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 transmitter dopamine reduces electrotonic coupling between retinal horizontal cells and increases their sensitivity to glutamate. Since in other systems single afferents establish mixed electrotonic and chemical excitatory synapses with their targets, dopamine might be expected there to depress one component of excitation while enhancing the other. This hypothesis was tested by applying dopamine locally in the vicinity of the lateral dendrite of the goldfish Mauthner cell (M cell) and monitoring the composite electrotonic and chemical excitatory postsynaptic potentials and currents evoked by ipsilateral eighth nerve stimulation. Dopamine produces persistent enhancements of both components of the postsynaptic response while it also increases input conductance. All these dopamine actions are prevented by superfusing the brain with saline containing the dopamine D1 receptor antagonist SCH-23390. Postsynaptic injections of the cAMP-dependent protein kinase inhibitor (Walsh inhibitor, or PKI5-24) block the dopamine-induced changes in synaptic transmission, implicating a cAMP-dependent mechanism. Furthermore, there is a dopaminergic innervation of the M cell, as demonstrated immunohistochemically with antibodies against dopamine and the rate-limiting enzyme in its synthetic pathway, tyrosine hydroxylase. Varicose immunoreactive fibers lie in the vicinity of the distal part of the lateral dendrite between the large myelinated club endings that establish the mixed synapses. As determined with electron microscopy, the dopaminergic fibers contain small vesicles, and they do not have synaptic contacts with either the afferents or the M cell, remaining instead in the synaptic bed. Taken together, these results suggest that dopamine released at a distance from these terminals increases the gain of this primary sensory input to the M cell, most likely through a phosphorylation mechanism.
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PMID:Dopamine enhances both electrotonic coupling and chemical excitatory postsynaptic potentials at mixed synapses. 133 56

We have investigated the role of protracted phosphatase inhibition and the consecutive protracted protein phosphorylation on neuronal viability. We found that in primary cultures of cerebellar granule neurons, the protracted (24-h) inhibition of the serine/threonine protein phosphatases 1 and 2A (EC 3.1.3.16) by treatment of the cultures with okadaic acid (OKA; 5-20 nM) caused neurotoxicity that could be inhibited by the protein kinase inhibitor 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7) or by the previous down-regulation of the neuronal protein kinase C (PKC; ATP:protein phosphotransferase; EC 2.7.1.37). PKC was down-regulated by exposure of the cultures for 24 h to 100 nM phorbol 12-myristate 13-acetate (TPA). The effect of the drugs used in the viability studies on the pattern of protein phosphorylation was measured by quantitative autoradiography. In particular, the 50- and 80-kDa protein bands showed dramatic changes in the degree of phosphorylation: increase by OKA and brief TPA treatment; decrease by H7 or 24 h of TPA treatment; and inhibition of the OKA-induced increase by H7 or 24 h of TPA treatment. The results suggest that the protracted phosphorylation, in particular that mediated by PKC, may lead to neuronal death and are in line with our previous suggestion that prolonged PKC translocation is operative in glutamate neurotoxicity.
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PMID:Pathological phosphorylation causes neuronal death: effect of okadaic acid in primary culture of cerebellar granule cells. 140 5

Our present work characterized the role of hormone-mediated signal transduction pathways in regulating hepatic reduced glutathione (GSH) synthesis. Cholera toxin, dibutyryl cAMP (DBcAMP), and glucagon inhibited GSH synthesis in cultured hepatocytes by 25-43%. Cellular cAMP levels exhibited a lower threshold for stimulation of the GSH efflux than inhibition of its synthesis. The effect of DBcAMP was independent of the type of sulfur amino acid precursor and cellular ATP levels and unassociated with increased GSH mixed disulfide formation or altered GSH/oxidized glutathione ratio. In liver cytosols, addition of DBcAMP and cAMP-dependent protein kinase (A-kinase) inhibited GSH synthesis from substrates (cysteine, ATP, glutamate, and glycine) by approximately 20% which was prevented by the A-kinase inhibitor. However, if only substrates of the second step in GSH synthesis were used (gamma-glutamylcysteine, glycine, and ATP), DBcAMP and A-kinase exerted no inhibitory effect. Phenylephrine, vasopressin, and phorbol ester also inhibited GSH synthesis in cultured cells by approximately 20%, and depleted cell GSH independent of the type of sulfur amino acid precursor. Cellular cysteine level was unchanged despite the significant fall in GSH after glucagon or phenylephrine treatment. Pretreatment with either staurosporine, C-kinase inhibitor, or calmidazolium, a calmodulin inhibitor, partially prevented but, together, completely prevented the inhibitory effect of phenylephrine. The same combination had no effect on the inhibitory effect of glucagon. The effects of hormones were confirmed in both the intact perfused liver and after in vivo administration. Thus, two classes of hormones acting through distinct signal transduction pathways may down-regulate hepatic GSH synthesis by phosphorylation of gamma-glutamylcysteine synthetase.
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PMID:Hormone-mediated down-regulation of hepatic glutathione synthesis in the rat. 164 17

