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PubMed1990

cAMP analogs and selective inhibitors used to study low Km Mucor rouxii cAMP phosphodiesterase.

1. The sensitivity of partially purified low Km phosphodiesterase (PDE) from Mucor rouxii to pharmacological agents and cAMP analogs was studied. The IC50 obtained were compared with those reported for PDEs from higher eukaryotes. 2. The best inhibitors of the hydrolysis of 1 microM cAMP were SQ 65.442 (IC50 c 10 microM), dipyridamol and CI 930. cGMP was not an inhibitor (IC50 greater than 1000 microM). 3. The cAMP analogs were tested as inhibitors of the hydrolysis of 0.1 microM cAMP. 8-Aminohexylamino cAMP was the best inhibitor with an IC50 of c 1 microM. 4. A sedimentation profile of Mucor PDE was assayed in the presence of several pharmacological inhibitors and cAMP analogs. No isoforms with different sensitivity towards the inhibitors were detected. Forms with slightly different behaviour towards some cAMP analogs were observed.

1-Methyl-3-isobutylxanthine↗

PubMed1991

Suppression of malignancy targeting the intracellular signal transducing proteins of cAMP: the use of site-selective cAMP analogs, antisense strategy, and gene transfer.

An hypothesis has been presented suggesting that two isoforms of cAMP receptor proteins are crucial effectors in tumorigenesis. The evidence in support of this hypothesis shows that: (1) cAMP transduces dual controls, both positive and negative, on cell growth and differentiation. (2) Such dual controls are respectively governed by two isoforms of cAMP receptor proteins, the type I and type II regulatory subunits of cAMP-dependent protein kinase. (3) In normal physiology, the functional balance of these cAMP receptor isoforms is strictly controlled to meet either stimulation or inhibition of cell growth as it is required, whereas such control is lost in cancer cells. (4) Cancer cells can also be made to differentiate and acquire growth control when the functional balance of these intracellular signal transducers of cAMP is restored by the use of site-selective cAMP analogs, antisense strategy, or gene transfer, suggesting new approaches to cancer therapy.

Animals↗

PubMed1994

Metabolic effect of PGE1 analogue 01206.alpha CD on nerve Na(+)-K(+)-ATPase activity of rats with streptozocin-induced diabetes is mediated via cAMP: possible role of cAMP in diabetic neuropathy.

We investigated the dose-dependent effects of prostaglandin E1 (PGE1) analogue, OP1206.alpha CD (OP), on motor nerve conduction velocity (MNCV), nerve blood flow (NBF) and Na(+)-K(+)-ATPase (ATPase) activity in streptozocin-induced diabetic rats. At 10 micrograms/kg/day, OP ameliorated MNCV and NBF, but no ATPase activity, whereas at 30 micrograms/kg/day it increased MNCV and ATPase activity, but not NBF. These results suggested a possible direct metabolic effect of OP, at least at a certain dose, on ATPase activity independent of NBF. Since PGE1 exerts an effect on nerve cAMP content, we conducted an in vitro study to clarify the relationship of cAMP to the modulation of ATPase activity in diabetic nerves. We studied sciatic nerves isolated from 53 rats with streptozocin-induced diabetes that had exhibited hyperglycemia for 6 wk. OP increased the activity of ATPase and the accumulation of cAMP in a dose-dependent manner. Dibutyryl cAMP, a cAMP analogue, and aminophyline, which increases nerve cAMP content, enhanced ATPase activity in a dose-dependent manner. In addition, the increased activity of ATPase in diabetic nerves produced by OP was suppressed by a protein kinase inhibitor, H8. These results suggest that ATPase activity in diabetic nerves might be regulated or modified by cAMP and, possibly, by protein kinase A, a finding that is important for clarifying the pathogenesis of diabetic neuropathy and for developing new approaches to treatment.

