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)

We have observed that soluble extracts from the extreme acidothermophilic archaebacterium Sulfolobus solfataricus contained protein phosphatase activity that was greatly stimulated by the divalent metal ions Mn2+, Mg2+, Ni2+, or Co2+. This activity apparently arose from a single enzyme since (a) stimulation by these divalent metal ions was not additive and (b) protein phosphatase activity eluted as a single peak from both a DE52 ion-exchange column and a Sephadex G-100 gel filtration column. Its apparent molecular mass was approximately 28,000 daltons. The enzyme dephosphorylated a variety of phosphoserine-containing substrates including casein, histone H2a, phosphorylase kinase, or glycogen phosphorylase. The enzyme would not dephosphorylate either histone H1 or a number of phosphotyrosine-containing compounds. It removed only half the phosphate bound to histone H2b, which is phosphorylated at two sites by the cAMP-dependent protein kinase. Protein phosphatase activity was inhibited by EDTA, Cu2+, Zn2+, NaF, inorganic phosphate, or pyrophosphate; but was unaffected by other potential activators and inhibitors such as microcystin, okadaic acid, vanadate, polyamines, or sulfhydryl modifying reagents. This enzyme represents the first protein phosphatase to be identified in any member of the third and oldest phylogenetic kingdom in nature, the archaebacteria.
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PMID:Identification of a serine/threonine-specific protein phosphatase from the archaebacterium Sulfolobus solfataricus. 838 14

The mechanism of dephosphorylation of multiphosphorylated proteins in the brain is not well understood. We have used the multiphosphorylated protein, phosvitin as a model substrate and undertaken the purification and characterization of brain phosphatases that preferentially dephosphorylate multiphosphorylated proteins. Two phosvitin phosphatase activities, termed Phosvitin Phosphatase 1 and 2 (PvP1, PvP2), which show acidic pH optima were resolved from the 33,000g supernatant fraction from rat brain by a procedure employing successive DEAE-cellulose, Sepharose 6B, second DEAE-cellulose and FPLC/Superose 6 chromatography steps. Following FPLC/Superose 6 size exclusion chromatography of PvP1 and PvP2, single peaks of phosvitin phosphatase activities were eluted in the range of 160-220 kDa with acidic pH optima. When FPLC/Sepharose 6 chromatography was performed in the presence of 0.5 M NaCl and 0.1% Triton X-100, low molecular mass protein phosphatase forms were produced in addition to the high-M, activity peak, ranging from 25 to 35 kDa (PvP1) and from 15 to 25 kDa (PvP2). Under these conditions, both high- and low-M, forms of PvP1 and PvP2 exhibited neutral pH optima. Both phosphatases dephosphorylate also (i) phosphorylase a, (ii) the alpha and beta subunits of phosphorylase kinase, and (iii) the microtubule-associated protein tau, phosphorylated by cAMP-dependent protein kinase. The present results suggest that two forms of protein phosphatases, displayed molecular and biochemical characteristics both similar and distinct from type 1 and type 2A protein phosphatases, are present in rat brain.
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PMID:Partial purification and characterization of two phosvitin phosphatases from rat brain. 862 49

To better understand the physical interaction between glycogen phosphorylase-b (P-b) and its only known kinase, phosphorylase kinase (PbK) and the relationship of this interaction to the activation of PbK, direct binding studies are necessary. By utilizing an enzyme-linked immunosorbent assay, a method was developed for measuring the binding of PbK to immobilized P-b under a variety of experimental conditions. A monoclonal antibody specific for the alpha subunit of PbK that had no effect on the phosphorylation of P-b by PbK or on the interaction of PbK with known effectors was used to detect PbK bound to plated P-b. Hyperbolic binding curves were obtained regardless of whether the concentration of Pbk or P-b was varied, and the assay detected changes in relative affinity caused by certain effectors of the kinase. The allosteric effector ADP, alkaline pH, and phosphorylation by cAMP-dependent protein kinase, all activators of PbK, did not cause significant changes in its relative affinity for P-b; however, Ca2+ and Mg2+ ions, which also stimulate PbK, increased its affinity for P-b, with Mg2+ being more effective. Mn2+, which inhibits the P-b conversion activity of PbK, was found to be the most potent enhancer of its affinity for P-b, although divalent cations may enhance binding. Inclusion of ATP analogs in the binding assay with Ca2+ and Mg2+ to stimulate catalytic assay conditions did not further affect the apparent affinity for P-b, which is consistent with the previously reported rapid equilibrium random bi-bi kinetic mechanism for P-b conversion.
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PMID:Divalent cations but not other activators enhance phosphorylase kinase's affinity for glycogen phosphorylase. 866 94

