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:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A protein inhibitor of HMG-CoA reductase phosphatase activity from rat liver was purified to homogeneity. The protein was purified 4,000-fold with an overall yield of 4%. The purified protein had a molecular mass of 31 kDa. This spontaneously active protein is thermostable and acid-resistant. The protein inhibitor is phosphorylated by glycogen synthase kinase-3 and cAMP-dependent protein kinase without change in its inhibitory activity. The inhibition caused by this inhibitor on phosphatases 1 and 2A is similar to that of inhibitor-2 from rabbit skeletal muscle using hydroxymethylglutaryl-CoA reductase as substrate. The regulation properties of this inhibitor towards phosphatase 1 together with another protein inhibitor of phosphatase 2A in cholesterol metabolism are discussed.
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PMID:Purification and characterization of a protein inhibitor from rat liver that inhibits type 1 protein phosphatase when 3-hydroxy-3-methylglutaryl CoA reductase is the substrate. 216 23

A heat-stable protein inhibitor of the hydroxymethylglutaryl-CoA reductase phosphatase 2A activity has been identified and purified to homogeneity, as judged by polyacrylamide gel electrophoresis. The apparent molecular mass was 20,000 Da. The protein lost its inhibitory properties when incubated with trypsin or treated with ethanol. The inhibitor protein does not inhibit type 1 phosphatase when either phosphorylase or hydroxymethylglutaryl-CoA reductase is the substrate. In contrast, this protein inhibitor inhibits the rat liver type 2A phosphatase activity when hydroxymethylglutaryl-CoA reductase is the substrate but not when phosphorylase a is the substrate. The inhibitor protein is not activated by incubation with ATP and cyclic AMP-dependent protein kinase and it is not phosphorylated by glycogen synthase kinase-3. These results, together with those of the kinetic experiments, suggest that the reductase phosphatase inhibitor is distinct from protein phosphatase inhibitor-1 and inhibitor-2.
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PMID:Purification and properties of a protein inhibitor that inhibits phosphatase 2A activity when hydroxymethylglutaryl coenzyme A reductase is the substrate. 254 29

A calcium-activated and phospholipid-dependent protein kinase (protein kinase C) catalyzes the phosphorylation of both insoluble microsomal (Mr approximately 100,000) and purified soluble (Mr = 53,000) 3-hydroxy-3-methylglutaryl coenzyme A reductase. The phosphorylation and concomitant inactivation of enzymic activity of HMG-CoA reductase was absolutely dependent on Ca2+, phosphatidylserine, and diolein. Dephosphorylation of phosphorylated HMG-CoA reductase was associated with the loss of protein bound radioactivity and reactivation of enzymic activity. Maximal phosphorylation of purified HMG-CoA reductase was associated with the incorporation of 1.05 +/- 0.016 mol of phosphate/mol of native form of HMG-CoA reductase (Mr approximately 100,000). The apparent Km for purified HMG-CoA reductase and histone H1 was 0.08 mg/ml, and 0.12 mg/ml, respectively. The tumor-promoting phorbol ester, phorbol 12-myristate 13-acetate stimulated the protein kinase C-catalyzed phosphorylation of HMG-CoA reductase. Increased phosphorylation of HMG-CoA reductase by phorbol 12-myristate 13-acetate suggests a possible in vivo protein kinase C-mediated mechanism for the short-term regulation of HMG-CoA reductase activity. The identification of the protein kinase C system in addition to the reductase kinase-reductase kinase kinase bicyclic cascade systems for the modulation of the enzymic activity of HMG-CoA reductase may provide new insights into the molecular mechanisms involved in the regulation of cholesterol biosynthesis.
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PMID:Phosphorylation of hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase and modulation of its enzymic activity by calcium-activated and phospholipid-dependent protein kinase. 315 37

A protein kinase which phosphorylates and inactivates acetyl-CoA carboxylase has been purified to apparent homogeneity from rat liver. The kinase was found to exist in two forms: bound to carboxylase in a complex or in a free form that is in different stages of aggregation over a wide range of molecular weights. The purification of the kinase involved first partial purification of acetyl-CoA carboxylase through polyethylene glycol precipitation and DEAE-cellulose chromatography. The kinase was then separated from acetyl-CoA carboxylase by Sepharose 2B chromatography. The molecular weight of the kinase subunit was 170,000 as determined by sodium dodecyl sulfate-gel electrophoresis. The incorporation of 1 mol of phosphate/mole of carboxylase subunit caused complete inactivation of the carboxylase. Acetyl-CoA carboxylase, inactivated by the kinase, can be dephosphorylated and reactivated when incubated with phosphorylase phosphatase. The Km values of the kinase for acetyl-CoA carboxylase and ATP are 90 nM and 20 microM, respectively. The kinase was found to be cyclic AMP-independent, but activated by CoA. The protein kinase can phosphorylate acetyl-CoA carboxylase, protamine, and histones, but could not act on hydroxymethylglutaryl-CoA reductase or phosphorylase b.
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PMID:Purification and properties of a kinase which phosphorylates and inactivates acetyl-CoA carboxylase. 612 Jan 70