Alprostadil↗

PubMed1992

Glucose-induced activation of plasma membrane H(+)-ATPase in mutants of the yeast Saccharomyces cerevisiae affected in cAMP metabolism, cAMP-dependent protein phosphorylation and the initiation of glycolysis.

Addition of glucose-related fermentable sugars or protonophores to derepressed cells of the yeast Saccharomyces cerevisiae causes a 3- to 4-fold activation of the plasma membrane H(+)-ATPase within a few minutes. These conditions are known to cause rapid increases in the cAMP level. In yeast strains carrying temperature-sensitive mutations in genes required for cAMP synthesis, incubation at the restrictive temperature reduced the extent of H(+)-ATPase activation. Incubation of non-temperature-sensitive strains, however, at such temperatures also caused reduction of H(+)-ATPase activation. Yeast strains which are specifically deficient in the glucose-induced cAMP increase (and not in basal cAMP synthesis) still showed plasma membrane H(+)-ATPase activation. Yeast mutants with widely divergent activity levels of cAMP-dependent protein kinase displayed very similar levels of activation of the plasma membrane H(+)-ATPase. This was also true for a yeast mutant carrying a deletion in the CDC25 gene. These results show that the cAMP-protein kinase A signaling pathway is not required for glucose activation of the H(+)-ATPase. They also contradict the specific requirement of the CDC25 gene product. Experiments with yeast strains carrying point or deletion mutations in the genes coding for the sugar phosphorylating enzymes hexokinase PI and PII and glucokinase showed that activation of the H(+)-ATPase with glucose or fructose was completely dependent on the presence of a kinase able to phosphorylate the sugar. These and other data concerning the role of initial sugar metabolism in triggering activation are consistent with the idea that the glucose-induced activation pathways of cAMP-synthesis and H(+)-ATPase have a common initiation point.

Cell Membrane↗

PubMed1988

Site-directed mutagenesis of the cAMP-binding sites of the recombinant type I regulatory subunit of cAMP-dependent protein kinase.

The type I regulatory subunit (R-I) of rat brain cAMP-dependent protein kinase was expressed in E. coli and site-directed mutagenesis was used to substitute amino acids in the putative cAMP-binding sites. The wild-type recombinant R-I bound 2 mol of cAMP/mol subunit, while two mutant R-Is with a single amino acid substitution in one of the two intrachain cAMP-binding sites (clone N153:a glutamate for Gly-200, and clone C254:an aspartate for Gly-324) bound 1 mol of cAMP/mol subunit. When these two substitutions were made in one mutant, cAMP did not bind to this mutant, indicating that binding of cAMP to N153 or C254 was to their nonmutated sites. Competition experiments with site-selective analogs and dissociation of bound cAMP from mutant R-Is provided evidence for strong intrachain interactions between the two classes of cAMP-binding sites in R-I.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1996

Calcium oscillations in melanotrope cells of Xenopus laevis are differentially regulated by cAMP-dependent and cAMP-independent mechanisms.

Intracellular Ca2+ oscillations play an important role in the induction of alpha-MSH release from pituitary melanotrope cells of Xenopus laevis. Oscillatory, secretory and adenylyl cyclase activities are all inhibited by dopamine, neuropeptide Y (NPY) and baclofen (a GABAB receptor agonist) and stimulated by sauvagine. In this study, we test the hypothesis that these neural messengers regulate the Ca2+ oscillations via a cAMP/protein kinase A (PKA)-dependent mechanism. To this end, video-imaging microscopy was applied to single Xenopus melanotropes loaded with the Ca2+ indicator Fura-2. The cAMP-dependent PKA inhibitor H89 blocked Ca2+ oscillations as well as the stimulatory actions of 8-Br-cAMP and sauvagine. Treatment of cells inhibited by baclofen with either 8-Br-cAMP or sauvagine led to a reappearance of Ca2+ oscillations. A similar result was found for cells inhibited by NPY. Neither 8-Br-cAMP nor sauvagine induced Ca2+ oscillations in cells inhibited by dopamine. Depolarizing dopamine-inhibited cells with high potassium also failed to induce oscillations, but combining 8-Br-cAMP with membrane depolarization induced oscillations. It is concluded that sauvagine, baclofen and NPY work primarily through a cAMP/PKA-pathway while dopamine inhibits Ca2+ oscillations in a dual fashion, namely via both a cAMP-dependent and a cAMP-independent mechanism, the latter probably involving membrane hyperpolarization.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed2002

