Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.11.11 (AMPK)
 12,425 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

cDNAs encoding PDE3 [cGMP-inhibited cyclic nucleotide phosphodiesterase (cGI PDE)] isoforms, cGIP1 and cGIP2, have been cloned from rat (R) and human (H) cDNA libraries. The deduced amino acid sequences of RcGIP1 and HcGIP2 are very similar in their conserved catalytic domains but differ in their N-terminal regulatory domains [Meacci, E., et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 3721-3725; Taira, M., et al. (1993) J. Biol. Chem. 268, 18573-18579]. cDNAs encoding both rat adipocyte RcGIP1 and human myocardial HcGIP2 (full-length forms and truncated forms lacking much of the putative N-terminal domain) were expressed in NIH 3006 fibroblasts and in Sf9 insect cells. The recombinant proteins exhibited the expected subunit molecular mass, immunologic reactivities, and characteristics of native membrane-associated forms of the enzymes, e.g., high affinity for cAMP (Km), sensitivity to the selective cGI PDE inhibitors OPC 3689 and OPC 3911 and to cGMP. The full-length recombinants were predominantly particulate, whereas the truncated HcGIP2 forms were cytosolic suggesting that N-terminal domains contain structural determinants important for membrane association. Both fibroblast RcGIP1 and authentic adipocyte cGI PDE were phosphorylated in vitro by cAMP-dependent protein kinase; tryptic [32P]peptides released from rat adipocyte 32P-cGI PDE and 32P-RcGIP1 exhibited identical electrophoretic profiles suggesting that the same peptides are phosphorylated in both.
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PMID:Characterization of two recombinant PDE3 (cGMP-inhibited cyclic nucleotide phosphodiesterase) isoforms, RcGIP1 and HcGIP2, expressed in NIH 3006 murine fibroblasts and Sf9 insect cells. 875 84

Incubation of cultured bovine vascular smooth muscle cells (VSMC) with forskolin increased cAMP as measured by an increase in cAMP-dependent protein kinase (PKA) activation (PKA ratio). Forskolin also produced a concentration- and time-dependent increase in activity (3-5-fold within 15 min) of a PDE4 (cAMP-specific cyclic nucleotide phosphodiesterase). The increase in PDE4 activity was not affected by cycloheximide and thus not likely due to increased synthesis of the enzyme. Activation, which was preserved during partial purification of the enzyme by chromatography on Sephacryl S-200 and MonoQ, was most likely due to a covalent modification. Incubation of cell homogenates with the catalytic subunit of PKA (PKA(c)) induced a approximately 5-fold activation of PDE4 with a time course similar to that in intact cells after forskolin addition. The forskolin-mediated activation was reversed during incubation of homogenates at room temperature for two hours. Addition of PKA(c) resulted in rapid reactivation of PDE4. These data are consistent with the hypothesis that rapid, reversible activation of PDE4 in cultured VSMC is mediated by PKA.
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PMID:Protein kinase A-dependent activation of PDE4 (cAMP-specific cyclic nucleotide phosphodiesterase) in cultured bovine vascular smooth muscle cells. 909 92

The responsiveness of melanophores of the medaka fish (wild type, Oryzias latipes) to a neurotransmitter and hormones is changed differentially after long-term adaptation to a black or white background. In the present study, we further examined whether this phenomenon involved some change in the intracellular signaling system. Using a permeabilized melanophore model, in which pigment granules could be dispersed by exogenously applied cAMP, the requirement of cAMP for pigment-dispersing reaction was revealed to be higher in melanophores of fish adapted to a black background (B cells) than in those of white background-adapted fish (W cells). Specific inhibitors of cAMP-dependent protein kinase (PKA) and cyclic nucleotide phosphodiesterase did not reduce the difference in the pigment dispersion level between B and W cells. A similar result was obtained with the free catalytic subunit of PKA. In contrast, the inhibition of protein phosphatase activity by okadaic acid diminished the difference in the responsiveness between B and W cells. These results suggest that the activity of protein phosphatase in B cell is higher than that in W cells, and that the change in the melanophore responsiveness by long-term chromatic adaptation to a background involves the change in the enzyme activity in the intracellular signaling system.
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PMID:Regulation of melanophore responsiveness in the background-adapted medaka, Oryzias latipes: change in the intracellular signaling system. 929 5

