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)

Isolation of cDNA clones from lambda gt11 phage libraries by functional screening is limited by the low amount of lacZ-cDNA-encoded fusion protein synthesized in an isolated phage plaque. The amount of specific cDNA-encoded protein can be significantly enhanced by expression in bacterial colonies rather than phage plaques. Escherichia coli was lysogenized with a lambda gt11 cDNA expression library from Dictyostelium discoideum. Bacteria were selected for the presence of the lambda gt11 prophage by elimination of nonlysogenic parental cells with a lambda cI phage. The usefulness of the lysogen library was demonstrated by immuno-screening and functional screening with two different radiolabeled ligands. cDNA clones encoding a well-characterized D. discoideum protein, the regulatory subunit of the cAMP-dependent protein kinase, were isolated by screening the lysogen library with antibodies. Clones encoding this protein could also be identified by functional screening with [3H]cAMP, demonstrating that the limit of detection of positive clones by ligand screening is at least an order of magnitude lower for the lysogen library than for the corresponding phage library. We have subsequently used the lysogen library to isolate cDNA clones encoding calmodulin-binding protein(s) from D. discoideum by functional screening with [125I]calmodulin. For these clones, screening of the corresponding phage library had previously been found unsuccessful.
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PMID:Prophage lambda libraries for isolating cDNA clones by functional screening. 214 39

cDNA clones coding for the regulatory subunit (RII beta) of type II cAMP-dependent protein kinase were isolated from a bovine brain cDNA expression library in lambda gt11. The cDNA codes for a protein of 418 amino acids which is 98% homologous to the rat and human RII beta proteins. A series of expression vectors coding for truncated RII beta proteins were constructed in pATH plasmids and fusion proteins were expressed in Escherichia coli. Polyclonal and monoclonal antibodies made against purified bovine brain RII were immunoreactive with the fusion proteins on Western blots. The expressed RII beta-fusion proteins were used in overlay assays to identify the region in RII beta which binds to microtubule-associated protein 2 (MAP2) and to the 75,000-dalton calmodulin-binding protein (P75) (Sarkar, D., Erlichman, J., and Rubin, C.S. (1984) J. Biol. Chem. 259, 9844-9846) in bovine brain. Fusion protein containing amino acids 1-50 of the RII beta NH2 terminus (RII beta(1-50)] bound to both MAP2 and P75 immobilized on nitrocellulose filters. A pATH11-directed fusion protein containing the 31 amino acid RII-binding site of the human MAP2 protein (MAP2(31)) (Rubino, H.M., Dammerman, M., Shafit-Zagardo, B., and Erlichman, J. (1989) Neuron 3, 631-638) also bound RII beta-fusion proteins containing RII beta amino acids 1-50. Three fusion proteins, RII beta(1-25), RII beta(25-96), and RII beta(1-265,25-96 deleted) did not bind to MAP2(31) nor P75. The results showed that the binding domain for MAP2 and P75 was located within the NH2-terminal 50 amino acids of RII beta. Preincubation of bovine heart protein kinase II alpha and RII beta(1-50) with MAP2(31) prevented their binding to both P75 and MAP2(31) that were immobilized on nitrocellulose, suggesting that the binding sites for MAP2 and P75 are located near each other or that the same site on RII was binding to both proteins.
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PMID:Identification of the MAP2- and P75-binding domain in the regulatory subunit (RII beta) of type II cAMP-dependent protein kinase. Cloning and expression of the cDNA for bovine brain RII beta. 225 32

A major protein with a molecular weight of 17,000, designated as MP17, has been identified in mammalian eye lens plasma membranes. Hydrophobic photolabeling experiments revealed that MP17 is a genuine intrinsic membrane protein. By using monoclonal antibodies we demonstrated that MP17 is not detectable in liver, heart, muscle, spleen and kidney, and thus can be considered, like MP26, as a lens-specific membrane protein. Furthermore, we showed that MP17 is a substrate for cAMP-dependent protein kinase and that it is a calmodulin-binding protein.
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PMID:MP17, a fiber-specific intrinsic membrane protein from mammalian eye lens. 337 Oct 69

