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:2.7.11.2 (PDK1)
2,238 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Recent evidence from this laboratory indicates that at least two isoenzymic forms of pyruvate dehydrogenase kinase (PDK1 and PDK2) may be involved in the regulation of enzymatic activity of mammalian pyruvate dehydrogenase complex by phosphorylation (Popov, K.M., Kedishvili, N.Y., Zhao, Y., Gudi, R., and Harris, R.A. (1994) J. Biol. Chem. 269, 29720-29724). The present study was undertaken to further explore the diversity of the pyruvate dehydrogenase kinase gene family. Here we report the deduced amino acid sequences of three isoenzymic forms of PDK found in humans. In terms of their primary structures, two isoenzymes identified in humans correspond to rat PDK1 and PDK2, whereas a third gene (PDK3) encodes for a new isoenzyme that shares 68% and 67% of amino acid identities with PDK1 and PDK2, respectively. PDK3 cDNA expressed in Eschierichia coli directs the synthesis of a polypeptide with a molecular mass of approximately 45,000 Da that possesses catalytic activity toward kinase-depleted pyruvate dehydrogenase. PDK3 appears to have the highest specific activity among the three isoenzymes tested as recombinant proteins. Tissue distribution of all three isoenzymes of human PDK was characterized by Northern blot analysis. The highest amount of PDK2 mRNA was found in heart and skeletal muscle, the lowest amount in placenta and lung. Brain, kidney, pancreas, and liver expressed an intermediate amount of PDK2 (brain > kidney = pancreas > liver). The tissue distribution of PDK1 mRNA differs markedly from PDK2. The message for PDK1 was expressed predominantly in heart with only modest levels of expression in other tissues (skeletal muscle > liver > pancreas > brain > placenta = lung > kidney). In contrast to PDk1 and PDK2, which are expressed in all tissues tested, the message for PDK3 was found almost exclusively in heart and skeletal muscle, indicating that PDK3 may serve specialized functions characteristic of muscle tissues. In all tissues tested thus far, the level of expression of PDK2 mRNA was essentially higher than that of PDK1 and PDK3, consistent with the idea that PDK2 is a major isoenzyme responsible for regulation of pyruvate dehydrogenase in human tissues.
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PMID:Diversity of the pyruvate dehydrogenase kinase gene family in humans. 749 31

Four mitochondrial protein kinases have been cloned. These proteins represent a new family of protein kinases, related by sequence to the bacterial protein kinases but by function to the eukaryotic serine protein kinases. Arg288 is required for recognition by BCKDK of the phosphorylation site on the E1alpha subunit of the BCKDH complex. BCKDK inhibits the dehydrogenase activity of the BCKDH complex by introducing a negative charge into the active-site pocket of the E1 component. Protein starvation of rats induces an increase in the amount of BCKDK bound to the BCKDH complex. This causes inactivation of the BCKDH complex and conserves branched-chain amino acids for protein synthesis in the protein-starved state. Expression of the different PDK isoenzymes is tissue specific, and the different PDK isoenzymes are unique with respect to kinetic parameters for ATP and ADP and sensitivity to allosteric effectors (NADH, NAD+, coenzyme A, acetyl-CoA, pyruvate, and dichloroacetate). Preliminary experiments indicate that an increased amount of PDK2 protein partly explains the increase in PDK activity that occurs in rat liver in response to chemically induced diabetes.
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PMID:Mitochondrial alpha-ketoacid dehydrogenase kinases: a new family of protein kinases. 934 45

Tissue distribution and kinetic parameters for the four isoenzymes of pyruvate dehydrogenase kinase (PDK1, PDK2, PDK3 and PDK4) identified thus far in mammals were analysed. It appeared that expression of these isoenzymes occurs in a tissue-specific manner. The mRNA for isoenzyme PDK1 was found almost exclusively in rat heart. The mRNA for PDK3 was most abundantly expressed in rat testis. The message for PDK2 was present in all tissues tested but the level was low in spleen and lung. The mRNA for PDK4 was predominantly expressed in skeletal muscle and heart. The specific activities of the isoenzymes varied 25-fold, from 50nmol/min per mg for PDK2 to 1250nmol/min per mg for PDK3. Apparent Ki values of the isoenzymes for the synthetic analogue of pyruvate, dichloroacetate, varied 40-fold, from 0.2 mM for PDK2 to 8 mM for PDK3. The isoenzymes were also different with respect to their ability to respond to NADH and NADH plus acetyl-CoA. NADH alone stimulated the activities of PDK1 and PDK2 by 20 and 30% respectively. NADH plus acetyl-CoA activated these isoenzymes nearly 200 and 300%. Under comparable conditions, isoenzyme PDK3 was almost completely unresponsive to NADH, and NADH plus acetyl-CoA caused inhibition rather than activation. Isoenzyme PDK4 was activated almost 2-fold by NADH, but NADH plus acetyl-CoA did not activate above the level seen with NADH alone. These results provide the first evidence that the unique tissue distribution and kinetic characteristics of the isoenzymes of PDK are among the major factors responsible for tissue-specific regulation of the pyruvate dehydrogenase complex activity.
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PMID:Evidence for existence of tissue-specific regulation of the mammalian pyruvate dehydrogenase complex. 940 93

