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)

In purified preparations of voltage-sensitive sodium channels, the alpha subunit is selectively phosphorylated by cAMP-dependent protein kinase (Costa, M. R. C., Casnellie, J. E., and Catterall, W. A. (1982) J. Biol. Chem., 7918-7921). We have developed methods to measure sodium channel phosphorylation in both lysed synaptosomal membranes and intact synaptosomes. Incubation of lysed synaptosomal membranes with exogenously added catalytic subunit of cAMP-dependent kinase and [gamma-32P]ATP resulted in rapid phosphorylation of the alpha subunit as detected by specific immuno-precipitation, sodium dodecyl sulfate-gel electrophoresis, and autoradiography. Analysis of tryptic phosphopeptides revealed five major sites of reaction. The level of phosphorylation of these sites on the sodium channel in intact synaptosomes was monitored using a rephosphorylation method in which those sites not phosphorylated in situ were labeled with [gamma-32P]ATP and exogenously added protein kinase after lysis of the synaptosomes. Incubation of synaptosomes with 8-Br-cAMP completely blocked labeling of the alpha subunit in rephosphorylation indicating marked stimulation of phosphorylation of the sites on the sodium channel in situ. Phosphorylation was complete in 15 s and all four of the tryptic phosphopeptides detected under these conditions could be phosphorylated in situ. These results show that the sodium channel can be rapidly phosphorylated by endogenous cAMP-dependent protein kinase in intact synaptosomes. In addition, since ATP and protein kinase are only available inside the synaptosomes, they also show that the alpha subunit is a transmembrane polypeptide exposed on both sides of the synaptosomal membrane. The functional consequences of 8-Br-cAMP-stimulated phosphorylation were examined using ion flux and neurotoxin-binding methods. Binding of saxitoxin and scorpion toxin were unaffected, but neurotoxin-activated 22Na+ influx mediated by the sodium channel was reduced 16 to 26% (P less than 0.01) under various experimental conditions. The potential physiological significance of this action is considered.
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PMID:Cyclic AMP-dependent phosphorylation of the alpha subunit of the sodium channel in synaptic nerve ending particles. 633 Jan 3

Plectin (Mr = 300,000) was found to be an abundant polypeptide component of Chinese hamster ovary cells accounting for up to 1% of cellular protein. Seventy-five per cent of the plectin were present in cytoskeletons prepared by extraction of attached cells with 0.15% Triton X-100. As shown by immunofluorescence microscopy, plectin's spatial arrangement within these cytoskeletal preparations appeared well preserved, though slightly more filamentous compared to nonextracted cells. Upon in situ incubation of cytoskeletons with [gamma-32P] ATP followed by solubilization and immunoprecipitation, plectin was identified as one of the major phosphoacceptors. A basic phosphorylation of the protein was accomplished by a type I cAMP-independent protein kinase, while a cAMP-dependent protein kinase enhanced its phosphorylation up to 2-fold. Peptide mapping revealed that the two kinases phosphorylated different molecular sites. Peptide maps generated from cytoskeletal plectin phosphorylated in vitro using [gamma-32P]ATP and plectin phosphorylated in vivo using 32Pi were virtually identical demonstrating that the in situ phosphorylation of plectin in preparations of cytoskeletons was specific. Moreover, the specific radioactivity of cytoskeletal plectin was three times higher than that of detergent-extracted plectin, suggesting that phosphorylation is important for the protein's association with the cytoskeleton.
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PMID:Specific in situ phosphorylation of plectin in detergent-resistant cytoskeletons from cultured Chinese hamster ovary cells. 635 25

