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)

During the evolution of mammals, the endometrium has developed for one reason only: to implant an embryo in the uterus. In higher primates, should an oocyte fail to be fertilized, then the endometrial layer is sloughed off during menses and the menstrual cycle starts again with a new round of endometrial differentiation. This stromal differentiation process is called decidualization and is accompanied in vivo by sustained high levels of intracellular cAMP. The present study was conducted to determine whether manipulation of cAMP-phosphodiesterase (PDE) activities in cultured human endometrial stromal cells could positively influence the decidualization process. The combination of relaxin treatment with inhibition of PDE4 by the specific inhibitor rolipram induced a strong increase in relaxin-mediated cAMP production, both acutely, after 20 min, and after long-term treatment for 3 days, to promote a sustained intracellular cAMP concentration. Moreover, there was a dramatic synergistic effect on the decidualization phenotype, characterized both morphologically and by increased production of prolactin and insulin-like growth factor binding protein-1 gene transcripts. The observations that expression of PDE4D transcripts were selectively increased by cAMP and that inhibition of protein kinase A by H89 to potentially block negative feedback regulation enhanced the relaxin/rolipram-mediated cAMP accumulation lead to a complex picture of cAMP regulation in these cells. There appears to be a coordinated contribution by relaxin and PDE4 at different levels to promote a sustained increased cAMP concentration during decidualization, and thus to provide an adequate maternal interface for the implanting blastocyst.
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PMID:Relaxin and phosphodiesterases collaborate during decidualization. 1565 33

Sustained activation of adenylyl cyclase in vascular smooth muscle cells (VSMCs) results in the activation of a series of complex regulatory systems designed to desensitize these cells to further cAMP-mediated events. Although an increase in phosphodiesterase (PDE) 4-mediated hydrolysis of cAMP forms an integral part of this desensitization program in both "contractile/quiescent" and "synthetic/activated" VSMCs, distinct PDE4D gene variants coordinate these events in these phenotypically distinct cells. Using a combination of pharmacological, biochemical, and molecular biological approaches, and both in vivo and in vitro systems, we have identified the molecular basis underlying this VSMC phenotype-selective expression of PDE4D in response to cAMP-elevating agents in these cells. Thus, whereas the protein kinase A/cAMP response element-binding protein/cAMP response element signaling cascade regulates PDE4D expression in each VSMC phenotype, elevated levels of histone acetylation of the intronic promoter regulating PDE4D1 and PDE4D2 expression allows selective cAMP-mediated induction of expression of these PDE4D variants in synthetic/activated VSMCs. In contrast, the newly described EPAC1/Rap1A cAMP-dependent signaling cascade plays no role in regulating PDE4D expression in either VSMC phenotype. Our data are presented in the context of PDE4-mediated desensitization to cAMP-elevating agents in VSMCs and with the recognition that cAMP-elevating agents are being considered as adjunctive pharmacotherapy in percutaneous coronary interventions, including stenting.
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PMID:Vascular smooth muscle cell phenotype-dependent phosphodiesterase 4D short form expression: role of differential histone acetylation on cAMP-regulated function. 1595 94

PDE4B and PDE4D provide >90% of PDE4 cAMP phosphodiesterase activity in human embryonic kidney (HEK293B2) cells. Their selective small interference RNA (siRNA)-mediated knockdown potentiates isoprenaline-stimulated protein kinase A (PKA) activation. Whereas endogenous PDE4D co-immunoprecipitates with beta arrestin, endogenous PDE4B does not, even upon PDE4D knockdown. Ectopic overexpression of PDE4B2 confers co-immunoprecipitation with beta arrestin. Knockdown of PDE4D, but not PDE4B, amplifies isoprenaline-stimulated phosphorylation of the beta2-adrenergic receptor (beta2-AR) by PKA and activation of extracellular signal-regulated kinase (ERK) through G(i). Isoform-selective knockdown identifies PDE4D5 as the functionally important species regulating isoprenaline stimulation of both these processes. Ht31-mediated disruption of the tethering of PKA to AKAP scaffold proteins attenuates isoprenaline activation of ERK, even upon PDE4D knockdown. Selective siRNA-mediated knockdown identifies AKAP79, which is constitutively associated with the beta2-AR, rather than isoprenaline-recruited gravin, as being the functionally relevant AKAP in this process. Isoprenaline-stimulated membrane recruitment of PDE4D is ablated upon beta arrestin knockdown. A mutation that compromises interactions with beta arrestin prevents catalytically inactive PDE4D5 from performing a dominant negative role in potentiating isoprenaline-stimulated ERK activation. Beta arrestin-recruited PDE4D5 desensitizes isoprenaline-stimulated PKA phosphorylation of the beta2-AR and the consequential switching of its signaling to ERK. The ability to observe a cellular phenotype upon PDE4D5 knockdown demonstrates that other PDE4 isoforms, expressed at endogenous levels, are unable to afford rescue in HEK293B2 cells.
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PMID:RNA silencing identifies PDE4D5 as the functionally relevant cAMP phosphodiesterase interacting with beta arrestin to control the protein kinase A/AKAP79-mediated switching of the beta2-adrenergic receptor to activation of ERK in HEK293B2 cells. 1603 21

