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)

We have characterized the strongly homologous GPP1/RHR2 and GPP2/HOR2 genes, encoding isoforms of glycerol 3-phosphatase. Mutants lacking both GPP1 and GPP2 are devoid of glycerol 3-phosphatase activity and produce only a small amount of glycerol, confirming the essential role for this enzyme in glycerol biosynthesis. Overproduction of Gpp1p and Gpp2p did not significantly enhance glycerol production, indicating that glycerol phosphatase is not rate-limiting for glycerol production. Previous studies have shown that expression of both GPP1 and GPP2 is induced under hyperosmotic stress and that induction partially depends on the HOG (high osmolarity glycerol) pathway. We here show that expression of GPP1 is strongly decreased in strains having low protein kinase A activity, although it is still responsive to osmotic stress. The gpp1Delta/gpp2Delta double mutant is hypersensitive to high osmolarity, whereas the single mutants remain unaffected, indicating GPP1 and GPP2 substitute well for each other. Transfer to anaerobic conditions does not affect expression of GPP2, whereas GPP1 is transiently induced, and mutants lacking GPP1 show poor anaerobic growth. All gpp mutants show increased levels of glycerol 3-phosphate, which is especially pronounced when gpp1Delta and gpp1Delta/gpp2Delta mutants are transferred to anaerobic conditions. The addition of acetaldehyde, a strong oxidizer of NADH, leads to decreased glycerol 3-phosphate levels and restored anaerobic growth of the gpp1Delta/gpp2Delta mutant, indicating that the anaerobic accumulation of NADH causes glycerol 3-phosphate to reach growth-inhibiting levels. We also found the gpp1Delta/gpp2Delta mutant is hypersensitive to the superoxide anion generator, paraquat. Consistent with a role for glycerol 3-phosphatase in protection against oxidative stress, expression of GPP2 is induced in the presence of paraquat. This induction was only marginally affected by the general stress-response transcriptional factors Msn2p/4p or protein kinase A activity. We conclude that glycerol metabolism plays multiple roles in yeast adaptation to altered growth conditions, explaining the complex regulation of glycerol biosynthesis genes.
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PMID:The yeast glycerol 3-phosphatases Gpp1p and Gpp2p are required for glycerol biosynthesis and differentially involved in the cellular responses to osmotic, anaerobic, and oxidative stress. 1105 91

Sequence analysis of mitochondrial and nuclear candidate genes of complex I in children with deficiency of this complex and exhibiting Leigh-like syndrome has revealed, in one of them, a novel mutation in the NDUFS4 gene encoding the 18 kDa subunit. Phosphorylation of this subunit by cAMP-dependent protein kinase has previously been found to activate the complex. The present mutation consists of a homozygous G-->A transition at nucleotide position +44 of the coding sequence of the gene, resulting in the change of a tryptophan codon to a stop codon. Such mutation causes premature termination of the protein after only 14 amino acids of the putative mitochondrial targeting peptide. Fibroblast cultures from the patient exhibited severe reduction of the rotenone-sensitive NADH-->UQ oxidoreductase activity of complex I, which was insensitive to cAMP stimulation. Two-dimensional electrophoresis showed the absence of detectable normally assembled complex I in the inner mitochondrial membrane. These findings show that the expression of the NDUFS4 gene is essential for the assembly of a functional complex I.
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PMID:A nonsense mutation in the NDUFS4 gene encoding the 18 kDa (AQDQ) subunit of complex I abolishes assembly and activity of the complex in a patient with Leigh-like syndrome. 1118 77

