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:2.7.11.11 (AMPK)
12,425 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The catalytic (C) subunit of cAMP-dependent protein kinase (cAPK) is more stable by several criteria when it is part of a holoenzyme complex. By measuring the thermal stability of the free C subunit in the presence and absence of nucleotides and/or divalent metal ions, it was found that most of the stabilizing effects associated with the type I holoenzyme could be attributed to the nucleotide. The specific requirements for this enhanced stability were further dissected: Adenosine stabilized the C subunit up to 5 degrees C; however, divalent cations (i.e., Mg2+, Ca2+, and Mn2+) do not increase heat stability in combination with adenosine and adenine (1). Divalent cations as well as ATP and ADP have no effect by themselves (2). The enhanced stability derived from both ATP and ADP requires divalent cations. MnATP (12 degrees C) shows a much stronger effect than CaATP (7 degrees C) and MgATP (5 degrees C) (3). In the holoenzyme complex or the protein kinase inhibitor/C subunit complex, metal/ATP is also required for enhanced stability; neither the RI or RII subunits nor PKI alone stabilize the C subunit significantly (4). For high thermal stability, the occupation of the second, low-affinity metal-binding site is necessary (5). From these results, we concluded that the adenine moiety works independently from the metal-binding sites, stabilizing the free C subunit by itself. When the beta- and gamma-phosphates are present, divalent metals are required for positioning these phosphates, and two metals are required to achieve thermostability comparable to adenosine alone. The complex containing two metals is the most stable. A comparison of several conformations of the C subunit derived from different crystal structures is given attributing open and closed forms of the C subunit to less and more thermostable enzymes, respectively.
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PMID:Dissection of the nucleotide and metal-phosphate binding sites in cAMP-dependent protein kinase. 1032 Mar 66

Adenosine (Ado) is a potent anti-inflammatory agent acting on a variety of cell functions. However, its effects on human monocytes have been less well characterized. We investigated the effect of Ado and its receptor-specific analogs on NADPH oxidase activity with the use of luminol-enhanced chemiluminescence (CL). Adenosine inhibited fMLP-triggered NADPH oxidase activity with a maximal inhibition of 55+/-5%. IB-MECA, a selective A3 Ado receptor agonist reduced fMLP triggered NADPH oxidase activity more potently than the A2 receptor agonist CGS 2180 HCl (CGS) and the A1 Ado receptor agonist N-2-phenylethyl-adenosine (R-PIA). The inhibitory effect of Ado was reversed by neither the A1 Ado receptor antagonist 1,3-dipropyl-8(2-amino-4chlorophenyl)-xanthine (PACPX) nor the A2 Ado receptor antagonist 3,7-dimethyl-1-(2-propynyl)xanthine (DMPX). It was significantly reversed by the A1/A3 Ado receptor antagonist xanthine amine congener (XAC). Pretreatment of monocytes by cytochalasin B reversed the effect of Ado but not of dibutyryl cAMP (dBcAMP) on fMLP-CL response. KT 5720, a specific cAMP-dependent protein kinase inhibitor completely counteracted the inhibition of NADPH oxidase activity by dBcAMP but not by Ado. Using flow cytometry, we observed that Ado did not inhibit intracellular oxidative metabolism, whereas dBcAMP did. Furthermore, the inhibition of NADPH oxidase activity by Ado was not mediated by changes in cytosolic calcium. These results demonstrated that Ado inhibited NADPH oxidase activity via A3 Ado receptor independently of cAMP elevation or changes in calcium mobilization.
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PMID:Inhibition of fMLP-triggered respiratory burst of human monocytes by adenosine: involvement of A3 adenosine receptor. 1049 21

