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At least 307 records · Page 17Linked to original sources
PubMed2005

Dexamethasone represses cAMP rapid upregulation of TRH gene transcription: identification of a composite glucocorticoid response element and a cAMP response element in TRH promoter.

Hypothalamic proTRH mRNA levels are rapidly increased (at 1 h) in vivo by cold exposure or suckling, and in vitro by 8Br-cAMP or glucocorticoids. The aim of this work was to study whether these effects occurred at the transcriptional level. Hypothalamic cells transfected with rat TRH promoter (-776/+85) linked to the luciferase reporter showed increased transcription by protein kinase (PK) A and PKC activators, or by dexamethasone (dex), but co-incubation with dex and 8Br-cAMP decreased their stimulatory effect (as observed for proTRH mRNA levels). These effects were also observed in NIH-3T3-transfected cells supporting a characteristic of TRH promoter and not of hypothalamic cells. Transcriptional regulation by 8Br-cAMP was mimicked by noradrenaline which increased proTRH mRNA levels, but not in the presence of dex. PKA inhibition by H89 avoided 8Br-cAMP or noradrenaline stimulation. TRH promoter sequences, cAMP response element (CRE)-like (-101/-94 and -59/-52) and glucocorticoid response element (GRE) half-site (-210/-205), were analyzed by electrophoretic mobility shift assays with nuclear extracts from hypothalamic or neuroblastoma cultures. PKA stimulation increased binding to CRE (-101/-94) but not to CRE (-59/-52); dex or 12-O-tetradecanoylphorbol-13-acetate (TPA) increased binding to GRE, a composite site flanked by a perfect and an imperfect activator protein (AP-1) site in the complementary strand. Interference was observed in the binding of CRE or GRE with nuclear extracts from cells co-incubated for 3 h with 8Br-cAMP and dex; from cells incubated for 1 h, only the binding to GRE showed interference. Rapid cross-talk of glucocorticoids with PKA signaling pathways regulating TRH transcription constitutes another example of neuroendocrine integration.

Animals↗
PubMed1999

Identification of a cAMP response element within the glucose- 6-phosphatase hydrolytic subunit gene promoter which is involved in the transcriptional regulation by cAMP and glucocorticoids in H4IIE hepatoma cells.

The expression of a luciferase reporter gene under the control of the human glucose 6-phosphatase gene promoter was stimulated by both dexamethasone and dibutyryl cAMP in H4IIE hepatoma cells. A cis-active element located between nucleotides -161 and -152 in the glucose 6-phosphatase gene promoter was identified and found to be necessary for both basal reporter-gene expression and induction of expression by both dibutyryl cAMP and dexamethasone. Nucleotides -161 to -152 were functionally replaced by the consensus sequence for a cAMP response element. An antibody against the cAMP response element-binding protein caused a supershift in gel-electrophoretic-mobility-shift assays using an oligonucleotide probe representing the glucose 6-phosphatase gene promoter from nucleotides -161 to -152. These results strongly indicate that in H4IIE cells the glucose 6-phosphatase gene-promoter sequence from -161 to -152 is a cAMP response element which is important for the regulation of transcription of the glucose 6-phosphatase gene by both cAMP and glucocorticoids.

Base Sequence↗
PubMed1991

The cAMP-regulated enhancer-binding protein ATF-1 activates transcription in response to cAMP-dependent protein kinase A.

Many promoters respond transcriptionally to elevated levels of cAMP through the cAMP-responsive enhancer (CRE). Several proteins have been characterized which bind to the CRE and presumably modulate CRE-dependent transcription. Of these CRE-binding proteins, only CREB has been shown to be activated by cAMP-dependent protein kinase A (PKA), and as such, CREB represents the only basis for our understanding of cAMP-regulated transcriptional activity. In this report, we describe the complete cDNA sequence of another CRE-binding protein, ATF-1. This protein contains a consensus phosphorylation site for PKA and shares extensive homology with CREB in the region surrounding and carboxyl-terminal to the PKA site. ATF-1 does not contain sequences homologous to the glutamine-rich amino-terminal domain found in CREB, however. ATF-1, like CREB, is expressed in a wide variety of cell types, and ATF-1 is capable of dimerizing with CREB. Both ATF-1 homodimers and ATF-1/CREB heterodimers bind to the CRE but not to the related phorbol ester response element. ATF-1 is as active as CREB in its ability to mediate the transcriptional effects of PKA, and, because ATF-1 has a smaller effect on basal expression, it is actually more responsive than CREB to cAMP. These findings indicate that CREB is not unique in its ability to mediate cAMP-dependent transcriptional regulation.