Human NADH CoQ oxidoreductase is composed of a total of 43 subunits and has been demonstrated to be a major site for the production of superoxide by mitochondria. Incubation of rat heart mitochondria with ATP resulted in the phosphorylation of two mitochondrial membrane proteins, one with a M(r) of 6 kDa consistent with the NDUFA1 (MWFE), and one at 18kDa consistent with either NDUFS4 (AQDQ) or NDUFB7 (B18). Phosphorylation of both subunits was enhanced by cAMP derivatives and protein kinase A (PKA) and was inhibited by PKA inhibitors (PKAi). When mitochondrial membranes were incubated with pyruvate dehydrogenase kinase, phosphorylation of an 18kDa protein but not a 6kDa protein was observed. NADH cytochrome c reductase activity was decreased and superoxide production rates with NADH as substrate were increased. On the other hand, with protein kinase A-driven phosphorylation, NADH cytochrome c reductase was increased and superoxide production decreased. Overall there was a 4-fold variation in electron transport rates observable at the extremes of these phosphorylation events. This suggests that electron flow through complex I and the production of oxygen free radicals can be regulated by phosphorylation events. In light of these observations we discuss a potential model for the dual regulation of complex I and the production of oxygen free radicals by both PKA and PDH kinase.
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PMID:Control of oxygen free radical formation from mitochondrial complex I: roles for protein kinase A and pyruvate dehydrogenase kinase. 1186 82

Compromised SERCA 2a activity is a key malfunction leading to the Ca(2+) cycling alterations in failing human myocardium. SERCA 2a activity is regulated by the Ca(2+)/calmodulin-dependent protein kinase (CaM-kinase) but alterations of the CaM-kinase pathway regarding SERCA 2a in heart failure are unresolved. Therefore we investigated the CaM-kinase and phosphatase calcineurin mediated regulation of SERCA 2a in failing and non-failing human myocardium. We studied human myocardial preparations from explanted hearts from non-failing organ donors (NF, n=8) and from patients with terminal heart failure undergoing cardiac transplantation (dilated cardiomyopathy, DCM, n=8). SERCA 2a activity was determined using a NADH-coupled enzyme assay [expressed in nmol ATP/(mg protein x min)] and by(45)Ca(2+) uptake. Protein expression of SERCA 2a, phospholamban, calsequestrin and calcineurin was assessed by Western blotting (expressed as densitometric units/microg protein); phosphorylation of cardiac proteins was detected with specific phospho-antibodies for phospholamban at threonine-17 (PT17) or by incorporation of [gamma -(32)P] (expressed as pmol(32)P/mg). Maximal(45)Ca(2+) uptake (in pmol/mg/min) (NF: 3402+/-174; DCM: 2488+/-189) and maximal SERCA 2a activity were reduced in DCM compared to NF (V(max): NF: 125+/-9; DCM: 98+/-5). The V(max) reduction could be mimicked by calcineurin in vitro in NF (NF(control): 72.1+/-3.7; NF(+calcineurin): 49.8+/-2.9) and restored in DCM by CaM-kinase in vitro (DCM(control): 98+/-5; DCM(+CaM-kinase): 120+/-6). Protein expression of SERCA 2a, phospholamban and calsequestrin remained similar, but calcineurin expression was significantly increased in failing human hearts (NF: 11.6+/-1.5 v DCM: 17.1+/-1.6). Although the capacity of endogenous CaM-kinase to phosphorylate PT17 was significantly higher in DCM (DCM(control): 128+/-36; DCM(+endogenous CaM-kinase): 205+/-20) compared to NF myocardium (NF(control): 273+/-37; NF(+endogenous CaM-kinase): 254+/-31), net phosphorylation at threonine-17 phospholamban was significantly lower in DCM (DCM 130+/-11 v NF 170+/-11). A calcineurin-dependent dephosphorylation of phospholamban could be mimicked in vitro by incubation of NF preparations with calcineurin (NF(control) 80.7+/-4.4 v NF(+calcineurin) 30.7+/-4.1, P<0.05). In human myocardium, the V(max) of SERCA 2a and the phosphorylation of phospholamban is modulated by CaM-kinase and calcineurin, at least in vitro. In failing human myocardium, despite increased CaM-kinase activity, calcineurin dephosphorylation leads to decreased net phosphorylation of threonine-17 phospholamban in vivo. Increased calcineurin activity contributes to the impaired V(max) of SERCA 2a in failing human myocardium and the disorder in Ca(2+)-handling in heart failure.
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PMID:Evidence for calcineurin-mediated regulation of SERCA 2a activity in human myocardium. 1194 24