This study investigated whether conditions known to alter the activity and phosphorylation state of the pyruvate dehydrogenase complex have specific effects on the levels of isoenzymes of pyruvate dehydrogenase kinase (PDK) in rat heart. Immunoblot analysis revealed a remarkable increase in the amount of PDK4 in the hearts of rats that had been starved or rendered diabetic with streptozotocin. Re-feeding of starved rats and insulin treatment of diabetic rats very effectively reversed the increase in PDK4 protein and restored PDK enzyme activity to levels of chow-fed control rats. Starvation and diabetes also markedly increased the abundance of PDK4 mRNA, and re-feeding and insulin treatment reduced levels of the message to that of controls. In contrast with the findings for PDK4, little or no changes in the amounts of PDK1 and PDK2 protein and the abundance of their messages occurred in response to starvation and diabetes. The observed shift in the relative abundance of PDK isoenzymes probably explains previous studies of the effects of starvation and diabetes on heart PDK activity. The results indicate that control of the amount of PDK4 is important in long-term regulation of the activity of the pyruvate dehydrogenase complex in rat heart.
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PMID:Starvation and diabetes increase the amount of pyruvate dehydrogenase kinase isoenzyme 4 in rat heart. 940 94

Two maize cDNAs were isolated and sequenced that had open reading frames with approximately 37% amino acid identity to mammalian pyruvate dehydrogenase kinases. Both maize kinase sequences contain the five domains with conserved signature residues typical of procaryotic two-component histidine kinases. Sequence comparisons identified six other highly conserved motifs that are proposed to be specific to pyruvate dehydrogenase kinases. In addition, specific Trp and Cys residues are also invariant in these sequences. The maize cDNAs are 1332 (PDK1) and 1602 (PDK2) nucleotides in length, encoding polypeptides with calculated molecular masses of 38,867 and 41,327 Da that share 77% amino acid identity. Reverse transcriptase-polymerase chain reaction analysis with oligonucleotide-specific primers revealed a differential expression pattern for the two isoforms. PDK1 and PDK2 were expressed in Escherichia coli with N-terminal His6 tags to facilitate purification. The recombinant proteins migrated at 44 and 48 kDa, respectively, during SDS-polyacrylamide gel electrophoresis. Anti-PDK1 antibodies immunoprecipitated 75% of pyruvate dehydrogenase kinase activity from a maize mitochondrial matrix fraction, and recognized a matrix protein of 43 kDa. Recombinant PDK2, expressed as a fusion with the maltose-binding protein, inactivated kinase-depleted maize pyruvate dehydrogenase complex when incubated with MgATP, coincident with incorporation of 32P from [gamma-32P]ATP into the alpha subunit of pyruvate dehydrogenase.
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PMID:Molecular analysis of two pyruvate dehydrogenase kinases from maize. 975 1

