Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.11 (AMPK)
 12,425 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The predominant 40 S ribosomal protein S6 kinase in skeletal muscle extracts from insulin-treated rats was purified over 10,000-fold to near homogeneity with approximately 4.5% recovery of starting activity. This S6 kinase was resolved from the catalytic subunit of cAMP-dependent protein kinase only by the seventh and final column chromatography step. The purified S6 kinase migrated as a tight doublet of approximately 31 kDa on an SDS-polyacrylamide gel, and it was eluted from gel filtration columns with a similar apparent M(r), which indicated that the enzyme exists as a monomer. This S6 kinase was immunologically distinct from the other known insulin-activated S6 kinases, i.e. p70S6K and p90rsk. It was inhibited by [ethylenebis(oxyethylenenitrilo)]tetraacetic acid and beta-glycerophosphate at concentrations routinely used to stabilize p70S6K and p90rsk. In addition to S6, phosvitin was also a substrate, whereas myelin basic protein, casein, protamine, and histones were poorly phosphorylated if at all by the purified S6 kinase. The purified enzyme was inactivated upon incubation with serine/threonine-specific protein phosphatase 2A, which indicated that it may be an intermediary component in a cascade of insulin-activated protein kinases.
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PMID:Purification and characterization of a novel ribosomal S6 kinase from skeletal muscle of insulin-treated rats. 812 8

The human platelet cilostamide- and cGMP-inhibited cAMP phosphodiesterase (cGI-PDE) was rapidly purified approximately 19,000-fold to apparent homogeneity using single step affinity chromatography on the isothiocyanate derivative of cilostamide coupled to aminoethyl agarose. Within 24 h, 30 micrograms of enzyme protein was obtained from 20 ml of packed platelets. Vmax for cAMP and cGMP was 6.1 and 0.9 mumol/min per mg protein, respectively. Several polypeptides (110/105, 79, 62, 55/53 kDa) were identified after SDS-PAGE, all of which were immunologically related to cGI-PDE and represented approx. 5, 20, 50 and 20% of the total protein, respectively. Limited proteolysis of the cGI-PDE with chymotrypsin produced a major fragment of approximately 47 kDa (and at least two smaller peptides) with catalytic activity and sensitivity to cGMP and OPC 3911 similar to controls. Phosphorylation of the cGI-PDE by cAMP-dependent protein kinase (A-kinase) resulted in maximal incorporation of 0.6-1.8 mol of 32P/mol 110/105 and 79 kDa polypeptides; much lower and variable amounts of phosphate were incorporated into the 62 and 55/53 kDa polypeptides. After digestion of cGI-PDE with several proteinases a number of peptides were isolated and sequenced. Most of the peptide sequences obtained could be aligned within the carboxy terminal domain of the deduced sequence of the human cardiac cGI-PDE. These and other results suggest that the subunit size of the intact platelet cGI-PDE is 110 kDa and that proteolytic fragments of 79, 62 and 55/53 kDa are produced during purification. The smaller fragments (62 and 55/53 kDa) contain the catalytic domain; the larger fragments (110 and 79 kDa) also contain the regulatory domain with phosphorylation sites for A-kinase.
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PMID:Single-step affinity purification, partial structure and properties of human platelet cGMP inhibited cAMP phosphodiesterase. 815 97

The effects of amylin and insulin on the phosphorylation of glycogen synthase and phosphorylase were investigated using rat diaphragms incubated with 32Pi. Muscles were incubated with insulin (200 nM) or amylin (200 nM) for 30 min before extracts were prepared. The 32P contents of the enzymes were determined after immunoprecipitation and SDS-polyacrylamide gel electrophoresis. Amylin increased both the activity ratio (-AMP/+AMP) and the 32P content of phosphorylase by approximately 2-fold. Insulin alone was without significant effect on phosphorylase, but insulin blocked the effect of amylin on increasing the phosphorylation of phosphorylase. Insulin increased the glycogen synthase activity ratio (low glucose-6-P/high glucose-6-P) by approximately 80%. Amylin decreased this ratio from 0.14 to 0.08 and increased the phosphorylation of synthase by approximately 40%. To investigate changes in phosphorylation of different sites in the synthase, the enzyme was subjected to exhaustive proteolysis with trypsin, and 32P-labeled fragments were separated by reverse phase high performance liquid chromatography. Insulin decreased the 32P contents of sites 3(a+b+c) and 2(a+b), which appears to account for the increase in synthase activity. Amylin increased phosphorylation of sites 1a, 1b, and 3(a+b+c), but not sites 2(a+b). With insulin plus amylin, phosphorylation of none of the sites was significantly changed. The results indicate that the effects of amylin on glycogen synthase must involve more than activation of cAMP-dependent protein kinase, as this kinase phosphorylates site 2 and does not phosphorylate sites 3(a+b+c).
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PMID:Control of glycogen synthase and phosphorylase by amylin in rat skeletal muscle. Hormonal effects on the phosphorylation of phosphorylase and on the distribution of phosphate in the synthase subunit. 815 93