Cyclic nucleotide signaling functions as a negative modulator of inflammatory cell responses, and type 4 phosphodiesterases (PDE4) are important regulators of this pathway. In this study, we provide evidence that only one of the three PDE4 genes expressed in mouse peritoneal macrophages is involved in the control of TLR signaling. In these cells, LPS stimulation of TLR caused a major up-regulation of PDE4B but not the paralogs PDE4A or PDE4D. Only ablation of PDE4B impacted LPS signaling and TNF-alpha production. TNF-alpha mRNA and protein were decreased by >50% in PDE4B-/-, but not in PDE4A-/- or PDE4D-/- macrophages. The PDE4 selective inhibitors rolipram and roflumilast had no additional inhibitory effect in macrophages deficient in PDE4B, but suppressed the TNF-alpha response in the other PDE4 null cells. The inhibition of TNF-alpha production that follows either genetic ablation or acute inhibition of PDE4B is cAMP-dependent and requires protein kinase A activity. However, no global changes in cAMP concentration were observed in the PDE4B-/- macrophages. Moreover, ablation of PDE4B protected mice from LPS-induced shock, suggesting that altered TLR signaling is retained in vivo. These findings demonstrate the highly specialized function of PDE4B in macrophages and its critical role in LPS signaling. Moreover, they provide proof of concept that a PDE4 inhibitor with subtype selectivity retains useful pharmacological effects.
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PMID:Specific role of phosphodiesterase 4B in lipopolysaccharide-induced signaling in mouse macrophages. 1603 90

Interleukin-6 (IL-6), and the related cytokines IL-11, leukemia inhibitory factor (LIF) and oncostatin M (OSM), are potent stimulators of osteoclastic bone resorption. In the present study, we have addressed the possibility that the neuropeptide vasoactive intestinal peptide (VIP) may regulate the production of and/or sensitivity to the IL-6 family of cytokines in mouse calvarial osteoblasts. VIP stimulated IL-6 mRNA expression and protein release in a time- and concentration-dependent manner, whereas mRNA expression of the IL-6 receptor, as well as mRNA expressions of IL-11, LIF, OSM and their cognate receptors, were unaffected by VIP. In cells transfected with the IL-6 promoter coupled to luciferase, VIP increased transcriptional activity. The effects of VIP were shared by the related neuropeptide PACAP-38, belonging to the same superfamily of neuropeptides, whereas secretin did not have any effect, indicating that the effects were mediated by VPAC2 receptors. The effects of VIP were potentiated by the cyclic AMP phosphodiesterase inhibitor rolipram and mimicked by forskolin, indicating the involvement of the cyclic AMP/protein kinase A pathway. This was further demonstrated by the facts that the stimulatory effect of VIP on luciferase activity could be reversed by the PKA inhibitors H-89 and KT5720 and was mimicked by cyclic AMP analogues selective for PKA, but not by those selective for Epac. In addition, VIP enhanced the phosphorylation of CREB, as assessed by both immunocytochemical analysis and Western blot. The DNA binding activity of nuclear extracts to C/EBP was increased by VIP, whereas binding to AP-1 was decreased. In contrast, DNA binding to NF-kappaB, as well as nuclear translocation of NF-kappaB and C/EBP, were unaffected by VIP. The mRNA expressions of C/EBPbeta, C/EBPdelta, C/EBPgamma, c-Jun, JunB, c-Fos, Fra-1 and IkappaBalpha and protein level of IkappaBalpha were all unaffected by VIP. These observations, together, demonstrate that VIP stimulates IL-6 production in osteoblasts by a mechanism likely to be mediated by VPAC2 receptors and dependent on cyclic AMP/protein kinase A/CREB activation and also involving the transcription factors C/EBP and AP-1.
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PMID:Increased expression of interleukin-6 by vasoactive intestinal peptide is associated with regulation of CREB, AP-1 and C/EBP, but not NF-kappaB, in mouse calvarial osteoblasts. 1608 72

