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

Recently, we have reported that the inhibition of mitochondrial respiration by nitric oxide (NO) leads to an up-regulation of glycolysis and affords cytoprotection against energy failure through the stimulation of AMPK (5'-AMP-activated protein kinase) [Almeida, Moncada and Bolanos (2004) Nat. Cell Biol. 6, 45-51]. To determine whether glucose transport contributes specifically to this effect, we have now investigated the possible role of NO in modulating glucose uptake through GLUT3, a facilitative high-affinity glucose carrier that has been suggested to afford cytoprotection against hypoglycaemic episodes. To do so, GLUT3-lacking HEK-293T cells (human embryonic kidney 293T cells) were transformed to express a plasmid construction encoding green fluorescent protein-tagged GLUT3 cDNA. This carrier was preferentially localized to the plasma membrane, was seen to be functionally active and afforded cytoprotection against low glucose-induced apoptotic death. Inhibition of mitochondrial respiration by NO triggered a rapid, cGMP-independent enhancement of GLUT3-mediated glucose uptake through a mechanism that did not involve transporter translocation. Furthermore, the functional disruption of AMPK by the RNA interference strategy rendered cells unable to respond to NO by activating GLUT3-mediated glucose uptake. These results suggest that the inhibition of mitochondrial respiration by NO activates AMPK to stimulate glucose uptake, thereby representing a novel survival pathway during pathophysiological conditions involving transient reductions in the supply of cellular glucose.
 ...
PMID:Inhibition of mitochondrial respiration by nitric oxide rapidly stimulates cytoprotective GLUT3-mediated glucose uptake through 5'-AMP-activated protein kinase. 1532 Aug 70

The function of beta-adrenergic receptor (betaAR) is modulated by the activity status of alpha1-adrenergic receptors (alpha1ARs) via molecular crosstalk, and this becomes evident when measuring cardiac contractile responses to adrenergic stimulation. The molecular mechanism underlying this crosstalk is unknown. We have previously demonstrated that overexpression of alpha1B-adrenergic receptor (alpha1BAR) in transgenic mice leads to a marked desensitization of betaAR-mediated adenylyl cyclase stimulation which is correlated with increased levels of activated protein kinase C (PKC) beta, delta and [J. Mol. Cell. Cardiol. 30 (1998) 1827]. Therefore, we wished to determine which PKC isoforms play a role in heterologous betaAR desensitization and also which isoforms of the betaAR were the molecular target(s) for PKC. In experiments using constitutively activated PKC expression constructs transfected into HEK 293 cells also expressing the beta2AR, constitutively active (CA)-PKC overexpression was first confirmed by immunoblots using specific anti-PKC antibodies. We then demonstrated that the different PKC subtypes lead to a decreased maximal cAMP accumulation following isoproterenol stimulation with a rank order of PKCalpha > or = PKCzeta>PKC>PKCbetaII. However, a much more dramatic desensitization of adenylyl cyclase stimulation was observed in cells co-transfected with different PKC isoforms and beta1AR. Further, the modulation of beta1AR by PKC isoforms had a different rank order than for the beta2AR: PKCbetaII>PKCalpha>PKC>PKCzeta. PKC-mediated desensitization was reduced by mutating consensus cAMP-dependent protein kinase (PKA)/PKC sites in the third intracellular loop and/or the carboxy-terminal tail of either receptor. Our results demonstrate therefore that the beta1AR is the most likely molecular target for PKC-mediated heterologous desensitization in the mammalian heart and that modulation of adrenergic receptor activity in any given cell type will depend on the complement of PKC isoforms present.
 ...
PMID:Role of specific protein kinase C isoforms in modulation of beta1- and beta2-adrenergic receptors. 1545 Oct 24