Post-translational modification has long been recognized as a way in which the properties of proteins may be subtly altered after synthesis of the polypeptide chain is complete. Amongst the moieties most commonly encountered covalently attached to proteins are oligosaccharides, phosphate, acetyl, formyl and nucleosides. Protein phosphorylation and dephosphorylation is one of the most prevalent and best understood modifications employed in cellular regulation. The bovine heart calmodulin-dependent cyclic nucleotide phosphodiesterase (CaMPEDE) can be phosphorylated by cAMP-dependent protein kinase, resulting in a decrease in the enzyme's affinity for Ca2+ and calmodulin (CaM). The phosphorylation of CaMPDE is blocked by Ca2+ and CaM and reversed by the CaM-dependent phosphatase (calcineurin). The dephosphorylation is accompanied by an increase in the affinity of the phosphodiesterase for CaM. Analysis of the complex regulatory properties of CaMPDE has led to the suggestion that fluxes of cAMP and Ca2+ during cell activations are closely coupled and that the CaMPDE play a key role in the signal coupling phenomenon. The high molecular weight calmodulin binding protein (HMWCaMBP) was phosphorylated by cAMP-dependent protein kinase. Phosphorylation of HMWCBP was higher in the absence of Ca2+/CaM then in the presence of Ca2+/CaM and reversed by the CaM-dependent phosphatase. Recently, it has become apparent that the binding of myristate to proteins is also widespread in eukaryotic cells and viruses and certainly is of great importance to the correct functioning of an organism. Myristoyl CoA:protein N-myristoyltransferase (NMT) catalyses the attachment of myristate to the amino-terminal glycine residue of various signal transduction proteins. Cardiac tissue express high levels of cAMP-dependent protein kinase whose catalytic subunit is myristoylated. The subcellular localization of bovine cardiac muscle NMT indicated a majority of the activity was localized in cytoplasm. Under native conditions the enzyme exhibited an apparent molecular mass of 50 kDa. Recovery of NMT activity, from both cytosol and particulate fractions, was found to be higher than the total activity in crude homogenates, suggesting that particulate fraction may contain an inhibitory activity towards NMT. Research in our laboratory has been focusing on the covalent modification of proteins and regulation of various signal transduction proteins. This special review is designed to summarize some aspects of the current work on co- and post-translational modification of proteins in cardiac muscle.
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PMID:Biological significance of phosphorylation and myristoylation in the regulation of cardiac muscle proteins. 940 55

High molecular weight calmodulin binding protein (HMWCaMBP) is one of the major proteins expressed in bovine cardiac muscle. In this study, we report the phosphorylation and dephosphorylation of HMWCaMBP in vitro with a view to understand the function of this protein. The HMWCaMBP was phosphorylated by cAMP-dependent protein kinase with the incorporation of 2.30 mol of phosphate/mol of protein in the presence of EGTA. When phosphorylation was carried out in the presence of Ca2+/calmodulin (CaM), the incorporation of phosphate was reduced to 1.40 mol of phosphate/mol of protein. The decrease in the stoichometry of phosphorylation by Ca2+/CaM appears to be substrate directed i.e. due to the interaction of Ca2+/CaM with HMWCaMBP. The phosphorylated HMWCaMBP was unable to compete for free CaM in a CaM-dependent cyclic nucleotide phosphodiesterase (CaMPDE) assay. These results suggest that the phosphorylation sites may reside in or in proximity to the CaM-binding domain on HMWCaMBP since phosphorylated HMWCaMBP did not inhibit CaMPDE activity. HMWCaMBP was dephosphorylated by CaM-dependent phosphatase, calcineurin.
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PMID:In vitro phosphorylation of bovine cardiac muscle high molecular weight calmodulin binding protein by cyclic AMP-dependent protein kinase and dephosphorylation by calmodulin-dependent phosphatase. 945 Jun 65

