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:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Hypertonic-induced cell shrinkage increases glucose release in H-4-II-E rat hepatoma cells. This is paralleled by a concomitant increase in the mRNA levels of the rate-limiting enzymes of the pathway of gluconeogenesis, phosphoenolpyruvate carboxykinase (PCK) and fructose-1,6-bisphosphatase (FBP), of seven- and fivefold, respectively. In contrast, hypotonic-induced swelling of the cells results in a transient decrease in PCK and FBP mRNAs to 15% and 39% of control levels. The antagonistic effects of hyper- and hypotonicity mimic the counteracting effects of adenosine 3',5'-cyclic monophosphate (cAMP) and insulin on PCK and FBP mRNA levels. The hypertonic-induced increase in mRNA levels is due to an enhanced transcriptional rate, whereas the decrease in mRNAs caused by hypotonicity results from a decrease in transcription as well as mRNA stability. The inductive effect of hypertonicity does not require ongoing protein synthesis and acts independently of the cAMP-dependent protein kinase and protein kinase C pathways. These results suggest that cell volume changes in liver cells may play an important role in regulating hepatic glucose metabolism by altered gene expression.
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PMID:Cell volume regulates liver phosphoenolpyruvate carboxykinase and fructose-1,6-bisphosphatase genes. 953 Jan 52

The alpha isoform of CCAAT/enhancer-binding protein (C/EBPalpha) is a transcription factor that regulates expression of genes linked to adipose differentiation and hepatic nutrient metabolism. Recently, our laboratory has characterized a role for C/EBPalpha in mediating hormonal responsiveness. For example, the cAMP responsiveness of the phosphoenolpyruvate carboxykinase gene promoter in liver requires synergism among the cAMP response element-binding protein (CREB), C/EBPalpha, and activator protein-1. In the present study, we show that C/EBPalpha can functionally substitute for CREB in this cAMP response unit, i.e. cAMP responsiveness can occur in the absence of CREB. This observation is physiologically relevant since both CREB and C/EBPalpha have been shown to bind with high affinity to the cAMP response element in this particular promoter. Structure/function analysis of C/EBPalpha identified specific mutations that differentially affected its constitutive and protein kinase A-inducible activities. This finding suggests that the mechanism whereby C/EBPalpha mediates constitutive transactivation is distinct from that whereby it mediates cAMP responsiveness. These data support the hypothesis that C/EBPalpha plays a critical role in metabolism, in part by participating in the hormonal regulation of expression of metabolically important genes.
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PMID:Characterization of CCAAT/enhancer-binding protein alpha as a cyclic AMP-responsive nuclear regulator. 961

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

Transcription of the phosphoenolpyruvate carboxykinase (PEPCK) gene is induced by glucagon, acting through cAMP and protein kinase A, and this induction is inhibited by insulin. Conflicting reports have suggested that insulin inhibits induction by cAMP by activating the Ras/mitogen-activated protein kinase (MAPK) pathway or by activating the phosphatidylinositol 3-kinase (PI3-kinase), but not MAPK, pathway. Insulin activated PI3-kinase phosphorylates lipids that activate protein kinase B (PKB) and Ca2+/diacylglycerol-insensitive forms of protein kinase C (PKC). We have assessed the roles of these pathways in insulin inhibition of cAMP/PKA-induced transcription of PEPCK by using dominant negative and dominant active forms of regulatory enzymes in the Ras/MAPK and PKB pathways and chemical inhibitors of PKC isoforms. Three independently acting inhibitory enzymes of the Ras/MAPK pathway, blocking SOS, Ras, and MAPK, had no effect upon insulin inhibition. However, dominant active Ras prevented induction of PEPCK and also stimulated transcription mediated by Elk, a MAPK target. Insulin did not stimulate Elk-mediated transcription, indicating that insulin did not functionally activate the Ras/MAPK pathway. Inhibitors of PI3-kinase, LY294002 and wortmannin, abolished insulin inhibition of PEPCK gene transcription. However, inhibitors of PKC and mutated forms of PKB, both of which are known downstream targets of PI3-kinase, had no effect upon insulin inhibition. Dominant negative forms of PKB did not interfere with insulin inhibition and a dominant active form of PKB did not prevent induction by PKA. Phorbol ester-mediated inhibition of PEPCK transcription was blocked by bisindole maleimide and by staurosporine, but insulin-mediated inhibition was unaffected. Thus, insulin inhibition of PKA-induced PEPCK expression does not require MAPK activation but does require activation of PI3-kinase, although this signal is not transmitted through the PKB or PKC pathways.
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PMID:Assessment of the roles of mitogen-activated protein kinase, phosphatidylinositol 3-kinase, protein kinase B, and protein kinase C in insulin inhibition of cAMP-induced phosphoenolpyruvate carboxykinase gene transcription. 966 48

