Involvement of the CDC25 gene product in the signal transmission pathway of the glucose-induced RAS-mediated cAMP signal in the yeast Saccharomyces cerevisiae.

Addition of glucose or related fermentable sugars to derepressed cells of the yeast Saccharomyces cerevisiae triggers a RAS-protein-mediated cAMP signal, which induces a protein phosphorylation cascade. Yeast strains without a functional CDC25 gene were deficient in basal cAMP synthesis and in the glucose-induced cAMP signal. Addition of dinitrophenol, which in wild-type strains strongly stimulates in vivo cAMP synthesis by lowering intracellular pH, did not enhance the cAMP level. cdc25 disruption mutants, in which the basal cAMP level was restored by the RAS2val19 oncogene or by disruption of the gene (PDE2) coding for the high-affinity phosphodiesterase, were still deficient in the glucose- and acidification-induced cAMP responses. These results indicate that the CDC25 gene product is required not only for basal cAMP synthesis in yeast but also for specific activation of cAMP synthesis by the signal transmission pathway leading from glucose to adenyl cyclase. They also show that intracellular acidification stimulates the pathway at or upstream of the CDC25 protein. When shifted to the restrictive temperature, cells with the temperature sensitive cdc25-5 mutation lost their cAMP content within a few minutes. After prolonged incubation at the restrictive temperature, cells with this mutation, and also those with the temperature sensitive cdc25-1 mutation, arrested at the 'start' point (in G1) of the cell cycle, and subsequently accumulated in the resting state G0. In contrast with cdc25-5 cells, however, the cAMP level did not decrease and normal glucose- and acidification-induced cAMP responses were observed when cdc25-1 cells were shifted to the restrictive temperature.(ABSTRACT TRUNCATED AT 250 WORDS)     

Crystal structure of the GAF-B domain from human phosphodiesterase 10A complexed with its ligand, cAMP

Cyclic nucleotide phosphodiesterases (PDEs) catalyze the degradation of the cyclic nucleotides cAMP and cGMP, which are important second messengers. Five of the 11 mammalian PDE families have tandem GAF domains at their N termini. PDE10A may be the only mammalian PDE for which cAMP is the GAF domain ligand, and it may be allosterically stimulated by cAMP. PDE10A is highly expressed in striatal medium spiny neurons. Here we report the crystal structure of the C-terminal GAF domain (GAF-B) of human PDE10A complexed with cAMP at 2.1-angstroms resolution. The conformation of the PDE10A GAF-B domain monomer closely resembles those of the GAF domains of PDE2A and the cyanobacterium Anabaena cyaB2 adenylyl cyclase, except for the helical bundle consisting of alpha1, alpha2, and alpha5. The PDE10A GAF-B domain forms a dimer in the crystal and in solution. The dimerization is mainly mediated by hydrophobic interactions between the helical bundles in a parallel arrangement, with a large buried surface area. In the PDE10A GAF-B domain, cAMP tightly binds to a cNMP-binding pocket. The residues in the alpha3 and alpha4 helices, the beta6 strand, the loop between 3(10) and alpha4, and the loop between alpha4 and beta5 are involved in the recognition of the phosphate and ribose moieties. This recognition mode is similar to those of the GAF domains of PDE2A and cyaB2. In contrast, the adenine base is specifically recognized by the PDE10A GAF-B domain in a unique manner, through residues in the beta1 and beta2 strands.     

The large N-terminal domain of Cdc25 protein of the yeast Saccharomyces cerevisiae is required for glucose-induced Ras2 activation

The Saccharomyces cerevisiae CDC25 gene encodes a guanine nucleotide exchange factor for Ras proteins whose catalytic domain is highly homologous to Ras-guanine nucleotide exchange factors from higher eukaryotes. In this study, glucose-induced Ras activation and cAMP response were investigated in mutants lacking the N-terminal domain of Cdc25 or where the entire CDC25 coding sequence was substituted by an expression cassette for a mammalian guanine nucleotide exchange factor catalytic domain. Our results suggest that an unregulated, low Ras guanine nucleotide exchange factor activity allows a normal glucose-induced cAMP signal that appears to be mediated mainly by the Gpr1/Gpa2 system, but it was not enough to sustain the glucose-induced increase of Ras2-GTP normally observed in a wild-type strain.     

The overexpression of the 3' terminal region of the CDC25 gene of Saccharomyces cerevisiae causes growth inhibition and alteration of purine nucleotides pools.

