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At least 289 records · Page 16Linked to original sources
PubMed1976

Sequential alterations in the hepatic content and metabolism of cyclic AMP and cyclic GMP induced by DL-ethionine: evidence for malignant transformation of liver with a sustained increase in cyclic AMP.

There is evidence than adenosine 3',5'-monophosphate (cAMP) and guanosine 3',5'-monophosphate (cGMP) may have antagonistic actions on cell growth, with cAMP inhibiting and cGMP stimulating this process. However, reductions in cAMP and increases in cGMP are not charactersitic of all neoplastic tissues. Thus, benign and malignant tissues from hepatoma-bearing rats exposed to the hepatic carcinogen DL-ethionine have elevated rather than depressed cAMP, compared to control liver, and parenteral administration of this drug increases hepatic cAMP within hours. In the present study, the effects of ethionine ingestion on the hepatic content and metabolism of both cAMP and cGMP were examined sequentially in rats at 2 and then 6 wk intervals, from the initiation of drug administration until the development of hepatomas. After 2 wk, cAMP content of quick-frozen liver from rats receiving ethionine (E) was significantly increased (826 +/- 91 pmole/g wet weight) above that of liver from pair-fed controls (C, 415 +/- 44), whether calculated by tissue wet weight, protein, or DNA content. In benign tissue from E, higher cAMP was still evident after in vitro incubations of slices with 2 mM 1-methyl-3-iso-butylxanthine (MIX) and was associated with enhanced adenylate cyclase and unchanged high or low Km cAMP-phosphodiesterase activities. These findings are compatible with accelerated cAMP generation in liver from E. Protein kinase activity ratios were significantly increased in frozen liver from E (0.52 +/- 0.04 versus 0.36 +/- 0.03 in C), and the percent glycogen synthetase in the I form was clearly reduced (19% +/- 2% in E versus 47% +/- 5% in c). incubation of hepatic slices from E or C with MIX and/or 10 muM glucagon further increased cAMP and protein kinase activity ratios, data which imply higher effective, as well as total, cellular cAMP in E. Changes in cAMP metabolism and action observed at 2 wk persisted throughout the 38-wk period of drug ingestion. Adenylate cyclase activity, cAMP content, and protein kinase activity ratios of ethionine-induced hepatomas exceeded those of both the surrounding liver from tumor-bearing rats and that of control liver, but alterations in these parameters were qualitatively similar in both tissues from E. By contrast, while cGMP in quick-frozen surrounding liver from tumor-bearing rats (36 +/- 4 pmole/g wet weight) did not differ from that of control liver (30 +/- 3), cGMP in the hepatomas was increased. This change was evident in both frozen tumor (89 +/- 10) and in tumor slices incubated in vitro with MIX (C, 90 +/- 11; surrounding liver, 85 +/- 10; hepatoma 231 +/- 29). These results indicate that malignant conversion can occur in liver with a sustained elevation of both total and effective cAMP during the premalignant phase. The increase in cGMP detected in ethionine-induced hepatomas could also be a key determinant of malignant transformation in the model, although premalignant changes in cGMP were not apparent.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1992

Near ultraviolet circular dichroism study of the cyclic AMP receptor protein, its NH2-terminal domain and their interaction with cyclic AMP.

Circular dichroism in the near ultraviolet wavelength range was employed to examine conformational features of CRP (a dimer with a chain of 209 amino acids) and of its subtilisin core -alpha CRP- which retains the cAMP binding site (a dimer spanning the sequence 1-117). Binding of the ligand cAMP (allosteric activator), as well as cGMP was also investigated. The well resolved transitions could be assigned to the various classes of aromatic amino acid residues in the two proteins. In addition to signals which are attributable to the missing aromatic residues (Phe-136 and Tyr-206) the difference spectrum (CRP minus alpha CRP) shows a significant perturbation of a tryptophanyl contribution centred at 296 nm. From the available X-ray structure of the cAMP-CRP complex we are led to conclude that a conformational reorganisation takes place in the alpha CRP. A very large negative maximum is observed at 255 nm when cAMP binds to CRP and to alpha CRP. The maximum effect is observed in both cases at a ratio of one ligand bound per protomer. In the 280-300 nm wavelength range a smaller but significant perturbation affects specifically the spectra and reveals different cAMP-induced conformational changes in the two proteins. We propose that the major (255 nm) contribution to the perturbation spectrum of bound cAMP, and the qualitatively similar signal for cGMP, reflects an immobilisation of the sugar and adenine moieties of the bound ligand in an almost anti-conformation for both CRP and alpha CRP.

