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)

The transcription factor CREB (cAMP responsive element binding protein) is activated by protein kinase A (PKA) phosphorylation of a single serine residue. To investigate possible mechanisms of CREB regulation by phosphorylation, we initiated a structural and biophysical characterization of the full-length, wild-type CREB protein, an altered CREB protein (CREB/SER) in which the three cysteine residues in the DNA-binding domain were replaced with serine residues and a truncated protein (ACT265) which encompasses the entire activation domain of CREB. Circular dichroism (CD) reveals that CREB and CREB/SER have identical secondary structures and contain approximately 20% alpha-helix, 9% beta-strand, 34% beta-turn, and 37% random coil structures. PKA phosphorylation does not alter the CD spectra, and therefore the secondary structure, of CREB or of CREB bound to DNA. Protease cleavage patterns indicate that PKA phosphorylation does not induce a global conformational change in CREB. Furthermore, PKA phosphorylation does not change the DNA binding affinity of CREB for either canonical or non-canonical CRE sequences as measured by a fluorescence anisotropy DNA binding assay. Since PKA phosphorylation of CREB results in its specific binding to the transcriptional co-activators CREB-binding protein and p300, we suggest that the PKA activation of CREB occurs by the production of specific, complementary interactions with these proteins, rather than through the previously proposed mechanisms of a phosphorylation-dependent conformational change or increased DNA binding affinity.
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PMID:Analysis of the structural properties of cAMP-responsive element-binding protein (CREB) and phosphorylated CREB. 866 19

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

The experiments presented herein were designed to understand the molecular mechanism(s) by which membrane Ig (mIg)-dependent signals are integrated at the level of the junB promoter to induce gene transcription. Functional studies using chloramphenicol acetyltransferase reporter gene constructs that contained deleted 5' flanking region junB sequences identified a region located between -194 and -87 that contains an Ets binding site and a putative cAMP response element binding site (CRE-like). Point mutagenesis of the CRE-like site blocked junB promoter activation in response to mIg cross-linking in mature Bal17 B cells. Nuclear extract binding activity to a synthetic oligonucleotide containing the junB CRE-like site was detected in unstimulated B cells and was increased in response to mIg cross-linking. Binding activity was competed with unlabeled oligonucleotides that contained the junB CRE-like site or the somatostatin CRE consensus motif, the latter observation suggests that members of the activating transcription factor/CRE binding protein (CREB) family may mediate mIg-dependent junB transcription. Consistent with this interpretation, recombinant CREB and activating transcription factor proteins bound the junB CRE-like site, but did not interact with a mutant CRE-like site. Expression of a dominant negative CREB protein blocked mIg-mediated transcription from a junB CRE-like site-chloramphenicol acetyltransferase reporter gene. CRE-like nucleoprotein complexes from Bal17 B cells contained constitutively bound CREB-1, which was phosphorylated on serine 133 in response to mIg cross-linking. Activating transcription factor-1 protein was also constitutively expressed in CRE-like nucleoprotein complexes. Collectively, these results suggest that components of the protein kinase A signaling pathway are recruited by mIg to induce junB transcription.
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PMID:Transcriptional regulation of the junB promoter in mature B lymphocytes. Activation through a cyclic adenosine 3',5'-monophosphate-like binding site. 868 8

The capacity of various growth factors to induce c-fos expression is diminished with senescence. Because adenosine 3',5'-cyclic monophosphate (cAMP)-mediated responses are also blunted with aging, we wondered whether cAMP-induced c-fos gene expression might be impaired with senescence. Using IMR fibroblasts, we found that prostaglandin E1 (PGE1) and forskolin, stimulators of cAMP accumulation in young and senescent cells, increased abundance of c-fos and junB mRNA more in young than senescent cells. The abundance of the cAMP response element binding protein (CREB), a transcription factor which enhances gene expression when phosphorylated by protein kinase A, was markedly decreased in both whole cell and nuclear extracts of senescent cells, in both Western blotting and in gel retardation assays. Also, PGE1-induced phosphorylation of CREB by protein kinase A was markedly attenuated in senescent cells. There is a marked decrement in expression of CREB with senescence, and the results suggest the possibility that the diminished expression of CREB may contribute to altered cAMP-mediated regulation of gene expression with senescence.
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PMID:Impaired cAMP-mediated gene expression and decreased cAMP response element binding protein in senescent cells. 876 66

