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:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The thrombin-induced platelet shape change was blocked by nitric oxide (NO), as revealed by scanning electron microscopy, light transmission, and resistive-particle volume determination. The inhibitory effect of NO was accompanied by an increase in levels of both cyclic guanosine monophosphate (cGMP) and cyclic adenosine monophosphate (cAMP) and phosphorylation of the vasodilator-stimulated phosphoprotein (VASP). However, the inhibition of the shape change was only mimicked by cAMP analogs (Sp-5,6-DClcBIMPS, 8-AHA-cAMP, and 8-CPT-cAMP) and not by cGMP analogs (8-Br-PET-cGMP, 8-Br-cGMP, and 8-pCPT-cGMP). The effect of NO on the thrombin-induced shape change was prevented by the protein kinase A (PKA) antagonists Rp-8-Br-cAMPS and Rp-cAMPS. The protein kinase G (PKG) antagonist Rp-8-CPT-cGMPS strongly inhibited PKG-mediated 46-kDa VASP Ser239 phosphorylation, but did not inhibit the thrombin-induced shape change or the PKA-mediated VASP Ser157 phosphorylation. Whereas an inhibitor of cyclic nucleotide phosphodiesterase (PDE) 3A (milrinone) mimicked the effect of NO, inhibitors of PDE2 (erythro-9-(2-hydroxy-3-nonyl)adenine) and PDE5 (dipyridamole) were poorly effective. We concluded that (1) NO was a potent and reversible inhibitor of the platelet shape change, (2) the shape change was reversible, (3) the inhibitory effect of NO was mediated through activation of PKA, (4) the onset of the NO effect coincided with VASP Ser157 phosphorylation, and (5) removal of NO and platelet shape change coincided with VASP Ser157 dephosphorylation. These findings are compatible with elevation of cGMP by NO in a compartment close to PDE3A, PKA, and VASP, leading to a local increase of cAMP able to block thrombin-induced shape change.
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PMID:Protein kinase A mediates inhibition of the thrombin-induced platelet shape change by nitric oxide. 1526 92

PDE3A (phosphodiesterase 3A) was identified as a phosphoprotein that co-immunoprecipitates with endogenous 14-3-3 proteins from HeLa cell extracts, and binds directly to 14-3-3 proteins in a phosphorylation-dependent manner. Among cellular stimuli tested, PMA promoted maximal binding of PDE3A to 14-3-3 proteins. While p42/p44 MAPK (mitogen-activated protein kinase), SAPK2 (stress-activated protein kinase 2)/p38 and PKC (protein kinase C) were all activated by PMA in HeLa cells, the PMA-induced binding of PDE3A to 14-3-3 proteins was inhibited by the non-specific PKC inhibitors Ro 318220 and H-7, but not by PD 184352, which inhibits MAPK activation, nor by SB 203580 and BIRB0796, which inhibit SAPK2 activation. Binding of PDE3A to 14-3-3 proteins was also blocked by the DNA replication inhibitors aphidicolin and mimosine, but the PDE3A-14-3-3 interaction was not cell-cycle-regulated. PDE3A isolated from cells was able to bind to 14-3-3 proteins after in vitro phosphorylation with PKC isoforms. Using MS/MS of IMAC (immobilized metal ion affinity chromatography)-enriched tryptic phosphopeptides and phosphospecific antibodies, at least five sites on PDE3A were found to be phosphorylated in vivo, of which Ser428 was selectively phosphorylated in response to PMA and dephosphorylated in cells treated with aphidicolin and mimosine. Phosphorylation of Ser428 therefore correlated with 14-3-3 binding to PDE3A. Ser312 of PDE3A was phosphorylated in an H-89-sensitive response to forskolin, indicative of phosphorylation by PKA (cAMP-dependent protein kinase), but phosphorylation at this site did not stimulate 14-3-3 binding. Thus 14-3-3 proteins can discriminate between sites in a region of multisite phosphorylation on PDE3A. An additional observation was that the cytoskeletal cross-linker protein plectin-1 coimmunoprecipitated with PDE3A independently of 14-3-3 binding.
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PMID:Phosphodiesterase 3A binds to 14-3-3 proteins in response to PMA-induced phosphorylation of Ser428. 1615 82