Activating Transcription Factors↗
PubMed1990

The effect of depolarization on expression of the human proenkephalin gene is synergistic with cAMP and dependent upon a cAMP-inducible enhancer.

Membrane depolarization is a critical component of neural signaling; in recent years there also has been a great deal of evidence that membrane depolarization can regulate neural gene expression. Therefore, excitatory neurotransmission may be an important mechanism of neural plasticity. We have investigated the intracellular pathways and DNA regulatory elements through which membrane depolarization activates expression of the neural gene encoding human proenkephalin. In PC12 and C6-glioma cells, depolarization-induced expression of a transfected proenkephalin fusion gene was proportional to extracellular calcium concentration and was inhibited by verapamil. Activation of the gene by KCl-induced depolarization or the calcium ionophore A23187 was dependent upon and synergistic with cAMP in PC12 and C6-glioma cells, but neither depolarization nor treatment with A23187 affected cAMP levels. Trifluoperazine and W7 inhibited depolarization-induced gene expression but did not affect expression induced by the adenylyl cyclase activator forskolin. At the level of the DNA, depolarization-induced activation is conferred on the proenkephalin gene by a previously characterized cAMP-inducible enhancer. Multiple copies of a single component element of that enhancer, containing the CGTCA sequence motif characteristic of cAMP regulatory elements, can reconstitute the entire repertoire of responses to both cAMP and depolarization. These data suggest a model in which membrane depolarization activates gene expression through a calcium-dependent pathway, potentially involving calmodulin, and in which the transcriptional responses to both cAMP and calcium are transduced by the same DNA element.

Base Sequence↗
PubMed1985

Evidence for negative control of cya transcription by cAMP and cAMP receptor protein in intact Escherichia coli cells.

The transcriptional regulation of cya by cAMP and its receptor protein (CRP) has been studied by S1 nuclease and RNA dot blot assays. The crp- Escherichia coli cells were shown to produce about 5-fold more cya mRNA than do the wild type cells. The effect of cAMP and CRP on the cya transcription was directly examined by introducing a crp plasmid into the cells and/or by adding cAMP exogenously. The levels of cya mRNA in crp+ cells decreased with increasing concentrations of cAMP in the growth medium. The repressive effect of cAMP on cya transcription was strongly enhanced in cells carrying a multicopy crp plasmid. These results indicate that the cya transcription is negatively regulated by cAMP-CRP complex in intact cells.

Base Composition↗
PubMed1987

Parathyroid hormone-activated calcium channels in an osteoblast-like clonal osteosarcoma cell line. cAMP-dependent and cAMP-independent calcium channels.

Changes in free cytosolic calcium were measured in UMR-106 cells in response to parathyroid hormone (PTH) stimulation. Bovine PTH-(1-34) induced an increase in [Ca2+]i with the contour of the rise in [Ca2+]i occurring in three successive phases: a rapid increase in [Ca2+]i occurring within seconds, rapid decrement in [Ca2+]i to near-resting levels within 1 min, and slow increment in [Ca2+]i. Phase one and phase three increases in [Ca2+]i were dependent on medium calcium. The phase one rise in [Ca2+]i was inhibitable by the calcium channel blockers lanthanum and verapamil. Only the phase one rise in [Ca2+]i was blocked by preincubation of the cells with the phorbol ester, phorbol 12-myristate 13-acetate. This channel was also blocked when cellular cAMP levels were increased prior to PTH stimulation. The phase two decrement of [Ca2+]i was due to the rapid inactivation of the phase one calcium channel. The phase three rise in [Ca2+]i was mediated by cellular cAMP levels. This cAMP-dependent Ca2+ channel was insensitive to pretreatment of the cells with phorbol diesters and showed low sensitivity to Ca2+ channel blockers. It is concluded that UMR-106 cells respond to PTH stimulation by the activation of a cAMP-independent Ca2+ channel. This channel rapidly inactivates. The subsequent PTH-dependent increase in cellular cAMP is followed by activation of a cAMP-dependent Ca2+ channel resulting in a slow rise in [Ca2+]i.

