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)

In boar spermatozoa, the capacitating agent bicarbonate has been shown to induce rapid changes both in plasma membrane lipid architecture and in motility; in each case, a PKA-dependent pathway is involved. Early bicarbonate-induced changes in protein phosphorylation were probed using a commercial antibody against the phosphorylated form of the consensus substrate site for cyclic AMP-dependent protein kinase. The antibody detected relatively few bands in sperm extracts, of which only a small number showed incubation-dependent changes. While the quantitative response varied between boar ejaculates, in general terms bicarbonate induced phosphorylation increases in bands of 96, 64, and 59 kDa within 80 sec. The changes reached a maximum after about 160 sec, declined somewhat thereafter, and then increased again slowly as incubation progressed further (up to 21 min). The bicarbonate-induced increases were strongly dependent on the presence of BSA in the incubation medium. They were inhibited by H89 (PKA inhibitor) but not by GF (PKC inhibitor), and were enhanced by papaverine (phosphodiesterase inhibitor) and by calyculin (protein phosphatase inhibitor). The cyclic AMP analogue cBIMPS was able to mimic bicarbonate action though its effect was less dramatic. Stearated Ht31, a permeable inhibitor of PKA's binding to A-kinase anchoring protein, did not affect either the intensity or the specificity of the bicarbonate-induced phosphorylation changes, though it blocked motility entirely. Immunocytochemical studies revealed marked bicarbonate-dependent phosphorylation changes in the post-acrosomal region of the head and in the neck, midpiece, and anterior regions of the tail. Fractionation of stimulated spermatozoa showed that all bands detectable with the antibody were bound to heads and to midpieces and associated large tail fragments; no bands were detected in either small tail or membrane fragments or in the cytoplasmic fraction. Differential extraction of the midpiece/large tail fraction revealed two protein bands with closely similar electrophoretic mobilities to the 96- and 59-kDa phosphorylated bands; MALDI-TOF analyses of these bands revealed both to be members of the Odf2 family.
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PMID:Rapid PKA-catalysed phosphorylation of boar sperm proteins induced by the capacitating agent bicarbonate. 1473 95

Sustained cardiac pressure overload induces hypertrophy and pathological remodeling, frequently leading to heart failure. Genetically engineered hyperstimulation of guanosine 3',5'-cyclic monophosphate (cGMP) synthesis counters this response. Here, we show that blocking the intrinsic catabolism of cGMP with an oral phosphodiesterase-5A (PDE5A) inhibitor (sildenafil) suppresses chamber and myocyte hypertrophy, and improves in vivo heart function in mice exposed to chronic pressure overload induced by transverse aortic constriction. Sildenafil also reverses pre-established hypertrophy induced by pressure load while restoring chamber function to normal. cGMP catabolism by PDE5A increases in pressure-loaded hearts, leading to activation of cGMP-dependent protein kinase with inhibition of PDE5A. PDE5A inhibition deactivates multiple hypertrophy signaling pathways triggered by pressure load (the calcineurin/NFAT, phosphoinositide-3 kinase (PI3K)/Akt, and ERK1/2 signaling pathways). But it does not suppress hypertrophy induced by overexpression of calcineurin in vitro or Akt in vivo, suggesting upstream targeting of these pathways. PDE5A inhibition may provide a new treatment strategy for cardiac hypertrophy and remodeling.
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PMID:Chronic inhibition of cyclic GMP phosphodiesterase 5A prevents and reverses cardiac hypertrophy. 1569 88