2'-Deoxyadenosine 3'-tetraphosphate (2'-deoxy-3'-A4P) and 2', 5'-dideoxyadenosine 3'-tetraphosphate (2',5'-dideoxy-3'-A4P) were synthesized, and their effects were tested on crude and purified forms of native adenylyl cyclases isolated from brain. Syntheses combined the method of alkoxide activation with the use of tribromoethyl phosphoromorpholino-chloridate as an initial phosphorylating agent. Inhibition of adenylyl cyclase was rapid in onset. With 2'-d-3'-A4P or 2',5'-dd-3'-A4P inhibition of a purified native enzyme conformed to a linear noncompetitive behavior with respect to substrate, metal-5'ATP. Order of potency was 2', 5'-dideoxy- > 2'-deoxyadenosine and 3'-tetraphosphate > 3'-triphosphate. Both mechanism of inhibition and rank order of potency were consistent with inhibition via the 3'-nucleotide-(P)-site on adenylyl cyclase. Neither 2',5'-dd-3'-ATP nor 2',5'-dd-3'-A4P had any effect on the activities of other adenosine nucleotide binding proteins such as Ca2+/calmodulin-sensitive cyclic nucleotide phosphodiesterase, Na+/K+-ATPase, or cAMP-dependent protein kinase. With purified adenylyl cyclase from bovine brain 2',5'-dd-3'-A4P and 2'-d-3'-A4P gave, respectively, IC50 values of 9.3 and 15 nM and Ki values of 23 and 53 nM. These 3'-nucleotides are the most potent regulators described for adenylyl cyclases.
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PMID:Adenine nucleoside 3'-tetraphosphates are novel and potent inhibitors of adenylyl cyclases. 973 5

Clinical trials of beta-adrenergic receptor agonists and cyclic nucleotide phosphodiesterase inhibitors in heart failure have demonstrated a reduction in survival in treated patients despite initial inotropic responses. These findings have led many to infer that activation of the mechanisms through which contractility is increased has deleterious effects on failing myocardium. It should be remembered, however, that these agents act proximately by raising intracellular cyclic adenosine monophosphate (cAMP) content and stimulating protein phosphorylation by cAMP-dependent protein kinase, and that the proteins whose phosphorylation contributes to the inotropic responses may be different from the proteins whose phosphorylation contributes to the reduction in survival. Evidence in support of the latter interpretation is presented, and potential therapeutic approaches through which the phosphorylation of different proteins might be selectively affected are considered.
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PMID:Beta-adrenergic receptor agonists and cyclic nucleotide phosphodiesterase inhibitors: shifting the focus from inotropy to cyclic adenosine monophosphate. 1044 Jan 39

Heterotrimeric GTP-binding proteins (G protein) are known to participate in the transduction of signals from ligand activated receptors to effector molecules to elicit cellular responses. Sustained activation of cAMP-G protein signaling system by agonist results in desensitization of the pathway at receptor levels, however it is not clear whether such receptor responses induce other changes in post-receptor signaling path that are associated with maintenance of AMP levels, i.e. cAMP-forming adenylate cyclase (AC), cAMP-degrading cyclic nucleotide phosphodiesterase (PDE) and cAMP-dependent protein kinase (PKA). Experiments were performed to determine the expression of AC, PDE, and PKA isoforms in SH-SY5Y neuroblastoma cells, in which cAMP system was activated by expressing a constitutively activated mutant of stimulatory G protein (Q227L Gsalpha). Expression of ACI mRNA was increased, but levels of ACVIII and ACIX mRNA were decreased. All of the 4 expressed isoforms of PDE (PDE1C, PDE2, PDE 4A, and PDE4B) were increased in mRNA expression; the levels of PKA RIalpha, RIbeta, and RIIbeta were increased moderately, however, those of RIIalpha and Calpha were increased remarkably. The activities of AC, PDE and PKA were also increased in the SH-SY5Y cells expressing Q227L Gsalpha. The similar changes in expression and activity of AC, PDE and PKA were observed in the SH-SY5Y cells treated with dbcAMP for 6 days. Consequently, it is concluded that the cAMP system adapts at the post-receptor level to a sustained activation of the system by differential expression of the isoforms of AC, PDE, and PKA in SH-SY5Y neuroblastoma. We also showed that an increase in cellular cAMP concentration might mediate the observed changes in the cAMP system.
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PMID:Adaptation of cAMP signaling system in SH-SY5Y neuroblastoma cells following expression of a constitutively active stimulatory G protein alpha, Q227L Gsalpha. 1132 85

