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At least 73 records · Page 4Linked to original sources
PubMed2004

Crystal structures of RIalpha subunit of cyclic adenosine 5'-monophosphate (cAMP)-dependent protein kinase complexed with (Rp)-adenosine 3',5'-cyclic monophosphothioate and (Sp)-adenosine 3',5'-cyclic monophosphothioate, the phosphothioate analogues of cAMP.

Cyclic adenosine 5'-monophosphate (cAMP) is an ancient signaling molecule, and in vertebrates, a primary target for cAMP is cAMP-dependent protein kinase (PKA). (R(p))-adenosine 3',5'-cyclic monophosphothioate ((R(p))-cAMPS) and its analogues are the only known competitive inhibitors and antagonists for cAMP activation of PKA, while (S(p))-adenosine 3',5'-cyclic monophosphothioate ((S(p))-cAMPS) functions as an agonist. The crystal structures of a Delta(1-91) deletion mutant of the RIalpha regulatory subunit of PKA bound to (R(p))-cAMPS and (S(p))-cAMPS were determined at 2.4 and 2.3 A resolution, respectively. While the structures are similar to each other and to the crystal structure of RIalpha bound to cAMP, differences in the dynamical properties of the protein when (R(p))-cAMPS is bound are apparent. The structures highlight the critical importance of the exocyclic oxygen's interaction with the invariant arginine in the phosphate binding cassette (PBC) and the importance of this interaction for the dynamical properties of the interactions that radiate out from the PBC. The conformations of the phosphate binding cassettes containing two invariant arginine residues (Arg209 on domain A, and Arg333 on domain B) are somewhat different due to the sulfur interacting with this arginine. Furthermore, the B-site ligand together with the entire domain B show significant differences in their overall dynamic properties in the crystal structure of Delta(1-91) RIalpha complexed with (R(p))-cAMPS phosphothioate analogue ((R(p))-RIalpha) compared to the cAMP- and (S(p))-cAMPS-bound type I and II regulatory subunits, based on the temperature factors. In all structures, two structural solvent molecules exist within the A-site ligand binding pocket; both mediate water-bridged interactions between the ligand and the protein. No structured waters are in the B-site pocket. Owing to the higher resolution data, the N-terminal segment (109-117) of the RIalpha subunit can also be traced. This strand forms an intermolecular antiparallel beta-sheet with the same strand in an adjacent molecule and implies that the RIalpha subunit can form a weak homodimer even in the absence of its dimerization domain.

Binding Sites↗
PubMed1986

cAMP induction of prespore and prestalk gene expression in Dictyostelium is mediated by the cell-surface cAMP receptor.

Extracellular adenosine 3',5'-cyclic monophosphate (cAMP) is required for cell-type-specific gene expression in developing Dictyostelium discoideum. We have developed a microassay for the expression of these genes, using antibodies directed against their protein products. To characterize the transduction mechanism, we have used in this assay cAMP analogues that preferentially activate either the cell-surface cAMP receptor or the internal cAMP-dependent protein kinase. N6-(aminohexyl) cAMP activates the Dictyostelium cAMP-dependent protein kinase but does not bind to the cell-surface cAMP receptor and does not cause cell-type-specific gene expression. 2'-Deoxy-cAMP does not activate the cAMP-dependent protein kinase but binds to the receptor and causes cell-type-specific gene expression. Cyclic AMP-induced accumulation of prestalk mRNA in shaking cultures still occurs in the presence of caffeine, which blocks the receptor-coupled activation of adenyl cyclase. This suggests that the extracellular cAMP induction of cell-type-specific gene expression in developing Dictyostelium cells is mediated by the cell-surface cAMP receptor and that activating adenyl cyclase by this receptor is not essential. Using the N6-(aminohexyl) cAMP to competitively inhibit phosphodiesterase, we show that 30 nM cAMP is sufficient to induce prestalk or prespore gene expression.