The AMP-activated protein kinase is responsible for the regulation of fatty acid synthesis by phosphorylation of acetyl-CoA carboxylase. It may also regulate cholesterol synthesis via phosphorylation and inactivation of hormone-sensitive lipase and hydroxymethylglutaryl-CoA reductase. We have purified the AMP-activated protein kinase 14,000-fold from porcine liver. The 63-kDa catalytic subunit co-purifies with two proteins of 40 and 38 kDa that may function as subunits. Partial amino acid sequence of the 63-kDa subunit revealed a striking homology with the catalytic domain of the yeast protein kinase transcriptional regulator Snf1 and its plant homologs. The Snf1 (72 kDa) and Snf4 (36 kDa) complex was also purified and found to phosphorylate the AMP-activated protein kinase peptide substrate, HMRSAMSGLHLVKRR-amide, but was not activated by AMP. Both Snf1/4 and the AMP-activated protein kinase phosphorylate and inactivate yeast acetyl-CoA carboxylase in vitro. These results indicate that during evolution the catalytic domain sequences of the Snf1 protein kinase subfamily have been exploited in the control of mammalian lipid metabolism and raise the possibilities that the AMP-activated protein kinase may have other substrates involved in regulating gene expression pathways, as well as Snf1 homologs participating in the control of lipid metabolism in many eukaryotic organisms.
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PMID:Mammalian AMP-activated protein kinase shares structural and functional homology with the catalytic domain of yeast Snf1 protein kinase. 790 77

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

The activity of Pseudomonas mevalonii HMG-CoA reductase (EC 1.1.1.88) is not regulated by phosphorylation, presumably due to the absence of a suitable target serine and protein kinase recognition motif. We have engineered P. mevalonii HMG-CoA reductase to a form whose activity, like that of mammalian HMG-CoA reductases, is regulated by phosphorylation/dephosphorylation. We substituted serine for arginine 387, the residue that corresponds to the regulatory serine of the HMG-CoA reductases of higher eukaryotes. A recognition motif for cAMP-dependent protein kinase was added by replacing leucine 384 by histidine (enzyme L384H/R387S) and also valine 391 by leucine (enzyme L384H/R387S/V391L). The activity of P. mevalonii HMG-CoA reductase mutant enzymes L384H/R387S and L384H/R387S/V391L was attenuated by phosphorylation. Restoration of activity accompanied subsequent dephosphorylation catalyzed by lambda protein phosphatase. Incorporation and subsequent release of phosphate paralleled the attenuation and restoration of catalytic activity. Incorporation of 0.5 mol of phosphate per subunit was accompanied by an approximately 50% decrease in initial activity. As in the analogous Syrian hamster mutant enzyme S871D, P. mevalonii mutant enzyme R387D exhibited 10% wild-type activity, suggesting that the attenuation of activity that accompanies phosphorylation results at least in part from the introduction of negative charge. Engineering of P. mevalonii HMG-CoA reductase to forms whose activity is reversibly regulated by phosphorylation/dephosphorylation provides an attractive model for future structure-based mechanistic studies. Solution of the X-ray structure of phosphorylated and dephosphorylated forms of engineered P. mevalonii HMG-CoA reductase should then reveal interactions of the active site phosphoseryl residue that result in attenuation of catalytic activity.
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PMID:Protein engineering of the HMG-CoA reductase of Pseudomonas mevalonii. Construction of mutant enzymes whose activity is regulated by phosphorylation and dephosphorylation. 904 17

