Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.11.11 (AMPK)
 12,425 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The ability of Pit-1 to mediate transcriptional responses to cAMP has been explored. To test the ability of Pit-1 to mediate transcriptional responses to cAMP, an expression vector was prepared for a mutant Pit-1 in which the major sites of phosphorylation by the cAMP-dependent protein kinase were eliminated. Before using the mutant Pit-1 to study transcriptional regulation, we first examined the ability of the protein to be phosphorylated in vivo in response to cAMP. Transfection and in vivo labeling experiments confirmed that the mutant Pit-1 did not support cAMP-inducible phosphorylation. The ability of the wild type or mutant Pit-1 to mediate transcriptional responses to cAMP was assessed in cotransfection experiments using reporter genes containing either the proximal region of the rat PRL gene or seven copies of a Pit-1 binding site placed upstream of a minimal promoter. Surprisingly, the wild type and mutant Pit-1 expression vectors supported similar responses to cAMP. To further assess the ability of Pit-1 to mediate responses to cAMP, a GAL4-Pit-1 fusion gene was prepared. Although a GAL4-cAMP response element binding protein fusion gene was found to permit transcriptional responses to cAMP, the GAL4-Pit-1 gene was unresponsive. These findings demonstrate that although Pit-1 can facilitate the ability of the PRL promoter to respond to cAMP, phosphorylation of Pit-1 is not required for this response. It seems likely that additional factors that interact with Pit-1 binding sites are important for mediating transcriptional responses to cAMP.
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PMID:Pit-1 binding sites mediate transcriptional responses to cyclic adenosine 3',5'-monophosphate through a mechanism that does not require inducible phosphorylation of Pit-1. 787 13

We report that the small tumor (small-t) antigen of simian virus 40 (SV40) forms complexes with nuclear protein phosphatase 2A (PP2A) and regulates the phosphorylation and transcriptional transactivation function of the cyclic AMP (cAMP)-regulatory element binding protein (CREB). PP2A coimmunoprecipitated with small t from nuclear extracts from HepG2 cells expressing small t or from rat liver nuclear extracts to which recombinant small t was added. Protein phosphatase 1 was not detected in small-t immunoprecipitates. In HepG2 cells expressing small t, dibutyryl-cAMP (Bt2cAMP) stimulated the phosphorylation of CREB 65-fold, whereas CREB phosphorylation was stimulated only 5- to 8-fold by Bt2cAMP in cells not expressing small t. Small t also inhibited the dephosphorylation of cAMP-dependent protein kinase (PKA)-phosphorylated CREB in rat liver nuclear extracts. In cells expressing small t, Bt2cAMP-stimulated transcription from the phosphoenolpyruvate carboxykinase (PEPCK) gene promoter was enhanced over the level of transcription from the PEPCK promoter in cells not expressing small t. Small t also enhanced Bt2cAMP-stimulated transcription from a Gal4-responsive promoter in cells expressing a chimeric protein containing the Gal4 DNA-binding domain linked to the CREB transactivation domain. However, small t did not stimulate transcription either from a 5' deletion mutant of the PEPCK promoter that is not able to bind CREB or from the Gal4-responsive promoter in the absence of the Gal4-CREB protein. These data suggest that small t enhances Bt2cAMP-stimulated gene transcription by inhibiting the dephosphorylation of PKA-phosphorylated CREB by nuclear PP2A. These findings support previous observations that nuclear PP2A is the primary phosphatase that dephosphorylates PKA-phosphorylated CREB.
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PMID:Simian virus 40 small tumor antigen inhibits dephosphorylation of protein kinase A-phosphorylated CREB and regulates CREB transcriptional stimulation. 806 21

We previously reported that microinjection of purified Ras protein stimulated DNA synthesis in quiescent Wistar rat thyrocytes and that TSH (TSH)-stimulated DNA synthesis was Ras-dependent. In contrast to these results, microinjection of cellular or oncogenic Ras significantly reduced TSH-stimulated thyroglobulin (Tg) expression, a marker of thyrocyte differentiation. Microinjection of a dominant inhibitory Ras mutant had no effect on TSH-stimulated Tg expression. As the Tg promoter is cAMP-responsive and Ras was previously reported to interfere with entry of catalytic (C) subunit of the cAMP-dependent protein kinase into the nucleus, experiments were performed to assess the effects of Ras on cAMP-mediated signaling. Microinjection of either cellular or oncogenic Ras had no effect on TSH-stimulated entry of C subunit into the nucleus. Consistent with these data, Ras did not reduce TSH-stimulated cAMP response element binding protein phosphorylation, or cAMP response element-regulated gene expression. These results demonstrate that Ras exerts differential effects on TSH signaling; Ras increases TSH-stimulated DNA synthesis and decreases TSH-induced Tg expression. Moreover, the mechanism through which Ras induces Tg expression lies distal to entry of C subunit into the nucleus, cAMP response element binding protein phosphorylation, and cAMP response element-regulated gene expression.
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PMID:Ras inhibits thyroglobulin expression but not cyclic adenosine monophosphate-mediated signaling in Wistar rat thyrocytes. 853 48