Adenosine acting via A2a receptors (A2aR) is a potent cerebral vasodilator that relaxes vascular smooth muscle cells (VSMCs) by a mechanism attributed to activation of cAMP-dependent protein kinase (cAK). We examined effects of adenosine and its mechanism of action on L-type Ca2+ channels in native VSMCs from rat basilar artery. Reverse transcription-polymerase chain reaction and immunofluorescence imaging confirmed transcription and expression of A2aR, and in situ hybridization confirmed presence of mRNA for L-type Cav1.2b channels. In patch-clamp experiments, adenosine down-regulated Ca2+ channel currents in a concentration-dependent manner, with receptor-subtype-specific antagonists [4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo-[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM-241385) versus 1,3-dipropyl-8-cyclopentyl-1,3-dipropylxanthine] showing that this was caused by action of A2aR. Down-regulation of channel currents was mimicked by stimulation of cGMP-dependent protein kinase (cGK; 8-Br-cGMP) and by inhibition of tyrosine kinase (AG-18) but not by stimulation of cAK [forskolin and 8-bromo-cAMP (8-Br-AMP)]. Down-regulation of currents by the A2aR agonist 2-[p-(2-carboxyeth yl)phenylethylamino]-5'-N-ethyolcarboxamidoadenosine (CGS-21680) was blocked by inhibiting protein tyrosine phosphatase (PTP; orthovanodate and dephostatin), but not by inhibiting cGK (KT-5823 and H-7). Western blots of lysate or of immunoisolated Ca2+ channels from arterial segments incubated with CGS-21680 showed 1) increased phosphorylation of vasodilator-stimulated phosphoprotein that was blocked by inhibiting cAK (KT-5720), consistent with activation of cAK by A2aR; and 2) decreased tyrosine phosphorylation of immunoisolated alpha1c subunit of the Ca2+ channel. Our data show that cAK, although activated, was not germane to down-regulation of Ca2+ channel activity by A2aR, and they delineate a novel signaling mechanism involving reduced tyrosine phosphorylation of Ca2+ channels by A2aR probably caused by PTP activation.
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PMID:Adenosine-A2a receptor down-regulates cerebral smooth muscle L-type Ca2+ channel activity via protein tyrosine phosphatase, not cAMP-dependent protein kinase. 1292 Feb

Adenosine A(2B) receptors have been suggested to influence cell differentiation and proliferation. Human adenosine A(2B) receptors expressed in Chinese hamster ovary cells mediate phosphorylation and activation of the extracellular signal-regulated kinase (ERK1/2). Already low concentrations of agonists such as 5'-N-ethylcarboxamidoadenosine (NECA) are effective. Phosphorylation of the stress-activated protein kinase p38 was also potently induced by NECA (EC(50) 18.5 nM). These NECA-induced effects were mimicked by forskolin and 8-Br-cAMP. Inhibition of cAMP-dependent protein kinase (PKA) using H89 (N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide)) blocked phosphorylation of the cAMP response element-binding protein (CREB) and p38, but did not decrease NECA-induced ERK1/2 phosphorylation. NECA activated the small GTPase Rap1, and this was also not blocked by H89. Inhibition of phosphatidylinositol-3'-kinase (PI3K) by wortmannin inhibited adenosine A(2B) receptor-mediated ERK1/2 phosphorylation and activation of Rap1, without affecting CREB and p38 phosphorylation. A(2B) receptor-stimulated protein kinase B phosphorylation was sensitive to wortmannin, but not to H89. Thus, stimulation of adenosine A(2B) receptors activates both ERK1/2 and p38 via cAMP, but the downstream pathways are markedly different. ERK1/2 activation was dependent on PI3K but not on PKA. p38 activation by NECA was instead independent of PI3K but required cAMP and PKA. The potent activation of both MAPKs suggests a physiological role.
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PMID:The G(s)-coupled adenosine A(2B) receptor recruits divergent pathways to regulate ERK1/2 and p38. 1451 97