Phosphorylation of G protein-coupled receptors is a critical step in the rapid termination of G protein signaling. In rod cells of the vertebrate retina, phosphorylation of rhodopsin is mediated by GRK1. In cone cells, either GRK1, GRK7, or both, depending on the species, are speculated to initiate signal termination by phosphorylating the cone opsins. To compare the biochemical properties of GRK1 and GRK7, we measured the K(m) and V(max) of these kinases for ATP and rhodopsin, a model substrate. The results demonstrated that these kinases share similar kinetic properties. We also determined that cAMP-dependent protein kinase (PKA) phosphorylates GRK1 at Ser(21) and GRK7 at Ser(23) and Ser(36) in vitro. These sites are also phosphorylated when FLAG-tagged GRK1 and GRK7 are expressed in HEK-293 cells treated with forskolin to stimulate the endogenous production of cAMP and activation of PKA. Rod outer segments isolated from bovine retina phosphorylated the FLAG-tagged GRKs in the presence of dibutyryl-cAMP, suggesting that GRK1 and GRK7 are physiologically relevant substrates. Although both GRKs also contain putative phosphorylation sites for PKC and Ca(2+)/calmodulin-dependent protein kinase II, neither kinase phosphorylated GRK1 or GRK7. Phosphorylation of GRK1 and GRK7 by PKA reduces the ability of GRK1 and GRK7 to phosphorylate rhodopsin in vitro. Since exposure to light causes a decrease in cAMP levels in rod cells, we propose that phosphorylation of GRK1 and GRK7 by PKA occurs in the dark, when cAMP levels in photoreceptor cells are elevated, and represents a novel mechanism for regulating the activities of these kinases.
 ...
PMID:Phosphorylation of GRK1 and GRK7 by cAMP-dependent protein kinase attenuates their enzymatic activities. 1594 41

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.
 ...
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

NR2C-containing N-methyl-D-aspartate (NMDA) receptors are highly expressed in cerebellar granule cells where they mediate the majority of current in the adult. NMDA receptors composed of NR1/NR2C exhibit a low conductance and reduced sensitivity to Mg(2+), compared with the more commonly studied NR2A- and NR2B-containing receptors. Despite these interesting features, very little is known about the regulation of NR2C function. Here we investigate the role of phosphorylation of NR2C in regulating NMDA receptor trafficking and ion channel properties. We identify a phosphorylation site, serine 1244 (Ser(1244)), near the extreme COOH terminus of NR2C, which is phosphorylated by both cAMP-dependent protein kinase and protein kinase C. This residue is located adjacent to the consensus PDZ ligand, a region that regulates protein-protein interactions and receptor trafficking in NR2A and NR2B. We show that Ser(1244) on NR2C is phosphorylated in vitro, in heterologous cells, and in neurons. Moreover, we demonstrate for the first time that NR2C interacts with the PSD-95 family of PDZ domain-containing proteins but that phosphorylation of Ser(1244) does not influence this PDZ interaction. Furthermore, Ser(1244) phosphorylation does not regulate surface expression of NR1/NR2C receptors. However, we find that this site does regulate the kinetics of the ion channel: a phosphomimetic mutation at Ser(1244) accelerates both the rise and decay of NMDA-evoked currents in excised patches from HEK-293 cells. Therefore, phosphorylation of Ser(1244) does not regulate trafficking but unexpectedly affects ion channel function, suggesting that phosphorylation of Ser(1244) on NR2C may be important in defining the functional properties of NMDA receptor-mediated currents in the cerebellum.
 ...
PMID:Regulation of NR1/NR2C N-methyl-D-aspartate (NMDA) receptors by phosphorylation. 1660 16

Challenge of the beta(2)Ar (beta(2)-adrenergic receptor) with isoprenaline in HEK-293beta(2) cells (human embryonic kidney cells stably overexpressing a FLAG- and green fluorescent protein-tagged beta(2)Ar) results in the PKA (cAMP-dependent protein kinase) phosphorylation of GRK2 (G-protein receptor kinase-2). This response was enhanced when PDE4 (phosphodiesterase-4) activity was attenuated using either rolipram, a PDE4-selective inhibitor, or with siRNA (small interfering RNA) knockdown of both PDE4B and PDE4D. Rolipram also facilitated GRK2 recruitment to the membrane and phosphorylation of the beta(2)Ar by GRK2 in response to isoprenaline challenge of cells. In resting cells, rolipram treatment alone is sufficient to promote PKA phosphorylation of GRK2, with consequential effects on GRK2 translocation and GRK2 phosphorylation of the beta(2)Ar. Similar effects are observed in cardiac myocytes. We propose that PDE4 activity protects GRK2 from inappropriate phosphorylation by PKA in resting cells that might have occurred through fluctuations in basal cAMP levels. Thus PDE4 gates the action of PKA to phosphorylate GRK2.
 ...
PMID:Phosphodiesterase-4 gates the ability of protein kinase A to phosphorylate G-protein receptor kinase-2 and influence its translocation. 1685 36