Animals↗
PubMed1986

Structure-function analysis of three cAMP-independent forms of the cAMP receptor protein.

cAMP receptor protein (CRP)-dependent operon expression in Escherichia coli requires the CRP X cAMP complex form of wild-type CRP. One class of crp mutants (crp*) activates CRP-dependent promoters in strains (cya) incapable of endogenous cAMP synthesis. Of fundamental interest is the difference in regulatory properties exhibited by crp* mutant strains, some of which exhibit glucose-mediated repression of beta-galactosidase synthesis, some of which do not. To gain a better understanding of the mechanisms of cAMP-independent promoter activation and repression we have: determined through cloning and DNA sequence analysis the primary structure of three CRP* forms of CRP; purified the mutant proteins; characterized the effect of these mutations on CRP secondary structure; and studied CRP*-activated lac promoter regulation in a purified in vitro transcription system. The results of this study provide strong evidence that mutations in crp alter the conformation of CRP and result in cAMP-independent activation of CRP-dependent promoters in vitro. In addition, a CRP allele-specific inhibition of CRP* activity by spermidine was observed in vitro that parallels crp* strain-specific sensitivity to glucose-mediated repression of CRP-dependent enzyme synthesis in vivo. This observation provides evidence that catabolite repression in cells lacking cAMP may be mediated through a mechanism that inhibits CRP* activity.

Bacterial Proteins↗
PubMed1983

Changes in ribosome function by cAMP-dependent and cAMP-independent phosphorylation of ribosomal protein S6.

Alterations in ribosomal function were examined following phosphorylation of 40 S ribosomal subunits by the cAMP-dependent protein kinase and two cAMP-independent protein kinases, protease-activated kinases I and II. The cAMP-dependent protein kinase incorporated 2.0 mol of phosphate/mol of 40 S ribosomal subunits; ribosomal protein S6 was the sole phosphate acceptor. Phosphorylation of 40 S ribosomal subunits by the cAMP-dependent protein kinase inhibited the binding of AUG by 41% and poly(A,U,G) by 25% when compared with nonphosphorylated 40 S ribosomal subunits. In addition, phosphorylation of 40 S ribosomal subunits by the cAMP-dependent protein kinase inhibited translation of poly(A,U,G) by 30% in a reconstituted protein-synthesizing system. Protease-activated kinase II incorporated an average of 2.5 mol of phosphate/mol of 40 S ribosomal subunits which was distributed in equimolar amounts in derivatives of S6 containing one to four phosphates. Phosphorylation of 40 S ribosomal subunits by protease-activated kinase II increased the binding of AUG and poly(A,U,G) by 26 and 42%, respectively. Poly(A,U,G)-directed translation was stimulated 15% over that observed with nonphosphorylated ribosomes and 45% over that observed with ribosomes phosphorylated by the cAMP-dependent protein kinase. Protease-activated kinase I incorporated 1.0 mol of phosphate/mol of 40 S ribosomal subunits into ribosomal protein S10. Phosphorylation of 40 S ribosomal subunits by protease-activated kinase I did not alter the binding of AUG or poly(A,U,G). The effects of phosphorylation of 40 S ribosomal subunits by protease-activated kinase I on protein synthesis could not be examined due to the rapid release of phosphate from S10 in the reconstituted translation system.

Animals↗
PubMed1989

cAMP-independent and cAMP-dependent protein phosphorylations by isolated goldfish xanthophore cytoskeletons: evidence for the association of cytoskeleton with a carotenoid droplet protein.