Cyclic AMP plays an important role in regulating sperm motility and acrosome reaction through activation of cAMP-dependent protein kinase A (PKA). Phosphodiesterases (PDEs) modulate the levels of cyclic nucleotides by catalyzing their degradation. Although PDE inhibitors specific to PDE1 and PDE4 are known to alter sperm motility and capacitation in humans, little is known about the role or subcellular distribution of PDEs in spermatozoa. The localization of PKA is regulated by A-kinase anchoring proteins (AKAPs), which may also control the intracellular distribution of PDE. The present study was undertaken to investigate the role and localization of PDE4 during sperm capacitation. Addition of Rolipram or RS25344, PDE4-specific inhibitors significantly increased the progressive motility of bovine spermatozoa. Immunolocalization techniques detected both PDE4A and AKAP3 (formerly known as AKAP110) in the principal piece of bovine spermatozoa. The PDE4A5 isoform was detected primarily in the Triton X-100-soluble fraction of caudal epididymal spermatozoa. However, in ejaculated spermatozoa it was seen primarily in the SDS-soluble fraction, indicating a shift in PDE4A5 localization into insoluble organelles during sperm capacitation. AKAP3 was detected only in the SDS-soluble fraction of both caudal and ejaculated sperm. Immunoprecipitation experiments using COS cells cotransfected with AKAP3 and either Pde4a5 or Pde4d provide evidence that PDE4A5 but not PDE4D interacts with AKAP3. Pulldown assays using sperm cell lysates confirm this interaction in vitro. These data suggest that AKAP3 binds both PKA and PDE4A and functions as a scaffolding protein in spermatozoa to regulate local cAMP concentrations and modulate sperm functions.
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PMID:AKAP3 selectively binds PDE4A isoforms in bovine spermatozoa. 1617 23

Phosphodiesterases (PDEs) regulate the local concentration of 3',5' cyclic adenosine monophosphate (cAMP) within cells. cAMP activates the cAMP-dependent protein kinase (PKA). In patients, PDE inhibitors have been linked to heart failure and cardiac arrhythmias, although the mechanisms are not understood. We show that PDE4D gene inactivation in mice results in a progressive cardiomyopathy, accelerated heart failure after myocardial infarction, and cardiac arrhythmias. The phosphodiesterase 4D3 (PDE4D3) was found in the cardiac ryanodine receptor (RyR2)/calcium-release-channel complex (required for excitation-contraction [EC] coupling in heart muscle). PDE4D3 levels in the RyR2 complex were reduced in failing human hearts, contributing to PKA-hyperphosphorylated, "leaky" RyR2 channels that promote cardiac dysfunction and arrhythmias. Cardiac arrhythmias and dysfunction associated with PDE4 inhibition or deficiency were suppressed in mice harboring RyR2 that cannot be PKA phosphorylated. These data suggest that reduced PDE4D activity causes defective RyR2-channel function associated with heart failure and arrhythmias.
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PMID:Phosphodiesterase 4D deficiency in the ryanodine-receptor complex promotes heart failure and arrhythmias. 1621 10