A complete biochemical understanding of the mechanisms by which hyperglycemia causes vascular functional and structural changes associated with the diabetic milieu still eludes us. In recent years, the numerous biochemical and metabolic pathways postulated to have a causal role in the pathogenesis of diabetic vascular disease have been distilled into several unifying hypotheses. These involve either increased reductive or oxidative stress to the cell, or the activation of numerous protein kinase pathways, particularly protein kinase C and mitogen-activated protein kinases. As detailed below, there is tremendous crosstalk between these competing hypotheses. We propose that increased tissue glucose levels alter cytosolic coenzyme balance by increased flux of glucose through the sorbitol pathway increasing free cytosolic NADH levels. Increased NADH levels can generate reactive oxygen species via numerous mechanisms, lead to the formation of intracellular advanced glycation end products, and induce growth factor expression via mechanisms involving protein kinase C activation. The elevation in growth factors, particularly vascular endothelial growth factor (VEGF), is responsible for the vascular dysfunction via numerous mechanisms reported here in detail.
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PMID:Diabetic vascular dysfunction: links to glucose-induced reductive stress and VEGF. 1211 45

Evidence is accumulating that the activity of spinach (Spinacia oleracea L.) leaf NADH:nitrate reductase (NR) is modulated both in vitro and in vivo by protein phosphorylation. From the present study we report the partial purification of the two protein factors needed for NR inactivation. We identified NR-protein kinase (NR-PK) as a calcium-dependent and metabolite-regulated protein kinase and have provided additional evidence that phosphorylation of NR is necessary but not sufficient to inactivate the enzyme. The inhibitor protein required for inactivation of phospho-NR was purified 625-fold by polyethylene glycol fractionation and sequential column chromatography. Using partially purified inhibitor protein and NR-PK, we characterized NR inactivation (increased sensitivity to Mg2+ inhibition) in a reconstituted in vitro system. NR-PK activity was inhibited by a variety of metabolic phosphate esters including di-hydroxyacetone phosphate, glucose-6-phosphate, and fructose-1,6-bisphosphate. Light-to-dark transition experiments with a starchless tobacco (Nicotiana sylvestris) mutant, which accumulates phosphate esters during the photoperiod, indicated that NR inactivation in vivo might, indeed, be down-regulated by metabolites. Additionally, we postulate that cytosolic free calcium could play an important role in the regulation of NR activity in vivo.
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PMID:Partial Purification and Characterization of a Calcium-Dependent Protein Kinase and an Inhibitor Protein Required for Inactivation of Spinach Leaf Nitrate Reductase. 1222 29

ADR1 and CAT8 encode carbon source-responsive transcriptional regulators that cooperatively control expression of genes involved in ethanol utilization. These transcription factors are active only after the diauxic transition, when glucose is depleted and energy-generating metabolism has shifted to the aerobic oxidation of non-fermentable carbon sources. The Snf1 protein kinase complex is required for activation of their downstream target genes described previously. Using DNA microarrays, we determined the extent to which these three factors collaborate in regulating the expression of the yeast genome after glucose depletion. The expression of 108 genes is significantly decreased in the absence of ADR1. The importance of ADR1 during the diauxic transition is illustrated by the observation that expression of almost one-half of the 40 most highly glucose-repressed genes is ADR1-dependent. ADR1-dependent genes fall into a variety of functional classes with carbon metabolism containing the largest number of members. Most of the genes in this class are involved in the oxidation of different non-fermentable carbon sources. These microarray data show that ADR1 coordinates the biochemical pathways that generate acetyl-CoA and NADH from non-fermentable substrates. Only a small number of ADR1-dependent genes are also CAT8-dependent. However, nearly one-half of the ADR1-dependent genes are also dependent on the Snf1 protein kinase for derepression. Many more genes are SNF1-dependent than are either ADR1- or CAT8-dependent suggesting that SNF1 plays a broader role in gene expression than either ADR1 or CAT8. The largest class of SNF1-dependent genes encodes regulatory proteins that could extend SNF1 dependence to additional pathways.
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PMID:Multiple pathways are co-regulated by the protein kinase Snf1 and the transcription factors Adr1 and Cat8. 1267 48