To assess whether the cAMP-dependent protein kinase-A and/or the diacylglycerol-dependent protein kinase C (PKC) pathways play important roles in the activation of CRF neurons in vivo under physiological conditions, we tested the effect of microinjection of 8-bromo-cAMP (8-Br-cAMP) or 12-O-tetradecanoyl phorbol 13-acetate (TPA) into both paraventricular nuclei (PVN) of the hypothalamus in conscious rats. Both 8-Br-cAMP and TPA increased plasma ACTH concentrations and the POMC messenger RNA (mRNA) concentrations in the anterior pituitary. While injection of 8-Br-cAMP also increased CRF mRNA concentrations in hypothalamic tissue containing the PVN, TPA injection had no effect on CRF mRNA concentrations there. During insulin-induced hypoglycemia, which stimulates CRF gene expression and release, c-fos and c-jun mRNA increases in the hypothalamic tissue preceded the increase in the CRF mRNA level after insulin-induced hypoglycemia. Antisense oligodeoxyribonucleotides (oligos) directed against c-fos, c-jun, or the cAMP response element binding protein (CREB) mRNA were injected into both PVN before insulin-induced hypoglycemia to assess whether activator protein-1 or CREB mediates transcriptional activation of CRF during hypoglycemia. Only antisense oligo against CREB mRNA reduced the CRF mRNA level after insulin-induced hypoglycemia. These results suggest that protein kinase A may transduce intracellular signals in CRF neurons under physiological conditions and raises the possibility that CREB may be involved in stress-induced CRF gene expression.
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PMID:Major role of 3',5'-cyclic adenosine monophosphate-dependent protein kinase A pathway in corticotropin-releasing factor gene expression in the rat hypothalamus in vivo. 864 Nov 91

Activity-mediated gene expression is thought to play an important role in many forms of neuronal plasticities. We have used pentylenetetrazol-induced seizure that produces synchronous and sustained neuronal activity as a model to examine the mechanism(s) of gene activation. The transcription factor CREB (Ca2+/cAMP response element-binding protein) is thought to be necessary for long-term memory formation both in invertebrates and vertebrates. When phosphorylated on Ser133 either by cAMP-dependent protein kinase and/or Ca2+/calmodulin-dependent protein kinases, CREB increases transcription of genes containing the CRE (cAMP response element) sequence. Using an antibody that detects Ser133-phosphorylated CREB protein, we show that CREB phosphorylation is maximal between 3 and 8 min after the onset of seizure activity and declines slowly both in the hippocampus and the cortex. The total amount of CREB protein did not change at the time points examined. The increased phosphorylation of CREB protein is preceded by an increase in the amount of cAMP, suggestive of cAMP-dependent protein kinase activation, in the hippocampus and activation of Ca2+/calmodulin-dependent protein kinases in the cortex. Subsequent to CREB phosphorylation, the expression of the CRE-containing gene, c-fos, and the AP-1 complexes (heterodimers of Fos and Jun family members) is increased. These findings support the role of CREB-mediated gene expression in activity-dependent neuronal plasticities.
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PMID:Neuronal activity increases the phosphorylation of the transcription factor cAMP response element-binding protein (CREB) in rat hippocampus and cortex. 866 77

Recent studies from our laboratory (Endocrinology 136:4762-4768, 1995) demonstrating that the expression of cAMP-dependent nuclear transcription factor CREB (cAMP response element binding protein) is lost following ovulation in macaques has revealed a novel mechanism by which the cytoplasmic and nuclear actions the cAMP-protein kinase A (PKA) intracellular signaling system may be regulated independently. Implicit in this hypothesis is the assumption that PKA activity is maintained throughout the luteal phase of the menstrual cycle, yet to date there have been no published reports regarding PKA activity in the primate corpus luteum. PKA activity was assessed by the incorporation of 32P from radiolabeled ATP into a PKA-specific peptide substrate (kemptide) in the presence or absence of cAMP. Luteal cytosolic fractions were obtained from corpora lutea collected during the spontaneous luteal phase (days 3-5, 7-8, 10-11, 13-15, and postmenses) or obtained from animals on days 11 or 16 of the luteal phase after the animals received seven days of exogenous human CG (hCG) treatment. Examination of PKA activity in luteal slices from various aged CL maintained in short-term organ culture in the presence or absence of recombinant cynomolgus monkey LH was also performed. There were no significant differences in basal or cAMP-stimulated PKA activities in corpora lutea collected throughout the spontaneous luteal phase. Further, Western immunoblot analyses of the catalytic subunit of PKA (PKA C alpha) in corpora lutea collected throughout the luteal phase revealed immunoreactive protein bands with similar intensities. In vitro addition of recombinant cynomolgus LH and dibutyryl cAMP stimulated PKA activity in corpora lutea collected during the early, mid, and late luteal phases. In corpora lutea obtained from animals treated with hCG during the midluteal phase, basal PKA activity was decreased 65% as compared with untreated day 11 controls and in late luteal phase, hCG-exposed CL basal PKA activity was decreased 30% as compared with untreated day 16 controls. However, there were no measurable differences in cAMP-stimulated PKA activity in CL exposed to prior hCG treatment in vivo and Western immunoblot analyses for PKA C alpha in these tissues revealed immunoreactive protein bands that were comparable with corpora lutea collected from untreated animals. Further, immunoblot analyses for CREB in corpora lutea collected from hCG-treated animals revealed that CREB immunoreactivity remained undetectable following a treatment regimen with hCG that mimics early pregnancy. These results demonstrate that, although CREB expression ceases following ovulation, PKA activity is maintained throughout the luteal phase, which provides a mechanism by which the acute steroidogenic actions of LH may be separated from longer term trophic actions that may rely the transcriptional activity of CREB.
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PMID:Cyclic adenosine monophosphate signaling in the primate corpus luteum: maintenance of protein kinase A activity throughout the luteal phase of the menstrual cycle. 923