cAMP plays crucial roles in cardiac remodeling and the progression of heart failure. Recently, we found that expression of cAMP hydrolyzing phosphodiesterase 3A (PDE3A) was significantly reduced in human failing hearts, accompanied by up-regulation of inducible cAMP early repressor (ICER) expression. Angiotensin II (Ang II) and the beta-adrenergic receptor agonist isoproterenol (ISO) also induced persistent PDE3A down-regulation and concomitant ICER up-regulation in vitro, which is important in Ang II- and ISO-induced cardiomyocyte apoptosis. We hypothesized that interactions between PDE3A and ICER may constitute an autoregulatory positive feedback loop (PDE3A-ICER feedback loop), and this loop would cause persistent PDE3A down-regulation and ICER up-regulation. Here, we demonstrate that ICER induction repressed PDE3A gene transcription. PDE3A down-regulation activated cAMP/PKA signaling, leading to ICER up-regulation via PKA-dependent stabilization of ICER. With respect to Ang II, the initiation of the PDE3A-ICER feedback loop depends on activation of Ang II type 1 receptor (AT1R), classical PKC(s), and CREB (cAMP response element binding protein). We further show that the PDE3A-ICER feedback loop is essential for Ang II-induced cardiomyocyte apoptosis. ISO and PDE3 inhibitors also induced the PDE3A-ICER feedback loop and subsequent cardiomyocyte apoptosis, highlighting the importance of this PDE3A-ICER feedback loop and cAMP signaling in cardiomyocyte apoptosis. Our findings may provide a therapeutic paradigm to prevent cardiomyocyte apoptosis and the progression of heart failure by inhibiting the PDE3A-ICER feedback loop.
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PMID:A positive feedback loop of phosphodiesterase 3 (PDE3) and inducible cAMP early repressor (ICER) leads to cardiomyocyte apoptosis. 1620 75

We investigated the effects of cilostazol, a potent inhibitor of cGMP-inhibited cAMP phosphodiesterase, on mechanical activity of isolated pressurized rabbit cerebral penetrating arterioles with special reference to the function of the endothelium. Both cilostazol and milrinone, another inhibitor of cAMP phosphodiesterase, produced vasodilation of the cerebral penetrating arterioles in a dose-dependent manner. Pretreatment with selective inhibitors of cyclooxygenase or nitric oxide synthase, or chemical denudation of the endothelial cells caused no significant effect on the cilostazol-mediated vasodilation of the cerebral arterioles. A selective large-conductance calcium-activated potassium channel inhibitor, iberiotoxin, and a selective protein kinase A inhibitor, H-89, caused no significant effect on the cilostazol-mediated vasodilation. In the cerebral arterioles, low concentration (10(-6)M) of cilostazol or milrinone caused a significant shift of the dose-vasodilatory response curve for adenosine to the left. These findings suggest that cilostazol produces vasodilation independent of the presence of the endothelium or activation of endogenous vasodilative prostaglandins, nitric oxide, calcium-activated potassium channel and protein kinase A. In conclusion, the vasodilator action of cilostazol may, in part, contribute to the beneficial effect of preventing lacunar cerebral infarction in patients with functional damage of the endothelium in cerebral penetrating arterioles.
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PMID:Cilostazol, an inhibitor of type 3 phosphodiesterase, produces endothelium-independent vasodilation in pressurized rabbit cerebral penetrating arterioles. 1628 83

The squamous cell carcinoma HeLa cell line and an epithelial cell line hTERT-RPE with a nonmalignant phenotype were interrogated for HeLa cell selectivity in response to 1267 annotated compounds representing 56 pharmacological classes. Selective cytotoxic activity was observed for 14 of these compounds dominated by cyclic adenosine monophosphate (cAMP) selective phosphodiesterase (PDE) inhibitors, which tended to span a representation of the chemical descriptor space of the library. The PDE inhibitors induced delayed cell death with features compatible with classical apoptosis. The PDE inhibitors were largely inactive when tested against a cell line panel consisting of hematological and nonsquamous epithelial phenotypes. In a genome-wide DNA microarray analysis, PDE3A and PDE2A were found to be significantly increased in HeLa cells compared to the other cell lines. The pathway analysis software PathwayAssist was subsequently used to extract a list of proteins and small molecules retrieved from Medline abstracts associated with the hit compounds. The resulting list consisted of major parts of the cAMP-protein kinase A pathway linking to ERK, P38, and AKT. This molecular network may provide a basis for further exploitation of novel candidate targets for the treatment of squamous cell carcinoma.
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PMID:Phenotype-based screening of mechanistically annotated compounds in combination with gene expression and pathway analysis identifies candidate drug targets in a human squamous carcinoma cell model. 1692 83

