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 mammalian brain, physiological signals carried by cAMP seem to be targeted to intraneuronal sites by the association of cAMP-dependent protein kinase II beta with anchoring proteins that bind the regulatory subunit (RII beta) of the enzyme. Previously, an RII beta-binding domain was characterized in a large (Mr approximately 150,000) candidate anchor protein, rat brain P150 (Bregman, D. B., Bhattacharyya, N., and Rubin, C. S. (1989) J. Biol. Chem. 264, 4648-4656). RII beta-binding proteins with Mr values of 65,000-80,000 were detected in the brains of other species. Since little was known about the structural features of these lower Mr proteins, we undertook the characterization of bovine brain P75 as a prototype. A cDNA encoding 258 amino acid residues at the C terminus of P75 was cloned by probing a lambda gt11 expression library with 32P-RII beta. The cDNA insert was ligated into the pET-3b expression plasmid, and large amounts of the partial P75 polypeptide (designated P47) were produced in Escherichia coli. A purification scheme that yielded 9 mg of soluble P47 from a 1-liter bacterial culture was devised. Antibodies directed against the P47 polypeptide revealed that P75 is expressed almost exclusively in brain. The sequence of 117 amino acid residues at the C terminus of P75 contains the RII beta-binding site and is 80% identical to the corresponding region of P150. In contrast, a lower level of identity (36%) between P75 and P150 at a more N-terminal region indicates that the two RII beta-binding proteins are related, but distinct proteins. P75 is not homologous to microtubule-associated protein 2, an RII alpha-selective binding protein, or any other previously studied proteins. C-terminal truncation analysis disclosed that the final 26 residues in P75 are essential for binding RII beta.
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PMID:Molecular characterization of bovine brain P75, a high affinity binding protein for the regulatory subunit of cAMP-dependent protein kinase II beta. 201 23

The Mr = 38,300 polypeptide of the purified recombinant rat DNA polymerase beta served as an excellent substrate for protein kinase C (PKC) in vitro but not for the catalytic subunit of cAMP-dependent protein kinase. The phosphorylation by PKC resulted in inactivation of DNA polymerase beta activity, and recovery was achieved by dephosphorylation with alkaline phosphatase. Since the phosphorylated DNA polymerase beta was retained with use of a single-stranded DNA-cellulose column, inactivation might occur at a site different from that for the DNA binding. Amino acid sequence analysis of the phosphopeptides revealed that the phosphorylated sites were 2 serine residues at positions 44 and 55 from the NH2 terminus, either or both of which might be involved in the catalytic activity of DNA polymerase beta. Thus, the inactivation of the DNA repair enzyme, DNA polymerase beta, by PKC may be an important process in the modification of DNA metabolism in the nucleus through signal transduction processes.
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PMID:Inactivation of DNA polymerase beta by in vitro phosphorylation with protein kinase C. 204 Jun 2

A number of recent studies have suggested that phosphorylation of the gamma-aminobutyric acid A (GABAA) receptor could modulate receptor function. Activators of protein kinase C and cAMP-dependent protein kinase have been shown to influence GABAA receptor function. In addition, Sweetnam et al. [Sweetnam, P. M., Lloyd, J., Gallombardo, P., Malison, R. T., Gallager, D. W., Tallman, J. F. & Nestler, E. J. (1988) J. Neurochem. 51, 1274-1284] have reported that a kinase associated with a partially purified preparation of the receptor could phosphorylate the alpha subunit of the receptor. Moreover, Kirkness et al. [Kirkness, E. F., Bovenkerk, C. F., Ueda, T. & Turner, A. J. (1989) Biochem. J. 259, 613-616] have recently shown that cAMP-dependent protein kinase could phosphorylate a muscimol binding polypeptide of the GABAA receptor. To explore the issue further, we have examined the ability of specific kinases to catalyze significant phosphorylation of the GABAA receptor that has been purified to near homogeneity. The GABAA receptor was purified as previously described using benzodiazepine affinity chromatography. The purified receptor possessed no detectable kinase activity. Protein kinase C and cAMP-dependent protein kinase catalyzed the phosphorylation of the beta and alpha subunits of the receptor. However, most of the phosphate incorporation was associated with the beta subunit. Two muscimol binding polypeptides designated beta 58 (Mr 58,000) and beta 56 (Mr 56,000) were present in the preparation. The higher molecular weight polypeptide, beta 58, was phosphorylated specifically by cAMP-dependent protein kinase. beta 56 was phosphorylated specifically by protein kinase C. beta 58 and beta 56 gave distinct patterns in a one-dimensional phosphopeptide analysis. The stoichiometry of phosphorylation (mol of phosphate/mol of muscimol binding) catalyzed by cAMP-dependent protein kinase was 0.52 and that catalyzed by protein kinase C was 0.38. Taken together these data confirm that there are two forms of the beta subunit of the GABAA receptor and suggest that these two forms of the beta subunit are phosphorylated by distinct kinases.
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PMID:Protein kinase C and cAMP-dependent protein kinase phosphorylate the beta subunit of the purified gamma-aminobutyric acid A receptor. 215 39

