Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.4.1 (phosphodiesterase)
18,767 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The mitosis inhibitory pentapeptide, pGlu-Glu-Asp-Ser-GlyOH (EPP), which was isolated from mouse epidermis extracts, belongs to a group of growth inhibitory peptides that all have pyroglutamyl at the N-terminal end. Earlier experiments with crude or partially purified skin extracts have shown that the inhibitory effect could be enhanced by beta-receptor agonists and by dibutyryl cAMP, and that beta-receptor blockade could neutralise it. We now show that treatment with the beta receptor blocker propranolol before or after EPP treatment of hairless mice significantly modifies the effect of EPP on mouse epidermal cell proliferation, as estimated by using a metaphase-arrest technique (Colcemid) to estimate the G2-M cell flux. The interaction between propranolol and EPP is complex; only the EPP-induced inhibition of the G2-M cell flux was modified by beta-receptor blockade, while the late (18-21 h) inhibition of the mitotic rate was unaltered. Propranolol alone was followed by a dose-related and transient increase in the epidermal mitotic rate. The phosphodiesterase inhibitor caffeine had no effect on its own on epidermal cell proliferation but counter-acted the late (18-21 h) EPP-induced inhibition.
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PMID:Beta-receptor blockade by propranolol modifies the effect of the inhibitory, endogenous epidermal pentapeptide on epidermal cell flux at the G2-M transition but not at the G1-S transition. 819 66

The amino acid sequences of all known cGMP-binding phosphodiesterases (PDEs) contain internally homologous repeats (a and b) that are 80-90 residues in length and are arranged in tandem within the putative cGMP-binding domains. In the bovine lung cGMP-binding, cGMP-specific PDE (cGB-PDE or PDE5A), these repeats span residues 228-311 (a) and 410-500 (b). An aspartic acid (residue 289 or 478) that is invariant in repeats a and b of all known cGMP-binding PDEs was changed to alanine by site-directed mutagenesis of cGB-PDE, and wild type (WT) and mutant cGB-PDEs were expressed in COS-7 cells. Purified bovine lung cGB-PDE (native) and WT cGB-PDE displayed identical cGMP-binding kinetics, with approximately 1.8 microM cGMP required for half-maximal saturation. The D289A mutant showed decreased affinity for cGMP (Kd > 10 microM) and the D478A mutant showed increased affinity for cGMP (Kd approximately 0.5 microM) as compared to WT and native cGB-PDE. WT and native cGB-PDE displayed an identical curvilinear profile of cGMP dissociation which was consistent with the presence of distinct slowly dissociating (koff = 0.26 h-1) and rapidly dissociating (koff = 1.00 h-1) sites of cGMP binding. In contrast, the D289A mutant displayed a single koff = 1.24 h-1, which was similar to the calculated koff for the fast site of WT and native cGB-PDE, and the D478A mutant displayed a single koff = 0.29 h-1, which was similar to that calculated for the slow site of WT and native cGB-PDE. These results were consistent with the loss of a slow cGMP-binding site in repeat a of the D289A mutant cGB-PDE, and the loss of a fast site in repeat b of the D478A mutant, suggesting that cGB-PDE possesses two distinct cGMP-binding sites located at repeats a and b, with the invariant aspartic acid being crucial for interaction with cGMP at each site.
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PMID:An essential aspartic acid at each of two allosteric cGMP-binding sites of a cGMP-specific phosphodiesterase. 853 May 5

Stimulation of rat adipocytes with insulin and isoproterenol results in serine phosphorylation and activation of the adipocyte cGMP-inhibited phosphodiesterase (cGI PDE), events believed to be important in the antilipolytic action of insulin (Degerman, E., Smith, C.J., Tornqvist, H., Vasta, V., Manganiello, V.C., and Belfrage, P. (1990) Proc. Natl. Acad. Sci. U.S.A. 87,533-537). Here we demonstrate, by two-dimensional phosphopeptide mapping, that the major phosphopeptide generated by trypsin, or trypsin followed by Asp-N protease digestion of [32P]cGI PDE phosphorylated in adipocytes in response to isoproterenol and/or insulin, in each case co-migrates with the phosphopeptide released by the same treatment of M297FRRPS(P)LPCISREQ310. This peptide was synthesized based on the deduced sequence of the cloned rat adipocyte cGI PDE and phosphorylated by cAMP-dependent protein kinase (protein kinase A). Radiosequencing of authentic and synthetic tryptic 32P-peptides showed that a single site in cGI PDE (Ser302) was phosphorylated in adipocytes incubated with isoproterenol and/or insulin. The more than additive phosphorylation and activation of cGI PDE in response to the two hormones found in this report and previously (Smith, C.J., Vasta, V., Degerman, E., Belfrage, P., and Manganiello, V.C. (1991) J. Biol. Chem. 266, 13385-13390) is proposed to reflect cross-talk between their respective signal transduction pathways at the level of the cGI PDE serine protein kinase or upstream regulatory component(s).
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PMID:Identification of the site in the cGMP-inhibited phosphodiesterase phosphorylated in adipocytes in response to insulin and isoproterenol. 862 20