Bacterial Proteins↗
PubMed2002

Regulation of cyclic AMP in rat pulmonary microvascular endothelial cells by rolipram-sensitive cyclic AMP phosphodiesterase (PDE4).

We report here studies on the regulation of the metabolism of adenosine 3',5'-monophosphate (cAMP) in established and primary cultures of rat pulmonary microvascular endothelial cells (RPMVEC). Inhibition by rolipram, a selective inhibitor of cAMP phosphodiesterase (PDE) of the PDE4 gene family, was required to achieve maximal cAMP accumulation induced by direct or receptor-mediated adenylate cyclase activation when measured by [3H]-adenine prelabeling. Rolipram increased cAMP accumulation more effectively than did forskolin, isoproterenol, or adenosine derivatives alone, although extensive synergy was seen with combined agents. High-affinity PDE4 inhibitors, but not low-affinity or non-selective inhibitors, were effective inducers of cAMP accumulation in intact cells. The maximum effects (i.e. intrinsic activities) of these agents in the intact cell did not correlate with their in vitro PDE4 inhibitory affinities. RPMVEC were shown to express almost exclusively the PDE4 gene family isoforms A6 and B3. Guanosine 3',5'-monophosphate hydrolysis, observed in other types of endothelial cells was not found in early or late passage RPMVEC. Reverse transcription-polymerase chain reaction identification of mRNAse supported these conclusions with the exception that PDE2 and PDE4D mRNA isoform transcripts were present. These studies also support the conclusion that the mechanism of rolipram reversal of rat lung ischemia-reperfusion-induced permeability involves PDE4 inhibition in the microvascular endothelial cells of the lung.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1998

Cyclic AMP-specific phosphodiesterase inhibitor rolipram and RO-20-1724 promoted apoptosis in HL60 promyelocytic leukemic cells via cyclic AMP-independent mechanism.

Phosphodiesterases (PDEs) are responsible for the hydrolysis of cAMP and cGMP which act as intracellular second messengers in a variety of cellular functions. In this paper we report that PDE3 and PDE4 were two dominant classes of PDEs expressed in HL60 cells. The influence of specific PDE inhibitors on apoptosis in HL60 cells was studied. The non-specific inhibitor IBMX and PDE3 specific inhibitors (milrinone and trequinsin) did not promote apoptosis. They inhibited apoptosis induced by paclitaxel or thapsigargin. However, PDE4 specific inhibitors (rolipram and RO-20-1724) promoted apoptosis within 5 h. In HL60 cells, other cAMP-eliciting reagents (8-bromo-cAMP, Sp-cAMP and forskolin) also inhibited apoptosis, while cell-permeable cGMP analogs did not affect apoptosis. Therefore, IBMX and PDE3 specific inhibitors may prevent HL60 cells from apoptosis by increasing intracellular cAMP. However, apoptosis induced by PDE4 specific inhibitors is not likely due to increased cAMP level. These results suggest that rolipram and RO-20-1724 promoted apoptosis in HL60 cells through cAMP-independent mechanism.

1-Methyl-3-isobutylxanthine↗
PubMed2003

Occupancy of the catalytic site of the PDE4A4 cyclic AMP phosphodiesterase by rolipram triggers the dynamic redistribution of this specific isoform in living cells through a cyclic AMP independent process.

