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 present work was undertaken to determine the action of methylmalonic acid (MMA), a metabolite, which accumulates in high amounts in methylmalonic acidemia, on the endogenous phosphorylating system associated with the cytoskeletal fraction proteins of cerebral cortex of young rats. We demonstrated that pre-treatment of cerebral cortex slices of young rats with 2.5 mM buffered methylmalonic acid (MMA) is effective in decreasing in vitro incorporation of [32P]ATP into neurofilament subunits (NF-M and NF-L) and alpha- and beta-tubulins. Based on the fact that this system contains cAMP-dependent protein kinase (PKA), Ca2+/calmodulin-dependent protein kinase II (CaMKII) and protein phosphatase 1 (PP1), we first tested the effect of MMA on the kinase activities by using the specific activators cAMP and Ca2+/calmodulin or the inhibitors PKAI or KN-93 for PKA and CaMKII, respectively. We observed that MMA totally inhibited the stimulatory effect of cAMP and interfered with the inhibitory effect of PKAI. In addition, the metabolite partially prevented the stimulatory effect of Ca2+/calmodulin and interfered with the effect of KN-93. Furthermore, in vitro dephosphorylation of neurofilament subunits and tubulins was totally inhibited in brain slices pre-treated with MMA. Taken together, these results suggest that MMA, at the same concentrations found in tissues of methylmalonic acidemic children, inhibits the in vitro activities of PKA, CaMKII and PP1 associated with the cytoskeletal fraction of the cerebral cortex of rats, a fact that may be involved with the pathogenesis of the neurological dysfunction characteristic of methylmalonic acidemia.
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PMID:Methylmalonic acid reduces the in vitro phosphorylation of cytoskeletal proteins in the cerebral cortex of rats. 929 63

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

In the present study we demonstrate that propionic acid (PA), a metabolite that accumulates in large amounts in propionic acidemia, is able to decrease in vitro incorporation of [32P]ATP into neurofilament subunits (NF-M and NF-L) and alpha- and beta-tubulin. Considering that the endogenous phosphorylating system associated with the cytoskeletal fraction contains cAMP-dependent protein kinase (PKA), Ca2+/calmodulin protein kinase II (CaMKII), and protein phosphatase 1 (PP1), we first assayed the effect of the acid on the kinase activities by using the specific activators cAMP and Ca2+/calmodulin or the inhibitors PKAI or KN-93 for PKA and CaMKII, respectively. Results demonstrated that the acid totally inhibited the stimulatory effect of cAMP and interfered with the inhibitory effect of PKAI. In addition, PA partially prevented the stimulatory effect of Ca2+/calmodulin and interfered with the effect of KN-93. In addition, we demonstrated that PA totally inhibited in vitro dephosphorylation of neurofilament subunits and tubulins mediated by PP1 in brain slices pretreated with the acid. Taken together, these results demonstrate that PA inhibits the in vitro activities of PKA, CaMKII, and PP1 associated with the cytoskeletal fraction of the cerebral cortex of rats. This study suggests that PA at the same concentrations found in tissues from propionic acidemic children may alter phosphorylation of cytoskeletal proteins, which may contribute to the neurological dysfunction characteristic of propionic acidemia.
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PMID:In vitro phosphorylation of cytoskeletal proteins in the rat cerebral cortex is decreased by propionic acid. 934 49

The signal transduction pathway underlying the cAMP-dependent modulation of rat striatal N-methyl-D-aspartate (NMDA) responses was investigated by using the two-electrode voltage-clamp technique. In oocytes injected with rat striatal poly(A)+ mRNA, activation of cAMP-dependent protein kinase (PKA) by forskolin potentiated NMDA responses. Inhibition of protein phosphatase 1 (PP1) and/or protein phosphatase 2A (PP2A) by the specific inhibitor calyculin A occluded the PKA-mediated potentiation of striatal NMDA responses, suggesting that the PKA effect was mediated by inhibition of a protein phosphatase. Coinjection of oocytes with striatal mRNA and antisense oligodeoxynucleotides directed against the protein phosphatase inhibitor DARPP-32 dramatically reduced the PKA enhancement of NMDA responses. NMDA responses recorded from oocytes injected with rat hippocampal poly(A)+ mRNA were not affected by stimulation of PKA. When oocytes were coinjected with rat hippocampal poly(A)+ mRNA plus complementary RNA coding for DARPP-32, NMDA responses were potentiated after stimulation of PKA. The results provide evidence that DARPP-32, which is enriched in the striatum, may participate in the signaling between the two major afferent striatal pathways, the glutamatergic and the dopaminergic projections, by the cAMP-dependent regulation of striatal NMDA currents.
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PMID:The phosphoprotein DARPP-32 mediates cAMP-dependent potentiation of striatal N-methyl-D-aspartate responses. 940 4