CRE-mediated transcriptional activation is involved in cAMP protection of T-cell receptor-induced apoptosis but not in cAMP potentiation of glucocorticoid-mediated programmed cell death.

Apoptosis of thymic cells induced by glucocorticoids (GC) and T-cell receptor (TCR) engagement are mutually antagonistic. We demonstrate that cAMP enhances GC and antagonizes TCR (anti-CD3) apoptosis on the same cell (DO-11.10 and 2B4.11 T-cell hybridomas). We analyzed the activity of several transcription factors in this cAMP dual, stimulus-dependent, regulatory action. Anti-CD3 increases kB-activity which is inhibited by CPTcAMP or dexamethasone (DEX), supporting the proapoptotic role of NFkB on TCR-induced apoptosis. Anti-CD3 not only increases kB- but diminishes GC response element (GRE)-activity induced by DEX, suggesting that TCR-mediated blockade of GC-induced apoptosis involves not only the proposed antiapoptotic action of NF-kB on GC, but also the inhibition of GRE-regulated proapoptotic genes. To test the involvement of CRE-driven transcription in the cAMP dual apoptotic regulation, cells were transfected with a CRE decoy DNA oligomer. Blockade of CRE transactivation with decoy targeting of CRE completely blocked the protection of TCR-induced apoptosis by cAMP, while it did not modify the enhancement by cAMP on GC-induced apoptosis. We show that CRE-binding factors have a definite role in T-cell apoptosis: they are involved in cAMP protection of TCR- but not in cAMP potentiation of GC-induced apoptosis.

Animals↗

PubMed1993

Fluorescence resonance energy transfer within a heterochromatic cAMP-dependent protein kinase holoenzyme under equilibrium conditions: new insights into the conformational changes that result in cAMP-dependent activation.

Previous studies of the ligand regulation of the cAMP-dependent protein kinase have demonstrated the cAMP-mediated dissociation of the holoenzyme by using nonequilibrium techniques; i.e., gel filtration, ion-exchange chromatography, and differential centrifugation. While physically mild, these could have caused weakly associated species to dissociate, thereby providing a potentially flawed interpretation of the mechanism of activation of the protein kinase. To assess this, the activation of the cAMP-dependent protein kinase has been monitored under equilibrium conditions using dipolar fluorescence energy transfer to measure changes in the proximity relations between the catalytic (C) and regulatory (R) subunits that compose the holoenzyme. Specifically, we prepared a heterochromatically labeled protein kinase type II holoenzyme, with the regulatory and catalytic subunits labeled with sulforhodamine and carboxyfluorescein, respectively, and monitored the exchange of electronic excitation energy between the C and R subunits by both donor lifetime and steady-state fluorescence. Biochemically, the heterochromatic holoenzyme was closely identical to the native protein with regard to cAMP-induced increase in catalytic activity, reassociation of C and R subunits, inhibition of catalytic activity by the specific protein kinase inhibitor (PKI), and observed dissociation examined by gel filtration upon cAMP addition. However, under equilibrium conditions, the energy-transfer measurements revealed that the addition of cAMP to this heterochromatic reporter complex promoted an estimated 10-A increase in the distance between the derivatization sites on C and R but not a dissociation of these subunits. Addition of PKI plus cAMP promoted full dissociation of the two subunits.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1991

Comparison of cAMP receptor protein (CRP) and a cAMP-independent form of CRP by Raman spectroscopy and DNA binding.