The CDC25 gene is transcribed at a very low level in S. cerevisiae cells. We have studied the effects of an overexpression of this regulatory gene by cloning either the whole CDC25 open reading frame (pIND25-2 plasmid) or its 3' terminal portion (pIND25-1 plasmid) under the control of the inducible strong GAL promoter. The strain transformed with pIND25-2 produced high levels of CDC25 specific mRNA, induced by galactose. This strain does not show any apparent alteration of growth, both in glucose and in galactose. Instead the yeast cells transformed with pIND25-1, that overexpress the 3' terminal part of CDC25 gene, grow very slowly in galactose medium, while they grow normally in glucose medium. The nucleotides were extracted from transformed cells, separated by HPLC and quantitated. The ATP/ADP and GTP/GDP ratios were almost identical in control and in pIND25-2 transformed strains growing in glucose and in galactose, while the strain that overexpresses the 3' terminal portion of CDC25 gene showed a decrease of ATP/ADP ratio and a partial depletion of the GTP pool. The disruption of RAS genes was only partially able to 'cure' this phenotype. A ras2-ts1, ras1::URA3 strain, transformed with pIND25-1 plasmid, was able to grow in galactose at 36 degrees C. These results suggest that the carboxy-terminal domain of the CDC25 protein could stimulate an highly unregulated GTPase activity in yeast cells by interacting not only with RAS gene products but also with some other yeast G-proteins.     

Identification of the cofilin-binding sites in the large cytoplasmic domain of Na,K-ATPase

Na,K-ATPase, an alpha, beta heterodimer, is found in the plasma membrane of all animal cells. The alpha chain is believed to have 10 transmembrane regions and a large cytoplasmic domain between the 4th and 5th transmembrane regions (H4-H5). In our previous report, the large (3rd) cytoplasmic domains of the alpha1 and alpha2 isoform were found to interact with cofilin, an actin-modulating protein, by the yeast two-hybrid system. Here we show that cofilin interacts only with the 3rd cytoplasmic domain of the alpha2 subunit but not with the 2nd, 4th, and 5th cytoplasmic domains or the cytoplasmic region of the beta subunit of Na,K-ATPase. We also demonstrate that cofilin interacts with the large cytoplasmic domains of the alpha1, alpha2 and alpha3 isoforms of Na,K-ATPase, but not with those of glucose transporter 1, glucose transporter 4, cystic fibrosis transmembrane conductance regulator and plasma membrane Ca-ATPase. We introduced 10 mutations into the 3rd cytoplasmic domain of Na,K-ATPase to identify the binding sites with cofilin. Eight of these mutants were single amino acid substitutions (R417Q, K470Q, K654G, D672A, K691A, R700G, R700A and D710G) and two were double mutant (K654GR700G and K719AK720A). Analysis of the activity of the reporter gene of these mutants shows that residues D672 and R700 of the 3rd cytoplasmic domain of Na,K-ATPase are involved in the interaction with cofilin.     

Age-related changes in the control of hepatic cyclic AMP levels by alpha 1- and beta 2-adrenergic receptors in male rats

Hepatocytes from juvenile male rats (80-110 g) showed a 12-fold elevation of cAMP in response to epinephrine, which was mediated by beta 2-adrenergic receptors. In these cells, either alpha 1- or beta 2-adrenergic stimulation alone activated phosphorylase and glucose release although the alpha 1-phosphorylase response was 10-fold more sensitive to epinephrine and resulted in more rapid (by 10-20 s) activation of the enzyme. This suggests that the beta 2-adrenergic response is functionally unimportant for glycogenolysis, even in juvenile rats. beta 2-Adrenergic stimulation did, however, produce an increase in the rate of gluconeogenesis from [U-14C] lactate in these cells. Aging in the male rat was associated with attenuation of the beta 2-adrenergic cAMP response coupled with the emergence of an alpha 1-receptor-mediated accumulation of cAMP. The order of potency displayed by the alpha 1-adrenergic/cAMP system to adrenergic agonists and antagonists was identical with that of the alpha 1-adrenergic/Ca2+ system. These data suggest that, in maturity, hepatic alpha 1-receptors become linked to 2 separate transduction mechanisms, namely Ca2+ mobilization and cAMP generation. Calcium depletion of hepatocytes from adult, but not juvenile, male rats increased the alpha 1-component of the cAMP response to epinephrine, but under these conditions, alpha 1-activation of phosphorylase occurred more slowly than in calcium-replete cells. Blockade of alpha 2-adrenergic receptors did not significantly modify catecholamine effects on hepatocyte cAMP or phosphorylase a levels in male rats at any age studied, suggesting a lack of functional significance for these receptors in the regulation of glycogenolysis.     