cGMP-inhibited cAMP phosphodiesterase 3A (PDE3A) is expressed in mouse oocytes, and its function is indispensable for meiotic maturation as demonstrated by genetic ablation. Moreover, PDE3 activity is required for insulin/insulin-like growth factor-1 stimulation of Xenopus oocyte meiotic resumption. Here, we investigated the cAMP-dependent protein kinase B (PKB)/Akt regulation of PDE3A and its impact on oocyte maturation. Cell-free incubation of recombinant mouse PDE3A with PKB/Akt or cAMP-dependent protein kinase A catalytic subunits leads to phosphorylation of the PDE3A protein. Coexpression of PDE3A with constitutively activated PKB/Akt (Myr-Akt) increases PDE activity as well as its phosphorylation state. Injection of pde3a mRNA potentiates insulin-dependent maturation of Xenopus oocytes and rescues the phenotype of pde3(-/-) mouse oocytes. This effect is greatly decreased by mutation of any of the PDE3A serines 290-292 to alanine in both Xenopus and mouse. Microinjection of myr-Akt in mouse oocytes causes in vitro meiotic maturation and this effect requires PDE3A. Collectively, these data indicate that activation of PDE3A by PKB/Akt-mediated phosphorylation plays a role in the control of PDE3A activity in mammalian oocytes.
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PMID:Protein kinase B/Akt phosphorylation of PDE3A and its role in mammalian oocyte maturation. 1712 99

Substantial evidence suggests that the progressive loss of cardiomyocytes caused by apoptosis significantly contributes to the development of heart failure. beta-Adrenergic receptor activation and subsequent persistent phosphodiesterase 3A (PDE3A) downregulation and concomitant inducible cAMP early repressor (ICER) upregulation (PDE3A/ICER feedback loop) has been proposed to play a key role in the pathogenesis of cardiomyocyte apoptosis. In contrast, insulin-like growth factor-1 can activate cell survival pathways, providing protection against cell death and restoring muscle function. In this study, we found that insulin-like growth factor-1 activates extracellular signal-regulated kinase 5 (ERK5) and inhibits PDE3A/ICER feedback loop. Insulin-like growth factor-1 normalized isoproterenol-mediated PDE3A downregulation and ICER upregulation via ERK5/MEF2 activation, and also inhibited isoproterenol-induced myocyte apoptosis. To determine the physiological relevance of ERK5 activation in regulating PDE3A/ICER feedback loop, we investigated the PDE3A/ICER expression and cardiomyocyte apoptosis in transgenic mice with cardiac specific expression of a constitutively active form of mitogen-activated protein (MAP)/extracellular signal-regulated protein kinase (ERK) kinase 5alpha (MEK5alpha) (CA-MEK5alpha-Tg). In wild-type mice, pressure overload- or doxorubicin-induced significant reduction of PDE3A expression and subsequent ICER induction. Cardiac specific expression of CA-MEK5alpha rescued pressure overload- or doxorubicin-mediated PDE3A downregulation and ICER upregulation and inhibited myocyte apoptosis as well as subsequent cardiac dysfunction in vivo. These data suggest that preventing the feedback loop of PDE3A/ICER by ERK5 activation could inhibit progression of myocyte apoptosis as well as cardiac dysfunction. These data suggest a new therapeutic paradigm for end stage of heart failure by inhibiting the PDE3A/ICER feedback loop via activating ERK5.
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PMID:Activation of extracellular signal-regulated kinase 5 reduces cardiac apoptosis and dysfunction via inhibition of a phosphodiesterase 3A/inducible cAMP early repressor feedback loop. 1727 11