The secondary structures of the cAMP receptor protein (CRP), a complex of CRP and cAMP, and a cAMP-independent receptor protein mutant (CRP*141 gln) were examined by using Raman spectroscopy. Spectra were obtained from CRP and CRP*141 gln dissolved in 0.3 M NaCl and 30 mM sodium phosphate at protein concentrations of 30-40 mg/mL. CRP and CRP.cAMP1 were compared at lower protein concentrations (10-12 mg/mL) in a solvent of 0.35 M NaCl and 20 mM sodium phosphate. Raman analysis indicates that CRP structural changes induced by one bound cAMP or by the Gly to Gln mutation at residue 141 are small. Spectra of the three CRP samples are essentially identical from 400 to 1900 cm-1. This result differs from the Raman spectroscopy study of CRP and CRP.cAMP2 cocrystals [DeGrazia et al. (1990) Biochemistry 29, 3557]. The latter work showed spectral differences between CRP and CRP.cAMP2 consistent with alterations in the protein conformation. These studies indicate that CRP and CRP.cAMP1 in solution are similar in structure and differ from CRP.cAMP2 cocrystals. Protease digestion and a DNA binding assay were also employed to characterize the wild-type and mutant proteins. CRP*141 gln exhibited the same conformational characteristics of previously reported cAMP-independent mutant proteins. It was sensitive to proteolytic attack in the absence of cAMP, or upon addition of cGMP. In the absence of cAMP, both wild-type and mutant CRPs bound noncooperatively to a 62 bp lac promoter DNA. The equilibrium constants were approximately 10(6) M-1 in 0.1 M Na+. CRP*141 gln had a 2-4-fold higher affinity for the 62 bp DNA than CRP.(ABSTRACT TRUNCATED AT 250 WORDS)

Cyclic AMP↗

PubMed1990

Three new potential cAMP affinity labels. Inactivation of human platelet low Km cAMP phosphodiesterase by 8-[(4-bromo-2,3-dioxobutyl)thio]adenosine 3',5'-cyclic monophosphate.

Three new analogues of cAMP have been synthesized and characterized: 2-[(4-bromo-2,3-dioxobutyl)thio]adenosine 3',5'-cyclic monophosphate (2-BDB-TcAMP), 2-[(3-bromo-2-oxopropyl)thio]-adenosine 3',5'-cyclic monophosphate (2-BOP-tcAMP), and 8-[(4-bromo-2,3-dioxobutyl)thio]adenosine 3',5'-cyclic monophosphate (8-BDB-TcAMP). The bromoketo moiety has the ability to react with the nucleophilic side chains of several amino acids, while the dioxobutyl group can interact with arginine. These cAMP analogues were tested for their ability to inactivate the low Km (high affinity) cAMP phosphodiesterase from human platelets. The 2-BDB-TcAMP and 2-BOP-TcAMP were competitive inhibitors of cAMP hydrolysis by the phosphodiesterase with Ki values of 0.96 +/- 0.12 and 0.70 +/- 0.12 microM, respectively. However, 2-BDB-TcAMP and 2-BOP-TcAMP did not irreversibly inactivate the phosphodiesterase at pH values from 6.0 to 7.5 and at concentrations up to 10 mM. These results indicate that although the 2-substituted TcAMP analogues bind to the enzyme, there are no reactive amino acids in the vicinity of the 2-position of the cAMP binding site. In contrast, incubation of the platelet low Km cAMP phosphodiesterase with 8-BDB-TcAMP resulted in a time-dependent, irreversible inactivation of the enzyme with a second-order rate constant of 0.031 +/- 0.009 min-1 mM1. Addition of the substrates, cAMP and cGMP, and the product, AMP, to the reaction mixture resulted in marked decreases in the inactivation rate, suggesting that the inactivation was due to reaction at the active site of the phosphodiesterase.(ABSTRACT TRUNCATED AT 250 WORDS)

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed1997

Mapping conformational changes in a protein: application of a protein footprinting technique to cAMP-induced conformational changes in cAMP receptor protein.