We studied the impact of an NO-cGMP dependent signalling pathway on the high-voltage-activated (HVA) Ca(2+) current in identified neurons of the pulmonate snail, Helix pomatia, using Ba(2+) as charge carrier. The 3',5'-cyclic guanosine monophosphate (cGMP) analogues, dibutyryl-cGMP and 8-bromo-cGMP, consistently induced a biphasic response, consisting of an increase superseded by a decline of the Ba(2+) current. The NO donor, sodium nitroprusside (SNP), modulated only in a minority of neurons the Ba(2+) current. Blockade of protein kinase activity with 1-[5-isoquinolinesulfonyl]-2 methyl piperazine (H 7), a nonselective protein kinase inhibitor, or Rp-8-pCPT-cGMP, a selective protein kinase G (PKG) inhibitor, decreased, whereas Rp-cAMP, a selective protein kinase A (PKA) inhibitor, increased the Ba(2+) current upon application of cGMP analogues or SNP. Okadaic acid or calyculin, inhibitors of protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A), augmented the Ba(2+) current. Under these conditions, cGMP analogues or SNP had an additive-enhancing effect on the Ba(2+) current. When neurons were exposed to the nonselective phosphodiesterase (PDE) inhibitor 3-isobutyl-1-methylxanthine (IBMX), cGMP analogues induced a persistent increase of the Ba(2+) current, whereas SNP induced a biphasic response. These data suggest coexistence of cGMP-PKG and cGMP-PDE pathways as well as crosstalk between cGMP and 3',5'-cyclic adenosine monophosphate (cAMP) pathways, which converge on HVA Ca channels in Helix neurons. In this model, augmentation of the Ba(2+) current through HVA Ca channels is accomplished by PKA and PKG, whereas attenuation is mediated by PDEs, which prevent activation of protein kinases via hydrolysis of cyclic nucleotides.
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PMID:A cGMP-dependent cascade enhances an L-type-like Ca2+ current in identified snail neurons. 1568 Sep 43

Ionotropic nicotinic acetylcholine (ACh) receptors have been shown to be modulated by protein kinase-mediated phosphorylation in vitro. Here we demonstrate that 5-hydroxytryptamine (5-HT) can downregulate postsynaptic nicotinic ACh responses, elicited in an identified arthropod motoneuron in situ, by a mechanism dependent on protein kinase activity. Serotonergic modulation can be mimicked by perfusion with membrane-permeable analogues of either adenine (cAMP) or guanine (cGMP) cyclic nucleotides, and is prolonged in the presence of phosphodiesterase inhibitors. Furthermore, suppression of the ACh response by 5-HT is blocked by specific competitive inhibitors of protein kinase A and G, as well as the broad specificity protein kinase inhibitor staurosporine. The protein phosphatase inhibitor cantharidin similarly blocks recovery of the ACh response from suppression mediated by 5-HT. Thus, it appears that the nicotinic ACh response is modulated by a cAMP-mediated phosphorylation-dependent intracellular signalling pathway that is distinct from the direct block of mammalian nicotinic ACh receptors by 5-HT previously reported in vitro.
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PMID:Indirect phosphorylation-dependent modulation of postsynaptic nicotinic acetylcholine responses by 5-hydroxytryptamine. 1581 27

Phosphatidic acid (PA) has been recognized as a lipid second messenger, yet few cellular targets for PA have been identified. Previous work demonstrated PA as a potent and noncompetitive tight-binding inhibitor of the catalytic subunit (gamma isoform) of protein phosphatase-1 (PP1c gamma) in vitro. The high potency of inhibition, coupled with high specificity for PA over other phospholipids, suggested the presence of a high-affinity PA binding domain on PP1c gamma. In the current study, quantification of the binding interaction and identification of the binding domain were pursued. Surface plasmon resonance was employed to quantitate the interaction between PP1c gamma and immobilized mixed lipid vesicles of PA/phosphatidylcholine (PC) or PC alone. The data disclosed a high-affinity interaction with a KD measured in the low (1-40) nanomolar range, consistent with the range of Ki previously obtained from in vitro enzymatic assays. Next, identification of the segment of PP1 necessary for PA binding was determined using a deletion mutagenesis strategy. Binding assays revealed that PP1c gamma residues between 274 and 299 were required for the interaction with the lipid. When fusions of PP1c gamma fragments with green fluorescent protein (GFP) were generated, it was then determined that PP1c gamma residues 286-296 were sufficient to confer PA binding to GFP, a protein that does not interact with PA. The minimal PA binding domain of PP1c gamma lacked similarity to the previously described PA binding segments of Raf-1 kinase and cyclic-AMP phosphodiesterase 4A1. When these results were taken together with the known crystallographic structure of PP1, they identified a novel PA binding region on PP1c gamma that contains a unique loop-strand structural fold responsible for the interaction with PA.
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PMID:Identification of a novel phosphatidic acid binding domain in protein phosphatase-1. 1620 49