Exposing primary cultures of cerebellar granule neurons to 100 nM phorbol 12-myristate 13-acetate (PMA) for 24 hr decreases the Ca2+/phosphatidylserine/diolein-dependent protein kinase C (PKC; ATP:protein phosphotransferase, EC 2.7.1.37) by approximately 90% in the 100,000 x g supernatant and pellet fractions of neuronal culture homogenates. Immunoblot analysis of the homogenates with polyclonal antibodies raised against either the beta-type PKC peptide or total rat brain PKC reveals a virtual loss of 78-kDa PKC immunoreactivity in the supernatant and a marked decrease of PKC immunoreactivity in the pellet. Exposure of the cultures to 50 microM glutamate for 15 min (no Mg2+) induces the translocation of supernatant PKC immunoreactivity to the pellet. Such translocation persists after glutamate withdrawal and is followed by a progressive increase in neuronal death, which begins 2 hr later. Neuronal death approaches completion in about 24 hr. PMA-induced down-regulation of PKC decreases glutamate-elicited neurotoxicity. Yet, the culture exposure to 100 nM PMA fails to decrease the high-affinity binding of [3H]glutamate to neuronal membranes and does not reduce glutamate-induced activation of ionotropic or metabolotropic receptors (assayed as total membrane current measured in whole-cell voltage-clamped neurons, 45Ca2+ uptake in intact monolayers, inositolphospholipid hydrolysis, and transcriptional activation and translation of c-fos mRNA). Moreover, the immediate cell-body swelling and activation of spectrin proteolysis elicited by glutamate remain unchanged. On the other hand, PMA-induced PKC down-regulation reduces any increase in 45Ca2+ uptake or Ca2(+)-dependent proteolysis (measured as spectrin degradation) after glutamate withdrawal. These results support the view that PKC translocation is operative in glutamate-induced destabilization of cytosolic ionized Ca2+ homeostasis and neuronal death.
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PMID:Down-regulation of protein kinase C protects cerebellar granule neurons in primary culture from glutamate-induced neuronal death. 168 50

"Charged-to alanine" scanning mutagenesis of the catalytic subunit of the Saccharomyces cerevisiae cAMP-dependent protein kinase (C1) identified three glutamate residues, E171, E214, and E274, that are involved in the recognition of a peptide substrate, kemptide (Leu1Arg2Arg3Ala4Ser5Leu6Gly7). These glutamate residues are conserved or conservatively substituted with asparate in the serine/threonine protein kinases that have a requirement for basic residues on the N-terminal side of their phosphorylation sites. Alanine replacement mutants in C1 were subjected to kinetic analysis using alanine-substituted peptides as substrates. The additivity or nonadditivity of the effects of the alanine substitutions on the catalytic efficiency (kcat/Km) was analyzed. This allowed the identification of electrostatic interactions between the three glutamate residues in the enzyme and the two arginine residues present in the peptide substrate. The data suggest that E171 interacts with Arg2 in the substrate and that E214 and E274 both interact with Arg3. This may be a general method for identifying simple intermolecular interactions involving proteins when there is no three-dimensional structure available of the complex of interacting species. The identification of these interactions provides the potential for rational protein engineering of enzymes with alternative specificities.
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PMID:Identification of electrostatic interactions that determine the phosphorylation site specificity of the cAMP-dependent protein kinase. 203

The type I regulatory subunit (R-I) of rat brain cAMP-dependent protein kinase was expressed in E. coli and site-directed mutagenesis was used to substitute amino acids in the putative cAMP-binding sites. The wild-type recombinant R-I bound 2 mol of cAMP/mol subunit, while two mutant R-Is with a single amino acid substitution in one of the two intrachain cAMP-binding sites (clone N153:a glutamate for Gly-200, and clone C254:an aspartate for Gly-324) bound 1 mol of cAMP/mol subunit. When these two substitutions were made in one mutant, cAMP did not bind to this mutant, indicating that binding of cAMP to N153 or C254 was to their nonmutated sites. Competition experiments with site-selective analogs and dissociation of bound cAMP from mutant R-Is provided evidence for strong intrachain interactions between the two classes of cAMP-binding sites in R-I.
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PMID:Site-directed mutagenesis of the cAMP-binding sites of the recombinant type I regulatory subunit of cAMP-dependent protein kinase. 283 71