Oxidative metabolism of glucose is regulated by pyruvate dehydrogenase (PDH) that can be inhibited by isoforms of PDH kinase (PDK). Recently, increased PDK activity has been implicated in the pathogenesis of insulin resistance and non-insulin-dependent diabetes mellitus (NIDDM) in obese subjects. Using quantitative RT-PCR, we measured mRNA of PDK2 and PDK4 isoforms in skeletal muscle biopsies from nondiabetic Pima Indians, a population with a high prevalence of NIDDM associated with obesity. PDK2 and PDK4 mRNAs were positively correlated with fasting plasma insulin concentration, 2-h plasma insulin concentration in response to oral glucose, and percentage body fat, whereas both isoforms were negatively correlated with insulin-mediated glucose uptake rates. Measurements of PDK2 and PDK4 mRNA during the hyperinsulinemic-euglycemic clamp and of PDK2 in cell culture indicated that both transcripts decrease in response to insulin. Increased fatty acid (FA) oxidation has been traditionally viewed as the cause for increased PDK activity contributing to insulin resistance in obese subjects. In contrast, our data indicate that insufficient downregulation of PDK mRNA in insulin-resistant individuals could be a cause of increased PDK expression leading to impaired glucose oxidation followed by increased FA oxidation.
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PMID:Insulin downregulates pyruvate dehydrogenase kinase (PDK) mRNA: potential mechanism contributing to increased lipid oxidation in insulin-resistant subjects. 978 10

Type 1 von Willebrand disease (VWD) is a common inherited disorder characterized by mild to moderate bleeding and reduced levels of von Willebrand factor (VWF). An animal model for human type 1 VWD, the RIIIS/J mouse strain, exhibits a prolonged bleeding time and reduced plasma VWF levels. We have previously mapped the defect in RIIIS/J to distal mouse Chr 11, distinct from the Vwf locus on Chr 6. This locus, Mvwf, was localized to an approximately 0.5-cM interval, tightly linked to Gip, distal to Ngfr, and proximal to Hoxb. We have now used these genetic markers to construct a contig of yeast and bacterial artificial chromosomes and bacteriophage P1 clones spanning the approximately 300-kb Mvwf nonrecombinant interval. In a comparative mapping approach, mouse homologues of mapped human expressed sequence tags (ESTs) were localized relative to the candidate interval. Twenty-one sequence-tagged sites and ESTs from the corresponding human syntenic region 17q21.3 were ordered using the high-resolution Stanford TNG3 radiation hybrid panel. Based on the resulting radiation hybrid map and our mouse genetic and physical maps, the order of human and mouse genes in a >0.7-cM region appears to be conserved. Six genes localized to the Mvwf nonrecombinant interval by comparative mapping included orthologs of GNGT2, ATP6N1, and a nuclear domain protein. Seven other genes or ESTs were excluded from the candidate interval, including orthologs of PHB, PDK2, a speckle-type protein, and a UDP-galactose transporter. Using exon trapping, 10 additional putative expressed sequences were identified within the Mvwf nonrecombinant interval, including a previously cloned murine glycosyltransferase as well as exons showing sequence similarity to genes for Caenorhabditis elegans and Saccharomyces cerevisiae predicted proteins, an Arabidopsis thaliana ubiquitin-conjugating enzyme, and a Gallus gallus mRNA zipcode-binding protein. Further characterization of these putative genes could identify the dominant mutation responsible for low plasma VWF levels in RIIIS/J mice. These data may also aid in the localization of other disease loci mapped to this region, including the gene for tricho-dento-osseous syndrome and a murine locus for susceptibility to ozone-induced acute lung injury.
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PMID:Comparative mapping of distal murine chromosome 11 and human 17q21.3 in a region containing a modifying locus for murine plasma von Willebrand factor level. 980 26

The serine-threonine kinase Akt is a downstream target of phosphoinositide 3-kinase (PI 3-kinase); it is activated by the phosphoinositide 3-phosphate-dependent kinases PDK1 and PDK2. Certain mutated forms of Akt induce oncogenic transformation in chicken embryo fibroblast cultures and hemangiosarcomas in young chickens. This ability to transform cells depends on localization of Akt at the plasma membrane and on the kinase activity of Akt. A transdominant negative form of Akt interferes with oncogenic transformation induced by the p3k oncogene, which codes for an activated form of PI 3-kinase. Akt is therefore an essential mediator of p3k-induced oncogenicity.
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PMID:The akt kinase: molecular determinants of oncogenicity. 984 96