1. Rat liver microsomal membranes were studied for the presence of protein kinases. Microsomal proteins solubilized with Triton X-100 were analyzed by means of ion exchange chromatography. 2. Protein kinase activity was detected in the column fractions using specific assays for cAMP-dependent protein kinase, cGMP-dependent protein kinase, protein kinase C, Ca2+/calmodulin-dependent protein kinase and casein kinases. 3. Fractions with protein kinase activity were further analyzed by SDS-polyacrylamide gel electrophoresis. 4. The results indicate that cAMP-dependent protein kinase type I and II, casein kinases I and II, protein kinase C proenzymes I and II and Ca2+/calmodulin kinase II are associated with the membranes of endoplasmic reticulum (ER).
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PMID:Rat liver endoplasmic reticulum protein kinases. 818 36

A 62 kd protein was purified from the Triton-insoluble fraction of porcine brain white matter. This protein formed 10nm filaments, in vitro. The phosphorylation of the 62 kd protein by cAMP-dependent protein kinase caused electrophoretic mobility to shift to 66 kd on SDS-PAGE and a complete loss of the filament forming ability ensued. Amino acid sequences of four peptide fragments obtained from the 62 kd protein by lysylendopeptidase were identical with that of a 66 kd rat brain alpha-internexin. Amino acid analyses of the phosphopeptide fragment derived from phosphorylated porcine alpha-internexin revealed that the phosphorylation sites by cAMP-dependent protein kinase located in the amino-terminal head domain of this protein. These results strongly suggest that alpha-internexin polymerizes into 10nm filaments in vitro and that phosphorylation of the amino-terminal domain of alpha-internexin controls its polymerizability.
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PMID:Phosphorylation of a 62 kd porcine alpha-internexin, a newly identified intermediate filament protein. 821 81

The participation of protein kinases in phosphorylation of nicotinic acetylcholine receptor (nAChR) in electric organ and muscle has been precisely investigated in vitro and in vivo whereas phosphorylation of neuronal nAChR is not yet fully characterized. Here, we first report the in vitro phosphorylation of brain nAChR. nAChR purified from rat brains was phosphorylated in vitro by cAMP-dependent protein kinase (PKA), immunoprecipitated with monoclonal antibody against the receptor, and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by autoradiography. PKA specifically phosphorylated nAChR on the alpha 4 subunits, and H8, an inhibitor of PKA, inhibited completely the phosphorylation. Under the conditions used, a maximal stoichiometry of the phosphorylation by PKA was near to 1 mol of phosphate/mol of the alpha 4 subunits. The 32P-labeled subunits were digested with S. aureas V8 protease followed by SDS-PAGE autoradiography and the resultant phosphopeptide maps revealed three distinct phosphopeptide bands, one major band and two minor bands. Phosphoamino acid analysis of the 32P-labeled alpha 4 subunits showed that serine residues were exclusively phosphorylated. Based on these results, participation of PKA in the regulation of neuronal nAChR is discussed.
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PMID:Phosphorylation of rat brain nicotinic acetylcholine receptor by cAMP-dependent protein kinase in vitro. 825 79