Lack of endothelial nitric oxide synthase (eNOS) may affect the sensitivity of cyclic GMP signaling through soluble guanylyl cyclase (sGC). We hypothesized that in eNOS knockout (eNOS-/-) mice, stimulation of guanylyl cyclase would have enhanced effects inhibiting cardiac contraction. We measured cell shortening and calcium transients in isolated ventricular myocytes from adult eNOS-/- and wild-type (WT) mice after stimulating particulate guanylyl cyclase (pGC) with C-type natriuretic peptide (CNP, 10(-8) and 10(-7) M) or sGC with S-nitroso-N-acetyl-penicillamine (SNAP, NO donor, 10(-6) and 10(-5) M). Although sGC activity was increased by +71% in eNOS-/-, SNAP had similar effects in the two groups (%shortening -39% control vs. -37% eNOS-/-), suggesting that the cyclic GMP pathway was desensitized in eNOS-/- myocytes. CNP had significantly smaller effects on cell contraction (%shortening -34% control vs. -14% eNOS-/-) and pGC activity was not changed in eNOS-/- myocytes. Similar effects were also produced by guanylin and carbon monoxide, stimulators of pGC and sGC. CNP's effects on Ca(2+) transients were also attenuated in eNOS-/- myocytes. SNAP did not alter Ca(2+) transients in eNOS-/- or control cells. In the eNOS-/- mice, cyclic GMP-dependent protein kinase and cyclic AMP phosphodiesterase activity were reduced. This study demonstrated that the downstream cyclic GMP pathway was attenuated in eNOS-/- mice and this was partially compensated for by increased sGC, but not pGC activity in ventricular myocytes.
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PMID:Alterations in ventricular myocyte contraction caused by C-type natriuretic peptide and nitric oxide in eNOS-/- mice. 1623 10

Membrane-recruitment of GRK2 (G-protein receptor kinase 2) provides a fundamental step in the desensitization process controlling GPCRs (G-protein-coupled receptors), such as the beta2AR (beta2-adrenergic receptor). In the present paper, we show that challenge of HEK-293beta2 [human embryonic kidney cells stably overexpressing the FLAG-tagged beta2AR-GFP (green fluorescent protein)] cells with the beta-adrenoceptor agonist, isoprenaline, causes GRK2 to become phosphorylated by PKA (cAMP-dependent protein kinase). This action is facilitated when cAMP-specific PDE4 (phosphodiesterase-4) activity is selectively inactivated, either chemically with rolipram or by siRNA (small interfering RNA)-mediated knockdown of PDE4B and PDE4D. PDE4-selective inhibition by rolipram facilitates the isoprenaline-induced membrane translocation of GRK2, phosphorylation of the beta2AR by GRK2, membrane translocation of beta-arrestin and internalization of beta2ARs. PDE4-selective inhibition also enhances the ability of isoprenaline to trigger the PKA phosphorylation of GRK2 in cardiac myocytes. In the absence of isoprenaline, rolipram-induced inhibition of PDE4 activity in HEK-293beta2 cells acts to stimulate PKA phosphorylation of GRK2, with consequential effects on GRK2 membrane recruitment and GRK2-mediated phosphorylation of the beta2AR. We propose that a key role for PDE4 enzymes is: (i) to gate the action of PKA on GRK2, influencing the rate of GRK2 phosphorylation of the beta2AR and consequential recruitment of beta-arrestin subsequent to beta-adrenoceptor agonist challenge, and (ii) to protect GRK2 from inappropriate membrane recruitment in unstimulated cells through its phosphorylation by PKA in response to fluctuations in basal levels of cAMP.
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PMID:Phosphodiesterase-4 influences the PKA phosphorylation status and membrane translocation of G-protein receptor kinase 2 (GRK2) in HEK-293beta2 cells and cardiac myocytes. 1635 65

The spatiotemporal regulation of cAMP can generate microdomains just beneath the plasma membrane where cAMP increases are larger and more dynamic than those seen globally. Real-time measurements of cAMP using mutant cyclic nucleotide-gated ion channel biosensors, pharmacological tools and RNA interference (RNAi) were employed to demonstrate a subplasmalemmal cAMP signaling module in living cells. Transient cAMP increases were observed upon stimulation of HEK293 cells with prostaglandin E1. However, pretreatment with selective inhibitors of type 4 phosphodiesterases (PDE4), protein kinase A (PKA) or PKA/A-kinase anchoring protein (AKAP) interaction blocked an immediate return of subplasmalemmal cAMP to basal levels. Knockdown of specific membrane-associated AKAPs using RNAi identified gravin (AKAP250) as the central organizer of the PDE4 complex. Co-immunoprecipitation confirmed that gravin maintains a signaling complex that includes PKA and PDE4D. We propose that gravin-associated PDE4D isoforms provide a means to rapidly terminate subplasmalemmal cAMP signals with concomitant effects on localized ion channels or enzyme activities.
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PMID:An anchored PKA and PDE4 complex regulates subplasmalemmal cAMP dynamics. 1664 35


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