Triton-insoluble cytoskeleton of nonpigment cells has bound protein kinase that phosphorylates, with or without added cAMP, tubulins and the intermediate filament proteins p60, p56, p53, and p45a to give multiple charge variants. In the absence of 8-Br-cAMP, Triton-insoluble cytoskeletons from xanthophores also phosphorylate p60, p56, and p45a, but not p53; tubulin phosphorylation may also be reduced. In the presence of 8-Br-cAMP, p53, as well as several other peptides, are phosphorylated. One of these latter peptides was identified as the carotenoid droplet (pigment organelle) protein p57, whose phosphorylation and dephosphorylation precede pigment dispersion and aggregation respectively (Lynch et al.: J. Biol. Chem. 261:4204-4211, 1986). The amount of pp57 produced depends on the state of pigment distribution in the xanthophores used to prepare the cytoskeletons for labeling. With cytoskeletons from xanthophores with aggregated pigment, pp57 is a major labeled phosphoprotein seen in two-dimensional gels. With cytoskeletons prepared from xanthophores with dispersed pigment, the yield of labeled pp57 is greatly reduced (by at least 90%). Together with earlier results, we propose that, in the aggregated state, p57 serves to bind carotenoid droplets to the cytoskeletons, most likely the microtubules. The significance of other cAMP-dependent phosphorylation reactions is unknown but may be related to cAMP-induced cytoskeleton rearrangement in intact xanthophores.

Animals↗
PubMed1983

Specific DNA binding of the cAMP receptor protein within the lac operon stabilizes double-stranded DNA in the presence of cAMP.

The effects of varying amounts of cAMP receptor protein (CRP) in the presence and absence of cAMP on the melting and differential melting curves of a 301-bp fragment containing the lac control region in 5 mM Na+ have been investigated. The native 301-bp fragment consists of three cooperatively melting thermalites. At 5 mM Na+, thermalite I (155 bp) has a Tm of 66.4 degrees C and the melting transitions of thermalites II (81 bp) and III (65 bp) are superimposed with a Tm of 61.9 degrees C. The specific DNA target site for CRP and the lac promotor are located within thermalite II. CRP alone exerts no specific effects on the melting of the 301-bp fragment, non-specific DNA binding of CRP resulting in a progressive stabilization of the double-stranded DNA by increasing the number of base pairs melting at a higher Tm in a non-cooperative transition. The cAMP-CRP complex, however, exerts a specific effect with a region of approximately 36 bp, comprising the specific CRP binding site and a neighbouring region of DNA, being stabilized. The appearance of this new cooperatively melting region, known as thermalite IV, is associated with a corresponding decrease in the area of thermalites II/III. The Tm of thermalite IV is 64.4 degrees C, 2.5 degrees C higher than that of thermalites II/III. With two or more cAMP-CRP complexes bound per 301-bp fragment, the stabilization also affects the remaining 110 bp now making up thermalites II/III whose Tm is increased by 1 degrees C to 62.9 degrees C. The implications of these findings for various models of the mode of action of the cAMP-CRP complex are discussed.

Cyclic AMP↗
PubMed1978

Studies of cAMP metabolism in cultured hepatoma cells: presence of functional adenylate cyclase despite low cAMP content and lack of hormonal responsiveness.

The ability of isoproterenol, glucagon, PGE1 and cholera toxin to stimulate the synthesis of cAMP and protein kinase activity in line of liver cells (BRL) and a line of rat hepatoma cells (H35) has been determined. The concentration of cAMP in BRL cells (approximately 10 pmoles/mg protein) is in the range reported for other cultured cell lines but H35 cells contain extraordinarily low amounts of this cyclic nucleotide (approximately 0.05 pmoles/mg protein). Isoproterenol and PGE1 caused an increase in cAMP content, and protein kinase activation in BRL cells, although glucagon was ineffective. H35 cells, in contrast, were completely insensitive to all hormonal agonists. Despite this fact, cholera toxin was able to produce a marked increase in cAMP content, adenylate cyclase activity and protein kinase activation in H35 cells. binding studies with [125 I]-iodohydroxybenzylpindolol, a specific beta-adrenergic receptor antagonist, revealed that each H35 cell possesses fewer than 10 beta-adrenergic receptors whereas BRL cells contain 2-5,000 receptors per cell. The low level of cAMP in H35 cells appears to result from a combination of totally unstimulated adenylate cyclase and apparently elevated phosphodiesterase activities.