Amino acids are not only important precursors for the synthesis of proteins and other N-containing compounds, but also participate in the regulation of major metabolic pathways. Glutamate and aspartate, for example, are components of the malate/aspartate shuttle and their concentrations control the rate of mitochondrial oxidation of glycolytic NADH. Glutamate also controls the rate of urea synthesis, not only as the precursor of ammonia and aspartate, but as substrate for synthesis of N-acetylglutamate, the essential activator of carbamoyl-phosphate synthase. This mechanism allows large variations in urea synthesis at relatively constant ammonia concentrations. Increases in intracellular amino acid concentration increase cell volume. Cell swelling per se has anabolic effects on protein, carbohydrate and lipid metabolism: enhanced synthesis of macromolecules compensates for increases in intracellular osmolarity. Mechanisms responsible for cell swelling-induced changes in pathway fluxes include changes in intracellular ion concentrations and in signal transduction. Specific amino acids (e.g., leucine) stimulate protein synthesis and inhibit (autophagic) protein degradation independent of changes in cell volume because they stimulate mTOR (mammalian target of rapamycin), a protein kinase, which is one of the components of a signal transduction pathway used by insulin. When the cellular energy state is low, stimulation of mTOR by amino acids is prevented by activation of AMP-dependent protein kinase. Amino acid-dependent signaling also promotes insulin production by beta-cells. This further adds to the anabolic properties of amino acids. It is concluded that amino acids are important regulators of major metabolic pathways.
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PMID:Amino acids as regulators and components of nonproteinogenic pathways. 1277 65

We had previously suggested that phosphorylation of proteins by mitochondrial kinases regulate the activity of NADH/CoQ oxidoreductase. Initial data showed that pyruvate dehydrogenase kinase (PDK) and cAMP-dependent protein kinase A (PKA) phosphorylate mitochondrial membrane proteins. Upon phosphorylation with crude PDK, mitochondria appeared to be deficient in NADH/cytochrome c reductase activity associated with increased superoxide production. Conversely, phosphorylation by PKA resulted in increased NADH/cytochrome c reductase activity and decreased superoxide formation. Current data confirms PKA involvement in regulating Complex I activity through phosphorylation of an 18 kDa subunit. Beef heart NADH/ cytochrome c reductase activity increases to 150% of control upon incubation with PKA and ATP-gamma-S. We have cloned the four human isoforms of PDK and purified beef heart Complex I. Incubation of mitochondria with PDK isoforms and ATP did not alter Complex I activity or superoxide production. Radiolabeling of mitochondria and purified Complex I with PDK failed to reveal phosphorylated proteins.
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PMID:Regulation of NADH/CoQ oxidoreductase: do phosphorylation events affect activity? 1511 79

Alcohol has long been thought to cause fatty liver by way of altered NADH/NAD(+) redox potential in the liver, which, in turn, inhibits fatty acid oxidation and the activity of tricarboxylic acid cycle reactions. More recent studies indicate that additional effects of ethanol both impair fat oxidation and stimulate lipogenesis. Ethanol interferes with DNA binding and transcription-activating properties of peroxisome proliferator-activated receptor-alpha (PPARalpha), as demonstrated with cultured cells and in ethanol-fed mice. Treatment of ethanol-fed mice with a PPARalpha agonist can reverse fatty liver even in the face of continued ethanol consumption. Ethanol also activated sterol regulatory element binding protein 1, inducing a battery of lipogenic enzymes. These effects may be due in part to inhibition of AMP-dependent protein kinase, reduction in plasma adiponectin, or increased levels of TNF-alpha in the liver. The understanding of these ethanol effects provides new therapeutic targets to reverse alcoholic fatty liver.
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PMID:Recent advances in alcoholic liver disease II. Minireview: molecular mechanisms of alcoholic fatty liver. 1519 57


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