In cells transfected to express wild-type PDE4A4 cAMP phosphodiesterase (PDE), the PDE4 selective inhibitor rolipram caused PDE4A4 to relocalise so as to form accretion foci. This process was followed in detail in living cells using a PDE4A4 chimera formed with Green Fluorescent Protein (GFP). The same pattern of behaviour was also seen in chimeras of PDE4A4 formed with various proteins and peptides, including LimK, RhoC, FRB and the V5-6His tag. Maximal PDE4A4 foci formation, occurred over a period of about 10 h, was dose-dependent on rolipram and was reversible upon washout of rolipram. Inhibition of protein synthesis, using cycloheximide, but not PKA activity with H89, inhibited foci generation. Foci formation was elicited by Ro20-1724 and RS25344 but not by either Ariflo or RP73401, showing that not all PDE4 selective inhibitors had this effect. Ariflo and RP73401 dose-dependently antagonised rolipram-induced foci formation and dispersed rolipram pre-formed foci as did the adenylyl cyclase activator, forskolin. Foci formation showed specificity for PDE4A4 and its rodent homologue, PDE4A5, as it was not triggered in living cells expressing the PDE4B2, PDE4C2, PDE4D3 and PDE4D5 isoforms as GFP chimeras. Altered foci formation was seen in the Deltab-LR2-PDE4A4 construct, which deleted a region within LRZ, showing that appropriate linkage between the N-terminal portion of PDE4A4 and the catalytic unit of PDE4A4 was needed for foci formation. Certain single point mutations within the PDE4A4 catalytic site (His505Asn, His506Asn and Val475Asp) were shown to ablate foci formation but still allow rolipram inhibition of PDE4A4 catalytic activity. We suggest that the binding of certain, but not all, PDE4 selective inhibitors to PDE4A4 induces a conformational change in this isoform by 'inside-out' signalling that causes it to redistribute in the cell. Displacing foci-forming inhibitors with either cAMP or inhibitors that do not form foci can antagonise this effect. Specificity of this effect for PDE4A4 and its homologue PDE4A5 suggests that interplay between the catalytic site and the unique N-terminal region of these isoforms is required. Thus, certain PDE4 selective inhibitors may exert effects on PDE4A4 that extend beyond simple catalytic inhibition. These require protein synthesis and may lead to redistribution of PDE4A4 and any associated proteins. Foci formation of PDE4A4 may be of use in probing for conformational changes in this isoform and for sub-categorising PDE4 selective inhibitors.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed2003

Steady-state and time-resolved fluorescence studies of conformational changes induced by cyclic AMP and DNA binding to cyclic AMP receptor protein from Escherichia coli.

cAMP receptor protein (CRP), allosterically activated by cAMP, regulates the expression of several genes in Escherichia coli. As binding of cAMP leads to undefined conformational changes in CRP, we performed a steady-state and time-resolved fluorescence study to show how the binding of the ligand influences the structure and dynamics of the protein. We used CRP mutants containing a single tryptophan residue at position 85 or 13, and fluorescently labeled with 1,5-I-AEDANS attached to Cys178. Binding of cAMP in the CRP-(cAMP)2 complex leads to changes in the Trp13 microenvironment, whereas its binding in the CRP-(cAMP)4 complex alters the surroundings of Trp85. Time-resolved anisotropy measurements indicated that cAMP binding in the CRP-(cAMP)2 complex led to a substantial increase in the rotational mobility of the Trp13 residue. Measurement of fluorescence energy transfer (FRET) between labeled Cys178 and Trp85 showed that the binding of cAMP in the CRP-(cAMP)2 complex caused a substantial increase in FRET efficiency. This indicates a decrease in the distance between the two domains of the protein from 26.6 A in apo-CRP to 18.7 A in the CRP-(cAMP)2 complex. The binding of cAMP in the CRP-(cAMP)4 complex resulted in only a very small increase in FRET efficiency. The average distance between the two domains in CRP-DNA complexes, possessing lac, gal or ICAP sequences, shows an increase, as evidenced by the increase in the average distance between Cys178 and Trp85 to approximately 20 A. The spectral changes observed provide new structural information about the cAMP-induced allosteric activation of the protein.