A protein that exhibits greater substrate specificity for cGMP-dependent protein kinase than for cAMP-dependent protein kinase has been purified 8,000-fold from cytosol of rabbit cerebellum to apparent homogeneity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The protein, termed G-substrate, is a monomer of 23,000 daltons. It is heterogeneous on isoelectric focusing, exhibiting three isoelectric forms over the pH range of 5.2-5.6 cGMP-dependent protein kinase catalyzes the incorporation of 2 mol of phosphate/mol of G-substrate, both into threonine residues. The protein has a high content of aspartate, glutamate, and proline. The hydrodynamic properties, heat stability, and acid solubility of this protein are consistent with an unfolded, nonglobular structure. G-substrate is localized primarily in the cytosol of cerebellum, although low concentrations of a phosphorylated protein with a similar molecular weight are detected in other brain regions.
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PMID:A specific substrate from rabbit cerebellum for guanosine 3':5'-monophosphate-dependent protein kinase. I. Purification and characterization. 625 70

DARPP-32 is a neuronal phosphoprotein of Mr = 32,000, originally identified in rat brain (Walaas, S.I., D.W. Aswad, and P. Greengard (1983) Nature 301: 69-72). This protein has now been identified in bovine caudate nucleus cytosol and purified 435-fold to apparent homogeneity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purification procedure involved acid extraction at pH 2, CM-cellulose chromatography, DEAE-cellulose chromatography, hydroxylapatite chromatography, and gel filtration on Ultrogel AcA 44. The purified catalytic subunit of cAMP-dependent protein kinase catalyzed the incorporation of 0.96 mol of phosphate/mol of purified DARPP-32. Phosphorylation occurred exclusively on threonine. The isoelectric point of dephospho-DARPP-32 was 4.7, and that of phospho-DARPP-32 was 4.6. The amino acid composition showed a high content of glutamate/glutamine and proline, and a low content of hydrophobic amino acids. DARPP-32 was found to have a Stokes radius of 34 A and a sedimentation coefficient of 2.05 S, indicating that it exists as an elongated monomer.
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PMID:DARPP-32, a dopamine- and adenosine 3':5'-monophosphate-regulated phosphoprotein enriched in dopamine-innervated brain regions. II. Purification and characterization of the phosphoprotein from bovine caudate nucleus. 631 28

Glutamate receptors are the major excitatory neurotransmitter receptors in the central nervous system. A variety of data has recently suggested that protein phosphorylation of glutamate receptors regulates their function. To examine at a molecular level the role of protein phosphorylation in the modification of glutamate receptors, we have transiently expressed the non-NMDA glutamate receptor subunit GluR1 (flop) in human embryonic kidney 293 cells. Using a polyclonal antipeptide antiserum directed specifically against GluR1, we have immunoprecipitated a 106 kDa phosphoprotein corresponding to the GluR1 subunit. Phosphoamino acid analysis and thermolytic peptide mapping demonstrate that this basal phosphorylation occurs exclusively on serine residues in two phosphopeptides. Application of activators of endogenous cAMP-dependent protein kinase or protein kinase C revealed no consistent changes in the phosphorylation of GluR1. However, co-expression of the GluR1 subunit with the well characterized protein tyrosine kinase v-src results in phosphorylation of GluR1 on tyrosine residues, in a single thermolytic phosphopeptide. These results suggest that GluR1 may be a substrate for protein serine/threonine kinases as well as protein tyrosine kinases in the central nervous system.
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PMID:Phosphorylation of recombinant non-NMDA glutamate receptors on serine and tyrosine residues. 768 38

The results presented here show that the metabotropic glutamate receptor agonist L(+)-2-amino-4-phosphonobutyric acid (L-AP4) is capable of markedly stimulating the survival of rat cerebellar granule cells in culture. This is the first demonstration of a neurotrophic role for metabotropic glutamate receptors. The survival promoting action of L-AP4 does not involve a large, rapid rise in [Ca2+]i which is seen with other neurotrophic agents in granule cells such as N-methyl-D-aspartate, ionomycin and high potassium. In addition, the survival-promoting effect of L-AP4 did not appear to be related to changes in cAMP levels. Survival due to L-AP4 was enhanced by pertussis toxin and by forskolin and was unaffected by inhibitors of cAMP-dependent protein kinase. Measurement of cAMP levels after long-term treatment with neurotrophic agents showed no clear relationship between cAMP concentration and granule cell survival. The mechanism of L-AP4 stimulated cell survival is unknown but seems unlikely to involve an acute rise in [Ca2+]i or modulation of cAMP levels. Survival induced by L-AP4 was not blocked by the antagonist (RS)-alpha-methyl-4-carboxyphenylglycine. Similarity in these properties with those of the mGLu7 receptor suggests that granule cell survival was stimulated by an mGlu7-like metabotropic receptor.
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PMID:Activation of metabotropic glutamate receptors by L-AP4 stimulates survival of rat cerebellar granule cells in culture. 770 63


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