Cyclic adenosine monophosphate (cAMP) stimulates transcription of somatostatin and other target genes with burst-attenuation kinetics. The kinetics of protein kinase (PK-A)-dependent cAMP response element binding protein (CREB) phosphorylation closely parallel the changes in transcription of cAMP-responsive genes by run-on assay. Nuclear translocation of PK-A, visualized by microinjection of fluorescently labeled PK-A holoenzyme, appears to represent the rate-limiting step in CREB phosphorylation and transcriptional activation. We and others have recently characterized a CREB-binding protein (CBP), which specifically recognizes sequences within the Ser133 phosphorylated form of CREB. CBP does not regulate the DNA binding, dimerization, or nuclear targeting properties of CREB, but binds selectively to the kinase-inducible 60 amino acid trans-activation domain (KID) of CREB, critical for PK-A-inducible transcription. We developed an antiserum directed against amino acid 634-648 within the CREB-binding domain of CBP. We detected a 265-kd polypeptide by Western blot as predicted from the cDNA, which coincided with the predominant phospho-CREB-binding activity in Hela nuclear extracts by "Far Western" blot assay. An identical phospho-CREB-binding activity was also found in NIH-3T3 cells. This phospho-CREB-binding protein appeared to be specific for Ser133-phosphorylated CREB, because no such band was detected with CREB labeled to the same specific activity at a nonregulatory phosphoacceptor site (Ser156) by casein kinase II (CKII). Following microinjection into nuclei of NIH-3T3 cells, a cAMP response element (CRE)-lacZ reporter was markedly induced by treatment with 8-Br cAMP plus isobutyl methyl xanthine (IBMX). Coinjection of CBP antiserum with the CRE-lacZ plasmid inhibited cAMP-dependent activity in a dose-dependent manner, but control immunoglobulin G (lgG) had no effect on this response. We can now begin reconstituting PK-A-dependent transcription in vitro, using well-characterized proteins such as CREB, TAF 110, and CBP. The assembly of such factors on cAMP-regulated promoters like somatostain may enable responsiveness to a variety of hormonal stimuli that employ cAMP as their second messenger.
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PMID:Regulation of somatostatin gene transcription by cyclic adenosine monophosphate. 876 68

While evidence has accumulated in favor of cAMP-associated genomic involvement in long-term synaptic plasticity, the mechanisms downstream of the activated nucleus that underlie these changes in neuronal function remain mostly unknown. Dendritic spines, the locus of excitatory interaction among central neurons, are prime candidates for long-term synaptic modifications. We now present evidence that links phosphorylation of the cAMP response element binding protein (CREB) to formation of new spines; exposure to estradiol doubles the density of dendritic spines in cultured hippocampal neurons, and concomitantly causes a large increase in phosphorylated CREB and in CREB binding protein. Blockade of cAMP-regulated protein kinase A eliminates estradiol-evoked spine formation, as well as the CREB and CREB binding protein responses. A specific antisense oligonucleotide eliminates the phosphorylated CREB response to estradiol as well as the formation of new dendritic spines. These results indicate that CREB phosphorylation is a necessary step in the process leading to generation of new dendritic spines.
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PMID:Morphological plasticity of dendritic spines in central neurons is mediated by activation of cAMP response element binding protein. 903 79