The C terminus of the catalytic gamma-subunit of phosphorylase kinase comprises a regulatory domain that contains regions important for subunit interactions and autoinhibitory functions. Monospecific antibodies raised against four synthetic peptides from this region, PhK1 (362-386), PhK5 (342-366), PhK9 (322-346), and PhK13 (302-326), were found to have significant effects on the catalytic activities of phosphorylase kinase holoenzyme and the gamma delta complex. Antibodies raised against the very C terminus of the gamma-subunit, anti-PhK1 and anti-PhK5, markedly activated both holoenzyme and the gamma delta complex, in the presence and absence of Ca2+. In the presence of Ca2+ at pH 8.2, anti-PhK1 activated the holoenzyme more than 11-fold and activated the gamma delta complex 2.5-fold. Activation of the holoenzyme and the gamma delta complex by anti-PhK5 was 50-70% of that observed with anti-PhK1. Prior phosphorylation of the holoenzyme by the cAMP-dependent protein kinase blocked activation by both anti-PhK1 and anti-PhK5. Antibodies raised against the peptides from the N terminus of the regulatory domain, anti-PhK9 and anti-PhK13, were inhibitory, with their greatest effects on the gamma delta complex. These data demonstrate that the binding of antibodies to specific regions within the regulatory domain of the gamma-subunit can augment or inhibit structural changes and subunit interactions important in regulating phosphorylase kinase activity.
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PMID:Activation and inhibition of phosphorylase kinase by monospecific antibodies raised against peptides from the regulatory domain of the gamma-subunit. 870 82

The 5'-AMP-activated protein kinase (AMPK) mediates several cellular responses to metabolic stress. Rat liver contains at least two isoforms of this enzyme, either alpha1 or alpha2 catalytic subunits together with beta and gamma noncatalytic subunits in a trimeric complex. The alpha1 isoform is purified using a peptide substrate affinity chromatography column with ADR1 (222-234)P229 (LKKLTRRPSFSAQ), corresponding to the cAMP-dependent protein kinase phosphorylation site in the yeast transcriptional activator of the ADH2 gene, ADR1. This peptide is phosphorylated at Ser230 by AMPK alpha1 with a Km of 3.8 microM and a Vmax of 4.8 micromol/min/mg compared to the commonly used rat acetyl-CoA carboxylase (73-87)A77R86-87 peptide substrate, HMRSAMSGLHLVKRR, with a Km of 33.3 microM and a Vmax of 8.1 micromol/min/mg. Thus, the AMPK exhibits some overlapping specificity with the cAMP-dependent protein kinase. The rat liver AMPK alpha1 isoform has a Kcat approximately 250-fold higher than the AMPK alpha2 isoform isolated from rat liver. The AMPK alpha1 isoform readily phosphorylates peptides corresponding to the reported AMPK phosphorylation sites in rat, chicken, and yeast acetyl-CoA carboxylase and rat hydroxymethylglutaryl-CoA reductase but not phosphorylase kinase. Based on previous peptide substrate specificity studies (Dale, S., Wilson, W. A., Edelman, A. M., and Hardie, G. (1995) FEBS Lett. 361, 191-195) using partially purified enzyme and variants of the peptide AMARAASAAALARRR, it was proposed that the AMPK preferred the phosphorylation site motif Phi(X, beta)XXS/TXXXPhi (Phi, hydrophobic; beta, basic). In good AMPK alpha1 peptide substrates, a hydrophobic residue at the P-5 position is conserved but not at the P+4 position. Oxidation of the Met residues in the rat acetyl-CoA carboxylase (73-87)A77R86-87 peptide increased the Km 6-fold and reduced the Vmax to 4% of the reduced peptide.
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PMID:Isoform-specific purification and substrate specificity of the 5'-AMP-activated protein kinase. 891 Apr 70