A number of protein kinases have been shown to undergo autophosphorylation, but few have demonstrated a coordinate increase or decrease in enzymatic activity as a result. Described here is a novel S6 kinase isolated from human placenta which autoactivates through autophosphorylation in vitro. This S6/H4 kinase, purified in an inactive state, exhibited a molecular mass of 60 kDa as estimated by SDS-polyacrylamide gel electrophoresis. The 60-kDa protein underwent autophosphorylation, was labeled by 8-azido-[alpha-32P]ATP, and reacted with an antibody to the conserved APE domain of the cAMP-dependent protein kinase. The protein did not cochromatograph with p70 S6 kinase and did not cross-react with an anti-p70 kinase antibody. The synthetic peptide S6-21, histone H4, and myelin basic protein were phosphorylated by the purified S6/H4 kinase. Mild digestion of the inactive S6/H4 kinase with trypsin generated a 40-kDa fragment, as determined by SDS-polyacrylamide gel electrophoresis. The trypsin treatment was necessary, but not sufficient, to fully activate the kinase. Subsequent incubation of the trypsin-treated S6 kinase with MgATP resulted in the rapid autophosphorylation of the 40-kDa fragment along with a coordinate increase in kinase activity. The autophosphorylation of the 40-kDa protein was positively correlated with MgATP incubation time and an increase in activity toward the S6-21 peptide, histone H4, and myelin basic protein. Taken together, these data support the hypothesis that this previously uncharacterized S6 kinase belongs to a unique family of protein kinases which utilize autophosphorylation as part of their in vivo activation mechanism.
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PMID:Activation of an S6 kinase from human placenta by autophosphorylation. 836 21

The cDNA of human amidophosphoribosyltransferase (EC 2.4.2.14, ATase), which is the supposed regulatory allosteric enzyme of de novo purine nucleotide biosynthesis, has been cloned from human hepatoma (HepG2) cDNA library. The predicted open reading frame encodes a protein of 517 amino acids with a deduced molecular weight (Mr) of 57,398, which is consistent with the molecular mass of 56 kDa on SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of the ATase subunit purified from human placenta. The derived amino acid sequence exhibits 93, 82, 41, 37, and 33% identity with the sequences of rat, chicken, Bacillus subtilis, Escherichia coli, and Saccharomyces cerevisiae ATases, respectively. Southern blot analysis suggested that the ATase gene exists as multiple copies. ATase mRNA (3.5 kb) is ubiquitously expressed in various human tissues. Comparison with rat and chicken ATases showed that two cysteine residues for an iron-sulfur cluster were conserved. Four consensus phosphorylation sites for cAMP-dependent protein kinase were found.
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PMID:Molecular cloning of human amidophosphoribosyltransferase. 838 Jun 92

The distribution and phosphoprotein band patterns of low Km, cGMP-inhibited cAMP phosphodiesterase (cGI PDE) activity were examined in cytosolic and microsomal fractions of human, canine, rabbit and guinea pig left ventricular myocardium following phosphorylation by cAMP-dependent protein kinase, immunoprecipitation with anti-cGI PDE antibodies and SDS-PAGE. The recovery of cGI PDE activity in cytosolic and microsomal fractions was comparable in all four species. Microsomal cGI PDE was comprised chiefly of a approximately 135 kDa phosphoprotein. Cytosolic cGI PDE was comprised solely of approximately 116 kDa and lower molecular weight phosphoproteins. The approximately 135 kDa phosphoprotein probably corresponds to the holoenzyme encoded by the recently cloned cDNA for human myocardial cGI PDE, whose predicted molecular weight is 126 kDa. The approximately 116 kDa phosphoprotein may result from deletion or removal of putative membrane-association domains from the N-terminal region of the holoenzyme. These results suggest that the cytosolic and sarcoplasmic reticulum-associated forms of mammalian myocardial cGI PDE are separate molecular species.
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PMID:Cytosolic and sarcoplasmic reticulum-associated low Km, cGMP-inhibited cAMP phosphodiesterase in mammalian myocardium. 838 Dec 78

The effect of androgen on protein kinase activities was studied in chromosomal proteins from female mouse submandibular gland. The protein kinase activities in the nuclei were stimulated 3 h after testosterone administration. The in vitro addition of cAMP in the nuclei resulted in the enhancement of the androgen-sensitive protein kinase activities. SDS-PAGE analysis revealed that nonhistone proteins having molecular weights of 20-30 kDa and around 43 kDa were phosphorylated after androgen treatment. The addition of cAMP stimulated phosphorylation of nonhistone proteins having molecular weights with 15-25 kDa, 30 kDa and 35 kDa and the intensity of phosphorylation of these nonhistone proteins was enhanced after androgen treatment. These results suggest that androgen-sensitive protein kinases including cAMP-dependent protein kinase were present and that phosphorylation of nonhistone proteins by these protein kinases may be involved in mediating androgen-induced gene activation.
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PMID:cAMP-dependent phosphorylation of nuclear proteins in mouse submandibular gland following testosterone administration. 839 74


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