Caffeine↗
PubMed2003

cAMP Dose-dependently prevents palmitate-induced apoptosis by both protein kinase A- and cAMP-guanine nucleotide exchange factor-dependent pathways in beta-cells.

Lipid accumulation in pancreatic beta-cells is thought to cause its dysfunction and/or destruction via apoptosis. Our studies show that incubation of the beta-cell line RINm5F with the saturated free fatty acids (FFA) palmitate caused apoptosis based on increases in caspase 3 activity, Annexin V staining, and cell death. Furthermore, exposure of RINm5F cells to cAMP-increasing agents, 3-isobutyl-1-methylxanthine (IBMX), and forskolin completely abolished palmitate-mediated caspase 3 activity and significantly inhibited Annexin V staining and cell death. The cyclic AMP analogs cpt-cAMP and dibutyryl-cAMP mimicked the protective effects of IBMX and forskolin, suggesting that cAMP is the mediator of the anti-apoptotic effects. The protective action of IBMX and forskolin was rapid and did not appear to require gene transcription or new protein synthesis. However, these protective effects were clearly independent of protein kinase A (PKA) because of the lack of inhibition by the PKA inhibitors H-89 and KT5720. In attempts to identify this PKA-independent mechanism, we found that the newly developed cAMP analog 8CPT-2Me-cAMP, which selectively activates the cAMP-dependent guanine nucleotide exchange factor (cAMP-GEF) pathway, mimicked the protective effects of IBMX and forskolin, suggesting that the cAMP-GEF pathway is involved. In addition, both glucagon-like peptide (GLP-1) and its receptor agonist, Exenatide, inhibited palmitate-mediated caspase 3 activation in a dose-dependent manner. Unexpectedly, H-89 partially reversed the protective effects of GLP-1 and Exenatide, suggesting that PKA may play a role in the protective effects of these incretins. To explain these seemingly conflicting results, we demonstrated that low concentrations of cAMP produced by GLP-1 and Exenatide preferentially activate the PKA pathway, whereas higher cAMP concentrations produced by IBMX and forskolin activate the more dominant cAMP-GEF pathway. Taken together, these results indicate that intracellular concentrations of cAMP may play a key role in determining divergent signaling pathways that lead to antiapoptotic responses.

1-Methyl-3-isobutylxanthine↗
PubMed1991

Attenuation of cAMP-mediated responses in MA-10 Leydig tumor cells by genetic manipulation of a cAMP-phosphodiesterase.

In order to assess the effect of increased cAMP degradation on the responsiveness on an endocrine cell, we have obtained stable transfectants of MA-10 Leydig tumor cells that overexpress a mammalian cAMP-phosphodiesterase. Two novel cell lines, designated MA-10(P+8) and MA-10(P+29), that express high levels of the transfected enzyme were characterized. Although the basal levels of cAMP in the mutant cell lines are comparable to those of the wild-type cells, the increase in cAMP accumulation elicited by human choriogonadotropin (hCG) is severely blunted. Further studies with MA-10(P+29) show that the ability of hCG to stimulate adenylyl cyclase activity is normal. The failure of MA-10(P+29) cells to accumulate cAMP in response to hCG can be correlated with a similar reduction in hCG-stimulated steroidogenesis. On the other hand, the maximal steroidogenic response of MA-10(P+29) cells to dibutyryl cAMP, a cAMP analogue that is fairly resistant to phosphodiesterase degradation, is normal. We also show that the ability of these cells to respond to hCG with increased cAMP accumulation and steroid synthesis can be restored with a specific phosphodiesterase inhibitor. These results demonstrate that overexpression of a cAMP-phosphodiesterase in MA-10 cells limits the levels of cAMP attained under hCG stimulation and supresses the steroidogenic response of these cells to hCG. Since gonadotropins increase the cAMP-phosphodiesterase activity in their target cells, these findings also provide evidence that this regulation plays a major role in the modulation of cell responsiveness. Last, these new cell lines should be valuable in the study of the actions of cAMP because they express a conditional and reversible cAMP-resistant phenotype.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1989

The roles of cAMP and cAMP-dependent protein kinase in the expression of cholesterol side chain cleavage and steroid 11 beta-hydroxylase genes in mouse adrenocortical tumor cells.

