation of Caco-2 cells with cAMP increased mit.HMG-CoA synthase mRNA levels 4-fold within 24 h. We have identified an active cAMP-response element (CRE) located 546 bp upstream of the mit. HMG-CoA synthase promoter that is necessary for the induction of expression by dibutyryl cAMP. Co-transfections of constructs, containing the CRE element of the mit.HMG-CoA synthase promoter fused to the gene for chloramphenicol acetyltransferase, with protein kinase A and a dominant-negative mutant of cAMP-response-element-binding protein (CREB) show that the response to cAMP is mediated by the transcription factor CREB. The CRE element confers responsiveness of protein kinase A to a heterologous promoter in transfection assays in Caco-2 cells. Gel-retardation assays revealed that the mit.HMG-CoA synthase CRE binds to recombinant CREB. The shifted band obtained with the putative mit. HMG-CoA synthase CRE sequence and nuclear proteins from Caco-2 cells competed with CRE sequences of other genes such as somatostatin and phosphoenolpyruvate carboxykinase. We conclude that the regulation of the expression of the gene for mit.HMG-CoA synthase in Caco-2 cells by cAMP is mediated by a CRE sequence in the promoter.
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PMID:Mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase promoter contains a CREB binding site that regulates cAMP action in Caco-2 cells. 1062 Apr 95

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

Increased renal catabolism of plasma glutamine during metabolic acidosis generates two ammonium ions that are predominantly excreted in the urine. They function as expendable cations that facilitate the excretion of acids. Further catabolism of alpha-ketoglutarate yields two bicarbonate ions that are transported into the venous blood to partially compensate for the acidosis. In rat kidney, this adaptation is sustained, in part, by the induction of multiple enzymes and various transport systems. The pH-responsive increases in glutaminase (GA) and phosphoenolpyruvate carboxykinase (PEPCK) mRNAs are reproduced in LLC-PK(1)-fructose 1,6-bisphosphatase (FBPase) cells. The increase in GA activity results from stabilization of the GA mRNA. The 3'-untranslated region of the GA mRNA contains a direct repeat of an eight-base AU sequence that functions as a pH-response element. This sequence binds zeta-crystallin/NADPH:quinone reductase with high affinity and specificity. Increased binding of this protein during acidosis may initiate the pH-responsive stabilization of the GA mRNA. In contrast, induction of PEPCK occurs at the transcriptional level. In LLC-PK(1)-FBPase(+) kidney cells, a decrease in intracellular pH leads to activation of the p38 stress-activated protein kinase and subsequent phosphorylation of transcription factor ATF-2. This transcription factor binds to cAMP-response element 1 within the PEPCK promoter and may enhance its transcription during metabolic acidosis.
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PMID:Mechanism of increased renal gene expression during metabolic acidosis. 1150 86

LLC-PK(1)-FBPase(+) cells are a gluconeogenic and pH-responsive renal proximal tubule-like cell line. On incubation with acidic medium (pH 6.9), LLC-PK(1)-FBPase(+) cells exhibit an increased rate of ammonia production as well as increases in glutaminase and phosphoenolpyruvate carboxykinase (PEPCK) mRNA levels and enzyme activities. The increase in PEPCK mRNA is due to an enhanced rate of transcription that is initiated in response to intracellular acidosis. The involvement of known MAPK activities (ERK1/2, SAPK/JNK, p38) in the associated signal transduction pathway was examined by determining the effects of specific MAPK activators and inhibitors on basal and acid-induced PEPCK mRNA levels. Transfer of LLC-PK(1)-FBPase(+) cultures to acidic medium resulted in specific phosphorylation, and thus activation, of p38 and of activating transcription factor-2 (ATF-2), respectively. Anisomycin (AI), a strong p38 activator, increased PEPCK mRNA to levels comparable to those observed with acid stimulation. AI also induced a time-dependent phosphorylation of p38 and ATF-2. SB-203580, a specific p38 inhibitor, blocked both acid- and AI-induced PEPCK mRNA levels. Western blot analyses revealed that the SB-203580-sensitive p38alpha isoform is strongly expressed. The octanucleotide sequence of the cAMP-response element-1 site of the PEPCK promotor is a perfect match to the consensus element for binding ATF-2. The specificity of ATF-2 binding was proven by ELISA. We conclude that the SB-203580-sensitive p38alpha-ATF-2 signaling pathway is a likely mediator of the pH-responsive induction of PEPCK mRNA levels in renal LLC-PK(1)-FBPase(+) cells.
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PMID:p38 MAPK mediates acid-induced transcription of PEPCK in LLC-PK(1)-FBPase(+) cells. 1221 59

Hepatic gluconeogenesis is essential for maintaining blood glucose levels during fasting and is the major contributor to postprandial and fasting hyperglycemia in diabetes. Gluconeogenesis is a classic cAMP/protein kinase A-dependent process initiated by glucagon, which is elevated in the blood during fasting and in diabetes. In this study, we have shown that p38 mitogen-activated protein kinase (p38) was activated in liver by fasting and in primary hepatocytes by glucagon or forskolin. Fasting plasma glucose levels were reduced upon blockade of p38 with either a chemical inhibitor or small interference RNA in mice. In examining the mechanism, inhibition of p38 suppressed gluconeogenesis in liver, along with expression of key gluconeogenic genes, including phosphoenolpyruvate carboxykinase and glucose-6-phosphatase. Peroxisome proliferator-activated receptor gamma coactivator 1alpha and cAMP-response element-binding protein have been shown to be important mediators of hepatic gluconeogenesis. We have shown that inhibition of p38 prevented transcription of the PPARgamma coactivator 1alpha gene as well as phosphorylation of cAMP-response element-binding protein. Together, our results from in vitro and in vivo studies define a model in which cAMP-dependent activation of genes involved in gluconeogenesis is dependent upon the p38 pathway, thus adding a new player to our evolving understanding of this physiology.
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PMID:p38 Mitogen-activated protein kinase plays a stimulatory role in hepatic gluconeogenesis. 1627 51

Vitamin A deficiency decreases hepatic phosphoenolpyruvate carboxykinase (PEPCK) gene expression in mice, and expression is restored with retinoic acid (RA) treatment in vivo. In the studies reported here, we examined changes in histone modification and coregulator association with the regulatory domains of the PEPCK gene in response to alterations in vitamin A status. We identified nuclear receptors that bind to retinoic acid response elements (RAREs) in the PEPCK promoter by electrophoretic mobility shift assay and verified these in vivo using chromatin immunoprecipitation in mouse liver. Hypothetically, nuclear receptors at PEPCK RAREs recruit specific coactivator molecules that contribute to the acetylation of core histones and/or serve as bridging molecules between nuclear receptors and basal transcription factors at the transcription start site. We identified 3 coactivator molecules, cAMP-response element binding protein (CBP), steroid receptor coactivator (SRC)-1, and peroxisome-proliferator activated receptor (PPAR)-gamma-coactivator (PGC)-1alpha, that bound in association with the PEPCK RAREs in vivo. Furthermore, there was differential binding of these coactivators in vitamin A-deficient mice. Related to this, specific lysine residues were acetylated on histones H3 and H4 at the 3 RAREs of the PEPCK promoter, consistent with the action of the above coactivators, and acetylation of certain lysines was significantly decreased with vitamin A deficiency. These results demonstrate the associated changes that occur in nuclear receptor binding, coactivator recruitment, and histone acetylation in response to vitamin A status, identified at specific RAREs in the PEPCK gene in vivo.
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PMID:Vitamin A status in mice affects the histone code of the phosphoenolpyruvate carboxykinase gene in liver. 1631 19


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