Based on a variety of single-cell measurements, the notion that cAMP microdomains exist in cells is being increasingly embraced. The cellular and molecular underpinnings of this organization are also steadily being revealed. A dependence of Ca(2+)-sensitive ACs (adenylate cyclases) in HEK-293 cells (human embryonic kidney cells) on capacitative Ca(2+) entry is enforced by their presence in lipid rafts and protein-protein interactions. In these cells, many of the participants in the cAMP cascade, including AC, phosphodiesterase 4, cAMP-dependent protein kinase [PKA (protein kinase A)] and protein phosphatase 2A, are now seen to be involved in higher order assemblies. Moreover, the presence of Na(+)/H(+) exchanger 1 in these domains creates a microclimate, protected against global swings in cellular pH. The Ca(2+)-stimulatable AC8, which is targeted to these regions, can sequester calmodulin for its own regulatory purposes. These devices are a sampling of the multiple layers of organization that are in place -- even in a simple cell -- to ensure faithful and economical communication of the cAMP message.
 ...
PMID:Layers of organization of cAMP microdomains in a simple cell. 1685 38

Phospholamban (PLN) is a key regulator of Ca(2+) homeostasis and contractility in the heart. Its regulatory effects are mediated through its interaction with the sarcoplasmic reticulum Ca(2+)-ATPase, (SERCA2a), resulting in alterations of its Ca(2+)-affinity. To identify additional proteins that may interact with PLN, we used the yeast-two-hybrid system to screen an adult human cardiac cDNA library. HS-1 associated protein X-1 (HAX-1) was identified as a PLN-binding partner. The minimal binding regions were mapped to amino acid residues 203-245 for HAX-1 and residues 16-22 for PLN. The interaction between the two proteins was confirmed using GST-HAX-1, bound to the glutathione-matrix, which specifically adsorbed native PLN from human or mouse cardiac homogenates, while in reciprocal binding studies, recombinant His-HAX-1 bound GST-PLN. Kinetic studies using surface plasmon resonance yielded a K(D) of approximately 1 muM as the binding affinity for the PLN/HAX-1 complex. Phosphorylation of PLN by cAMP-dependent protein kinase reduced binding to HAX-1, while increasing concentrations of Ca(2+) diminished the PLN/HAX-1 interaction in a dose-dependent manner. HAX-1 concentrated to mitochondria, but upon transient co-transfection of HEK 293 cells with PLN, HAX-1 redistributed and co-localized with PLN at the endoplasmic reticulum. Analysis of the anti-apoptotic function of HAX-1 revealed that the presence of PLN enhanced the HAX-1 protective effects from hypoxia/reoxygenation-induced cell death. These findings suggest a possible link between the Ca(2+) handling by the sarcoplasmic reticulum and cell survival mediated by the PLN/HAX-1 interaction.
 ...
PMID:Phospholamban interacts with HAX-1, a mitochondrial protein with anti-apoptotic function. 1724 41