Phosphatidylinositol 3-kinase (PI3-kinase) activates protein kinase B (also known as Akt), which phosphorylates and activates a cyclic nucleotide phosphodiesterase 3B. Increases in cyclic nucleotide concentrations inhibit agonist-induced contraction of vascular smooth muscle. Thus we hypothesized that the PI3-kinase/Akt pathway may regulate vascular smooth muscle tone. In unstimulated, intact bovine carotid artery smooth muscle, the basal phosphorylation of Akt was higher than that in cultured smooth muscle cells. The phosphorylation of Akt decreases in a time-dependent manner when incubated with the PI3-kinase inhibitor, LY-294002. Agonist (serotonin)-, phorbol ester (phorbol 12,13-dibutyrate; PDBu)-, and depolarization (KCl)-induced contractions of vascular smooth muscles were all inhibited in a dose-dependent fashion by LY-294002. However, LY-294002 did not inhibit serotonin- or PDBu-induced increases in myosin light chain phosphorylation or total O(2) consumption, suggesting that inhibition of contraction was not mediated by reversal or inhibition of the pathways that lead to smooth muscle activation and contraction. Treatment of vascular smooth muscle with LY-294002 increased the activity of cAMP-dependent protein kinase and increased the phosphorylation of the cAMP-dependent protein kinase substrate heat shock protein 20 (HSP20). These data suggest that activation of the PI3-kinase/Akt pathway in unstimulated smooth muscle may modulate vascular smooth muscle tone (allow agonist-induced contraction) through inhibition of the cyclic nucleotide/HSP20 pathway and suggest that cyclic nucleotide-dependent inhibition of contraction is dissociated from the myosin light chain contractile regulatory pathways.
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PMID:PI3-kinase/Akt modulates vascular smooth muscle tone via cAMP signaling pathways. 1156 68

Nitric oxide and endogenous nitrovasodilators regulate smooth muscle tone by elevation of cGMP and activation of cyclic GMP-dependent protein kinase (PKG). The amplitude and duration of the cGMP signal in smooth muscle is regulated in large part by cGMP-specific cyclic nucleotide phosphodiesterase (PDE5). Previous in vitro data have suggested that both cAMP-dependent protein kinase and PKG can regulate the activity of PDE5. To test if this type of regulation is important in the intact cell, we have generated phospho-PDE5-specific antisera and have utilized isolated smooth muscle cells from mice having a disruption in the PKG I gene as well as cells from normal human smooth muscle. The data show that in human smooth muscle cells, activation of PKG by 8-Br-cGMP led to phosphorylation and activation of PDE5. In the same cells, 8-Br-cAMP had no significant effect on PDE5 phosphorylation. Treatment of wild-type mouse aortic smooth muscle cells with 8-Br-cGMP also induced the phosphorylation of PDE5, whereas no phosphorylation was seen in smooth muscle cells isolated from mice in which the gene for PKG I had been disrupted. As with the human cells, no phosphorylation was seen in the mouse cells in response to 8-Br-cAMP. These results strongly suggest that a major regulatory pathway for control of PDE5 phosphorylation and activity in intact smooth muscle is via PKG-dependent phosphorylation of PDE5. Finally, experiments with calyculin A and okadaic acid suggest that PP1 phosphatase, the catalytic subunit of myosin phosphatase, can regulate PDE5 dephosphorylation. Together, the data suggest that phosphorylation and activation of PDE5 by PKG I and its subsequent dephosphorylation by myosin phosphatase may be key steps in the regulation of relaxation/contraction cycles of smooth muscle.
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PMID:Regulation of cGMP-specific phosphodiesterase (PDE5) phosphorylation in smooth muscle cells. 1172 16


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