C-protein, a component of the thick filament of striated muscles, becomes phosphorylated in response to beta-adrenergic receptor stimulation and dephosphorylated in response to cholinergic receptor stimulation in heart. We have purified C-protein in high yield from cardiac muscle (approximately 50% yield: 0.3 mg of C-protein/g of frozen chicken heart). C-protein has a molecular weight on sodium dodecyl sulfate polyacrylamide gels of 155,000 but the native protein migrates as a globular protein of 209,000 daltons in gel filtration on Sephacryl S-300, suggesting that it is an asymmetric molecule composed of a single 155,000-dalton polypeptide. C-protein from chicken cardiac muscle has an amino acid composition similar to that of C-proteins from other muscles. The purified protein contains approximately 0.2 mol of phosphate/mol of C-protein. The purified C-protein has no endogenous protein phosphatase activity but does exhibit protein kinase activity in the presence of calcium and calmodulin (approximately 160 pmol of phosphate incorporated/min/mg of C-protein). This endogenous kinase catalyzes the incorporation of approximately 1 mol of phosphate/mol of C-protein. C-protein is an excellent substrate for catalytic subunit of cAMP-dependent protein kinase (Km = 4 microM, Vmax = 18.6 mumol/min/mg). Phosphorylation by catalytic subunit of cAMP-dependent protein kinase exhibits a broad pH optimum between pH 8 and 9 and results in the incorporation of up to 3 mol of phosphate/mol of C-protein. Phosphate is incorporated into 3-5 different sites at both phosphothreonine and phosphoserine residues. The phosphorylated C-protein does not differ from unphosphorylated C-protein with regard to Stokes radius, migration on sodium dodecyl sulfate-polyacrylamide gels, or UV spectrum.
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PMID:Phosphorylation of purified cardiac muscle C-protein by purified cAMP-dependent and endogenous Ca2+-calmodulin-dependent protein kinases. 654 9

Inhibition of translation in hemin-containing reticulocyte lysates by catalytic subunit (cS) preparations of cAMP-dependent protein kinase from bovine heart, reported earlier by our group, is due to a highly active heat-stable protein contaminant (HS). The specific activity for translational inhibition goes up by a factor of 10 when cS is heated for 10 min at 80 degrees C, which completely destroys histone phosphorylation activity. HS has been purified to homogeneity from bovine heart. It consists of a single polypeptide chain (Mr approximately 68,000). HS inhibits translation with biphasic kinetics similar to those of hemin deficiency and induces pronounced phosphorylation of the alpha subunit of the eukaryotic initiation factor eIF-2. The inhibition is relieved by eIF-2 or GTP but not by high concentrations of double-stranded RNA, thus ruling out involvement of the double-stranded RNA-activated inhibitor. Judged by poly(U) translation, HS has no effect on chain elongation. When added to crude preparations of the proinhibitor form (proHCI) of the heme-controlled translational inhibitor (HCI), HS appears to produce an increase of the HCI-to proHCI ratio. The mode of action of HS is as yet unknown.
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PMID:Heat-stable inhibitor of translation in reticulocyte lysates. 695 64

The regulatory subunit of cAMP-dependent protein kinase I has been cleaved proteolytically into two structurally independent domains. The larger domain (35K with trypsin or thermolysin and 31K with chymotrypsin) corresponded to the COOH-terminal end of the polypeptide chain and retained the cAMP binding site(s). The smaller domain (11 to 12K with trypsin), corresponding to the NH2-terminal region of the regulatory subunit, contained the region of dimer interaction. In the absence of reducing reagent, the two protomers of the native regulatory subunit and of the smaller domain could be covalently cross-linked by a disulfide bond. In addition to the two major domains, a 15-residue peptide that links the two domains has been isolated and partially characterized. Two major sites on the type I regulatory subunit were susceptible to proteolytic degradation. Site 1, susceptible to cleavage by both trypsin and thermolysin, has the following sequence: LysArg-Arg-Gly-Ala-Ile-Ser-Ala-. Cleavage at this site generated a 35K cAMP-binding fragment. Site 2 contained a chymotryptic cleavage site as well as a secondary tryptic site. The sequence at Site 2 was Val-Arg-Arg-Val-Ile-Ala. Cleavage here generated a 31K cAMP-binding fragment. Both sites contained 2 consecutive basic amino acid residues similar to the corresponding sequence in the type II regulatory subunit; however, in the case of the type I regulatory subunit, the serine at Site 1 does not serve as a site of autophosphorylation. In contrast to the dissociated regulatory subunit, the holoenzyme is partially protected from proteolytic degradation.
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PMID:The structural domains of cAMP-dependent protein kinase I. Characterization of two sites of proteolytic cleavage and homologies to cAMP-dependent protein kinase II. 743 94