Phospholamban, the cardiac sarcoplasmic reticulum proteolipid, is phosphorylated by cAMP-dependent protein kinase, by Ca2+/phospholipid-dependent protein kinase, and by an endogenous Ca2+/calmodulin-dependent protein kinase, the identity of which remains to be defined. The aim of this study was therefore to characterize the latter kinase, called phospholamban kinase. Phospholamban kinase was purified approximately 42-fold with a yield of 11%. The purified fraction exhibits a specific activity of 6.5 nmol of phosphate incorporated into exogenous phospholamban per minute per milligram of protein. Phospholamban kinase appears to be a high molecular weight enzyme and presents a broad substrate specificity, synapsin-1, glycogen synthase, and smooth muscle myosin regulatory light chain being the best substrates. Phospholamban kinase phosphorylates synapsin-1 on a Mr 30 000 peptide. The enzyme exhibits an optimum pH of 8.6, a Km for ATP of 9 microM, and a requirement for Mg2+ ions. These data suggest that phospholamban kinase might be an isoenzyme of the multifunctional Ca2+/calmodulin-dependent protein kinase. Consequently we have searched for Mr 50 000-60 000 phosphorylatable subunits among cardiac sarcoplasmic reticulum proteins. A Mr 56 000 protein was found to be phosphorylated in the presence of Ca2+/calmodulin. Such phosphorylation alters the electrophoretic migration velocity of the protein. In addition, this protein that binds calmodulin was always found to be present in fractions containing phospholamban kinase activity. This Mr 56 000 protein is therefore a good candidate for being a subunit of phospholamban kinase. However, the Mr 56 000 calmodulin-binding protein and the Mr 53 000 intrinsic glycoprotein which binds ATP are two distinct entities.
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PMID:Characterization and partial purification of cardiac sarcoplasmic reticulum phospholamban kinase. 373 Mar 67

The association of regulatory subunits (RII) of Type II cAMP-dependent protein kinase from bovine cerebral cortex (RII-B) and bovine cardiac and skeletal muscle (RII-H) with specific binding proteins in bovine brain cytosol and purified brain microtubules was demonstrated using a solid phase binding assay. RII-binding proteins present in bovine cerebral cortex were immobilized on nitrocellulose filters after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Incubation of the filters with 32P-labeled regulatory subunits showed that both RII-B and RII-H interact with the 75,000-dalton calmodulin-binding protein (P75) and microtubule-associated protein 2 (MAP-2). However, significant differences in binding affinities and capacities were observed. RII-B displayed a higher affinity for P75 compared to RII-H while RII-H preferentially bound to MAP-2. Quantitation of radioactive RII bound to MAP-2 showed that MAP-2 bound 4-6 times more RII-H than RII-B. The differential binding affinities and capacities of RII-H and RII-B for MAP-2 were not affected by autophosphorylation since both phospho and dephospho forms of RII displayed the same binding characteristics. Competitive binding studies suggest that RII-H and RII-B bind to the same sites on MAP-2. The biochemical basis for the differential binding of RII-B and RII-H to the same sites of MAP-2 is unknown. However, other high affinity RII-binding proteins present in cerebral cortex (i.e. P75) might affect the affinity of RII-B for MAP-2. 32P-RI did not bind to P75 nor MAP-2 under the conditions used.
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PMID:Differential binding of the regulatory subunits (RII) of cAMP-dependent protein kinase II from bovine brain and muscle to RII-binding proteins. 394 17

Myosin light chain kinase and a fraction of type II cAMP-dependent protein kinase have been partially purified from bovine brain by affinity chromatography on calmodulin-Sepharose. The myosin kinase was purified approximately 3700-fold and has an estimated molecular weight of 130,000 +/- 10,000 by sodium dodecyl sulfate gel electrophoresis. A fraction of soluble cAMP-dependent protein kinase also bound to calmodulin-Sepharose and was purified 2300-fold. A fraction of this cAMP-dependent protein kinase after purification by glycerol gradient centrifugation was shown to contain the two subunits of calcineurin, a major calmodulin-binding protein in brain, and the two subunits of type II cAMP-dependent protein kinase in a ratio of 1:1:2:2. Its sedimentation coefficient was 8.1 S and 9.0 S when centrifuged in the absence or presence of calmodulin, suggesting the formation of a complex between calmodulin and protein kinase. Our results suggest the possibility that calcineurin may be involved in the interaction between the protein kinase and calmodulin. Furthermore, our studies imply that the regulatory subunit of the cAMP-dependent protein kinase, but not the catalytic subunit, is the site of interaction with calmodulin since the catalytic subunit of protein kinase was partially resolved from the complex by cAMP.
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PMID:Interaction of calmodulin with myosin light chain kinase and cAMP-dependent protein kinase in bovine brain. 626 40