Comparisons of the tertiary structures of the GDP-bound and guanosine 5'-O-(thiotriphosphate) (GTPgammaS)-bound forms of the alpha subunit of transducin (alphaT) indicate that there are three regions that undergo changes in conformation upon alphaT activation. Two of these regions, Switch I and Switch II, were originally identified in Ras, while Switch III appears to be unique to trimeric GTP-binding proteins (G proteins). We find that replacement of the Switch III region (aspartic acid 227 through asparagine 237) with a single alanine residue yields an alphaT subunit that fully binds and hydrolyzes GTP but no longer stimulates the activity of the cyclic GMP phosphodiesterase (PDE), the physiological target for transducin. We also show that changing glutamic acid 232 of alphaT to a leucine (E232L) had no effect on rhodopsin-stimulated GTP-GDP exchange nor on the GTP hydrolytic activity of alphaT. However, the GTPgammaS-bound form of the alphaTE232L mutant was unable to stimulate the activity of the cyclic GMP PDE. The lack of stimulation was not due to an inability of the alphaTE232L mutant to bind to the target. Taken together, these results indicate that glutamic acid 232 mediates a conformational coupling between Switch II and Switch III, which is essential for converting GTP-dependent G protein-target interactions into a stimulation of target/effector activity.
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PMID:Communication between switch II and switch III of the transducin alpha subunit is essential for target activation. 926 92

The regA and rdeA gene products of Dictyostelium are involved in the regulation of cAMP signaling. The response regulator, RegA, is composed of an N-terminal receiver domain linked to a C-terminal cAMP-phosphodiesterase domain. RdeA may be a phospho-transfer protein that supplies phosphates to RegA. We show genetically that phospho-RegA is the activated form of the enzyme in vivo, in that the predicted site of aspartate phosphorylation is required for full activity. We show biochemically that RdeA and RegA communicate, as evidenced by phospho-transfer between the two proteins in vitro. Phospho-transfer is dependent on the presumed phospho-accepting amino acids, histidine 65 of RdeA and aspartate 212 of RegA, and occurs in both directions. Phosphorylation of RegA by a heterologous phospho-donor protein activates RegA phosphodiesterase activity at least 20-fold. Our results suggest that the histidine phosphotransfer protein, RdeA, and the response regulator, RegA, constitute two essential elements in a eukaryotic His-Asp phospho-relay network that regulates Dictyostelium development and fruiting body maturation.
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PMID:The RdeA-RegA system, a eukaryotic phospho-relay controlling cAMP breakdown. 1048 68

Class I cyclic nucleotide phosphodiesterases (PDEs) share a catalytic domain containing 18 invariant residues. In cGMP-binding cGMP-specific PDE (PDE5), we showed previously that point mutation of nine of these profoundly decreases k(cat) when the assay is conducted in the presence of Mg(2+); seven of these are in the prototypical metal-binding motifs A and B (HX(3)HX(n)()E) that we identified earlier. Tandem arrangement of two of these metal-binding motifs in PDEs is novel, and whether residues within these motifs are involved in metal support of catalytic activity is a fundamental question in this field. This report shows that mutation of either His-607 (A motif) or His-643 (B motif) to alanine profoundly diminishes support of PDE catalysis by Mn(2+) or Mg(2+), but mutation of His-647 in B motif or of Glu in either motif does not. H607A and H643A mutants have much greater maximum catalytic rates supported by Mn(2+) than that by Mg(2+); catalytic activity of H603A mutant is supported weakly by either. In H607A and H643A, K(a)s for Mn(2+) and Mg(2+) are increased, but the effect of Mn(2+) is 2-fold greater than that of Mg(2+) in each. Mutation of any of the other conserved residues (Asn-604, Asp-644, His-675, Asp-714, and Asp-754) causes unremarkable changes in Mn(2+) or Mg(2+) support of catalysis. This study identifies specific residues in PDE5 that contribute to interactions with catalytically relevant metals. The combined data suggest that despite a high degree of sequence similarity between each HX(3)HX(n)()E motif in PDEs and certain metallo-endopeptidases, PDEs employ a distinct complement of residues for interacting with metals involved in catalysis.
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PMID:Histidine-607 and histidine-643 provide important interactions for metal support of catalysis in phosphodiesterase-5. 1092 56