[3H] Adenosine-3',5'-cyclic monophosphate (cAMP) could be entrapped efficiently into small unilamellar vesicles when bound to cAMP-dependent protein kinase. The leakage of [3H]cAMP protein kinase complex from liposomes was reduced by more than 60% as compared to free [3H]cAMP. Hyperosmolar mannitol increased the delivery of liposomally entrapped [3H]cAMP protein kinase to the brain with maximum uptake occurring at 10 min after mannitol administration. Optimal delivery to the brain was observed when vesicles composed of total brain lipids or phosphatidylcholine:cholesterol:sulfatides (7:2:1) were used. A slower clearance of liposomally entrapped material from brain tissue was seen under hyperosmolar conditions.
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PMID:Uptake of liposomally entrapped adenosine-3'-5'-cyclic monophosphate in mouse brain. 1500 42

Adenosine monophosphate (AMP)-activated protein kinase (AMPK) is a heterotrimeric complex that senses intracellular energy status and exerts rapid regulation on energy-demanding and -consuming metabolic pathways. Although alterations in the intracellular adenosine nucleotide pool are traditionally assumed to be the consequence of changes in energy metabolism, in this study we have addressed the question of whether extracellular adenosine contributes to AMPK regulation. In the intestinal rat epithelial cell line IEC-6, addition of adenosine rapidly increases AMP intracellular concentrations and upregulates alpha1AMPK, thus promoting phosphorylation of its downstream target acetyl-CoA carboxylase (ACC). The effect of adenosine on AMPK signaling is completely blocked by transducing IEC-6 cells with an adenoviral vector expressing a mutated alpha1 subunit, resulting in a dominant-negative effect on endogenous AMPK activity. These effects are blocked by 5'-iodotubercidine (5'-ITU), an inhibitor of adenosine kinase. Moreover, inhibition of adenosine transport through the concentrative adenosine plasma membrane transporter CNT2 with formycin B results in the blockade of adenosine-mediated AMPK signaling. Extracellular adenosine is equally able to activate AMPK and promote ACC phosphorylation in liver parenchymal cell models in a manner that is also inhibited by 5'-ITU. In summary, this study shows that adenosine, when added at physiological concentrations, activates AMPK and promotes ACC phosphorylation. Adenosine must be transported and phosphorylated to exert its action. Thus, nucleoside transporters might be novel players in the complex regulation of AMPK and energy metabolism.
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PMID:Extracellular adenosine activates AMP-dependent protein kinase (AMPK). 1656 64

Adenosine is known to modulate the function of neostriatal neurons. Adenosine acting on A(2A) receptors increases the phosphorylation of dopamine- and cAMP-regulated phosphoprotein of M(r) 32 kDa (DARPP-32) at Thr34 (the cAMP-dependent protein kinase [PKA] site) in striatopallidal neurons, and opposes dopamine D2 receptor signaling. In contrast, the role of adenosine A(1) receptors in the regulation of dopamine/DARPP-32 signaling is not clearly understood. Here, we investigated the effect of adenosine A(1) receptors on D(1), D(2) and A(2A) receptor signaling using mouse neostriatal slices. An A(1) receptor agonist, 2-chloro-N(6)-cyclopentyladenosine (100 nM), caused a transient increase, followed by a transient decrease, in DARPP-32 Thr34 phosphorylation. Our data support the following model for the actions of the A(1) receptor agonist. The A(1) receptor-induced early increase in Thr34 phosphorylation was mediated by presynaptic inhibition of dopamine release, and the subsequent removal of tonic inhibition by D(2) receptors of A(2A) receptor/G(olf)/cAMP/PKA signaling. The A(1) receptor-induced late decrease in Thr34 phosphorylation was mediated by a postsynaptic G(i) mechanism, resulting in inhibition of D(1) and A(2A) receptor-coupled G(olf)/cAMP/PKA signaling in direct and indirect pathway neurons, respectively. In conclusion, A(1) receptors play a major modulatory role in dopamine and adenosine receptor signaling.
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PMID:Role of adenosine A1 receptors in the modulation of dopamine D1 and adenosine A2A receptor signaling in the neostriatum. 1675 Aug 92