The PtdIns(3,4,5)P3-dependent activation of protein kinase B (PKB) by 3-phosphoinositide-dependent protein kinases-1 and -2 (PDK1 and PDK2 respectively) is a key event in mediating the effects of signals that activate PtdIns 3-kinase. The catalytic domain of serum- and glucocorticoid-regulated protein kinase (SGK) is 54% identical with that of PKB and, although lacking the PtdIns(3,4, 5)P3-binding pleckstrin-homology domain, SGK retains the residues that are phosphorylated by PDK1 and PDK2, which are Thr256 and Ser422 in SGK. Here we show that PDK1 activates SGK in vitro by phosphorylating Thr256. We also show that, in response to insulin-like growth factor-1 (IGF-1) or hydrogen peroxide, transfected SGK is activated in 293 cells via a PtdIns 3-kinase-dependent pathway that involves the phosphorylation of Thr256 and Ser422. The activation of SGK by PDK1 in vitro is unaffected by PtdIns(3,4,5)P3, abolished by the mutation of Ser422 to Ala, and greatly potentiated by mutation of Ser422 to Asp (although this mutation does not activate SGK itself). Consistent with these findings, the Ser422Asp mutant of SGK is activated by phosphorylation (probably at Thr256) in unstimulated 293 cells, and activation is unaffected by inhibitors of PtdIns 3-kinase. Our results are consistent with a model in which activation of SGK by IGF-1 or hydrogen peroxide is initiated by a PtdIns(3,4, 5)P3-dependent activation of PDK2, which phosphorylates Ser422. This is followed by the PtdIns(3,4,5)P3-independent phosphorylation at Thr256 that activates SGK, and is catalysed by PDK1. Like PKB, SGK preferentially phosphorylates serine and threonine residues that lie in Arg-Xaa-Arg-Xaa-Xaa-Ser/Thr motifs, and SGK and PKB inactivate glycogen synthase kinase-3 similarly in vitro and in co-transfection experiments. These findings raise the possibility that some physiological roles ascribed to PKB on the basis of the overexpression of constitutively active PKB mutants might be mediated by SGK.
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PMID:Activation of serum- and glucocorticoid-regulated protein kinase by agonists that activate phosphatidylinositide 3-kinase is mediated by 3-phosphoinositide-dependent protein kinase-1 (PDK1) and PDK2. 1019 Dec 62

The present study evaluated the substrate competition between fatty acids (FA) and glucose in the kidney in vivo in relation to the operation of the "glucose-FA" and "reverse glucose-FA" cycles. In fed rats, neither inhibition of adipocyte lipolysis by 5-methylpyrazole-3-carboxylic acid (MPCA) nor inhibition of mitochondrial long-chain FA oxidation by 2-tetradecylglycidate (TDG) influenced the renal ratio of free/acylated carnitine or the percentage of total renal pyruvate dehydrogenase complex (PDHC) in the active (dephosphorylated) form (PDHa). The additional provision of glucose, a precursor for the synthesis of malonyl-coenzyme A (coA), did not influence renal PDHa activity or the renal ratio of free to acylated carnitine, implying that FA oxidation is maximally suppressed in the fed state. A reverse glucose-FA cycle may therefore be important in suppressing renal FA oxidation in the fed state. After 48 hours of starvation, MPCA and TDG decreased short- and long-chain acylcarnitine concentrations (40% to 50%, P < .01) and elevated the renal ratio of free/acylated carnitine (2.5-fold, P < .001, and 3.3-fold, P < .001, respectively), indicating that FA oxidation is increased after starvation. Despite suppression of renal FA oxidation, renal PDHa activity in 48-hour starved rats was only partially restored by treatment with MPCA or TDG. The additional administration of glucose did not remedy this. The failure to reverse completely the effects of prolonged starvation in suppressing PDHC activity by acute inhibition of FA oxidation suggests additional regulatory mechanisms that dampen the PDHC response to acute changes in substrate supply. Estimations of PDH kinase (PDK) activity in renal mitochondria showed a significant 1.7-fold stable increase (P < .01) after 48 hours of starvation. Analysis of PDK pyruvate sensitivity in renal mitochondria incubated with respiratory substrate (5 mmol/L 2-oxoglutarate/0.5 mmol/L L-malate) showed that the pyruvate concentration required for 50% activation was substantially decreased by starvation. Enzyme-linked immunosorbent assay (ELISA) analysis over a range of PDHC activities demonstrated that increased PDK activity was concomitant with a significant (at least P < .01) 1.8-fold increase in the protein expression of the ubiquitously expressed PDK isoform, PDK2. We hypothesize that changes in protein expression and activity of individual PDK isoforms may dictate the renal response to incoming FA lesterification v oxidation) through modulation of the relationship between glycolytic flux and PDHC activity, and thus the provision of precursor for malonyl-coA production.
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PMID:Substrate interactions in the short- and long-term regulation of renal glucose oxidation. 1038 Nov 44


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