Expressed in intact cells and in vitro, PDE4B and PDE4C isoenzymes of cyclic nucleotide phosphodiesterase (PDE), in common with PDE4D isoenzymes, are shown to provide substrates for C-terminal catalytic unit phosphorylation by the extracellular signal-regulated kinase Erk2 (p42(MAPK)). In contrast, PDE4A isoenzymes do not provide substrates for C-terminal catalytic unit phosphorylation by Erk2. Mutant PDE4 enzymes were generated to show that Erk2 phosphorylation occurs at a single, cognate serine residue located within the C-terminal portion of the PDE4 catalytic unit. PDE4 long-form isoenzymes were markedly inhibited by Erk2 phosphorylation. The short-form PDE4B2 isoenzyme was activated by Erk2 phosphorylation. These functional changes in PDE activity were mimicked by mutation of the target serine for Erk2 phosphorylation to the negatively charged amino acid, aspartic acid. Epidermal growth factor (EGF) challenge caused diametrically opposed changes in cyclic AMP levels in COS1 cells transfected to express the long PDE4B1 isoenzyme compared to cells expressing the short PDE4B2 isoenzyme. We suggest that PDE4 enzymes may provide a pivotal point for integrating cyclic AMP and Erk signal transduction in cells with 4 genes encoding enzymes that are either insensitive to Erk2 action or may either be activated or inhibited. This indicates that PDE4 isoenzymes have distinct functional roles, giving credence to the notion that distinct therapeutic benefits may accrue using either PDE4 subfamily or isoenzyme-selective inhibitors.
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PMID:Sub-family selective actions in the ability of Erk2 MAP kinase to phosphorylate and regulate the activity of PDE4 cyclic AMP-specific phosphodiesterases. 1103 Jul 32

GAF domains are ubiquitous motifs present in cyclic GMP (cGMP)-regulated cyclic nucleotide phosphodiesterases, certain adenylyl cyclases, the bacterial transcription factor FhlA, and hundreds of other signaling and sensory proteins from all three kingdoms of life. The crystal structure of the Saccharomyces cerevisiae YKG9 protein was determined at 1.9 A resolution. The structure revealed a fold that resembles the PAS domain, another ubiquitous signaling and sensory transducer. YKG9 does not bind cGMP, but the isolated first GAF domain of phosphodiesterase 5 binds with K:(d) = 650 nM. The cGMP binding site of the phosphodiesterase GAF domain was identified by homology modeling and site-directed mutagenesis, and consists of conserved Arg, Asn, Lys and Asp residues. The structural and binding studies taken together show that the cGMP binding GAF domains form a new class of cyclic nucleotide receptors distinct from the regulatory domains of cyclic nucleotide-regulated protein kinases and ion channels.
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PMID:Structure of the GAF domain, a ubiquitous signaling motif and a new class of cyclic GMP receptor. 1103 96

Platelet-inhibitory drugs are of proven benefit to individuals who suffer from atherosclerotic cardiovascular disease. Despite substantial effort to identify more potent platelet-inhibitory agents, aspirin, an irreversible inhibitor of platelet cyclooxygenase activity, remains the standard against which other drugs are judged. Drugs that appear to be at least as efficacious as aspirin in specific clinical settings include the thienopyridines ticlopidine and clopidogrel, specific inhibitors of ADP-stimulated platelet function, and the phosphodiesterase 3 inhibitor cilostazol. Ligand binding to the platelet integrin alphaIIbbeta3 (GPIIb-IIIa), a prerequisite for platelet thrombus formation, has been a prominent target for drug development. Currently, three types of alphaIIbbeta3 antagonists are available: the monoclonal antibody Fab fragment abciximab, cyclic peptides based on the Arg-Gly-Asp (RGD) or related amino acid motifs, and RGD-based peptidomimetics. The efficacy of each type of alphaIIbbeta3 antagonist in the setting of acute coronary artery disease has been confirmed in multicenter clinical trials.
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PMID:Novel platelet inhibitors. 1116 Jul 73

The structure of cyclic GMP (cGMP)-binding (cGB), cGMP specific phosphodiesterase (PDE5) comprises several domains. We have used RT-PCR methods to clone the noncatalytic cGB domains of PDE5 from human colon cancer cell RNA and constructed glutathione-S-transferase (GST) fusion proteins to express and study the domains. One fragment showed 94% identity to bovine PDE5 and coded for the high affinity cGB domain of PDE5 (Val(156)-Asp(394), cGB-I). Another cloned fragment showed 92% identity to bovine PDE5 and coded for the phosphorylation site plus both high and low affinity cGB domains of PDE5 (Val(36)-Glu(529), cGB-II). Both fragments expressed as GST-cGB fusion proteins bound cGMP specifically, as determined by competitive [3H]-cGMP ligand binding. We found that cGB-I showed high affinity cGMP binding with K(d)=0.33 microM. cGB-II showed two cGMP binding sites with similar affinities and specificity to the native enzyme. cGB-II was phosphorylated by cGMP-dependent protein kinase (PKG) as reported for bovine PDE5. These data show that recombinant regulatory regions of PDE5 form cGB sites similar to native enzyme sites and confirm proposed domain functions. These results establish that recombinant fusion proteins of PDE5 domains may be used to further characterize the structure of PDE5.
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PMID:Specific cGMP binding by the cGMP binding domains of cGMP-binding cGMP specific phosphodiesterase. 1174 88


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