Adenylate Kinase↗
PubMed1980

Developmental pattern of cAMP, adenyl cyclase, and cAMP phosphodiesterase in the palate, lung, and liver of the fetal mouse: alterations resulting from exposure to methylmercury at levels inhibiting palate closure.

Exposure to methylmercury (MeHg: 10 mg Hg/kg maternal body weight) on 12(6) (days hours) of gestation significantly delays palate closure in the Swiss Webster CFW mouse. The cAMP content and activity of adenyl cyclase and phosphodiesterase (PDE) were measured in the tissues of control and MeHg-induced cleft palates between 13(6) and 17(6) of gestation. Lung and liver were investigated similarly to determine if MeHg affected the adenyl cyclase system of the palate in a unique manner. In control palatal tissue, cAMP levels increased sharply from 13(22) (undetectable) to 14(6) (maximum). PDE activity increased similarly up to 14(2), but decreased 50% between 14(2) and 14(6). Since it has been reported that cAMP induces the synthesis of PDE, the difference in cAMP/PDE from 13(22) to 14(2) and from 14(2) to 14(6) suggests the localization of relatively high levels of cAMP in at least two separate compartments. Between 14(6) and 14(10), the adenyl cyclase activity of control palates decreased significantly. This rapid decrease suggests relatively high adenyl cyclase activity in the medial edge epithelial cells which undergo autolysis prior to shelf fusion (centered at 14(15). Maternal MeHg administration at 12(6) delayed the median time of palatal shelf rotation (14(13)) by 5 hours, and significantly altered the developmental pattern of the adenyl cyclase system. Thus, the increase in cAMP between 14(2) and 14(6) was abolished and the decrease in adenyl cyclase activity between 14(6) and 14(10) was delayed by almost 20 hours. These changes may be manifestions of a MeHg-induced delay in medial edge epithelial cell differentiation. In a previous study, we observed that the fetal liver exhibits the highest MeHg concentration of all tissues. Since MeHg only slightly altered the adenyl cyclase system of the fetal liver compared to the lung and palate (in which MeHg uptake is considerably less), it may be that the effects of MeHg on palatal tissue are not due to a direct effect of MeHg on components of the adenyl cyclase system.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1996

Acyloxycoumarinylmethyl-caged cAMP, the photolabile and membrane-permeable derivative of cAMP that effectively stimulates pigment-dispersion response of melanophores.

Two acyloxycoumarinylmethyl-caged cAMPs (ACM- and PCM-cAMP) have been synthesized using a silver (I) oxide promoted method. Introduction of the acyloxy group to the 7-position on the coumarin ring not only enhanced the membrane permeability but diminished the photolability of the coumarin-cage. Because intracellular enzymatic hydrolysis of the 7-acyloxy group would produce the 7-hydroxy moiety which is more hydrophilic and photolabile, application of acyloxycoumarinylmethyl-caged cAMPs in biological studies would be expected to be efficient. Thus, the effect of extracellularly applied ACM- and PCM-cAMP had been investigated using the motile response of fish melanophores. After irradiation, a significant enhancement in the motility responses was observed. The observed magnitudes of the dispersions are comparable to that of Bt2cAMP/AM which is known as a membrane permeable cAMP derivative.

1-Methyl-3-isobutylxanthine↗
PubMed1989

Blastocoel expansion in the preimplantation mouse embryo: stimulation of sodium uptake by cAMP and possible involvement of cAMP-dependent protein kinase.

Elevating cAMP levels in mouse blastocysts increases the rate of blastocoel expansion (F. Manejwala, E. Kaji, and R. M. Schultz, 1986, Cell, 46, 95-103), which requires extracellular sodium (F. Manejwala, E. J. Crago, Jr., and R. M. Schultz, 1989, Dev. Biol. 133, 210-220). We report that cAMP analogs that can activate the cAMP-dependent protein kinase stimulate 22Na+ uptake by cavitating mouse blastocysts and that inhibitors of cAMP-dependent protein kinase activity inhibit the cAMP-stimulated increase in both the rate of blastocoel expansion and 22Na+ uptake.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1987

Site-selective cAMP analogs induce nuclear translocation of the RII cAMP receptor protein in Ha-MuSV-transformed NIH/3T3 cells.