Most enzymes involved in cell signaling, such as protein kinases, protein phosphatases, GTPases, and nucleotide cyclases catalyze nucleophilic substitutions at phosphorus. When possible, the mechanisms of such enzymes are most clearly described quantitatively in terms of how associative or dissociative they are. The mechanisms of cell signaling enzymes range from < or = 8% associative (cAMP-dependent protein kinase) to approximately 50% associative (G protein Gi alpha 1). Their catalytic powers range from 10(5.7) (p21ras) to 10(11.7) (lambda Ser-Thr protein phosphatase), usually comparable in magnitude with those of nonsignaling enzymes of the same mechanistic class. Exceptions are G proteins, which are 10(3)- to 10(5)-fold poorer catalysts than F1 and myosin ATPases. The lower catalytic powers of G proteins may be ascribed to the absence of general base catalysis, and additionally in the case of p21ras, to the absence of a catalytic Arg residue, which interacts with the transition state. From kinetic studies of mutant and metal ion substituted enzymes, the catalytic powers of cell signaling and related enzymes can be rationalized quantitatively by factors contributed by metal ion catalysis (> or = 10(5), general acid catalysis (approximately 10(3 +/- 1)), general base catalysis (approximately 10(3 +/- 1)), and transition-state stabilization by cationic and hydrogen bond donating residues (approximately 10(3 +/- 1)).
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PMID:Mechanisms of signaling and related enzymes. 940 38

A synthetic peptide corresponding to the autophosphorylation site of Ca2+/calmodulin-dependent protein kinase II (CaMKII) (residues 281-289) was conjugated to paramagnetic particles, and phosphorylated by a constitutively active CaMKII fragment. Using this phosphopeptide conjugate as a substrate, a calyculin A-insensitive, Mn(2+)-dependent, and poly-L-lysine-stimulated protein phosphatase activity was detected in the crude extract of rat brain. The protein phosphatase (designated as CaMKII phosphatase) (CaMKIIPase) was purified to near homogeneity from rat brain. CaMKIIPase showed apparent molecular weights of 54,000 and 65,000, on SDS-polyacrylamide gel electrophoresis and gel-filtration analysis, respectively. It was not inhibited by 100 nM calyculin A or 10 microM okadaic acid. Mn2+, but not Mg2+, was absolutely required for activity. CaMKIIPase was potently activated by polycations. Autophosphorylated CaMKII was dephosphorylated by CaMKIIPase, whereas phosphorylase kinase, mixed histones, myelin basic protein, and alpha-casein (which had been phosphorylated by cAMP-dependent protein kinase) and phosphorylase a (phosphorylated by phosphorylase kinase) were not significantly dephosphorylated. No other proteins than CaMKII in rat brain extract which had been phosphorylated by CaMKII were dephosphorylated. The stimulated Ca(2+)-independent activity of autophosphorylated CaMKII was reversed by the action of CaMKIIPase. Thus, CaMKIIPase appears to be a specialized protein phosphatase for the regulation of CaMKII.
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PMID:A novel protein phosphatase that dephosphorylates and regulates Ca2+/calmodulin-dependent protein kinase II. 944 23