We have used protein footprinting [Heyduk, E., & Heyduk, T. (1994) Biochemistry 33, 9643] to detect and map ligand-induced conformational changes in cAMP receptor protein (CRP). The binding of cAMP to CRP dramatically increases the specific DNA binding activity of the protein and, as has been previously shown, induces conformational changes in the protein. Protein footprinting experiments with the free CRP, the CRP-cAMP complex, and the CRP-cGMP complex were analyzed quantitatively. Binding of cAMP produced measurable differences in the susceptibility of CRP to the cleavage by Fe-EDTA. Almost all of these changes occurred in the C-terminal domain (DNA binding domain) of the protein. Additional changes were observed at the ends of the C alpha-helix, which is involved in intersubunit contacts in the CRP dimer, and in the hinge peptide, connecting N-terminal and C-terminal domains of the protein. The boundaries of the regions in the C-terminal domain, which exhibited changes in susceptibility to Fe-EDTA cleavage, almost exactly corresponded to D, E, and F alpha-helices which are involved directly in the recognition of DNA. The F alpha-helix, which provides all base-specific contacts in the CRP-DNA complex, became hypersensitive to Fe-EDTA-mediated cleavage, whereas the solvent exposure of D and E alpha-helices was decreased upon binding of cAMP. These results suggest that a significant part of cAMP-induced conformational change in CRP involves a movement of secondary structure elements in the C-terminal domain of the protein so that the recognition F alpha-helix becomes exposed to the solvent. In contrast to cAMP, binding of cGMP produced insignificant changes in susceptibility to Fe-EDTA-mediated cleavage. This is consistent with the inability of cGMP to induce functional conformational changes in CRP. The protein footprinting technique appears to be sufficiently sensitive for detection and mapping of ligand-induced conformational changes in proteins.

Binding Sites↗

PubMed2002

Comparison of the effects of cilostazol and milrinone on cAMP-PDE activity, intracellular cAMP and calcium in the heart.

We investigated the basis for the difference in the cardiotonic effects of the PDE3 inhibitors cilostazol and milrinone in the rabbit heart. Cilostazol displayed greater selectivity than milrinone for inhibition of cAMP-PDE activity in microsomal vs cytosolic fractions from rabbit heart. This difference was due to the inhibition of significantly less cytosolic cAMP-PDE activity by cilostazol compared to milrinone. A combination of cilostazol (>15 microM) and the PDE4 selective inhibitor, rolipram (5 microM), inhibited levels of cytosolic cAMP-PDE activity similar to those inhibited by milrinone on its own. This suggested that milrinone inhibited PDE4 in addition to PDE3 activity. In isolated rabbit cardiomyocytes, milrinone (>10 microM) caused greater elevations in intracellular cAMP and calcium than cilostazol. In the presence of rolipram, however, the cAMP and calcium elevating effects of cilostazol and milrinone were similar. Therefore, in rabbit heart, partial inhibition of PDE4 by milrinone contributed to greater increases in cardiomyocyte cAMP and calcium levels than cilostazol. PDE4 activity in failing human heart was lower than in rabbit heart and there was no significant difference in the inhibition of human cytosolic cAMP-PDE by cilostazol and milrinone. Our results suggest that in normal rabbit heart inhibition of PDE4 by milrinone may partly contribute to the greater cardiotonic effect of milrinone when compared to cilostazol. However, the lower level of PDE4 activity in failing human heart suggests that factors other than inhibition of PDE4 by milrinone may contribute to differences in cardiotonic action when compared to cilostazol.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed2002

Regulation of cAMP-specific phosphodiesterases type 4B and 4D (PDE4) splice variants by cAMP signaling in primary cortical neurons.