A 74-kDa delta/B" subunit was isolated by heparin-Sepharose column chromatography from human erythrocyte protein phosphatase 2A (PP2A) consisting of a 34-kDa catalytic subunit (alpha/C) and 63- and 74-kDa regulatory subunits (beta/A and delta/B") in a ratio of 1:1:1. The purified delta/B" was used as an immunogen in mice, to prepare specific antisera against delta/B". Immunoblot analyses with the antisera detected an immunoreactive 72-kDa protein in the cytosol from various rat tissues including erythrocytes, brain, lung, testis, adrenal gland, heart, spleen, kidney, and liver. The 72-kDa protein was highly abundant in brain and was distributed evenly in cerebral cortex, cerebellum, and brain stem. The 72-kDa protein was also detected in mitochondria and microsome fractions. An immunoreactive 68-kDa protein was detected mainly in nuclear and microsome fractions. The 72-kDa protein from rat brain cytosol copurified with phosphorylated H2B histone phosphatase activity during successive chromatographies on DEAE-Toyopearl, AH-Sepharose, Sephadex G-150, H1 histone-Toyopearl, TSK DEAE-5PW, protamine-Toyopearl, and TSK G3000SW columns. The purified enzyme migrated as a single protein band on nondenaturing PAGE and as three protein bands of 34, 63, and 72 kDa in a ratio of 1:1:1 on SDS-PAGE. The molecular weight of the enzyme was estimated to be 170,000 from the s20,W value of 7.2 +/- 0.3 S and the Stokes radius of 5.5 +/- 0.1 nm. The rat brain enzyme was classified as PP2A, based on the following properties; (1) an IC50 for okadaic acid of 10(-9) M; (2) its preferential dephosphorylation of the a subunit of phosphorylase kinase; (3) its insensitivity to protein inhibitor 2; and (4) its heterotrimeric subunit structure. The Km value and the molecular activity of the enzyme for phosphorylated H2B histone were 72.3 +/- 0.3 microM and 192 +/- 2 mol Pi released/min/mol enzyme, respectively, and were comparable to those of human erythrocyte PP2A (alpha1 beta1 delta1/ CAB"). The 72-kDa subunit in the purified rat brain PP2A was phosphorylated in vitro by cAMP-dependent protein kinase.
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PMID:Tissue and subcellular distributions, and characterization of rat brain protein phosphatase 2A containing a 72-kDa delta/B" subunit. 927 86

A synthetic peptide corresponding to the autophosphorylation site of Ca2+/calmodulin-dependent protein kinase II (CaMKII) (residues 281-289) was conjugated to paramagnetic particles, and phosphorylated by a constitutively active CaMKII fragment. Using this phosphopeptide conjugate as a substrate, a calyculin A-insensitive, Mn(2+)-dependent, and poly-L-lysine-stimulated protein phosphatase activity was detected in the crude extract of rat brain. The protein phosphatase (designated as CaMKII phosphatase) (CaMKIIPase) was purified to near homogeneity from rat brain. CaMKIIPase showed apparent molecular weights of 54,000 and 65,000, on SDS-polyacrylamide gel electrophoresis and gel-filtration analysis, respectively. It was not inhibited by 100 nM calyculin A or 10 microM okadaic acid. Mn2+, but not Mg2+, was absolutely required for activity. CaMKIIPase was potently activated by polycations. Autophosphorylated CaMKII was dephosphorylated by CaMKIIPase, whereas phosphorylase kinase, mixed histones, myelin basic protein, and alpha-casein (which had been phosphorylated by cAMP-dependent protein kinase) and phosphorylase a (phosphorylated by phosphorylase kinase) were not significantly dephosphorylated. No other proteins than CaMKII in rat brain extract which had been phosphorylated by CaMKII were dephosphorylated. The stimulated Ca(2+)-independent activity of autophosphorylated CaMKII was reversed by the action of CaMKIIPase. Thus, CaMKIIPase appears to be a specialized protein phosphatase for the regulation of CaMKII.
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PMID:A novel protein phosphatase that dephosphorylates and regulates Ca2+/calmodulin-dependent protein kinase II. 944 23