The regulation of glycogen synthase (GS) and glycogen phosphorylase (GP) activity by phosphorylation/ dephosphorylation has been proposed to be via changes in activities of several different protein (serine/threonine) phosphatases and kinases, including protein phosphatase (PP) 1/2A, PP2C, and cAMP-dependent protein kinase (PKA). In order to determine whether PP1/2A, PP2C, and/or PKA activities are related to GS and/or GP activities, these enzymes were measured in freeze-clamped liver biopsies obtained under basal fasting conditions from 16 obese monkeys. Four monkeys were normoglycemic and normoinsulinemic, five were hyperinsulinemic, and seven had type 2 diabetes (NIDDM). Liver glycogen and glucose 6-phosphate (G6P) contents were also determine. Basal enzyme activities and basal substrate concentrations were not significantly different between the three group of obese monkeys; however, there were several significant linear relationships observed when the monkeys were treated as one group. Therefore, multiple regression was used to determine the correlation between key variables. GS fractional activity was correlated to GP fractional activity (p < 0.05) and to PP2C activity (p = 0.005) (adjusted R2, 53%). GP independent activity was correlated to GS independent activity (p < 0.07) and to PKA fractional activity (p = 0.005) (adjusted R2, 64%). PP2C activity was correlated to GS fractional activity (p < 0.0005) and to PP1/2A activity (p < 0.0001) (adjusted R2, 83%). PKA fractional activity was correlated to GP total activity (p < 0.0005) and to age (p = 0.001) (adjusted R2, 82%). G6P content was correlated to glycogen content (p < 0.05) and to PP2C activity (p = 0.0005) (adjusted R2, 73%). In conclusion, PP2C and PKA are involved in the regulation of GS and GP activity in the basal state in liver of obese monkeys with a wide range of glucose tolerance.
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PMID:Relationship of glycogen synthase and glycogen phosphorylase to protein phosphatase 2C and cAMP-dependent protein kinase in liver of obese rhesus monkeys. 944 47

1. We examined the effects of noradrenaline on steady-state intracellular pH (pHi) and the recovery of pHi from internal acid loads imposed by the NH4+ prepulse technique in hippocampal CA1 neurones acutely dissociated from adult rats. 2. Under nominally HCO3--free conditions, acid extrusion was accomplished by a Na+-dependent mechanism, probably the amiloride-insensitive variant of the Na+-H+ exchanger previously characterized in both fetal and adult rat hippocampal neurones. In the presence of external HCO3-, acid extrusion appeared to be supplemented by a Na+-dependent HCO3--Cl- exchanger, the activity of which was dependent upon the absolute level of pHi. 3. Noradrenaline evoked a concentration-dependent and sustained rise in steady-state pHi and increased rates of pHi recovery from imposed intracellular acid loads. The effects of noradrenaline were not dependent upon the presence of external HCO3- but were blocked by substituting external Na+ with N-methyl-D-glucamine, suggesting that noradrenaline acts to increase steady-state pHi by increasing the activity of the Na+-H+ exchanger. 4. The effects of noradrenaline on steady-state pHi and on rates of pHi recovery from imposed acid loads were mimicked by beta1- and beta2-, but not alpha-, adrenoceptor agonists. The beta-adrenoceptor antagonist propranolol blocked the ability of noradrenaline to increase both steady-state pHi and rates of pHi recovery from acid loads. 5. The effects of noradrenaline on steady-state pHi and on pHi recovery rates following acid loads were not dependent on changes in [Ca2+]i. However, the effects of noradrenaline were blocked by pre-treatment with the adenylate cyclase inhibitor 2',5'-dideoxyadenosine and the cAMP-dependent protein kinase inhibitors Rp-adenosine-3',5'-cyclic monophosphorothioate (sodium salt; Rp-cAMPS) and N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulphonamide (H-89). 6. Forskolin, an activator of endogenous adenylate cyclase, and 3-isobutyl-1-methylxanthine, a phosphodiesterase inhibitor, mimicked the ability of noradrenaline to increase both steady-state pHi and rates of pHi recovery from imposed acid loads, as did Sp-cAMPS, a selective activator of cAMP-dependent protein kinase. The effect of forskolin on steady-state pHi was blocked by pre-treatment with Rp-cAMPS whereas the effect of Sp-cAMPS was enhanced by pre-treatment with the protein phosphatase inhibitor, okadaic acid. 7. Noradrenaline also increased steady-state pHi and rates of pHi recovery from imposed acid loads in cultured postnatal rat hippocampal neurones. In this preparation, the effects of noradrenaline were occluded by 18-24 h pre-treatment with cholera toxin. 8. We conclude that noradrenaline increases the activity of the Na+-H+ exchanger in rat hippocampal neurones, probably by inducing an alkaline shift in the pHi dependence of the antiport, thereby raising steady-state pHi. The effects of noradrenaline are mediated by beta-adrenoceptors via a pathway which involves the alpha-subunit of the stimulatory G-protein Gs (Gsalpha), adenylate cyclase, cAMP and the subsequent activation of cAMP-dependent protein kinase which, in turn, may phosphorylate the exchange mechanism.
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PMID:Effects of noradrenaline on intracellular pH in acutely dissociated adult rat hippocampal CA1 neurones. 976 38

