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)

Xenon elicits preconditioning of the myocardium via protein kinase C-epsilon. We determined the implication of (1) the mitochondrial adenosinetriphosphate dependent potassium (K(ATP)) channels and (2) the 3'phosphatidylinositol-dependent kinase-1 (PDK-1) in activating protein kinase C-epsilon. For infarct size measurements, anaesthetized rats were subjected to 25 min of coronary artery occlusion followed by 120 min of reperfusion. Rats received xenon 70% during three 5-min periods before ischaemia with or without the K(ATP) channel blocker 5-hydroxydecanoate or Wortmannin as PI3K/PDK-1 inhibitor. For Western blot, hearts were excised at five time points after xenon preconditioning (Control, 15, 25, 35, 45 min). Infarct size was reduced from 42+/-6% (mean+/-S.D.) to 27+/-8% after xenon preconditioning (P<0.05). Western blot revealed an increased activation of PKC-epsilon after 45 min and of PDK-1 after 25 min during xenon preconditioning. 5-hydroxydecanoate and Wortmannin blocked both effects. PKC-epsilon is activated downstream of mitochondrial K(ATP) channels and PDK-1. Both pathways are functionally involved in xenon preconditioning.
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PMID:Upstream signaling of protein kinase C-epsilon in xenon-induced pharmacological preconditioning. Implication of mitochondrial adenosine triphosphate dependent potassium channels and phosphatidylinositol-dependent kinase-1. 1671 95

PDK1 catalyzes phosphorylation of Thr in the conserved activation loop region of a number of its downstream AGC kinase family members. In addition to the consensus sequence at the site of phosphorylation, a number of PDK1 substrates contain a PIF sequence (PDK1-interacting fragment), which binds and activates the kinase domain of PDK1 (PDK1(deltaPH)). To gain further insight to PIF-dependent catalysis, steady-state kinetic and inhibition studies were performed for His6-PDK1(deltaPH)-catalyzed phosphorylation of PDK1-Tide (Tide), which contains an extended "PIF" sequence C-terminal to the consensus sequence for PDK1 phosphorylation. In two-substrate kinetics, a large degree of negative binding synergism was observed to occur on formation of the active ternary complex (alphaKd(ATP) = 40 microM and alphaKd(Tide) = 80 microM) from individual transitory binary complexes (Kd(ATP) = 0.6 microM and Kd(Tide) = 1 microM). On varying ATP concentrations, the ADP product and the (T/E)-PDK1-Tide product analog (p'Tide) behaved as competitive and noncompetitive inhibitors, respectively; on varying Tide concentrations, ADP and p'Tide behaved as noncompetitive and competitive inhibitors, respectively. Also, negative binding synergism was associated with formation of dead-end inhibited ternary complexes. Time progress curves in pre-steady-state studies under "saturating" or kcat conditions showed (i) no burst or lag phenomena, (ii) no change in reaction velocity when adenosine 5'-O-(thiotriphosphate) was used as a phosphate donor, and (iii) no change in reaction velocity on increasing relative microviscosity (0 < or = eta/eta0 < or = 3). Taken together, PDK1-catalyzed trans-phosphorylation of PDK1-Tide approximates a Rapid Equilibrium Random Bi Bi system, where motions in the central ternary complex are largely rate-determining.
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PMID:Steady-state kinetic mechanism of PDK1. 1673 71