Relaxin is a member of the insulin family of polypeptide hormones and is known to exert its biological effects on various parts of the mammalian reproductive system. Biologically active human relaxin has been chemically synthesized based on the nucleotide sequence obtained from an ovarian cDNA clone. In the present study synthetic human relaxin was radiolabled by phosphorylation with cAMP-dependent protein kinase and [gamma-32P]ATP to a specific activity of 5000 Ci/mmol. The phosphorylated relaxin was purified on cation exchange high performance liquid chromatography and was shown to co-migrate with relaxin on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Mass spectrometry revealed a single phosphorylated site on the B chain of relaxin. The 32P-relaxin was able to bind to a goat anti-relaxin antibody, and this binding could be displaced by unlabeled relaxin in a concentration-dependent manner. A comparison of the concentration responses of cellular cAMP production stimulated by relaxin and phosphorylated relaxin in a primary human uterine cell line showed that phosphorylation did not affect the in vitro biological efficacy of relaxin. This made it suitable for in situ autoradiographic localization of relaxin binding sites in rat uterine, cervical, and brain tissue sections. Displacement of the binding of 100 pM 32P-relaxin by 100, 10, and 3 nM unlabeled relaxin, but not by 100 nM insulin, insulin-like growth factor-I, and an insulin-like growth factor-I analog, demonstrated the high affinity and specificity of such binding. We conclude that 32P-labeled human relaxin is biologically and immunologically active and that this novel probe binds reversibly and with high affinity to classical (e.g. uterus) and unpredicted (e.g. brain) tissues.
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PMID:Preparation of biologically active 32P-labeled human relaxin. Displaceable binding to rat uterus, cervix, and brain. 216 Sep 76

The Ca2(+)-mobilizing second messenger D-myo-inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) is converted to the putative messenger D-myo-inositol 1,3,4,5-tetrakisphosphate by Ins(1,4,5)P3 3-kinase. We found that cAMP-dependent protein kinase and protein kinase C phosphorylate, and thereby modulate, the activity of Ins(1,4,5)P3 3-kinase. cAMP-dependent kinase introduced a stoichiometric amount of phosphate at serine 109 of the 53-kDa polypeptide and caused a 1.8-fold increase in Vmax, whereas the protein kinase C-dependent phosphorylation reduced the Vmax to one-fourth of that of the unphosphorylated enzyme. Upon prolonged incubation, protein kinase C introduced phosphate at multiple sites in Ins(1,4,5)P3 3-kinase, and the resulting inactivation of the enzyme appeared to be well-correlated with the simultaneous phosphorylation of two major sites, serine 109 and serine 175. The Km for Ins(1,4,5)P3 was not affected significantly after phosphorylation by either protein kinase. We propose, therefore, that the phosphorylation of Ins(1,4,5)P3 3-kinase by cAMP-dependent kinase and protein kinase C constitutes mechanisms of cross-talk between cellular signaling pathways that use various second messengers such as inositol phosphates, diacylglycerol, Ca2+, and cAMP.
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PMID:Regulation of D-myo-inositol 1,4,5-trisphosphate 3-kinase by cAMP-dependent protein kinase and protein kinase C. 216 47