Independent inhibition of DNA synthesis by protein kinase C, cyclic AMP and interferon alpha/beta in rabbit aortic smooth muscle cells

In quiescent cultures of rabbit aortic smooth muscle cells, whole blood serum-induced DNA synthesis was inhibited markedly by protein kinase C-activating 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and phorbol-12, 13-dibutyrate (PDBu), cyclic AMP-derivatives, such as dibutyryl cyclic AMP (Bt2cAMP) and 8-bromo-cyclic AMP, and interferon alpha/beta. Neither TPA nor interferon alpha/beta elevated the cellular cyclic AMP level. Neither Bt2cAMP nor interferon alpha/beta induced the phospholipase C-mediated hydrolysis of phosphoinositides. The down-regulation of protein kinase C by prolonged treatment with PDBu abolished the antiproliferative action of TPA but did not affect that of Bt2cAMP or interferon alpha/beta. TPA and Bt2cAMP inhibited the serum-induced DNA synthesis when added within 12 h after the addition of the serum, while interferon alpha/beta was active only when added within 6 h. These results suggest that there are at least three independent signaling systems, protein kinase C- and cyclic AMP-mediated systems and an unidentified system for interferon alpha/beta, which are involved in the antiproliferative mechanisms in rabbit aortic smooth muscle cells.     

The heterotrimeric G-protein GanB(alpha)-SfaD(beta)-GpgA(gamma) is a carbon source sensor involved in early cAMP-dependent germination in Aspergillus nidulans

The role of heterotrimeric G-proteins in cAMP-dependent germination of conidia was investigated in the filamentous ascomycete Aspergillus nidulans. We demonstrate that the G alpha-subunit GanB mediates a rapid and transient activation of cAMP synthesis in response to glucose during the early period of germination. Moreover, deletion of individual G-protein subunits resulted in defective trehalose mobilization and altered germination kinetics, indicating that GanB(alpha)-SfaD(beta)-GpgA(gamma) constitutes a functional heterotrimer and controls cAMP/PKA signaling in response to glucose as well as conidial germination. Further genetic analyses suggest that GanB plays a primary role in cAMP/PKA signaling, whereas the SfaD-GpgA (G betagamma) heterodimer is crucial for proper activation of GanB signaling sensitized by glucose. In addition, the RGS protein RgsA is also involved in regulation of the cAMP/PKA pathway and germination via attenuation of GanB signaling. Genetic epistatic analyses led us to conclude that all controls exerted by GanB(alpha)-SfaD(beta)-GpgA(gamma) on conidial germination are mediated through the cAMP/PKA pathway. Furthermore, GanB may function in sensing various carbon sources and subsequent activation of downstream signaling for germination.     

Domains of the Saccharomyces cerevisiae CDC25 gene controlling mitosis and meiosis

Summary The cell division cycle gene CDC25 was replaced by various disrupted and deleted mutant copies. Mutants disrupted at a central position of the gene, or lacking 532 residues within the amono-terminal half of the gene product grow normally in glucose, but not in acetate media, and they fail to sporulate as homozygous diploids. Disruptions or deletions within the carboxy-terminal half are lethal, except for the deletion of the 38 carboxy-terminal residues, which are required for sporulation but not for growth in glucose or acetate media. It is concluded that distinct domains of the CDC25 gene product are involved in the control of mitosis and/or meiosis.     

Mice with a deletion in the gene for CCAAT/enhancer-binding protein beta have an attenuated response to cAMP and impaired carbohydrate metabolism

Fifty percent of the mice homozygous for a deletion in the gene for CCAAT/enhancer-binding protein beta (C/EBP beta-/- mice; B phenotype) die within 1 to 2 h after birth of hypoglycemia. They do not mobilize their hepatic glycogen or induce the cytosolic form of phosphoenolpyruvate carboxykinase (PEPCK). Administration of cAMP resulted in mobilization of glycogen, induction of PEPCK mRNA, and a normal blood glucose; these mice survived beyond 2 h postpartum. Adult C/EBP beta-/- mice (A phenotype) also had difficulty in maintaining blood glucose levels during starvation. Fasting these mice for 16 or 30 h resulted in lower levels of hepatic PEPCK mRNA, blood glucose, beta-hydroxybutyrate, blood urea nitrogen, and gluconeogenesis when compared with control mice. The concentration of hepatic cAMP in these mice was 50% of controls, but injection of theophylline, together with glucagon, resulted in a normal cAMP levels. Agonists (glucagon, epinephrine, and isoproterenol) and other effectors of activation of adenylyl cyclase were the same in liver membranes isolated from C/EBP beta-/- mice and littermates. The hepatic activity of cAMP-dependent protein kinase was 80% of wild type mice. There was a 79% increase in the concentration of RI alpha and 27% increase in RII alpha in the particulate fraction of the livers of C/EBP beta-/- mice relative to wild type mice, with no change in the catalytic subunit (C alpha). Thus, a 45% increase in hepatic cAMP (relative to the wild type) would be required in C/EBP beta-/- mice to activate protein kinase A by 50%. In addition, the total activity of phosphodiesterase in the livers of C/EBP beta-/- mice, as well as the concentration of mRNA for phosphodiesterase 3A (PDE3A) and PDE3B was approximately 25% higher than in control animals, suggesting accelerated degradation of cAMP. C/EBP beta influences the regulation of carbohydrate metabolism by altering the level of hepatic cAMP and the activity of protein kinase A.     