The expression of the genes encoding cholesterol side chain cleavage enzyme (SCC) and steroid 11 beta-hydroxylase (11 beta-OHase) was examined in Y1 mouse adrenocortical tumor cells and in derivative cell lines defective in cAMP-dependent protein kinase activity. Y1 cells expressed both genes constitutively, and treatment with 8-bromo-cAMP (8-Br-cAMP) increased expression 5-10-fold. In three independent protein kinase mutants, expression of SCC and 11 beta-OHase was impaired to degrees dependent upon the severity of defect in cAMP-dependent protein kinase activity. In Kin-2, the least impaired mutant clone, basal expression of SCC was the same as in Y1 cells. Treatment of Kin-2 with 8-Br-cAMP increased SCC RNA to the levels seen in stimulated Y1 cells. In contrast, clone Kin-8, the most severe mutant, expressed markedly diminished basal and 8-Br-cAMP-stimulated levels of SCC mRNA. Kin-7 had basal and 8-Br-cAMP-stimulated levels of SCC mRNA which were intermediate to Kin-2 and Kin-8. None of the Kin mutants constitutively expressed detectable levels of 11 beta-OHase transcripts, and only Kin-2 responded to treatment with 8-Br-cAMP with increased expression of 11 beta-OHase; however, the time course of induction in Kin-2 was significantly delayed. The disparate patterns of expression of SCC and 11 beta-OHase in the Kin mutants suggest that these genes differ in their absolute requirement for cAMP-dependent protein kinase activity. Experiments also were performed in which Kin-7 and Kin-8 mutants were restored to cAMP-responsive states by transfection with genes encoding normal sub-units of cAMP-dependent protein kinase. These phenotypic revertants recovered 8-Br-cAMP-inducible expression of SCC and 11 beta-OHase. These results strongly support the hypothesis that impaired expression of steroidogenic enzymes in the Kin mutants results directly from defects in cAMP-dependent protein kinase activity.

Adrenal Cortex Neoplasms↗
PubMed1989

Expression and characterization of mutant forms of the type I regulatory subunit of cAMP-dependent protein kinase. The effect of defective cAMP binding on holoenzyme activation.

The mouse wild type and four mutant regulatory type I (RI) subunits were expressed in Escherichia coli and subjected to kinetic analyses. The defective RI subunits had point mutations in either cAMP-binding site A (G200/E), site B (G324/D, R332/H), or in both binding sites. In addition, a truncated form of RI which lacked the entire cAMP-binding site B was generated. All of the mutant RI subunits which bound [3H]cAMP demonstrated more rapid rates of cAMP dissociation compared to the wild type RI subunit. Dissociation profiles showed only a single dissociation component, suggesting that a single nonmutated binding site was functional. The mutant RI subunits associated with purified native catalytic subunit to form chromatographically separable holoenzyme complexes in which catalytic activity was suppressed. Each of these holoenzymes could be activated but showed varying degrees of cAMP responsiveness with apparent Ka values ranging from 40 nM to greater than 5 microM. The extent to which the mutated cAMP-binding sites were defective was also shown by the resistance of the respective holoenzymes to activation by cAMP analogs selective for the mutated binding sites. Kinetic results support the conclusions that 1) Gly-200 of cAMP-binding site A and Gly-324 or Arg-332 of site B are essential to normal conformation and function, 2) activation of type I cAMP-dependent protein kinase requires that only one of the cAMP-binding sites be functional, 3) mutational inactivation of site B (slow exchange) has a much more drastic effect than that of site A on increasing the Ka of the holoenzyme for cAMP, as well as in altering the rate of cAMP dissociation from the remaining site of the free RI subunit. The strong dependence of one cAMP-binding site on the integrity of the other site suggests a tight association between the two sites.