A monoclonal antibody designated as MAC-L1 immunoprecipitated [3H]PN200-110-labeled calcium channels of chick cardiac and skeletal muscle. On specific immunoprecipitation of 125I-labeled proteins, two large polypeptides (Mr 197,000 and 139,000 for heart, and 172,000 and 135,000 for skeletal muscle, under reducing conditions) were identified as the major components of these channels. Both polypeptides were found to exist together as a complex in 1% digitonin, but to become separated from each other in 1% Triton X-100. The 197 and 172 kDa peptides of cardiac and skeletal muscles, respectively, were photolabeled with [3H]azidopine. Under nonreducing conditions, the 139 kDa polypeptide of heart and the 135 kDa polypeptide of skeletal muscle took on larger molecular weights of 192,000 and 190,000, respectively. The 139 kDa but not the 197 kDa component of the heart was capable of binding to wheat germ agglutinin-Sepharose. Among the polypeptides specifically precipitated by MAC-L1, a 165 kDa peptide of skeletal muscle was phosphorylated by cAMP-dependent protein kinase. In contrast, a minor 99 kDa polypeptide, but not the major 197 kDa polypeptide, of the heart was phosphorylated by this kinase. These results suggest that the dihydropyridine-sensitive cardiac calcium channel has alpha 1 and alpha 2 subunits that are homologous but not identical to those of the skeletal muscle calcium channel.
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PMID:Molecular characterization of 1,4-dihydropyridine-sensitive calcium channels of chick heart and skeletal muscle. 216 21

The traffic of ions through the kainic acid (KA) receptor/channels present on chick cerebellar glia is modulated by intracellular events likely to involve phosphorylation reactions. The protein carrying the KA binding sites has been isolated from chick cerebellum and shown to be composed of a 49-kDa polypeptide. Its primary structure, established via cDNA cloning, shows the presence of two putative phosphorylation sites. We report here that this 49-kDa polypeptide is a substrate of the cAMP-dependent protein kinase which catalyzes the incorporation of up to 2 mol of phosphate/mol of KA binding site. KA prevents this phosphorylation reaction in a concentration range similar to that needed to activate the KA receptor/channels but higher by 3 orders of magnitude than that needed to saturate the KA binding sites. Kainatergic ligands produce similar effects to those of KA, but 1 mM N-methyl-D-aspartic acid and 1 mM quisqualic acid have no effect. However, 0.01 mM quisqualic acid prevents the inhibitory action of KA. These results raise the possibility that the phosphorylation of KA receptor/channels in their cellular environment is negatively regulated by KA.
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PMID:Phosphorylation of the 49-kDa putative subunit of the chick cerebellar kainate receptor and its regulation by kainatergic ligands. 217 71

The nematode Caenorhabditis elegans expresses substantial amounts of several forms (Mr values = 39,000-41,000) of the catalytic subunit (C) of cAMP-dependent protein kinase. Approximately 65% of the total cAMP-dependent phosphotransferase activity is recovered in particulate fractions of homogenates prepared from asynchronous populations of C. elegans. The C subunit is expressed at a low level in cytosolic and particulate compartments during embryogenesis. As the nematodes progress from late embryonic stages to the newly hatched, first larval (L1) stage, C subunit content increases 15-fold. High levels of C subunits are observed in several subsequent larval and adult stages of development. Since the relative abundance of C subunit mRNA changes little with development, it appears that control of C expression is exerted the translational and/or post-translational levels. cDNAs for two types of C have been cloned and sequenced. The derived amino acid sequence of a major isoform (CeCAT alpha, 358 residues) is highly homologous (82% identical) with the murine C alpha subunit. A second, novel C subunit (CeCAT alpha', 374 residues) has a unique 56-residue carboxyl-terminal region that is generated by the alternative splicing of the C pre-mRNA. The splicing process that yields CeCAT alpha' is unusual because it converts the central portion of an apparent 1-kilobase (kb) intron to an exon. The alternative exon introduces the novel carboxyl terminus and a new translation stop signal, while simultaneously converting the coding sequence for 40 carboxyl-terminal residues in CeCAT alpha into 3'-untranslated nucleotides. The 5' end of the C. elegans C subunit mRNA is produced by the trans-splicing of the C gene transcript to a 22-base pair C. elegans leader sequence originally described by Krause, M., and Hirsh, D. [1987) Cell 49, 753-761). The 20-kb C. elegans C gene is divided into seven exons by introns ranging in size from 54 to 8000 bp. The sizes of the C. elegans C subunit gene, cytoplasmic mRNA (2.5 kb), and subunit protein are similar to the sizes of the murine C alpha gene, mRNA, and polypeptide. However, the nematode and murine C genes differ significantly in the organization of their introns and exons.
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PMID:Cloning, characterization, and expression of the gene for the catalytic subunit of cAMP-dependent protein kinase in Caenorhabditis elegans. Identification of highly conserved and unique isoforms generated by alternative splicing. 232 4