This study examined the regulation of all known phosphodiesterase (PDE) type PDE4A, PDE4B and PDE4D splice variants in cortical neurons by cAMP signaling. Treatment with dibutyryl-cAMP (db-cAMP) caused the induction of two of the known splice variants, PDE4B2 and PDE4D1/PDE4D2. Although the splice variants PDE4A1, PDE4A5/PDE4A10, PDE4B3, PDE4B1, PDE4D3 and PDE4D4 were present in cortical neurons, their mRNA was not regulated at the transcriptional level by db-cAMP. To assess the increase in PDE4B2 and PDE4D1/D2 mRNA expression, the promoters containing these genes were characterized. Transcription from both promoters was stimulated by db-cAMP. Because chronic antidepressant treatment increases PDE4B, and not PDE4D, mRNA expression, we focused on the regulation of the PDE4B2 promoter by cAMP and CREB. Dominant negative mutants of CREB suppressed PDE4B2 promoter activity and a constitutively active form of CREB robustly stimulated it. These data demonstrate that in cortical neurons, a short PDE4B2 intronic promoter is regulated by CREB, confers cAMP responsitivity and directs PDE4B2 mRNA and protein expression.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed1982

The cAMP-binding domains of the regulatory subunit of cAMP-dependent protein kinase and the catabolite gene activator protein are homologous.

Comparison of the recently determined amino acid sequences of the regulatory subunit of cAMP-dependent protein kinase (RII) from bovine cardiac muscle and the Escherichia coli catabolite gene activator protein (CAP) shows significant homology. This homology extends over most of the amino-terminal domain in CAP and is particularly good for the region of the beta-roll structure. The RII sequence contains two adjacent and internally homologous regions, both of which have high resemblance to the cAMP-binding domain in CAP. This suggests that the protein kinase regulatory subunit contains two cAMP-binding domains in the carboxyl-terminal region, each having a beta-roll structure similar to that in CAP. The cAMP molecule is expected to bind to the RII within a pocket formed by residues from the beta-roll, as is the case with CAP. One cAMP molecule would interact with residues from about 163 to 220, and the other cAMP would interact with amino acids in the stretch 285-350 of the RII protein kinase sequence. As the carboxyl-terminal domain of CAP shows homologies to the DNA-binding domains of other regulatory proteins, the protein appears to be of modular construction: a DNA-binding domain joined to a cAMP-binding domain.

Amino Acid Sequence↗

PubMed1988

cAMP-mediated phosphorylation of the low-Km cAMP phosphodiesterase markedly stimulates its catalytic activity.

Treatment of intact human platelets with the adenylate cyclase agonist forskolin (100 microM) resulted in an increase in cAMP phosphodiesterase activity in freeze-thaw lysates. When the low-Km (high affinity), cGMP-inhibited cAMP phosphodiesterase was isolated from such lysates by blue dextran-Sepharose chromatography, the specific activity of the enzyme was increased an average of 11-fold over similarly processed control platelets. The increase in the low-Km, cGMP-inhibited cAMP phosphodiesterase activity was inhibited when platelets were incubated with the protein kinase inhibitor H-8 prior to treatment with forskolin, suggesting that the stimulation of cAMP phosphodiesterase activity involved a cAMP-dependent phosphorylation. When intact platelets that had been prelabeled with 32Pi were treated with forskolin and the low-Km, cGMP-inhibited phosphodiesterase was isolated by blue dextran-Sepharose chromatography, a protein of 110,000 kDa was phosphorylated. By using a monospecific antiserum to the purified phosphodiesterase, this protein was shown to be the low-Km, cGMP-inhibited cAMP phosphodiesterase by electrophoretic transfer blot (Western blot) analysis and by immunoprecipitation. The stable prostacyclin analog iloprost also stimulated the low-Km cAMP phosphodiesterase activity about 2-fold and caused phosphorylation of the enzyme. These results suggest that phosphorylation of the low-Km, cGMP-inhibited phosphodiesterase may be an important regulatory mechanism for this enzyme in platelets.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed1998