Anti-peptide antibodies specific for the neuronal calcium channel alpha 1E subunit (anti-CNE1 and anti-CNE2) were produced to study the biochemical properties and subcellular distribution of the alpha 1E polypeptide from rat brain. Immunoblotting identified a single size form of 245-255 kDa which was a substrate for phosphorylation by cAMP-dependent protein kinase, protein kinase C, cGMP-dependent protein kinase, and calcium/calmodulin-dependent protein kinase II. Ligand-binding studies of alpha 1E indicate that it is not a high affinity receptor for the dihydropyridine isradipine or the peptide toxins omega-conotoxin GVIA or omega-conotoxin MVIIC at concentrations which elicit high affinity binding to other channel types in the same membrane preparation. The alpha 1E subunit is widely distributed in the brain with the most prominent immunocytochemical staining in deep midline structures such as caudate-putamen, thalamus, hypothalamus, amygdala, cerebellum, and a variety of nuclei in the ventral midbrain and brainstem. Staining is primarily in the cell soma but is also prominent in the dendritic field of a discrete subset of neurons including the mitral cells of the olfactory bulb and the distal dendritic branches of the cerebellar Purkinje cells. Our observations indicate that the 245-255 kDa alpha 1E subunit is localized in cell bodies, and in some cases in dendrites, of a broad range of central neurons and is potentially modulated by multiple second messenger-activated protein kinase.
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PMID:Biochemical properties and subcellular distribution of the neuronal class E calcium channel alpha 1 subunit. 747 5

The interaction of the mammalian spermatozoon with the oocyte's extracellular matrix or zona pellucida is a critical first step leading to successful fertilization. In this cell-extracellular matrix interaction it is the carbohydrate of the zona pellucida which serves as the sperm receptor and the surface of the spermatozoon which provides the lectin-like adhesion molecules. To better understand sperm-zona pellucida binding we have analyzed one specific zona binding protein (ZBP). This study has determined the mRNA sequence encoding a mammalian testis and sperm specific protein of 16,891 Da, which we have designated Sp17. Analysis of Sp17 revealed that the mRNA is present in rabbit, mouse, and human testes but not in any somatic tissue tested. In the rabbit, Sp17 is the 17-kDa member of the rabbit sperm autoantigen family of sperm specific autoantigens and is encoded by two mRNAs of 0.9 and 1.1 kb. Each mRNA has a unique 5' untranslated region but both have identical coding regions. The deduced amino acid sequence of the Sp17 ZBP showed several interesting features, including a similarity to the N-terminal of human testis cAMP-dependent protein kinase. Localization of Sp17 on live spermatozoa using antibodies to recombinant Sp17 or to the Sp17 peptide, G22C, revealed that the peptide backbone of Sp17 is inaccessible until the acrosome reaction begins. However, on paraformaldehyde fixed, acrosome intact spermatozoa, the peptide backbone is accessible to the antibodies which localize Sp17 to the apical surface. In the rabbit as well as other similar species in which the corona radiata (granulosa) cells adhere tightly to the zona pellucida and synthesize zona glycoproteins, the fertilizing spermatozoon may have already begun the acrosome reaction within the cumulus oophorus. Thus, the rabbit sperm surface would be modified to expose the Sp17 polypeptide during the final phase of cumulus passage and consequently Sp17 would be available for initial zona binding. The present study has also demonstrated that recombinant Sp17 can bind zona pellucida, dextran, and dextran sulfate.
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PMID:Sequence of a rabbit sperm zona pellucida binding protein and localization during the acrosome reaction. 752 87