Allosteric Regulation↗
PubMed1996

Arginine 210 is not a critical residue for the allosteric interactions mediated by binding of cyclic AMP to site A of regulatory (RIalpha) subunit of cyclic AMP-dependent protein kinase.

The guanidinium groups of conserved arginines in the two intrachain cAMP-binding sites of regulatory (R) subunit of cAMP-dependent protein kinase have been implicated in the allosteric interactions by which cAMP binding leads to kinase activation. We have investigated the functional role of Arg-210, the conserved arginine in site A of murine type Ialpha R subunit, by analyzing the effects of nine different substitutions at this residue on cAMP binding and allosteric properties of bacterially expressed RIalpha subunits. All substitutions reduced the cAMP binding affinity of site A, but the magnitude of reduction varied from several hundredfold to 10(6)-fold. The differential effects of the different substitutions could not easily be rationalized by interactions with cAMP and might, in part, reflect interactions with other residues in the unoccupied cAMP-binding pocket. None of the Arg-210 substitutions appeared to disrupt the allosteric interaction by which occupation of site A slows dissociation of cAMP from site B, although the effect was difficult to elicit in full with mutations that had strong effects on cAMP binding. The two weakest substitutions, Arg-210 --> Ile and Arg-210 --> Thr, could be shown to have essentially no effect on the allosteric interaction by which occupation of site A reduces the affinity of R subunit for the catalytic subunit. The weaker mutations had a smaller effect on kinase activation by the suboptimal activator Rp-adenosine cyclic 3',5'-phosphorothioate than by cAMP, suggesting that the analog largely bypasses interactions with the guanidinium group of Arg-210.

Allosteric Regulation↗
PubMed2002

Differential signaling of cyclic AMP: opposing effects of exchange protein directly activated by cyclic AMP and cAMP-dependent protein kinase on protein kinase B activation.

The recent discovery of Epac, a novel cAMP receptor protein, opens up a new dimension in studying cAMP-mediated cell signaling. It is conceivable that many of the cAMP functions previously attributed to cAMP-dependent protein kinase (PKA) are in fact also Epac-dependent. The finding of an additional intracellular cAMP receptor provides an opportunity to further dissect the divergent roles that cAMP exerts in different cell types. In this study, we probed cross-talk between cAMP signaling and the phosphatidylinositol 3-kinase/PKB pathways. Specifically, we examined the modulatory effects of cAMP on PKB activity by monitoring the specific roles that Epac and PKA play individually in regulating PKB activity. Our study suggests a complex regulatory scheme in which Epac and PKA mediate the opposing effects of cAMP on PKB regulation. Activation of Epac leads to a phosphatidylinositol 3-kinase-dependent PKB activation, while stimulation of PKA inhibits PKB activity. Furthermore, activation of PKB by Epac requires the proper subcellular targeting of Epac. The opposing effects of Epac and PKA on PKB activation provide a potential mechanism for the cell type-specific differential effects of cAMP. It is proposed that the net outcome of cAMP signaling is dependent upon the dynamic abundance and distribution of intracellular Epac and PKA.

Base Sequence↗
PubMed1978

A new extragenic suppressor of cya mutation. Mutant cyclic AMP receptor protein with an increased affinity for cyclic AMP.

A strain bearing an extragenic suppressor of cya mutation was isolated as a second-site revertant of an adenylate cyclase deficient strain. The mutant was unable to synthesize cAMP but showed normal fermentation profiles and growth properties on a variety of carbon sources. The site of reversion was mapped in, or near, the structural gene for the cAMP receptor protein. Structural alteration of the protein was directly demonstrated by the following biochemical observations: (i) A 10-fold decrease in the dissociation constant for cAMP, (ii) an acidic shift in the isoelectric point, and (iii) the altered binding properties to lambdah80dlac ps DNA.