T cells are important effector cells in natural antiviral and anticancer immunity. It is important to reveal the cellular and molecular requirements for T cell differentiation and effector functions. We explored the idea that the final outcome of antigen receptor-driven immune processes is at least partially determined by physiologically abundant small signaling molecules in extracellular environment of lymphocytes in different tissues. Extracellular purines (ATP and adenosine) and their (purinergic) receptors were studied as an example of such molecules. Studies of functional effects of extracellular ATP and adenosine in immunoregulation have evolved in studies of individual molecules of purinergic receptors and of phosphorylation of extracellular domains of functionally important proteins. ATP-gated membrane pore, p2x 7(formerly p2z receptor) and A2a adenosine receptors are found to be predominantly expressed in T cells. The Gs-protein coupled A2a receptors activate cAMP-dependent protein kinase which was shown to have dual role in regulation of T cells functions. The results of our recent studies of adenosine receptors indicate that A2a receptors on T cell surface may play immunosuppressive role in conditions which lead to accumulation of extracellular adenosine. These conditions include pharmacological intervention with widely used anti-inflammatory drugs (methotrexate and sulfasalazine) and extracellular environment near large solid tumors. Hypoxic conditions in such tumors are known to cause accumulation of extracellular adenosine, which, in turn, as we have shown, could inhibit incoming antitumor cytotoxic T-lymphocytes from destroying the tumor. Normal development and functions of immune cells require adenosine deaminase (ADA) activity. Absence or low levels of ADA in humans result in severe combined immunodeficiency (SCID), which is characterized by hypoplastic thymus, T lymphocyte depletion, and autoimmunity. ADA SCID is currently explained only by intracellular lymphotoxicity of accumulated adenosine. We propose that T cell depletion, immunodeficiency, and autoimmunity could also be due to extracellular adenosine-induced signaling, which inhibits the antigen receptor (TCR) signaling and therefore affects the TCR-driven positive and negative selection of thymocytes. This, in turn, may lead to changes in antigen receptor repertoires and to immunodeficiency, Such properties of adenosine receptors suggest an expanded understanding of pathogenesis of ADA SCID as being due to two independent (intracellular and extracellular) mechanisms of adenosine action. It was conclusively demonstrated that functionally important T cell surface proteins including T cell receptor- are constitutively Ser/Thr phosphorylated on their ectodomains. We identified the major ecto-protein kinase activity in T-lymphocytes as casein kinase II-like (CKII-like) protein kinase. Consensus phosphorylation sites for serine and threonine protein kinases were found to be strongly evolutionary conserved in both alfa and beta TCR chains constant region. We have shown that ecto- or releasable by T-cells protein phosphatase has properties of PP1 and PP2a class protein phosphatase. Such covalent modifications of ectodomains may change T cells cognate interactions by e.g. affecting TCR-multimolecular complex formation and antigen binding affinity. It is suggested that TCR ectodomain phosphorylation could serve as a potential mechanism for regulation of TCR-mediated T-lymphocytes response.
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PMID:Extracellular purines and their receptors in immunoregulation. Review of recent advances. 980 87

Recently, the CLN3 gene associated with Batten disease (juvenile neuronal ceroid lipofuscinosis, JNCL), a recessively inherited, progressive, neurodegenerative disorder of childhood, has been identified. The CLN3 gene encodes a novel protein (battenin) of a predicted 438 amino acids containing several potential posttranslational modifications. We have expressed a full-length CLN3 protein as a C-terminal fusion with green fluorescent protein (GFP) to evaluate whether CLN3 protein is phosphorylated. By using in vivo labeling with 32P, detection with anti-phosphoamino acid antibodies, and phosphoamino acid analysis, we demonstrate that the CLN3 protein is phosphorylated on both serine and threonine residues. We also demonstrate that CLN3 protein is not modified by mannose 6-phosphate. Furthermore, we show that phosphorylation of CLN3 protein is carried out by protein kinase A (cAMP-dependent protein kinase, PKA), protein kinase G (cGMP-dependent protein kinase, PKG), and casein kinase II and that it is enhanced by inhibition of protein phosphatase 1 (PP 1) or protein phosphatase 2A (PP 2A).
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PMID:Evidence for phosphorylation of CLN3 protein associated with Batten disease. 987 58


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