The RIIbeta regulatory subunit of protein kinase A binds to cAMP response element: an alternative cAMP signaling pathway.

cAMP, through the activation of cAMP-dependent protein kinase (PKA), is involved in transcriptional regulation. In eukaryotic cells, cAMP is not considered to alter the binding affinity of CREB/ATF to cAMP-responsive element (CRE) but to induce serine phosphorylation and consequent increase in transcriptional activity. In contrast, in prokaryotic cells, cAMP enhances the DNA binding of the catabolite repressor protein to regulate the transcription of several operons. The structural similarity of the cAMP binding sites in catabolite repressor protein and regulatory subunit of PKA type II (RII) suggested the possibility of a similar role for RII in eukaryotic gene regulation. Herein we report that RIIbeta subunit of PKA is a transcription factor capable of interacting physically and functionally with a CRE. In contrast to CREB/ATF, the binding of RIIbeta to a CRE was enhanced by cAMP, and in addition, RIIbeta exhibited transcriptional activity as a Gal4-RIIbeta fusion protein. These experiments identify RIIbeta as a component of an alternative pathway for regulation of CRE-directed transcription in eukaryotic cells.

3T3 Cells↗

PubMed1995

Characterization of the human tryptophan hydroxylase gene promoter. Transcriptional regulation by cAMP requires a new motif distinct from the cAMP-responsive element.

We isolated and sequenced 2,117 nucleotides of the promoter region of the human tryptophan hydroxylase (TPH) gene. Transient transfection in pinealocyte cultures and PC12 cells was used to investigate the human TPH (hTPH) gene promoter activity and its regulation by the cAMP signaling pathway. A region of 2,117 base pairs upstream of the transcription initiation site of the hTPH gene efficiently directed the transcription of a luciferase reporter gene but not in a cell-specific manner. The hTPH promoter activity was significantly enhanced by a cyclic AMP analog in the two cell types. Deletion analysis showed that the promoter region from -73 to +2 is sufficient to direct cAMP-dependent transcription, although it does not contain a motif exhibiting a significant identity to the cAMP-responsive element (CRE) or AP-2 binding site. Following site-directed mutagenesis of the region between -73 and -51, an inverted CCAAT box motif was identified as essential for cAMP inducibility of the hTPH promoter. This sequence between -73 and -51 alone allowed cAMP enhancement of transcription when fused to a heterologous promoter. Additionally, electrophoretic mobility shift assays showed that a specific protein-DNA complex is formed between an oligonucleotide corresponding to the inverted CCAAT box motif and nuclear proteins from pinealocytes treated or not treated with cAMP. Thus cAMP responsiveness of hTPH gene expression is mediated by a cis-acting element, which shares strong identity with an inverted CCAAT box and which binds to a constitutively produced nuclear factor.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1996

Membrane localization of cAMP-dependent protein kinase amplifies cAMP signaling to the nucleus in PC12 cells.

The A126 cell line, in contrast to its PC12 parent, does not differentiate, accumulate nuclear cAMP-dependent protein kinase A (PKA) catalytic subunit, or transcribe cAMP-dependent promoters in response to cAMP. Total PKA is reduced by 50% and is partly resistant to cAMP-induced dissociation in vivo. Unlike PC12, where PKAII is membrane-associated, PKAII is exclusively cytosolic in A126. Cotransfection with the RII anchor protein (AKAP75) and the PKA catalytic subunit (C-PKA) restored cAMP-induced transcription to levels found in PC12. These data indicate that membrane-bound PKAII amplifies cAMP signaling to the nucleus and suggest that cAMP-mediated responses are specified by the type and cellular localization of the PKA isoform.

A Kinase Anchor Proteins↗