Transcriptional regulation of phosphoenolpyruvate carboxykinase (PEPCK), the rate-limiting enzyme in hepatic gluconeogenesis, by insulin was investigated with the use of adenovirus vectors encoding various mutant signaling proteins. Insulin inhibited transcription induced by dexamethasone and cAMP of a chloramphenicol acetyltransferase (CAT) reporter gene fused with the PEPCK promoter sequence in HL1C cells stably transfected with this construct. A dominant negative mutant of phosphoinositide (PI) 3-kinase blocked insulin inhibition of transcription of the PEPCK-CAT fusion gene, whereas a constitutively active mutant of PI 3-kinase mimicked the effect of insulin. Although a constitutively active mutant of Akt (protein kinase B) inhibited PEPCK-CAT gene transcription induced by dexamethasone and cAMP, a mutant Akt (Akt-AA) in which the phosphorylation sites targeted by insulin are replaced by alanine did not affect the ability of insulin to inhibit transcription of the fusion gene. Akt-AA almost completely inhibited insulin-induced activation of both endogenous and recombinant Akt in HL1C cells. Furthermore, neither a kinase-defective mutant protein kinase Clambda (PKClambda), which blocked insulin-induced activation of endogenous PKClambda, nor a dominant negative mutant of the small GTPase Rac prevented inhibition of PEPCK-CAT gene transcription by insulin. These data suggest that phosphoinositide 3-kinase is important for insulin-induced inhibition of PEPCK gene transcription and that a downstream effector of phosphoinositide 3-kinase distinct from Akt, PKClambda, and Rac may exist for mediating the effect of insulin.
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PMID:Dominant negative forms of Akt (protein kinase B) and atypical protein kinase Clambda do not prevent insulin inhibition of phosphoenolpyruvate carboxykinase gene transcription. 1040 89

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

Heme oxygenase (HO) catalyzes the rate-limiting enzymatic step of heme degradation and regulates the cellular heme content. The gene expression of the inducible isoform of HO, HO-1, is up-regulated in response to various agents causing oxidative stress. To investigate the regulatory role of protein phosphatases in the hepatic regulation of HO-1 gene expression, primary cultures of rat hepatocytes were treated with okadaic acid (OA), which specifically inhibits the serine threonine protein phosphatases 1 and 2A. Both protein synthesis and mRNA expression of HO-1 were induced by OA in cultured hepatocytes, but not in cultured tissue macrophages of rat liver. The HO-1 mRNA induction by OA occurred in a time- and concentration-dependent manner. Simultaneous treatment with OA plus dibutyryl cAMP caused a synergistic up-regulation of steady-state levels of HO-1 mRNA, and the specific protein kinase A inhibitor KT5720 markedly reduced the OA-dependent HO-1 mRNA induction. In contrast, the dibutyryl cAMP-dependent induction of the phosphoenolpyruvate carboxykinase mRNA expression and enzyme activity was inhibited by simultaneous treatment with OA in hepatocytes. The induction of the HO-1 gene expression by OA was transcriptional as determined by studies with actinomycin D, nuclear run-off assay, and measurement of the half-life of HO-1 mRNA. Luciferase reporter constructs containing DNA sequences of the rat HO-1 promoter 5'-flanking region were up-regulated by OA in transiently transfected hepatocytes. Mutation of the cAMP response element/activator protein-1 (-665/-654) site obliterated the OA-dependent induction, suggesting that this element is involved in the transcriptional induction of the rat HO-1 gene by OA.
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PMID:Transcriptional induction of heme oxygenase-1 gene expression by okadaic acid in primary rat hepatocyte cultures. 1069 3

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

Fifty percent of the mice homozygous for a deletion in the gene for CCAAT/enhancer-binding protein beta (C/EBP beta-/- mice; B phenotype) die within 1 to 2 h after birth of hypoglycemia. They do not mobilize their hepatic glycogen or induce the cytosolic form of phosphoenolpyruvate carboxykinase (PEPCK). Administration of cAMP resulted in mobilization of glycogen, induction of PEPCK mRNA, and a normal blood glucose; these mice survived beyond 2 h postpartum. Adult C/EBP beta-/- mice (A phenotype) also had difficulty in maintaining blood glucose levels during starvation. Fasting these mice for 16 or 30 h resulted in lower levels of hepatic PEPCK mRNA, blood glucose, beta-hydroxybutyrate, blood urea nitrogen, and gluconeogenesis when compared with control mice. The concentration of hepatic cAMP in these mice was 50% of controls, but injection of theophylline, together with glucagon, resulted in a normal cAMP levels. Agonists (glucagon, epinephrine, and isoproterenol) and other effectors of activation of adenylyl cyclase were the same in liver membranes isolated from C/EBP beta-/- mice and littermates. The hepatic activity of cAMP-dependent protein kinase was 80% of wild type mice. There was a 79% increase in the concentration of RI alpha and 27% increase in RII alpha in the particulate fraction of the livers of C/EBP beta-/- mice relative to wild type mice, with no change in the catalytic subunit (C alpha). Thus, a 45% increase in hepatic cAMP (relative to the wild type) would be required in C/EBP beta-/- mice to activate protein kinase A by 50%. In addition, the total activity of phosphodiesterase in the livers of C/EBP beta-/- mice, as well as the concentration of mRNA for phosphodiesterase 3A (PDE3A) and PDE3B was approximately 25% higher than in control animals, suggesting accelerated degradation of cAMP. C/EBP beta influences the regulation of carbohydrate metabolism by altering the level of hepatic cAMP and the activity of protein kinase A.
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PMID:Mice with a deletion in the gene for CCAAT/enhancer-binding protein beta have an attenuated response to cAMP and impaired carbohydrate metabolism. 1102 29

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


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