Carbohydrate Metabolism↗
PubMed2005

Role of basic region leucine zipper transcription factors cyclic AMP response element binding protein (CREB), CREB2, activating transcription factor 2 and CAAT/enhancer binding protein alpha in cyclic AMP response element-mediated transcription.

The transcription factor cAMP response element binding protein (CREB), a member of the basic region leucine zipper (bZIP) family of proteins, is the major cAMP response element (CRE) binding. Other bZIP proteins, including CREB2, activating transcription factor 2 (ATF2), or CAAT/enhancer binding protein (C/EBP) have been reported to transactivate CRE-containing genes or to interfere with transactivation by CREB. We have designed a simple transactivation assay using expression of either a constitutively active CREB mutant or a nuclear targeted mutant of the catalytic subunit of cAMP-dependent protein kinase. In both cases, a striking stimulation of transcription of CRE-containing reporter genes was observed in noradrenergic locus coeruleus-like CATH.a cells. In addition, a constitutively active mutant of ATF2 specifically transactivated a secretogranin II promoter/luciferase reporter gene, but had no effect on the tyrosine hydroxylase promoter. In contrast, CREB2 and C/EBPalpha did not transactivate CRE-containing reporter genes, indicating that these bZIP proteins target distinct genetic elements. Experiments involving dominant-negative bZIP mutants revealed that CREB does not heterodimerize with CREB2, ATF2, c-Jun or C/EBP. Rather, CREB and ATF2 compete for binding to the CRE, and are independently able to up-regulate transcription of genes containing CRE motifs in their regulatory regions.

Activating Transcription Factor 2↗
PubMed1997

Both the cyclic AMP response element and the activator protein 2 binding site mediate basal and cyclic AMP-induced transcription from the dominant promoter of the rat alpha 1B-adrenergic receptor gene in DDT1MF-2 cells.

cAMP markedly increases alpha 1B adrenergic receptor (alpha 1B-AR) expression in FRTL-5 and PC C13 rat thyroid cells, DDT1MF-2 smooth muscle cells, primary rat hepatocytes, and K9 rat liver cells. Here, we used DDT1MF-2 cells to evaluate further the mechanisms by which cAMP stimulates alpha 1B-AR expression. Receptor binding assays, Northern blotting, and nuclear run-on analyses demonstrated that forskolin (1 microM) in the presence of isobutylmethylxanthine (0.25 mM) increased alpha 1B-AR numbers, mRNA level, and gene transcription rate by 2.3 +/- 0.2-, 2.5 +/- 0.3-, and 3.5 +/- 0.2-fold over control, respectively. Dibutyryl cAMP (1 mM) plus isobutylmethylxanthine (0.25 mM) also enhanced alpha 1B-AR density by 2.7 +/- 0.1-fold over control. Further experiments demonstrated that the induction of alpha 1B-AR by forskolin requires new protein synthesis and is protein kinase A dependent. In DDT1MF-2 cells transfected with alpha 1B-AR gene P2 promoter/CAT constructs, both forskolin and dibutyryl cAMP significantly increased P2 promoter activity. The P2 promoter region of the rat alpha 1B-AR gene (-813 to -432) contains a cAMP response element (CRE) (-444 to -437) and an AP2 binding site (-647 to -638). Mutations in either one of these elements alone led to a decrease in both basal and cAMP-induced P2 promoter activity. Mutations in both elements caused a further inhibition of basal transcription and a complete block of cAMP-induced P2 promoter activity. Direct binding of purified activator protein 2 (AP2) to the AP2 element in the P2 promoter was reported previously. Gel mobility shift and super-shift assays using liver nuclear extracts from either rat liver or DDT1MF-2 cells demonstrated that the CRE in the alpha 1B-AR gene bound CRE binding protein. These data indicate that both the CRE and the AP2 element in the P2 promoter contribute to basal as well as cAMP-induced transcription of the alpha 1B-AR gene in DDT1MF-2 cells.

Animals↗