Animals↗
PubMed1985

cAMP-dependent protein kinase activation lowers hepatocyte cAMP.

Rat hepatocyte protein kinase was activated by incubating the cells with various cAMP analogs. Boiled extracts were then prepared and Sephadex G-25 chromatography was carried out. The G-25 procedure separated the analogs from cAMP since the resin had the unexpected property of binding cyclic nucleotides with differing affinities. Separation was necessary because the analogs would otherwise interfere with the sensitive protein kinase activation method developed for assay of cAMP. The cAMP analogs, but not 5'-AMP, lowered basal cAMP by 50-70%. The effect was rapid, analog concentration-dependent, and occurred parallel with phosphorylase activation, suggesting that the cAMP analogs act through cAMP-dependent protein kinase activation. A cAMP analog completely blocked the cAMP elevation produced by relatively low concentrations of glucagon, but did not block the phosphorylase response, indicating that the cAMP analog substitutes for cAMP as the intracellular activator of protein kinase. One implication of the results is that elevation of cAMP and protein kinase activity by hormones has a negative feedback effect on the cellular cAMP level.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1995

Differential regulation of human monocyte-derived TNF alpha and IL-1 beta by type IV cAMP-phosphodiesterase (cAMP-PDE) inhibitors.

Elevation of cAMP downregulates certain functions of inflammatory cells, including the release of TNF alpha and IL-1 beta by macrophages. Intracellular cAMP levels can be modulated pharmacologically by adding cell-permeable cAMP analogs, by stimulating adenylate cyclase or by inhibiting degradation of cAMP by cAMP-phosphodiesterases (cAMP-PDE). Multiple forms of cAMP-PDEs have been identified in various tissues and cells using both biochemical characterization and selective inhibitors. Therefore, we wanted to determine which of these different PDE isoforms was present in human monocytes and whether this isoform could regulate cytokine release from human monocytes by a mechanism similar to that seen with dbcAMP or PGE1. Our results demonstrate that selective inhibitors of type IV cAMP-PDE, such as rolipram and Ro20-1724, are clearly the most effective compounds at enhancing cAMP levels and inhibiting the release of TNF alpha and IL-1 beta in these cells. The type III cAMP-PDE-selective inhibitors C1930 and cilostamide and the nonselective PDE inhibitors IBMX and pentoxifylline were significantly less potent. In agreement with these data, cAMP-PDE activity in cytosolic extracts from human monocytes was also much more sensitive to inhibition by rolipram than by cilostamide. Additionally, rolipram dramatically reduced TNF alpha mRNA accumulation, which supports previous findings that cAMP regulates TNF alpha at the transcriptional level. Surprisingly, rolipram, rolipram, dbcAMP or PGE1 increased IL-1 beta was reduced, which indicates that cAMP can have both positive and negative effects on the regulation of IL-1 beta.(ABSTRACT TRUNCATED AT 250 WORDS)

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1994

The short-term activation of a rolipram-sensitive, cAMP-specific phosphodiesterase by thyroid-stimulating hormone in thyroid FRTL-5 cells is mediated by a cAMP-dependent phosphorylation.