In order to evaluate the importance of cAMP and cAMP-dependent protein kinase (cAMPdPK) in the regulation of chloride efflux via the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel, Caco-2, human colonic carcinoma cells were transfected with an expression vector encoding a mutant form of regulatory subunit of cAMPdPK under control of the mouse metallothionein 1 promoter. Four stable transformants were isolated that expressed the mutant subunit in a Zn(2+)-inducible manner and exhibited Zn(2+)-inducible inhibition of cAMPdPK activity. The parental and transformed Caco-2 cells were examined for their abilities to regulate chloride efflux in response to various secretagogues using a radioactive iodide-efflux assay. In the transformants, induction of the protein kinase mutation with ZnSO4 markedly decreased chloride efflux in response to forskolin, the 8-(4-chlorophenylthio) analog of cAMP, vasoactive intestinal polypeptide, prostaglandin E2 and isoproterenol, whereas Zn(2+)-treated parental cells remained responsive to these secretagogues. Treatment with carbachol, calcium ionophores or phorbol ester did not acutely affect chloride efflux. Together, these studies indicate that cAMP and cAMPdPK are essential components of secretagogue-regulated chloride channel activity in the Caco-2 cell line. In whole cell patch clamp recordings, induction of the cAMPdPK mutation inhibited anionic conductances indicative of the CFTR chloride channel, whereas purified catalytic subunit of cAMPdPK, added intracellularly, reversed the inhibition. These latter results demonstrate that the CFTR chloride channels in the protein kinase-defective transformants are normal and that the protein kinase mutation specifically affects their regulation, presumably by direct phosphorylation.
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PMID:Effects of mutations in cAMP-dependent protein kinase on chloride efflux in Caco-2 human colonic carcinoma cells. 752 38

The eukaryotic protein kinases that directly phosphorylate proteins are divided into two major classes: those that phosphorylate tyrosine and those that phosphorylate serine and threonine. Until recently, the similarities between these two classes of enzymes, which now total more than 400, were based primarily on sequence alignments. A recent report of the structure of the kinase domain (IRK) of the insulin receptor protein-tyrosine kinase now allows the features of these two families to be compared at the structural level. We review here this first tyrosine-specific protein kinase structure, and compare and contrast it to the structure of the serine/threonine-specific cAMP-dependent protein kinase. Although the general fold of the polypeptide backbone is conserved as predicted, unique features at the IRK active site provide a basis for understanding the differences in specificity for the phosphate acceptor amino acid. The structure of this inactive, dephosphorylated protein-tyrosine kinase also defines for the first time how activation might be achieved.
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PMID:How do protein kinases discriminate between serine/threonine and tyrosine? Structural insights from the insulin receptor protein-tyrosine kinase. 755 15

We report the long-term modulation of K+ channels by cAMP in cultured murine colliculi neurons. A short (1-2 s) application of 8-Br-cAMP induced a long-lasting broadening of the action potential, a loss of after-hyperpolarization, and a reduction in spike accommodation. In agreement with these changes, 8-Br-cAMP produced a long-lasting (2 hr) inhibition of a K+ current. These effects were also observed after a short activation of the pituitary adenylyl cyclase-activating polypeptide, beta-adrenergic, and 5-hydroxytryptamine type 4 (5-HT4) receptors, all known to increase cAMP. A transient activation of the cAMP-dependent protein kinase and a long-lasting inhibition of phosphatases (up to 2 hr) were detected. The blockade of the K+ current resulting from a brief application of 8-Br-cAMP or 5-hydroxytryptamine was prolonged from 2 to 4 hr when protein-serine/threonine phosphatases 1 and 2A were inhibited with 10 nM okadaic acid. The critical steps following the cAMP-dependent protein kinase activation and resulting in a long-term blockade of phosphatases are discussed in this report.
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PMID:cAMP-dependent, long-lasting inhibition of a K+ current in mammalian neurons. 760 46


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