Following its production by adenylyl cyclases, the second messenger cAMP is in involved in pleiotrophic signal transduction. The effectors of cAMP include the cAMP-dependent protein kinase (PKA), the guanine nucleotide exchange factor Epac (exchange protein activated by cAMP), and cAMP-dependent ion channels. In turn, cAMP signaling is attenuated by phosphodiesterase-catalyzed degradation. The association of cAMP effectors and the enzymes that regulate cAMP concentration into signaling complexes helps to explain the differential signaling initiated by members of the G(s)-protein coupled receptor family. The signal transduction complex formed by the scaffold protein mAKAP (muscle A kinase-anchoring protein) at the nuclear envelope of both striated myocytes and neurons contains three cAMP-binding proteins, PKA, Epac1, and the phosphodiesterase PDE4D3. In addition, the mAKAP complex also contains components of the ERK5 MAP kinase signaling pathway, the calcium release channel ryanodine receptor and the phosphatases PP2A as well as calcineurin. Analysis of the mAKAP complex illustrates how a macromolecular complex can serve as a node in the intracellular signaling network of cardiac myocytes to integrate multiple cAMP signals with those of calcium and MAP kinases to regulate the hypertrophic actions of several hormones.
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PMID:The mAKAP signaling complex: integration of cAMP, calcium, and MAP kinase signaling pathways. 1646 Aug 34

In Heteropneustes fossilis, in vitro incubation of postvitellogenic follicles with 2-hydroxyestradiol-17beta (2-OHE2, 5 micromol l(-1)) decreased significantly the total cAMP level, concomitant with germinal vesicle breakdown (GVBD). The incubation of the follicles with cAMP or cAMP-elevating drugs [phosphodiesterase (PDE) inhibitors], such as IBMX (3-isobutyl-1-methyl-xanthine), theophylline and caffeine, inhibited the 2-OHE2-induced GVBD in a concentration-dependent manner. The magnitude of the response varied: both cAMP and IBMX were effective at all concentrations (0.1-2.0 mmol l(-1)), followed by theophylline (0.5-2.0 mmol l(-1)) and caffeine (1-2.0 mmol l(-1)). The protein kinase A (PKA) inhibitor H89 stimulated oocyte maturation in a concentration-dependent manner. However, when co-incubated with 2-OHE2 for 24 h it produced a biphasic effect: low concentrations (0.1 and 1.0 micromol l(-1)) did not alter the 2-OHE2-induced GVBD, but high concentrations (5 and 10 micromol l(-1)) inhibited it. The incubation of the follicles with H89 lowered the inhibitory effect of IBMX on the 2-OHE2-induced GVBD. The incubation of the follicles with okadaic acid (OA), a protein phosphatase 1 and 2A inhibitor did not affect GVBD but when co-incubated with 2-OHE2, it enhanced the GVBD response. OA reversed the inhibitory effect of IBMX. The results suggest that OA may overcome the inhibition of 2-OHE2-induced GVBD by IBMX at a step distal to the cAMP-PKA pathway.
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PMID:2-Hydroxyestradiol-17beta-induced oocyte maturation: involvement of cAMP-protein kinase A and okadaic acid-sensitive protein phosphatases, and their interplay in oocyte maturation in the catfish Heteropneustes fossilis. 1678 39

Cyclic nucleotide monophosphate (cNMP) hydrolysis in bacteria and eukaryotes is brought about by distinct cNMP phosphodiesterases (PDEs). Since these enzymes differ in amino acid sequence and properties, they have evolved by convergent evolution. Cyclic NMP PDEs cleave cNMPs to NMPs, and the Rv0805 gene product is, to date, the only identifiable cNMP PDE in the genome of Mycobacterium tuberculosis. We have shown that Rv0805 is a cAMP/cGMP dual specificity PDE, and is unrelated in amino acid sequence to the mammalian cNMP PDEs. Rv0805 is a dimeric, Fe(3+)-Mn(2+) binuclear PDE, and mutational analysis demonstrated that the active site metals are co-ordinated by conserved aspartate, histidine and asparagine residues. We report here the structure of the catalytic core of Rv0805, which is distantly related to the calcineurin-like phosphatases. The crystal structure of the Rv0805 dimer shows that the active site metals contribute to dimerization and thus play an additional structural role apart from their involvement in catalysis. We also present the crystal structures of the Asn97Ala mutant protein that lacks one of the Mn(2+) co-ordinating residues as well as the Asp66Ala mutant that has a compromised cAMP hydrolytic activity, providing a structural basis for the catalytic properties of these mutant proteins. A molecule of phosphate is bound in a bidentate manner at the active site of the Rv0805 wild-type protein, and cacodylate occupies a similar position in the crystal structure of the Asp66Ala mutant protein. A unique substrate binding pocket in Rv0805 was identified by computational docking studies, and the role of the His140 residue in interacting with cAMP was validated through mutational analysis. This report on the first structure of a bacterial cNMP PDE thus significantly extends our molecular understanding of cAMP hydrolysis in class III PDEs.
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PMID:Structural and biochemical analysis of the Rv0805 cyclic nucleotide phosphodiesterase from Mycobacterium tuberculosis. 1705 28