The guanine nucleotide exchange factor CNrasGEF activates ras in response to cAMP and cGMP

Small GTPase proteins such as Ras are key regulators of cellular proliferation and are activated by guanine nucleotide exchange/releasing factors (GEFs/GRFs). Three classes of Ras GRFs have been identified to date, represented by Sos1/2, Ras-GRF1/2 and Ras-GRP. Here, we describe a novel candidate Ras activator, cyclic nucleotide rasGEF (CNrasGEF), which contains CDC25, Ras exchange motif (REM), Ras-association (RA), PDZ and cNMP (cAMP/cGMP) binding (cNMP-BD) domains, two PY motifs and a carboxy-terminal SxV sequence. CNrasGEF can activate Ras in vitro, and it binds cAMP directly via its cNMP-BD. In cells, CNrasGEF activates Ras in response to elevation of intracellular cAMP or cGMP, or treatment with their analogues 8-Br-cAMP or 8-Br-cGMP, independently of protein kinases A and G (PKA and PKG). This activation is prevented in CNrasGEF lacking its CDC25 domain or cNMP-BD. CNrasGEF can also activate the small GTPase Rap1 in cells, but this activation is constitutive and independent of cAMP. CNrasGEF is expressed mainly in the brain and is localized at the plasma membrane, a localization dependent on the presence of intact PDZ domain but not the SxV sequence. These results suggest that CNrasGEF may directly connect cAMP-generating pathways or cGMP-generating pathways to Ras.     

Gating of heteromeric retinal rod channels by cyclic AMP: role of the C-terminal and pore domains

Cyclic nucleotide-gated channels are tetramers composed of homologous alpha and beta subunits. C-terminal truncation mutants of the alpha and beta subunits of the retinal rod channel were expressed in Xenopus oocytes, and analyzed for cGMP- and cAMP-induced currents (single-channel records and macroscopic currents). When the alpha subunit truncated downstream of the cGMP-binding site (alpha D608stop) is co-injected with truncated beta subunits, the heteromeric channels present a drastic increase of cAMP sensitivity. A partial effect is observed with heteromeric alpha R656stop-containing channels, while alpha K665stop-containing channels behave like alpha wt/beta wt. The three truncated alpha subunits have wild-type activity when expressed alone. Heteromeric channels composed of alpha wt or truncated alpha subunits and chimeric beta subunits containing the pore domain of the alpha subunit have the same cAMP sensitivity as alpha-only channels. The results disclose the key role of two domains distinct from the nucleotide binding site in the gating of heteromeric channels by cAMP: the pore of the beta subunit, which has an activating effect, and a conserved domain situated downstream of the cGMP-binding site in the alpha subunit (I609-K665), which inhibits this effect.     

Pancreatic insulin content regulation by the estrogen receptor ER alpha

The function of pancreatic beta-cells is the synthesis and release of insulin, the main hormone involved in blood glucose homeostasis. Estrogen receptors, ER alpha and ER beta, are important molecules involved in glucose metabolism, yet their role in pancreatic beta-cell physiology is still greatly unknown. In this report we show that both ER alpha and ER beta are present in pancreatic beta-cells. Long term exposure to physiological concentrations of 17beta-estradiol (E2) increased beta-cell insulin content, insulin gene expression and insulin release, yet pancreatic beta-cell mass was unaltered. The up-regulation of pancreatic beta-cell insulin content was imitated by environmentally relevant doses of the widespread endocrine disruptor Bisphenol-A (BPA). The use of ER alpha and ER beta agonists as well as ER alphaKO and ER betaKO mice suggests that the estrogen receptor involved is ER alpha. The up-regulation of pancreatic insulin content by ER alpha activation involves ERK1/2. These data may be important to explain the actions of E2 and environmental estrogens in endocrine pancreatic function and blood glucose homeostasis.