The aim of this study was to assess the involvement of the adenylyl cyclase/cyclic AMP/protein kinase A pathway (AC) in endothelial cells (EC) exposed to different levels of mechanical strain. Bovine aortic EC were seeded to confluence on flexible membrane-bottom wells. The membranes were deformed with either 150 mm Hg (average 10% strain) or 37.5 mm Hg (average 6% strain) vacuum at 60 cycles per minute (0.5 s strain; 0.5 s relaxation) for 0-60 min. The results demonstrate that at 10% average strain (but not 6% average strain) there was a 1.5- to 2.2-fold increase in AC, cAMP, and PKA activity by 15 min when compared to unstretched controls. Further studies revealed an increase in cAMP response element binding protein in EC subjected to the 10% average strain (but not 6% average strain). These data support the hypothesis that cyclic strain activates the AC/cAMP/PKA signal transduction pathway in EC which may occur by exceeding a strain threshold and suggest that cyclic strain may stimulate the expression of genes containing cAMP-responsive promoter elements.
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PMID:Activation of the adenylyl cyclase/cyclic AMP/protein kinase A pathway in endothelial cells exposed to cyclic strain. 905 25

LAP/C/EBP beta is a member of the C/EBP family of transcription factors and is involved in hepatocyte-specific gene expression. Recently we showed that, besides its posttranscriptional regulation, LAP/C/EBP beta mRNA is modulated during liver regeneration. Therefore, in this study we investigated mechanisms which control LAP/C/EBP beta gene transcription. Deletion analysis of the 5'-flanking region, located upstream of the start site of transcription in the LAP/C/EBP beta gene, demonstrated that a small region in close proximity to the TATA box is important in maintaining a high level of transcription of the luciferase reporter gene constructs. In gel shift experiments two sites were identified which are important for specific complex formation within this region. Further analysis by cross-linking, super shift, and competition experiments was performed with liver cell nuclear extracts, hepatoma cell nuclear extracts, or recombinant CREB protein. These experiments conclusively demonstrated that CREB binds to both sites in the LAP/C/EBP beta promoter with an affinity similar to that with the CREB consensus sequence. Transfection experiments with promoter constructs where the CREB sites were mutated showed that these sites are important to maintain both basal promoter activity and LAP/C/EBP beta inducibility through CREB. Northern blot analysis and runoff transcription assays demonstrated that the protein kinase A pathway not only stimulated the activity of the luciferase reporter construct but also the transcription of the endogenous LAP/C/EBP beta gene in different cell types. Western blot analysis of rat liver cell nuclear extracts and runoff transcription assays of rat liver cell nuclei after two-thirds hepatectomy showed a functional link between the induction of CREB phosphorylation and LAP/C/EBP beta mRNA transcription during liver regeneration. These results demonstrate that the two CREB sites are important to control LAP/C/EBP beta transcription in vivo. As several pathways control CREB phosphorylation, our results provide evidence for the transcriptional regulation of LAP/C/EBP beta via CREB under different physiological conditions.
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PMID:CREB controls LAP/C/EBP beta transcription. 919 95

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

Activation of the cAMP signal transduction pathway results in the transcriptional induction of many genes. Several of them are induced with kinetics characteristic of the early response. One of these, the cAMP response element modulator (CREM) gene, is cAMP-inducible by virtue of an intronic promoter that directs the synthesis of the dominant negative inducible cAMP early repressor (ICER). ICER is involved in the down-regulation of its own promoter via an autoregulatory loop. Thus, while phosphorylation of cAMP response element binding protein (CREB) by the cAMP-dependent protein kinase A is the prerequisite for induction, it has been proposed that the following attenuation involves both CREB dephosphorylation and repression by the inducible repressor ICER. Here we show that ectopic expression of sense or antisense ICER in corticotroph AtT20 cells dramatically modifies the normal CREM inducibility profile. We have investigated the kinetics of CREM inducibility by recurrent stimulation of the cAMP-signaling pathway. We define the presence of a refractory phase that follows the first induction cycle. Accumulation of cAMP, protein kinase A activity, CREB/CREM phosphorylation, and ICER levels contribute to the refractory period. Strikingly, the length of the refractory period is determined by the length of the stimulation by cAMP responsible for the first cycle of induction.
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PMID:The dynamics of the transcriptional response to cyclic adenosine 3',5'-monophosphate: recurrent inducibility and refractory phase. 928 57


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