We have identified proteins in adult chicken skeletal muscle whose phosphorylation can be used as markers for the mature fast and slow muscle phenotype. These include phosphorylase, phosphorylase kinase, and a cyclic adenosine 3',5'-monophosphate (cAMP)-stimulated, calmodulin-inhibited 28-kDa band (markers for fast muscle), a calmodulin-stimulated 50-kDa band, and two cAMP-stimulated bands at 44 and 46 kDa (markers for slow muscle), and the relative concentrations of the regulatory subunits of cAMP-dependent protein kinase (RI and RII). After denervation the pattern of phosphorylation in fast muscle changed to resemble that of slow muscle: phosphorylation of the fast phenotype markers decreased; the slow phenotype markers, barely detectable in normal fast muscle, appeared as significant phosphoproteins; and the concentration of RII increased with no change in RI. This is consistent with denervation-induced changes observed using other phenotypic markers and indicates the potential for using these phosphoprotein markers in studies of muscle development and pathophysiology.
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PMID:Protein phosphorylation in fast and slow chicken skeletal muscles: effect of denervation. 953 85

The isolated catalytic subunit of cAMP-dependent protein kinase and smooth muscle myosin light chain kinase undergo interactions with the fluorescent dye 9-anthroylcholine (9AC) that are responsive to the two enzymes' associations with substrates and effectors. Additionally, the binding of 9AC is highly sensitive to subtle structural or functional differences among closely related protein kinases. Skeletal muscle myosin light chain kinase and the catalytically active chymotryptic fragment of the gamma-subunit of phosphorylase kinase do not associate with 9AC. The 1:1 fluorescent complex of the isolated catalytic subunit of cAMP-dependent protein kinase with 9AC exhibits a dissociation constant of 21 microM. The association of the catalytic subunit with either of the regulatory subunits, RI and RII, results in decreases in the observed 9AC fluorescence that are reversed upon the addition of cAMP. The effects of MgATP and of polypeptide substrates (Kemptide, troponin I, protamine) on the 9AC-catalytic subunit complex are consistent with a general noncompetitive model in which the interactions of 9AC and the other ligands with the enzyme are mutually antagonistic but not purely competitive.
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PMID:Binding of 9-anthroylcholine monitors the interactions of adenosine cyclic 3',5'-phosphate-dependent protein kinase with MgATP, substrates, and regulatory subunits. 985 61

AMP-activated protein kinase (AMPK) and cAMP-dependent protein kinase (cAMPK) have been reported to phosphorylate sites on phosphorylase kinase (PhK). Their target residues Ser 1018 and Ser 1020, respectively, are located in the so-called multi-phosphorylation domain in the PhK alpha subunit. In PhK preparations, only one of these serines is phosphorylated, but never both of them. The aim of this study was to determine whether phosphorylation by cAMPK or AMPK would influence subsequent phosphorylation by the other kinase. Surprisingly, employing four different PhK substrates, it could be demonstrated that, in contradiction to previous reports, PhK is not phosphorylated by AMPK.
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PMID:Muscle phosphorylase kinase is not a substrate of AMP-activated protein kinase. 1093 78


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