A single entity, the AMP-activated protein kinase (AMPK), phosphorylates and regulates in vivo hydroxymethylglutaryl-CoA reductase and acetyl-CoA carboxylase (key regulatory enzymes of sterol synthesis and fatty acid synthesis, respectively), and probably many additional targets. The kinase is activated by high AMP and low ATP via a complex mechanism, which involves allosteric regulation, promotion of phosphorylation by an upstream protein kinase (AMPK kinase), and inhibition of dephosphorylation. This protein-kinase cascade represents a sensitive system, which is activated by cellular stresses that deplete ATP, and thus acts like a cellular fuel gauge. Our central hypothesis is that, when it detects a 'low-fuel' situation, it protects the cell by switching off ATP-consuming pathways (e.g. fatty acid synthesis and sterol synthesis) and switching on alternative pathways for ATP generation (e.g. fatty acid oxidation). Native AMP-activated protein kinase is a heterotrimer consisting of a catalytic alpha subunit, and beta and gamma subunits, which are also essential for activity. All three subunits have homologues in budding yeast, which are components of the SNF1 protein-kinase complex. SNF1 is activated by glucose starvation (which in yeast leads to ATP depletion) and genetic studies have shown that it is involved in derepression of glucose-repressed genes. This raises the intriguing possibility that AMPK may regulate gene expression in mammals. AMPK/SNF1 homologues are found in higher plants, and this protein-kinase cascade appears to be an ancient system which evolved to protect cells against the effects of nutritional or environmental stress.
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PMID:The AMP-activated protein kinase--fuel gauge of the mammalian cell? 920 14

An increasing body of evidence suggests that an endogenous mammalian bufadienolide (BD) may be involved in the regulation of Na(+),K(+)-ATPase activity and the pathogenesis of arterial hypertension. We developed a purification scheme for marinobufagenin (MBG), an amphibian cardiotonic BD, and applied it to purify and characterize material in human plasma, culture medium conditioned by Y-1 adrenocortical cells, and rat adrenal tissue. MBG immunoreactivity purified from plasma and measured by ELISA showed important similarities (chromatography and antibody cross-reactivity) to material secreted into cell culture medium by Y-1 cells. This observation indicates that circulating mammalian BD may have an adrenocortical origin. Release of mammalian BD from adrenocortical cells grown in the absence of exogenous cholesterol was reduced by treatment of cultures with mevastatin, a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor. Supplementation of the serum and cholesterol-free cell culture medium with the LDL fraction of human plasma increased the production of MBG material in the presence of mevastatin, supporting its origin from cholesterol. We used Y-1 cell lines transfected with genes shown to inhibit steroidogenesis through cholesterol side-chain cleavage (Y-1/DAX and Y-1/RIAB) to investigate the dependence of MBG biosynthesis on side-chain cleavage. Our results indicate that the mammalian BD is synthesized in the adrenal cortex from cholesterol and shares important similarities with the amphibian BD MBG, that its biosynthesis is independent of transfer of cholesterol to the side-chain cleavage enzyme complex mediated by steroidogenic acute regulatory protein, and that neither cAMP nor protein kinase A appears to be a critical component of the pathway controlling its biosynthesis.
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PMID:Mammalian bufadienolide is synthesized from cholesterol in the adrenal cortex by a pathway that Is independent of cholesterol side-chain cleavage. 1098 79

A series of inhibitors were tested to determine the participation of de novo protein synthesis, protein kinase activity, extracellular Ca2+, and lipoxygenase activity in arachidonic acid elicitation of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) gene expression and sesquiterpene phytoalexin biosynthesis in potato (Solanum tuberosum L. cv Kennebec). Gene-specific probes were used to discriminate effects on the expression of two HMGR genes (hmg1 and hmg2) that respond differentially in tuber tissue following wounding or elicitor treatment. Inhibition of protein synthesis with cycloheximide completely blocked arachidonate-induced hypersensitive necrosis and browning, including HMGR gene induction and phytoalexin accumulation. This suggests that proteins necessary for coupling arachidonic acid reception to HMGR mRNA accumulation are either rapidly turned over or not present constitutively and are induced following elicitor treatment. Staurosporin, a potent inhibitor of protein kinases, and ethyleneglycol-bis([beta]-aminoethyl ether)-N,N[prime]-tetraacetic acid, a Ca2+ chelator, inhibited arachidonate-induction of hmg2 gene expression and phytoalexin accumulation but did not inhibit the wound-induced expression of hmg1. However, staurosporin inhibited arachidonate's suppression of hmg1 gene expression. Eicosatetraynoic acid, a lipoxygenase inhibitor that suppresses elicitor-induced phytoalexin accumulation, also inhibited arachidonate's suppression of hmg1 and induction of hmg2. The results indicate that arachidonate's suppression of hmg1 and activation of hmg2 depend on a common intermediate or set of intermediates whose generation is sensitive to the inhibitors tested.
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PMID:Involvement of de Novo Protein Synthesis, Protein Kinase, Extracellular Ca2+, and Lipoxygenase in Arachidonic Acid Induction of 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Genes and Isoprenoid Accumulation in Potato (Solanum tuberosum L.). 1223 62


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