Pyruvate dehydrogenase kinase isoforms (PDK1-4) are the molecular switch that down-regulates activity of the human pyruvate dehydrogenase complex through reversible phosphorylation. We showed previously that binding of the lipoyl domain 2 (L2) of the pyruvate dehydrogenase complex to PDK3 induces a "cross-tail" conformation in PDK3, resulting in an opening of the active site cleft and the stimulation of kinase activity. In the present study, we report that alanine substitutions of Leu-140, Glu-170, and Glu-179 in L2 markedly reduce binding affinities of these L2 mutants for PDK3. Unlike wildtype L2, binding of these L2 mutants to PDK3 does not preferentially reduce the affinity of PDK3 for ADP over ATP. The inefficient removal of product inhibition associated with ADP accounts for the decreased stimulation of PDK3 activity by these L2 variants. Serial truncations of the PDK3 C-terminal tail region either impede or abolish the binding of wild-type L2 to the PDK3 mutants, resulting in the reduction or absence of L2-enhanced kinase activity. Alanine substitutions of residues Leu-27, Phe-32, Phe-35, and Phe-48 in the lipoyl-binding pocket of PDK3 similarly nullify L2 binding and L2-stimulated PDK3 activity. Our results indicate that the above residues in L2 and residues in the C-terminal region and the lipoyl-binding pocket of PDK3 are critical determinants for the cross-talk between L2 and PDK3, which up-regulates PDK3 activity.
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PMID:Structural determinants for cross-talk between pyruvate dehydrogenase kinase 3 and lipoyl domain 2 of the human pyruvate dehydrogenase complex. 1684 21

Previous investigations show that intracerebroventricular administration of a potent inhibitor of fatty acid synthase, C75, increases the level of its substrate, malonyl-CoA, in the hypothalamus. The "malonyl-CoA signal" is rapidly transmitted to skeletal muscle by the sympathetic nervous system, increasing fatty acid oxidation, uncoupling protein-3 (UCP3) expression, and thus, energy expenditure. Here, we show that intracerebroventricular or intraperitoneal administration of C75 increases the number of mitochondria in white and red (soleus) skeletal muscle. Consistent with signal transmission from the hypothalamus by the sympathetic nervous system, centrally administered C75 rapidly (< or =2 h) up-regulated the expression (in skeletal muscle) of the beta-adrenergic signaling molecules, i.e., norepinephrine, beta3-adrenergic receptor, and cAMP; the transcriptional regulators peroxisomal proliferator activator regulator gamma coactivator 1alpha (PGC-1alpha) and estrogen receptor-related receptor alpha (ERRalpha); and the expression of key oxidative mitochondrial enzymes, including pyruvate dehydrogenase kinase, medium-chain length fatty acyl-CoA dehydrogenase, ubiquinone-cytochrome c reductase, cytochrome oxidase, as well as ATP synthase and UCP3. The role of PGC-1alpha in mediating these responses in muscle was assessed with C2C12 myocytes in cell culture. Consistent with the in vivo response, adenovirus-directed expression of PGC-1alpha in C2C12 muscle cells provoked the phosphorylation/inactivation and reduced expression of acetyl-CoA carboxylase 2, causing a reduction of the malonyl-CoA concentration. These effects, coupled with an increased carnitine palmitoyltransferase 1b, led to increased fatty acid oxidation. PGC-1alpha also increased the expression of ERRalpha, PPARalpha, and enzymes that support mitochondrial fatty acid oxidation, ATP synthesis, and thermogenesis, apparently mediated by an increased expression of UCP3.
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PMID:Hypothalamic malonyl-CoA triggers mitochondrial biogenesis and oxidative gene expression in skeletal muscle: Role of PGC-1alpha. 1703 Jul 88

The catalytic activities of eukaryotic protein kinases (EPKs) are regulated by movement of the C-helix, movement of the N and C lobes upon ATP binding, and movement of the activation loop upon phosphorylation. Statistical analysis of the selective constraints associated with AGC kinase functional divergence reveals conserved interactions between these regulatory regions and three regions of the C-terminal tail (C-tail): the N-lobe tether (NLT), the active-site tether (AST), and the C-lobe tether (CLT). The NLT serves as a docking site for an upstream kinase PDK1 and, upon activation, positions the C-helix within the ATP binding pocket. The AST directly interacts with the ATP binding pocket, and the CLT interacts with the interlobe linker and the alphaC-beta4 loop, which appears to serve as a hinge for C-helix movement. The C-tail is a hallmark of AGC functional divergence inasmuch as most of the conserved core residues that distinguish AGC kinases from other EPKs are associated with the NLT, AST, or CLT. Moreover, several AGC catalytic core conserved residues that interact with the C-tail strikingly diverge from the canonical residues observed at corresponding positions in nearly all other EPKs, suggesting that the catalytic core may have coevolved with the C-tail in AGC kinases. These observations, along with the fact that the C-tail is needed for catalytic activity suggests that the C-tail is a cis-acting regulatory module that can also serve as a regulatory "handle," to which trans-acting cellular components can bind to modulate activity.
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PMID:The hallmark of AGC kinase functional divergence is its C-terminal tail, a cis-acting regulatory module. 1722 59