AS160 (Akt substrate of 160 kDa) mediates insulin-stimulated GLUT4 (glucose transporter 4) translocation, but is widely expressed in insulin-insensitive tissues lacking GLUT4. Having isolated AS160 by 14-3-3-affinity chromatography, we found that binding of AS160 to 14-3-3 isoforms in HEK (human embryonic kidney)-293 cells was induced by IGF-1 (insulin-like growth factor-1), EGF (epidermal growth factor), PMA and, to a lesser extent, AICAR (5-aminoimidazole-4-carboxamide-1-b-D-ribofuranoside). AS160-14-3-3 interactions were stabilized by chemical cross-linking and abolished by dephosphorylation. Eight residues on AS160 (Ser318, Ser341, Thr568, Ser570, Ser588, Thr642, Ser666 and Ser751) were differentially phosphorylated in response to IGF-1, EGF, PMA and AICAR. The binding of 14-3-3 proteins to HA-AS160 (where HA is haemagglutinin) was markedly decreased by mutation of Thr642 and abolished in a Thr642Ala/Ser341Ala double mutant. The AGC (protein kinase A/protein kinase G/protein kinase C-family) kinases RSK1 (p90 ribosomal S6 kinase 1), SGK1 (serum- and glucocorticoid-induced protein kinase 1) and PKB (protein kinase B) displayed distinct signatures of AS160 phosphorylation in vitro: all three kinases phosphorylated Ser318, Ser588 and Thr642; RSK1 also phosphorylated Ser341, Ser751 and to a lesser extent Thr568; and SGK1 phosphorylated Thr568 and Ser751. AMPK (AMP-activated protein kinase) preferentially phosphorylated Ser588, with less phosphorylation of other sites. In cells, the IGF-1-stimulated phosphorylations, and certain EGF-stimulated phosphorylations, were inhibited by PI3K (phosphoinositide 3-kinase) inhibitors, whereas the RSK inhibitor BI-D1870 inhibited the PMA-induced phosphorylations. The expression of LKB1 in HeLa cells and the use of AICAR in HEK-293 cells promoted phosphorylation of Ser588, but only weak Ser341 and Thr642 phosphorylations and binding to 14-3-3s. Paradoxically however, phenformin activated AMPK without promoting AS160 phosphorylation. The IGF-1-induced phosphorylation of the novel phosphorylated Ser666-Pro site was suppressed by AICAR, and by combined mutation of a TOS (mTOR signalling)-like sequence (FEMDI) and rapamycin. Thus, although AS160 is a common target of insulin, IGF-1, EGF, PMA and AICAR, these stimuli induce distinctive patterns of phosphorylation and 14-3-3 binding, mediated by at least four protein kinases.
 ...
PMID:Regulation of multisite phosphorylation and 14-3-3 binding of AS160 in response to IGF-1, EGF, PMA and AICAR. 1761 58

The PDE4 (phosphodiesterase-4) enzyme family consists of a distinct array of N-terminal splice variant isoforms arising from four subfamily genes (4A, 4B, 4C and 4D). These all hydrolyse specifically the intracellular second messenger cAMP. Although identical in catalytic function, each isoform appears to serve a non-superfluous regulatory role. For example, a beta-arrestin-sequestered subpopulation of the PDE4D5 isoform specifically regulates the phosphorylation of the beta(2)-AR (beta(2)-adrenergic receptor) by PKA (protein kinase A; also called cAMP-dependent protein kinase). This was elucidated by the use of novel technologies, including dominant-negative approaches, siRNA (small interfering RNA) knockdown and spot-immobilized peptide array analyses. Functional phenotypes uncovered using these methodologies have shown that beta-arrestin-sequestered PDE4D5 shapes the spatial cAMP gradient around the membrane-bound beta(2)-AR, regulating its phosphorylation by PKA and its ability to activate ERK (extracellular-signal-regulated kinase) through G(i) in cardiomyocytes and HEK-293 (human embryonic kidney)-B2 cells. This approach has provided the very first identification of a non-redundant and specific role for a PDE isoform. The fact that phenotypes can be uncovered by displacing PDE4 isoforms from specific anchor sites using dominant-negative constructs and cell-permeable peptides points to novel means for developing therapeutics aimed at disrupting specifically sequestered PDE isoforms and even specifically sequestered subpopulations of individual isoforms.
 ...
PMID:cAMP-specific phosphodiesterase-4D5 (PDE4D5) provides a paradigm for understanding the unique non-redundant roles that PDE4 isoforms play in shaping compartmentalized cAMP cell signalling. 1795 50


<< Previous 1 2 3 4 5 Next >>