To elucidate the mechanism causing the transient accumulation of intracellular cAMP in the FRTL-5 thyroid cell line, the short-term effect of thyroid-stimulating hormone (TSH) on phosphodiesterase (PDE) activity was studied. Together with an increase in cAMP levels, TSH produced a significant increase in total PDE activity as early as 3 min, with a maximal stimulation reached after 15 min. This short-term increase in PDE activity was dependent on the TSH concentration (ED50 = 4 x 10(-11) M TSH). Forskolin and dibutyryl cAMP produced an even larger stimulation than that produced by TSH, suggesting that the effect of TSH is mediated by cAMP. To determine the properties of the PDE forms activated by TSH, antibodies specific for the cAMP-PDEs were used to immunoprecipitate the PDEs present in control cells, and cells incubated for 15 min in the presence of 10 nM TSH. Comparison of the activity recovered in the immunoprecipitation pellets demonstrated that TSH produced more than a 2.5-fold increase in the cAMP-PDE form(s) recognized by this antibody. Conversely, the activity remaining in the supernatants was not affected by the TSH treatment. Most of the activity recovered in the immunoprecipitation pellets (90%) was inhibited by 10 microM Rolipram, an inhibitor specific for the high affinity cAMP-PDEs. No TSH stimulation of the Rolipram-insensitive PDE activity could be observed under these conditions. Western blot analyses with two different cAMP-PDE specific antibodies showed that a 15-min stimulation with TSH induced the appearance of a new band with electrophoretic mobility slower than the polypeptide present in unstimulated cells. The appearance of this band did not require ongoing protein synthesis because it occurred in the presence of cycloheximide. Metabolic [32P]orthophosphate labeling of intact FRTL-5 cells indicated that the TSH treatment caused an increased 32P incorporation into a polypeptide that co-purified with the stimulated PDE activity and had an electrophoretic mobility identical to that of the cAMP-PDE. Okadaic acid, a potent inhibitor of protein phosphatase 1 and protein phosphatase 2A, elicited a potentiation of the TSH-stimulated PDE activity. The stimulating of a PDE with the same immunological properties and Rolipram sensitivity as the cAMP-PDE stimulated by TSH in the intact cells was reproduced, in a cell-free system, by incubating soluble extracts from FRTL-5 cells with the catalytic subunit of cAMP-dependent protein kinase. These data provide evidence that TSH produces a rapid activation of a cAMP-PDE in the FRTL-5 cells through a cAMP-dependent phosphorylation.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1991

Mechanisms of cAMP-mediated gene induction: examination of renal epithelial cell mutants affected in the catalytic subunit of the cAMP-dependent protein kinase.

The precise mechanistic role of the cAMP-dependent protein kinase (cAMP-PK) in cAMP-mediated gene induction remains unclear. Renal epithelial cell mutants were compared to the LLC-PK1 parental cell line for induction of the cAMP-responsive urokinase-type plasminogen activator (uPA) gene, as quantitated by the technique of mRNA solution hybridization. The FIB4 and FIB6 mutants, which possess less than 10% parental cAMP-PK catalytic (C) subunit activity, showed markedly diminished uPA mRNA induction in response to agents elevating intracellular cAMP such as the cAMP analogue 8-bromo-cAMP and the adenylate cyclase-stimulating hormones vasopressin and calcitonin. In contrast, the mutant cells responded to a similar or greater extent than the parental cells in terms of uPA mRNA induction following treatment with the Ca2+/phospholipid-dependent protein kinase activator phorbol 12-myristate 13-acetate (PMA). Elevation of intracellular cAMP was found to induce a translocation of the cAMP-PK C subunit from the perinuclear Golgi region to the nucleus in both parental and mutant cell lines, as shown by immunocytochemical techniques. Results argue for the role of the cAMP-PK C subunit activity and possibly nuclear translocation of the C subunit in cAMP-mediated uPA induction, which is mechanistically distinct from the PMA-stimulated response.

Animals↗
PubMed2005

The role of intracellular cAMP in renal gluconeogenesis in view of differential action of various cAMP analogues.