Due to the numerous kinases in the cell, many with overlapping substrates, it is difficult to find novel substrates for a specific kinase. To identify novel substrates of cAMP-dependent protein kinase (PKA), the PKA catalytic subunit was engineered to accept bulky N(6)-substituted ATP analogs, using a chemical genetics approach initially pioneered with v-Src (1). Methionine 120 was mutated to glycine in the ATP-binding pocket of the catalytic subunit. To express the stable mutant C-subunit in Escherichia coli required co-expression with PDK1. This mutant protein was active and fully phosphorylated on Thr(197) and Ser(338). Based on its kinetic properties, the engineered C-subunit preferred N(6)(benzyl)-ATP and N(6)(phenethyl)-ATP over other ATP analogs, but still retained a 30 microm K(m) for ATP. This mutant recombinant C-subunit was used to identify three novel PKA substrates. One protein, a novel mitochondrial ChChd protein, ChChd3, was identified, suggesting that PKA may regulate mitochondria proteins.
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PMID:Identification of ChChd3 as a novel substrate of the cAMP-dependent protein kinase (PKA) using an analog-sensitive catalytic subunit. 1724 5

AKT is a promising target for anticancer drug development. In this work, a bioinformatics approach was applied to search for AKT inhibitors based on the correlation analysis between phospho-Ser473 AKT expression level and the antiproliferative data of NCI small molecule compounds against NCI 60 cancer cell lines, the candidate compounds were then subject to AKT kinase assay. The possible effects of potent compound on PI3K/AKT, PDK1, and MAPK, its antiproliferative and apoptosis-inducing effects on breast cancer cells which have high-levels of AKT activation were assessed by Western blot analysis, cell viability assay, and apoptosis assay. One compound, CMEP (NSC632855, 9-chloro-2-methylellipticinium acetate) was identified with all three correlation algorithm, Pearson's, Sperman's, and Kendall's, showing a high-ranked correlation coefficient. CMEP inhibits only AKT, but does not inhibit PI3K, PDK1, or MAPK. CMEP also inhibits heregulin-induced AKT activation, does not inhibit heregulin-induced MAPK activation in MCF-7 breast cancer cells. Increased concentrations of ATP reverse the AKT inhibitory effect of CMEP. CMEP inhibits growth and induces apoptosis in breast cancer cells which have high-levels of AKT activation and lack functional PTEN; however, CMEP only shows a minimal activity in NIH3T3 cells which do not have AKT activation. In conclusion, a lead compound CMEP, as an AKT selective inhibitor has been identified started with a bioinformatics-based approach. CMEP inhibits growth and induces apoptosis in cancer cells which have high-levels of AKT activation and lack PTEN or harbor PTEN mutation.
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PMID:Bioinformatics-based discovery and characterization of an AKT-selective inhibitor 9-chloro-2-methylellipticinium acetate (CMEP) in breast cancer cells. 1729 30

Hypoxia-induced changes of rat skeletal muscle were investigated by two-dimensional difference in-gel electrophoresis (2D-DIGE) and mass spectrometry. The results indicated that proteins involved in the TCA cycle, ATP production, and electron transport are down-regulated, whereas glycolytic enzymes and deaminases involved in ATP and AMP production were up-regulated. Up-regulation of the hypoxia markers hypoxia inducible factor 1 (HIF-1alpha) and pyruvate dehydrogenase kinase 1 (PDK1) was also observed, suggesting that in vivo adaptation to hypoxia requires an active metabolic switch. The kinase protein, mammalian target of rapamycin (mTOR), which has been implicated in the regulation of protein synthesis in hypoxia, appears unchanged, suggesting that its activity, in this system, is not controlled by oxygen partial pressure.
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PMID:Metabolic modulation induced by chronic hypoxia in rats using a comparative proteomic analysis of skeletal muscle tissue. 1765 40