Ethanol impairs hormone-stimulated cAMP production in a number of cell types, yet the effects of ethanol on downstream responses mediated by cAMP-dependent protein kinase (PKA) are not understood. Here we have investigated the effects of ethanol feeding on cAMP-mediated inhibition of tumor necrosis factor-alpha (TNF-alpha) synthesis in rat Kupffer cells. Male Wistar rats were fed liquid diets containing 36% of calories as ethanol for 4 wk or were pair fed a control diet. Stimulation of cAMP production by the adenosine A2 receptor agonist 5'-(N-ethylcarboxamido)-adenosine (NECA), prostaglandin E2, or forskolin was decreased to 25% of control values in Kupffer cells isolated from ethanol-fed rats. This decrease was associated with a reduction in the quantity of immunoreactive Gsalpha protein in ethanol-fed rats, with no changes observed in Gialpha or Gbeta. TNF-alpha production was higher in ethanol-fed rats in response to stimulation with lipopolysaccharide or latex beads. Despite the profound reduction in the ability of hormone to increase cAMP production, NECA and prostaglandin E2 inhibited TNF-alpha production to an equivalent degree in Kupffer cells from ethanol- and pair-fed rats. Total activity and immuoreactive protein quantity of PKA did not differ between groups. Activation of PKA in response to a 15-min treatment with 1 microM NECA was reduced by 50% in ethanol-fed rats compared with control. Despite this reduction in activation, translocation of the catalytic subunit of PKA to the nucleus and phosphorylation of cAMP response element binding protein in response to activation were observed in Kupffer cells from both ethanol- and pair-fed rats. These data demonstrate that there is a dissociation between ethanol-induced desensitization of hormone-stimulated cAMP production in rat Kupffer cells and the downstream inhibition of TNF-alpha production mediated by cAMP.
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PMID:Ethanol dissociates hormone-stimulated cAMP production from inhibition of TNF-alpha production in rat Kupffer cells. 988 84

We report that the cAMP response element binding protein (CREB) undergoes cell-cycle-regulated phosphorylation. In human amnion FL cells, CREB was expressed as two forms with different molecular masses, 45 and 45.5 kDa. Although asynchronous cells contained predominantly the 45 kDa forms, this form shifted to 45.5 kDa when the cells were synchronized with the early S-phase. Furthermore the expression of the 45.5 kDa band was increased when cells were treated with okadaic acid, confirming that the 45.5 kDa band was a phosphorylated form of the 45 kDa band. Mutation analysis indicated that neither Ser133, the target of cAMP-dependent protein kinase and calcium calmodulin kinase, nor Ser129, the target of glycogen synthetase kinase 3, was responsible for the expression of the 45.5 kDa band, but that Ser108, Ser111 and Ser114, located in a region matching the consensus sequence for the casein kinase II target, were required. A mutant in which Ser111 and Ser114 were each replaced by a glutamic residue, mimicking a phosphorylated state, had a higher activation potential in cAMP response element-mediated transcription. These results strongly suggest that the casein kinase II target region is involved in cell cycle-regulated phosphorylation of the CREB protein and also in transcriptional enhancement.
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PMID:Cell-cycle-regulated phosphorylation of cAMP response element-binding protein: identification of novel phosphorylation sites. 993 Dec 97

In Drosophila, the catalytic subunit of cAMP-dependent protein kinase (PKA) is preferentially expressed in the brain and the male reproductive organs. Although the cAMP response element binding protein (CREB) is a major target of PKA in the brain, the target of PKA in the male reproductive organs has been unknown. In the present study, three cAMP-dependent phosphoproteins (referred to as pp45, pp20, and pp10) were detected in the lumen fluid of male accessory glands. They were tissue-specific secretory proteins that accumulated only after eclosion, and were transferred to females during mating as other secretory proteins of the accessory glands. Among them, the 45-kDa phosphoprotein was partially purified and characterized. The purified protein was phosphorylated in vitro by the catalytic subunit of PKA. The partial amino acid sequence of this 45-kDa phosphoprotein was identical to the predicted amino acid sequence of the Mst57Dc cDNA, which is a male accessory gland transcript.
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PMID:A 45-kDa cAMP-dependent phosphoprotein which is related to the product of Mst57Dc in Drosophila melanogaster. 1045 22

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


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