Effects of various cAMP analogues on gluconeogenesis in isolated rabbit kidney tubules have been investigated. In contrast to N(6),2'-O-dibutyryladenosine-3',5'-cyclic monophosphate (db-cAMP) and cAMP, which accelerate renal gluconeogenesis, 8-bromoadenosine-3',5'-cyclic monophosphate (Br-cAMP) and 8-(4-chlorophenylthio)-cAMP (pCPT-cAMP) inhibit glucose production. Stimulatory action of cAMP and db-cAMP may be evoked by butyrate and purinergic agonists generated during their extracellular and intracellular metabolism resulting in an increase in flux through fructose-1,6-bisphosphatase and in consequence acceleration of the rate of glucose formation. On the contrary, Br-cAMP is poorly metabolized in renal tubules and induces a fall of flux through glyceraldehyde-3-phosphate dehydrogenase. The contribution of putative extracellular cAMP receptors to the inhibitory Br-cAMP action is doubtful in view of a decline of glucose formation in renal tubules grown in the primary culture supplemented with forskolin. The presented data indicate that in contrast to hepatocytes, in kidney-cortex tubules an increased intracellular cAMP level results in an inhibition of glucose production.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed2003

cAMP-dependent protein kinase regulatory subunit type IIbeta: active site mutations define an isoform-specific network for allosteric signaling by cAMP.

cAMP-dependent protein kinase (cAPK) contains a regulatory (R) subunit dimer bound to two catalytic (C) subunits. Each R monomer contains two cAMP-binding domains, designated A and B. The sequential binding of two cAMPs releases active C. We describe here the properties of RIIbeta and two mutant RIIbeta subunits, engineered by converting a conserved Arg to Lys in each cAMP-binding domain thereby yielding a protein that contains one intact, high affinity cAMP-binding site and one defective site. Structure and function were characterized by circular dichroism, steady-state fluorescence, surface plasmon resonance and holoenzyme activation assays. The Ka for RIIbeta is 610 nM, which is 10-fold greater than its Kd(cAMP) and significantly higher than for RIalpha and RIIalpha. The Arg mutant proteins demonstrate that the conserved Arg is important for both cAMP binding and organization of each domain and that binding to domain A is required for activation. The Ka of the A domain mutant protein is 21-fold greater than that of wild-type and the Kd(cAMP) is increased 7-fold, confirming that cAMP must bind to the mutated site to initiate activation. The domain B mutant Ka is 2-fold less than its Kd(cAMP), demonstrating that, unlike RIalpha, cAMP can access the A site even when the B site is empty. Removal of the B domain yields a Ka identical to the Kd(cAMP) of full-length RIIbeta, indicating that the B domain inhibits holoenzyme activation for RIIbeta. In RIalpha, removal of the B domain generates a protein that is more difficult to activate than the wild-type protein.

Allosteric Site↗
PubMed2003

Cyclic adenosine monophosphate (cAMP) stimulation of the kit ligand promoter in sertoli cells requires an Sp1-binding region, a canonical TATA box, and a cAMP-induced factor binding to an immediately downstream GC-rich element.

Expression of Kit ligand (KL) mRNA is induced in primary prepuberal Sertoli cells by FSH and by other agents that increase cAMP levels. The cAMP effect is exerted at the transcriptional level and appears to be cell type specific, since it is not observed in other KL-expressing primary cells or cell lines. Deletion analysis of the 5'-flanking region of the mouse KL gene shows that the proximal promoter sequence between -88 and +8 from the transcriptional start site is necessary and sufficient to obtain the full cAMP responsiveness of the promoter in primary mouse Sertoli cells. In the -88/+8 promoter region, several cis-acting elements play a role in cAMP response. The -88/-56 sequence is necessary for full induction of the gene, since its removal causes a drastic decrease in cAMP responsiveness; however, cAMP-stimulated expression is still observed with the minimal promoter region between -55 and +8. A more detailed mutational analysis of the minimal promoter region shows that mutations in the canonical TATA box sequence and in an immediately downstream GC-rich element completely abolish cAMP responsiveness. DNA-binding experiments show that transcription factor Sp1 binds to the -88/-56 fragment of the KL proximal promoter in both control and cAMP-stimulated cells, whereas a new cAMP-induced complex is observed when the -55/+8 minimal promoter region is used as probe. The canonical TATA box sequence is essential for formation of the latter complex. We also show that the binding of an unknown nuclear factor (different from Sp1, Egr-1, Rnf6, and AP-2) to a GC-rich element between -19 and +8 increases after cAMP treatment, and this effect seems to be specific of primary Sertoli cells. Thus, cAMP-induced transcription from the KL gene promoter in primary mouse Sertoli cells is mediated by a complex interaction among a Sp1-binding region, factors recognizing the canonical TATA box sequence, and a not yet identified cAMP-induced factor binding a GC-rich sequence just downstream from it.