Three different phosphatases ("slow", "middle" and "fast") were found in Amoeba proteus (strain B) after PAGE and a subsequent gel staining in 1-naphthyl phosphate containing incubation mixture (pH 9.0). Substrate specificity of these phosphatases was determined in supernatants of homogenates using inhibitors of phosphatase activity. All phosphatases showed a broad substrate specificity. Of 10 tested compounds, p-nitrophenyl phosphate was a preferable substrate for all 3 phosphatases. All phosphatases were able to hydrolyse bis-p-nitrophenyl phosphate and, hence, displayed phosphodiesterase activity. All phosphatases hydrolysed O-phospho-L-tyrosine to a greater or lesser degree. Only little differences in substrate specificity of phosphatases were noticed: 1) "fast" and "middle" phosphatases hydrolysed naphthyl phosphates and O-phospho-L-tyrosine less efficiently than did "slow" phosphatase; 2) "fast" and "middle" phosphatases hydrolysed 2- naphthyl phosphate to a lesser degree than 1-naphthyl phosphate 3) "fast" and "middle" phosphatases hydrolysed O-phospho-L-serine and O-phospho-L-threonine with lower intensity as compared with "slow" phosphatase; 4) as distinct from "middle" and "slow" phosphatases, the "fast" phosphatase hydrolysed glucose-6-phosphate very poorly. The revealed broad substrate specificity of "slow" phosphatase together with data of inhibitory analysis and results of experiments with reactivation of this phosphatase by Zn2+-ions after its inactivation by EDTA strongly suggest that only the "slow" phosphatase is a true alkaline phosphatase (EC 3.1.3.1). The alkaline phosphatase of A. proteus is secreted into culture medium where its activity is low. The enzyme displays both phosphomono- and phosphodiesterase activities, in addition to supposed protein phosphatase activity. It still remains unknown, to which particular phosphatase class the amoeban "middle" and "fast" phosphatases (pH 9.0) may be assigned.
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PMID:[Substrate specifity in Amoeba proteus]. 1708 51

Thrombin-induced cyclic AMP (cAMP) reduction potentates several steps in platelet activation, including Ca(++) mobilization, cytoskeletal reorganization, and fibrinogen receptor conformation. We now reinvestigate the signaling pathways by which intracellular cAMP content is controlled after platelet activation by thrombin. When washed human platelets were stimulated with thrombin, cAMP-dependent phosphodiesterase (PDE3A) activity was significantly increased. A nonselective PDE inhibitor, 3-isobutyl-1-methylxanthine (IBMX), and the PDE3 selective inhibitors milrinone and cilostazol each suppressed thrombin-induced cAMP-dependent PDE responses, but not 2 different PDE2 inhibitors. Selective inhibition of PDE3A resulted in reversal of thrombin-induced cAMP reduction, indicating that thrombin activated PDE3A. In synergy with inhibition of adenylate cyclase by thrombin, activated PDE3A accelerates cAMP hydrolysis and maximally reduces the cAMP content. Thrombin-induced PDE3A activation was diminished concomitantly with dephosphorylation of PDE3A by protein phosphatase 1 (PP1). An Akt inhibitor blocked PDE3A activation and constrained thrombin-induced cAMP reduction. A P2Y(12) inhibitor also reduced thrombin-induced cAMP reduction. The combination of both reversed cAMP decrease by thrombin. Thrombin-mediated phosphorylated PDE3A was isolated by liquid chromatography, detected by a monoclonal antibody against Akt-phosphorylated substrate, and verified by immunoprecipitation study. The predominant isoform phosphorylated by Akt was the 136-kDa species. We suggest that activation/phosphorylation of PDE3A via Akt signaling pathway participates in regulating cAMP during thrombin activation of platelets.
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PMID:Thrombin regulates intracellular cyclic AMP concentration in human platelets through phosphorylation/activation of phosphodiesterase 3A. 1739 5


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