We previously demonstrated that protein kinases are useful probes of conformational changes that occur upon photoconversion of phytochrome [Wong, Y.-S., Cheng, H.-C., Walsh, D. A., & Lagarias, J. C. (1986) J. Biol. Chem. 261, 12089-12097]. Here we present phosphopeptide analyses of oat phytochrome phosphorylated by three mammalian protein kinases and by a polycation-stimulated, phytochrome-associated protein kinase. Phosphorylation of the Pr form by the cAMP-dependent protein kinase occurs predominantly on Ser17 while Ser598 is the preferred phosphorylation site on Pfr. The cGMP-dependent and Ca2(+)-activated, phospholipid-dependent protein kinases, which phosphorylate only the Pr form of phytochrome, recognize the same region on the phytochrome polypeptide as the cAMP-dependent protein kinase. Polycation-stimulated phytochrome phosphorylation reveals that, in contrast to the mammalian enzymes, the plant kinase recognizes the serine-rich, blocked N-terminus of phytochrome. The potential regulatory role of phytochrome phosphorylation, particularly in the structurally conserved serine/threonine-rich N-terminal region of the phytochrome polypeptide, is suggested by these results.
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PMID:Phosphopeptide mapping of Avena phytochrome phosphorylated by protein kinases in vitro. 235 57

Protein N-myristoylation refers to the covalent attachment of a myristoyl group (C14:0), via amide linkage, to the NH2-terminal glycine residue of certain cellular and viral proteins. Myristoyl-CoA:protein N-myristoyltransferase (NMT) catalyzes this cotranslational modification. We have developed a system for studying the substrate requirements and biological effects of protein N-myristoylation as well as NMT structure-activity relationships. Expression of the yeast NMT1 gene in Escherichia coli, a bacterium that has no endogenous NMT activity, results in production of the intact 53-kDa NMT polypeptide as well as a truncated polypeptide derived from proteolytic removal of its NH2-terminal 39 amino acids. Each E. coli-synthesized NMT species has fatty acid and peptide substrate specificities that are indistinguishable from those of NMT recovered from Saccharomyces cerevisiae, suggesting that the NH2-terminal domain of this enzyme is not required for its catalytic activity. By using a dual plasmid system, N-myristoylation of a mammalian protein was reconstituted in E. coli by simultaneous expression of the yeast NMT1 gene and a murine cDNA encoding the catalytic (C) subunit of cAMP-dependent protein kinase (PK-A). The fatty acid specificity of N-myristoylation was preserved in this system: [9,10(n)-3H]myristate but not [9,10(n)3H]palmitate was efficiently linked to Gly-1 of the C subunit. [13,14(n)-3H]10-Propoxydecanoic acid, a heteroatom-containing analog of myristic acid with reduced hydrophobicity but similar chain length, was an effective alternative substrate for NMT that also could be incorporated into the C subunit of PK-A. Such analogs have recently been shown to inhibit replication of certain retroviruses that depend upon linkage of a myristoyl group to their gag polyprotein precursors (e.g., the Pr55gag of human immunodeficiency virus type 1). A major advantage of the bacterial system over eukaryotic systems is the absence of endogenous NMT and substrates, providing a more straightforward way of preparing myristoylated, analog-substituted, and nonmyristoylated forms of a given protein for comparison of their structural and functional properties. The system should facilitate screening of enzyme inhibitors as well as alternative NMT fatty acid substrates for their ability to be incorporated into a specific target protein. Our experimental system may prove useful for recapitulating other eukaryotic protein modifications in E. coli so that structure-activity relationships of modifying enzymes and their substrates can be more readily assessed.
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PMID:Protein N-myristoylation in Escherichia coli: reconstitution of a eukaryotic protein modification in bacteria. 240 21


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