A homodimer of pyruvate dehydrogenase kinase (PDHK) is an integral part of pyruvate dehydrogenase complex (PDC) to which it is anchored primarily through the inner lipoyl-bearing domains (L2) of transacetylase component. The catalytic cycle of PDHK and its translocation over the PDC surface is thought to be mediated by the "symmetric" and "asymmetric" modes, in which the PDHK dimer binds to two and one L2-domain(s), respectively. Whereas the structure of the symmetric PDHK/L2 complex was reported, the structural organization and functional role of the asymmetric complex remain obscure. Here, we report the crystal structure of the asymmetric PDHK3/L2 complex that reveals several functionally important features absent from the previous structures. First, the PDHK3 subunits have distinct conformations: one subunit exhibits "open" and the other "closed" configuration of the putative substrate-binding cleft. Second, access to the closed cleft is additionally restricted by local unwinding of the adjacent alpha-helix. Modeling indicates that the target peptide might gain access to the PDHK active center through the open but not through the closed cleft. Third, the ATP-binding loop in one PDHK3 subunit adopts an open conformation, implying that the nucleotide loading into the active site is mediated by the inactive "pre-insertion" binding mode. Altogether our data suggest that the asymmetric complex represents a physiological state in which binding of a single L2-domain activates one of the PDHK protomers while inactivating another. Thus, the L2-domains likely act not only as the structural anchors but also modulate the catalytic cycle of PDHK.
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PMID:Crystal structure of an asymmetric complex of pyruvate dehydrogenase kinase 3 with lipoyl domain 2 and its biological implications. 1753 6

PPARalpha agonism impairs mitochondrial function, but the effect of PPARdelta agonism on mitochondrial function is equivocal. Furthermore, PPARalpha and delta agonism increases muscle fatty acid oxidation, potentially via activation of FOXO1 signalling and PDK4 transcription. Since FOXO1 activation has also been suggested to increase transcription of MAFbx and MuRF-1, and thereby the activation of ubiquitin-proteasome mediated muscle proteolysis, this raises the possibility that muscle fuel selection and the induction of a muscle atrophy programme could be regulated by a single common signalling pathway. We therefore investigated the effect of PPARdelta (delta) agonist, GW610742, administration on muscle mitochondrial function, fuel regulation, and atrophy and growth related signalling pathways in vivo. Twenty-four male Wistar rats received vehicle or GW610742 (5 and 100 mg per kg body mass (bm)) orally for 6 days. Soleus muscle was used to determine maximal rates of ATP production (MRATP) in isolated mitochondria, gene and protein expression, and enzyme activities. MRATP were unchanged by GW610742. Muscle PDK2 and PDK4 mRNA expression increased with GW610742 (100 mg (kg bm)(-1)) compared to vehicle (P<0.05), and was paralleled by a twofold increase in PDK4 protein expression (P<0.05). The activity of beta-hydroxyacyl-CoA dehydrogenase increased with GW610742 (P<0.05). Muscle MuRF1 and MAFbx mRNA expression was increased by GW610742 (100 mg (kg bm)(-1)) compared to vehicle (P<0.05), and was matched by increased protein expression (P<0.001), whilst Akt1 protein declined (P<0.05). There was no effect of GW610742 on 20S proteasome activity and mRNA expression, or the muscle DNA: protein ratio. GW610742 switched muscle fuel metabolism towards decreased carbohydrate use and enhanced lipid utilization, but did not induce mitochondrial dysfunction. Furthermore, GW610742 initiated a muscle atrophy programme, possibly via changes in the Akt1/FOXO/MAFbx and MuRF1 signalling pathway.
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PMID:PPARdelta agonism induces a change in fuel metabolism and activation of an atrophy programme, but does not impair mitochondrial function in rat skeletal muscle. 1754 Jul


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