Conjugates of oligoarginine peptides with adenine, adenosine, adenosine-5'-carboxylic acid, and 5-isoquinolinesulfonic acid were synthesized and characterized as bisubstrate-analog inhibitors of cAMP-dependent protein kinase. Adenosine and adenine derivatives were connected to the N- or C-terminus of peptides containing four to six L- or D-arginine residues via a linker with a length that had been optimized in structure-activity studies. The orientation of the peptide chain strongly affected the activity of compounds incorporating D-arginines. The biligand inhibitor containing Hidaka's H9 isoquinolinesulfonamide connected to the L-peptide had 65 times higher potency than the corresponding adenosine-containing conjugate, while both types of the conjugate comprising D-peptides had similar low nanomolar activity. Two of the most active adenosine- and H9-peptide conjugates were tested in the panel of 52 different kinases. At 1 microM concentration, both compounds showed strong (more than 95%) inhibition of several basophilic AGC kinases, including pharmaceutically important kinases ROCK II and PKB/Akt.
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PMID:Conjugation of adenosine and hexa-(D-arginine) leads to a nanomolar bisubstrate-analog inhibitor of basophilic protein kinases. 1712 67

Phosphorylation on the activation loop of AGC kinases is typically mediated by PDK1. The precise mechanism for this in-trans phosphorylation is unknown; however, docking of a hydrophobic (HF) motif in the C-tail of the substrate kinase onto the N-lobe of PDK1 is likely an essential step. Using a peptide array of PKA to identify other PDK1-interacting sites, we discovered a second AGC-conserved motif in the C-tail that interacts with PDK1. Since this motif [FD(X)(1-2)Y/F] lies in the active site tether region and in PKA contributes to ATP binding, we call it the Adenosine binding (Ade) motif. The Ade motif is conserved as a PDK1-interacting site in Akt and PRK2, and we predict it will be a PDK1-interacting site for most AGC kinases. In PKA, the HF motif is only recognized when the turn motif Ser338 is phosphorylated, possibly serving as a phosphorylation "switch" that regulates how the Ade and HF motifs interact with PDK1. These results demonstrate that the extended AGC C-tail serves as a polyvalent element that trans-regulates PDK1 for catalysis. Modeling of the PKA C-tail onto PDK1 structure creates two chimeric sites; the ATP binding pocket, which is completed by the Ade motif, and the C-helix, which is positioned by the HF motif. Together, they demonstrate substrate-assisted catalysis involving two kinases that have co-evolved as symbiotic partners. The highly regulated turn motifs are the most variable part of the AGC C-tail. Elucidating the highly regulated cis and trans functions of the AGC tail is a significant future challenge.
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PMID:A chimeric mechanism for polyvalent trans-phosphorylation of PKA by PDK1. 1953 Feb 48

This work evaluates adenosine effects on Sertoli cell functions, which are different to those resulting from occupancy of purinergic receptors. The effects of adenosine and N(6)-cyclohexyladenosine (CHA) - an A(1) receptor agonist resistant to cellular uptake - on Sertoli cell physiology were compared. Adenosine but not CHA increased lactate production, glucose uptake, GLUT1, LDHA and MCT4 mRNA levels, and stabilized ZO-1 protein at the cell membrane. These differential effects suggested a mechanism of action of adenosine that cannot be solely explained by occupancy of type A(1) purinergic receptors. Activation by adenosine but not by CHA of AMPK was observed. AMPK participation in lactate production and ZO-1 stabilization was confirmed by utilizing specific inhibitors. Altogether, these results suggest that activation of AMPK by adenosine promotes lactate offer to germ cells and cooperates in the maintenance of junctional complex integrity, thus contributing to the preservation of an optimum microenvironment for a successful spermatogenesis.
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PMID:Adenosine regulates Sertoli cell function by activating AMPK. 2072 79


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