Site-selective cAMP analogs, depending on the position of their substituents on the adenine ring, selectively bind to either site 1 or site 2 of the known cAMP binding sites of protein kinase. Treatment of Harvey murine sarcoma virus-transformed NIH/3T3 cells with such site-selective analogs results in growth inhibition and phenotypic reversion, and the combination of a C-8 thio or halogen analog (site 1 selective) with an N6 analog (site 2 selective) produces a synergistic effect. We report here that the growth inhibitory effect of the analogs correlates with the nuclear translocation of the RII cAMP receptor protein, the regulatory subunit of protein kinase type II. The transformed NIH/3T3 cells contained no detectable level of RII in the nucleus, whereas nontransformed NIH/3T3 cells exhibited a high level of nuclear RII. Within 30 min after treatment of the transformed cells with the site-selective analogs, immunofluorescence against the RII protein markedly increased in the cell nucleus. The nuclear translocation of the RII cAMP receptor protein is an early event in the reverse transformation of the fibroblasts treated with site-selective cAMP analogs.

Affinity Labels↗
PubMed1988

Opposite effects of adenosine on two types of cAMP-induced gene expression in Dictyostelium indicate the involvement of at least two different intracellular pathways for the transduction of cAMP signals.

Adenosine promotes the cAMP-induced increase of mRNAs, probed with the cDNAs D11 and D14, which are preferentially expressed in prestalk cells, while it inhibits cAMP-induced prespore gene expression. Half-maximal inhibition of prespore gene expression occurs at about 300 muM, while prestalk stimulation by adenosine occurs at about 100-fold lower concentrations and requires the presence of cAMP. These results indicate that adenosine interferes with the transduction to cAMP to gene expression and suggest the involvement of two different adenosine target sites. Our data furthermore indicate that the transduction of extracellular cAMP to prespore gene or prestalk gene expression occurs via divergent pathways.

Adenosine↗
PubMed1992

cAMP-CRP activator complex and the CytR repressor protein bind co-operatively to the cytRP promoter in Escherichia coli and CytR antagonizes the cAMP-CRP-induced DNA bend.

Initiation of transcription from the cytRP promoter in Escherichia coli is activated by the cAMP-CRP complex and negatively regulated by the CytR repressor protein. By combining gel retardation and footprinting assays, we show that cAMP-CRP binds to a single site centered at position -64 and induces a considerable bend in the DNA. CytR binds to a region immediately downstream from, and partially overlapping, the CRP site, and induces a modest bend into the DNA. In combination, cAMP-CRP and CytR bind co-operatively to cytRP forming a nucleoprotein complex in which the proteins directly interact with each other and bind to the same face of the DNA helix. CytR binding concomitantly antagonizes the cAMP-CRP-induced bend. This study indicates that the minimal DNA region required to obtain CytR regulation consists of a single binding site for each of cAMP-CRP and CytR. The case described here, in which a protein-induced DNA bend is modulated by a second protein, may illustrate a mechanism that applies to other regulatory systems.

Bacterial Proteins↗
PubMed1992

Role of cAMP in the functional interaction of carbachol with different cAMP elevating agents in rabbit atrium.

The muscarinic agonist carbachol antagonized positive inotropic responses of rabbit left atria to the beta-adrenoceptor agonist isoproterenol, the adenylate cyclase activator forskolin and the phosphodiesterase inhibitor IBMX. Carbachol also reduced cAMP levels elevated by isoproterenol, but had no significant effect on cAMP levels in the presence of either forskolin or IBMX. Pre-treatment of rabbits with a dose of pertussis toxin which completely blocked the reduction by carbachol of isoproterenol-induced increases in cAMP, also blocked the reversal by carbachol of positive inotropic responses to isoproterenol, but only partially attenuated the antagonism by carbachol of inotropic responses to forskolin and IBMX. These data suggest that antagonism by carbachol of forskolin and IBMX-induced increases in cAMP levels does not play an important role in the functional interaction of carbachol with these cAMP-elevating agents.

1-Methyl-3-isobutylxanthine↗