5' Flanking Region↗
PubMed1994

Differential regulation of chromogranin B and synapsin I gene promoter activity by cAMP and cAMP-dependent protein kinase.

cAMP has neutrotrophic effects in the nervous system. We have investigated whether there is a correlation between cAMP-induced neurite outgrowth and induction of chromogranin B and synapsin I gene expression. These genes encode marker proteins of distinct populations of vesicles in neurons, neuroendocrine and endocrine cells, and in addition, they contain a cAMP response element (CRE) in their upstream regions, making it likely that cAMP-induced neuronal differentiation might be accompanied by increased transcription of these genes. We increased intracellular cAMP levels in neuronal and neuroendocrine cells and analyzed the levels of chromogranin B and synapsin I mRNA. Our data revealed that, while chromogranin B mRNA was in fact induced following cAMP stimulation, synapsin I mRNA was not affected. To analyze the cis-acting sequences, we constructed hybrid genes containing the upstream region of the mouse chromogranin B gene fused to a reporter gene. Similar plasmids containing the synapsin I or the glucagon promoter were constructed. Transfections of neuronal and endocrine cells, together with deletion mutagenesis, revealed that the CRE of the chromogranin B gene mediated the effect of cAMP upon transcription. This effect was mimicked by overexpression of the catalytic subunit of the cAMP-dependent protein kinase. In addition, overexpression of the negative-acting CRE-binding protein CREB-2 revealed that the chromogranin B CRE functions as a bifunctional genetic regulatory element in that it mediates basal as well as cAMP-stimulated transcription. Synapsin I gene expression, however, was not induced by either elevated intracellular cAMP concentration or by overexpression of protein kinase A, although a similar pattern of proteins, including CREB, bound to the synapsin I and chromogranin B CRE in vitro. Thus while the CRE element in the chromogranin B gene promoter is responsive to cAMP, the same element, when present in the synapsin I promoter, does not confer cAMP inducibility.

1-Methyl-3-isobutylxanthine↗
PubMed2004

Steroidogenic factor-1 interacts with cAMP response element-binding protein to mediate cAMP stimulation of CYP1B1 via a far upstream enhancer.

CYP1B1 activates polycyclic aromatic hydrocarbon carcinogens in cAMP-regulated tissues such as the adrenal, ovary, and testis. A 27-fold cAMP stimulation of the CYP1B1-luciferase reporter in Y-1 adrenal cells depends entirely on a far upstream enhancer region (FUER; -5298 to -5110). Cooperative participation of multiple steroidogenic factor 1 (SF-1) elements with the downstream cAMP response element (CRE) in FUER is essential for both basal and cAMP-stimulated activities of FUER. Basal and induced activities were similarly lowered by DAX-1, an SF-1 suppressor, and raised by steroid receptor coactivator 1, an SF-1 coactivator. cAMP response element-binding protein (CREB)-binding protein (CBP) that interacts preferentially with the phosphorylated-CREB increased the cAMP-induced FUER. 10T1/2 cells and human embryonic kidney (HEK)293 cells do not express SF-1. Introduction of exogenous SF-1 generated cAMP stimulation of the FUER in 10T1/2 fibroblasts. The same transfection only increased basal activity of FUER in HEK293 cells, despite presence of active CREB in cells. HEK293 cells therefore remain deficient in additional factor(s) critical to the cAMP stimulation of CYP1B1. Mutations of the protein kinase A (PKA) and the mitogen-activated protein kinase phosphorylation sites (Ser-430 and Ser-203) on SF-1 had no effect on the SF-1-dependent FUER stimulation in Y-1 and 10T1/2 cells. This contrasts with loss of activity with mutation of CREB at PKA phosphorylation site (Ser-133). SF-1 phosphorylation at these sites is therefore not essential for the cAMP stimulation and the cooperation with CREB. cAMP-enhanced activation protein 1 (AP-1) and stimulatory protein 1 (Sp1) complexes in the proximal promoter region contributed substantially to both basal and cAMP-stimulated FUER activity. Chromatin immunoprecipitation from primary rat adrenal cells demonstrated cAMP stimulation of histone acetylation proximal to, respectively, the FUER and AP-1 sites of CYP1B1.