The neuronal protein neurogranin, also known as RC3, is a selective substrate for protein kinase C (PKC). We synthesized a peptide corresponding to the phosphorylation domain of neurogranin (amino acids 28-43) and characterized its properties as a PKC substrate. Neurogranin(28-43) was phosphorylated by purified PKC with a Km of 150 nM. No significant phosphorylation of the peptide by either cAMP-dependent protein kinase or by calcium/calmodulin-dependent protein kinase II could be detected. Thus, neurogranin(28-43) is a potent and selective substrate for PKC. We tested several peptide analogues of neurogranin(28-43) for their substrate potency and specificity as kinase substrates, in order to help elucidate the structural determinants involved in the phosphorylation of substrates by PKC. Substituting Arg36 with Ile caused a significant reduction in the affinity for PKC. Replacing Lys30 with Arg enhanced the catalytic efficiency (Vmax/Km) for PKC but diminished the selectivity of the substrate for PKC. These results support the generally held model that basic amino acids on both sides of the phosphorylated Ser are important structural determinants in PKC substrates. However, the data also suggest that the presence of particular basic amino acids (Arg vs Lys) can contribute to the degree of selectivity of a substrate for PKC. Replacement with Ala of Phe35, the amino acid adjacent to the Ser34 phosphorylation site, resulted in a peptide with greatly diminished potency as a PKC substrate. This finding indicates a critical role of Phe35 in modulating binding and phosphorylation of neurogranin-derived peptides by PKC.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Studies with synthetic peptide substrates derived from the neuronal protein neurogranin reveal structural determinants of potency and selectivity for protein kinase C. 842 32

Previous studies have shown that palmitoyl-carnitine is an anti-proliferative agent and a protein kinase C inhibitor. Two new palmitoyl-carnitine analogs were synthesized by replacing the ester bond with a metabolically more stable ether bond. An LD50 value in the nM range was found in anti-proliferative assays using HL-60 cells and was dependent on the alkyl-chain length. The inhibitory action of these water-soluble compounds on protein kinase C in vitro was greatly increased with respect to palmitoyl-carnitine and was dependent on the length of the alkyl chain. Its effect was mediated by an increase in the enzyme's requirement for phosphatidylserine. Inhibition of the in situ phosphorylation of a physiological platelet protein kinase C substrate and of phorbol ester-induced differentiation of HL-60 cells was also observed. Finally, to test for isoenzyme selectivity, several human recombinant protein kinase C isoforms were used. Only the Ca2+-dependent classic protein kinase Cs (alpha, betaIota, betaIotaIota and gamma) were inhibited by these compounds, yet the activities of casein kinase I, Ca2+/calmodulin-dependent kinase and cAMP-dependent protein kinase were unaffected. Thus, these novel inhibitors appear to be both protein kinase C and isozyme selective. They may be useful in assessing the individual roles of protein kinase C isoforms in cell proliferation and tumor development and may be rational candidates for anti-neoplasic drug design.
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PMID:Anti-proliferative effect of two novel palmitoyl-carnitine analogs, selective inhibitors of protein kinase C conventional isoenzymes. 1058 79

Hypothalamic inflammation has been known as a contributor to high-fat diet (HFD)-induced insulin resistance and obesity. Myeloid-specific sirtuin 1 (SIRT1) deletion aggravates insulin resistance and hypothalamic inflammation in HFD-fed mice. Neurogranin, a calmodulin-binding protein, is expressed in the hypothalamus. However, the effects of myeloid SIRT1 deletion on hypothalamic neurogranin has not been fully clarified. To investigate the effect of myeloid SIRT1 deletion on food intake and hypothalamic neurogranin expression, mice were fed a HFD for 20 weeks. Myeloid SIRT1 knockout (KO) mice exhibited higher food intake, weight gain, and lower expression of anorexigenic proopiomelanocortin in the arcuate nucleus than WT mice. In particular, KO mice had lower ventromedial hypothalamus (VMH)-specific neurogranin expression. However, SIRT1 deletion reduced HFD-induced hypothalamic neurogranin. Furthermore, hypothalamic phosphorylated AMPK and parvalbumin protein levels were also lower in HFD-fed KO mice than in HFD-fed WT mice. Thus, these findings suggest that myeloid SIRT1 deletion affects food intake through VMH-specific neurogranin-mediated AMPK signaling and hypothalamic inflammation in mice fed a HFD.
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PMID:Effects of myeloid sirtuin 1 deficiency on hypothalamic neurogranin in mice fed a high-fat diet. 3047 62