Smooth muscle of the gut undergoes rhythmic cycles of contraction and relaxation. Various constituents in the pathways that mediate muscle contraction could act to cross-regulate cAMP or cGMP levels and terminate subsequent relaxation. We have previously shown that cAMP levels are regulated by PKA-mediated phosphorylation of cAMP-specific phosphodiesterase 3A (PDE3A) and PDE4D5; the latter is the only PDE4D isoform expressed in smooth muscle. In the present study we have elucidated a mechanism whereby cholecystokinin (CCK) and, presumably, other contractile agonists capable of activating PKC can cross-regulate cAMP levels. Forskolin stimulated PDE4D5 phosphorylation and PDE4D5 activity. CCK significantly increased forskolin-stimulated PDE4D5 phosphorylation and activity and attenuated forskolin-stimulated cAMP levels. The effect of CCK on forskolin-induced PDE4D5 phosphorylation and activity and on cAMP levels was blocked by the inhibitors of PLC or PKC and in cultured muscle cells by the expression of Galpha(q) minigene. The effects of CCK on PDE4D5 phosphorylation, PDE4D5 activity, and cAMP levels were mimicked by low (1 nM) concentrations of okadaic acid, but not by a low (10 nM) concentration of tautomycin, suggesting involvement of PP2A. Purified catalytic subunit of PP2A but not PP1 dephosphorylated PDE4D5 in vitro. Coimmunoprecipitation studies demonstrated association of PDE4D5 with PP2A and the association was decreased by the activation of PKC. In conclusion, cAMP levels are cross-regulated by contractile agonists via a mechanism that involves PLC-beta-dependent, PKC-mediated inhibition of PP2A activity that leads to increase in PDE4D5 phosphorylation and activity and inhibition of cAMP levels.
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PMID:Stimulatory phosphorylation of cAMP-specific PDE4D5 by contractile agonists is mediated by PKC-dependent inactivation of protein phosphatase 2A. 1800

Phosphodiesterases (PDEs) are hydrolytic enzymes, which convert cyclic AMP (cAMP) and cyclic GMP (cGMP) into their corresponding monophosphates. PDE-dependent hydrolysis shape gradients of these second messengers in cells, which may form the basis of their compartmentation and play a key role in a vast number of physiological and pathological processes. Here, we present a novel approach for real-time monitoring of local cAMP and cGMP levels associated with particular PDEs. We used HEK 293 cells expressing genetic constructs encoding a PDE of interest (PDE3A, PDE4A1 or PDE5A) fused to cAMP and cGMP sensors, which allow to directly visualize changes in cyclic nucleotide concentrations in the vicinity of PDE molecules by fluorescence resonance energy transfer (FRET). FRET was detected by imaging of single cells on 96-well plates and demonstrated specific effects of PDE inhibitors on local cyclic nucleotide levels. In addition, this approach reported physiological regulation of PDE3A activity, its activation by PKA-dependent phosphorylation and inhibition by cGMP. In conclusion, our assay provides a unique and highly sensitive method to analyze PDE activity in living cells. It allows to sense cAMP gradients around particular PDE molecules and to study the pharmacological effects of selective inhibitors on localized cAMP signalling.
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PMID:Real-time monitoring of phosphodiesterase inhibition in intact cells. 1846 75

ADP-ribosylation factors (ARFs) have crucial roles in vesicular trafficking. Brefeldin A-inhibited guanine nucleotide-exchange proteins (BIG)1 and BIG2 catalyze the activation of class I ARFs by accelerating replacement of bound GDP with GTP. Several additional and differing actions of BIG1 and BIG2 have been described. These include the presence in BIG2 of 3 A kinase-anchoring protein (AKAP) domains, one of which is identical in BIG1. Proteins that contain AKAP sequences act as scaffolds for the assembly of PKA with other enzymes, substrates, and regulators in complexes that constitute molecular machines for the reception, transduction, and integration of signals from cAMP or other sources, which are initiated, propagated, and transmitted by chemical, electrical, or mechanical means. Specific depletion of HeLa cell PDE3A with small interfering RNA significantly decreased membrane-associated BIG1 and BIG2, which by confocal immunofluorescence microscopy were widely dispersed from an initial perinuclear Golgi concentration. Concurrently, activated ARF1-GTP was significantly decreased. Selective inhibition of PDE3A by 1-h incubation of cells with cilostamide similarly decreased membrane-associated BIG1. We suggest that decreasing PDE3A allowed cAMP to accumulate in microdomains where its enzymatic activity limited cAMP concentration. There, cAMP-activated PKA phosphorylated BIG1 and BIG2 (AKAPs for assembly of PKA, PDE3A, and other molecules), which decreased their GEP activity and thereby amounts of activated ARF1-GTP. Thus, PDE3A in these BIG1 and BIG2 AKAP complexes may contribute to the regulation of ARF function via limitation of cAMP effects with spatial and temporal specificity.
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PMID:Interaction of phosphodiesterase 3A with brefeldin A-inhibited guanine nucleotide-exchange proteins BIG1 and BIG2 and effect on ARF1 activity. 1933 78


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