Animals↗
PubMed1983

Facilitation of cAMP increments during ADCC mediated by monocytes pretreated with cAMP-elevating agents.

Addition of IgG-sensitized human erythrocytes to peripheral blood monocytes elicit a transient increment in monocyte cAMP levels. This increase in cAMP was facilitated when monocytes were preincubated with the phosphodiesterase inhibitors, isobutylmethylxanthine (IBMX) and theophylline, and the adenylate cyclase agonists, isoproterenol and prostaglandin E1 (PGE1). Although these cAMP elevating agents were able to inhibit monocyte ADCC, the degree of inhibition could not be anticipated from the cAMP levels achieved by these drugs since theophylline inhibited monocyte ADCC in doses not elevating cAMP and PGE1, isoproterenol and IBMX were less effective inhibitors of monocyte ADCC than theophylline when comparing their effects on cAMP levels. Both PGE1-induced elevation of cAMP levels and the further increments of cAMP after addition of IgG-sensitized erythrocytes to PGE1-treated monocytes were significantly correlated to the inhibition of beta-glucuronidase release during ADCC. Theophylline in doses of 0.5 mM did not elevate basal levels of monocyte cAMP but facilitated the ADCC-induced cAMP increment concomitant with inhibition of monocyte ADCC and degranulation. Possibly, facilitation of cAMP increments during ADCC by an inhibitory feedback mechanism may be responsible for the inhibition caused by cAMP-elevating agents.

1-Methyl-3-isobutylxanthine↗
PubMed1990

Characterization of the yeast low Km cAMP-phosphodiesterase with cAMP analogues. Applications in mammalian cells that express the yeast PDE2 gene.

The essential interactions between cAMP and the yeast low Km cAMP-phosphodiesterase have been analyzed using cAMP analogues and phosphodiesterase inhibitors. cAMP specificity is conferred by hydrogen bonding at the N-6 and N-7 positions. In contrast to the other yeast phosphodiesterase, (Rp)-adenosine 3',5'-monophosphorothioate is not hydrolyzed. Eleven standard phosphodiesterase inhibitors were not highly effective. In Chinese hamster ovary (CHO) cells that express the yeast cAMP-phosphodiesterase (PDE2) gene, cAMP levels cannot be raised by cholera toxin. cAMP analogues that are efficiently hydrolyzed by the yeast cAMP-phosphodiesterase had no effect on the growth of CHO cells that express the PDE2 gene, even though they block the growth and alter the morphology of control cells. cAMP analogues that are not hydrolyzed by the yeast enzyme inhibited the growth and changed the morphology of both control and PDE2 expressing CHO cells. We have developed a method for creating cell lines in which cAMP levels can be reduced by expression of an exogenous cAMP-phosphodiesterase gene. By employing cAMP analogues that are not hydrolyzed by this phosphodiesterase, the inhibitory effects of the enzyme can be bypassed.

3',5'-Cyclic-AMP Phosphodiesterases↗