Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.5.1.18 (glutathione S-transferase)
 22,582 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The dihydrolipoyl acetyltransferase (E2) component of the mammalian pyruvate dehydrogenase complex forms a 60-subunit core in which E2's inner domain forms a dodecahedron shaped structure surrounded by its globular outer domains that are connected to each other and the inner domain by 2-3-kDa mobile hinge regions. Two of the outer domains are approximately 10 kDa lipoyl domains, an NH2-terminal one, E2L1, and, after the first hinge region a second one, E2L2. The pyruvate dehydrogenase kinase binds tightly to the lipoyl domain region of the oligomeric E2 core and phosphorylates and inactivates the pyruvate dehydrogenase (E1) component. We wished to determine whether lipoyl domain constructs prepared by recombinant techniques from a cDNA for human E2 could bind the bovine E1 kinase and, that being the case, to pursue which lipoyl domain the kinase binds. We also wished to gain insights into how a molecule of kinase tightly bound to the E2 core can rapidly phosphorylate 20-30 molecules of the pyruvate dehydrogenase (E1) component which are also bound to an outer domain of the E2 core. We prepared recombinant constructs consisting of the entire lipoyl domain region or the individual lipoyl domains with or without the intervening hinge region. Constructs were made and used both as free lipoyl domains and fused to glutathione S-transferase (GST). Using GSH-Sepharose to selectively bind GST constructs, tightly bound kinase was shown to rapidly transfer in a highly preferential way from intact E2 core to GST constructs containing the E2L2 domain rather than to ones containing only the E2L1 domain. GST-E2L2-kinase complexes could be eluted from GSH-Sepharose with glutathione. Delipoylation of E2L2 by treatment with lipoamidase eliminated kinase binding supporting a direct role of the lipoyl prosthetic group in this association. Transfer to and selective binding of the kinase by E2L2 but not E2L1 was also demonstrated with free constructs using a sucrose gradient procedure to separate the large E2 core from the various lipoyl domain constructs. E2L2 but not E2L1 increased the activity of resolved kinase by up to 43%. We conclude that the kinase selectively binds to the inner lipoyl domain of E2 subunits and that this association involves its lipoyl prosthetic group. We further suggest that transfer of tightly bound kinase between E2L2 domains occurs by a direct interchange mechanism without formation of free kinase (model presented).(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Binding of the pyruvate dehydrogenase kinase to recombinant constructs containing the inner lipoyl domain of the dihydrolipoyl acetyltransferase component. 782 13

Serum and glucocorticoid-inducible kinase (SGK) is a novel member of the serine/threonine protein kinase family that is transcriptionally regulated. In this study, we have investigated the regulatory mechanisms that control SGK activity. We have established a peptide kinase assay for SGK and present evidence demonstrating that SGK is a component of the phosphoinositide 3 (PI 3)-kinase signaling pathway. Treatment of human embryo kidney 293 cells with insulin, IGF-1 or pervanadate induced a 3- to 12-fold activation of ectopically expressed SGK. Activation was completely abolished by pretreatment of cells with the PI 3-kinase inhibitor, LY294002. Treatment of activated SGK with protein phosphatase 2A in vitro led to kinase inactivation. Consistent with the similarity of SGK to other second-messenger regulated kinases, mutation of putative phosphorylation sites at Thr256 and Ser422 inhibited SGK activation. Cotransfection of PDK1 with SGK caused a 6-fold activation of SGK activity, whereas kinase-dead PDK1 caused no activation. GST-pulldown assays revealed a direct interaction between PDK1 and the catalytic domain of SGK. Treatment of rat mammary tumor cells with serum caused hyperphosphorylation of endogenous SGK, and promoted translocation to the nucleus. Both hyperphosphorylation and nuclear translocation could be inhibited by wortmannin, but not by rapamycin.
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PMID:Serum and glucocorticoid-inducible kinase (SGK) is a target of the PI 3-kinase-stimulated signaling pathway. 1035 15

Akt activation requires phosphorylation of Thr(308) and Ser(473) by 3-phosphoinositide-dependent kinase-1 and 2 (PDK1 and PDK2), respectively. While PDK1 has been cloned and sequenced, PDK2 has yet to be identified. The present study shows that phosphatidylinositol 3-kinase-dependent p38 kinase activation regulates Akt phosphorylation and activity in human neutrophils. Inhibition of p38 kinase activity with SB203580 inhibited Akt Ser(473) phosphorylation following neutrophil stimulation with formyl-methionyl-leucyl-phenylalanine, FcgammaR cross-linking, or phosphatidylinositol 3,4,5-trisphosphate. Concentration inhibition studies showed that Ser(473) phosphorylation was inhibited by 0.3 microm SB203580, while inhibition of Thr(308) phosphorylation required 10 microm SB203580. Transient transfection of HEK293 cells with adenoviruses containing constitutively active MKK3 or MKK6 resulted in activation of both p38 kinase and Akt. Immunoprecipitation and glutathione S-transferase (GST) pull-down studies showed that Akt was associated with p38 kinase, MK2, and Hsp27 in neutrophils, and Hsp27 dissociated from the complex upon activation. Active recombinant MK2 phosphorylated recombinant Akt and Akt in anti-Akt, anti-MK2, anti-p38, and anti-Hsp27 immunoprecipitates, and this was inhibited by an MK2 inhibitory peptide. We conclude that Akt exists in a signaling complex containing p38 kinase, MK2, and Hsp27 and that p38-dependent MK2 activation functions as PDK2 in human neutrophils.
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PMID:p38 Kinase-dependent MAPKAPK-2 activation functions as 3-phosphoinositide-dependent kinase-2 for Akt in human neutrophils. 1104 4

The dihydrolipoyl acetyltransferase (E2) has an enormous impact on pyruvate dehydrogenase kinase (PDK) phosphorylation of the pyruvate dehydrogenase (E1) component by acting as a mobile binding framework and in facilitating and mediating regulation of PDK activity. Analytical ultracentrifugation (AUC) studies established that the soluble PDK2 isoform is a stable dimer. The interaction of PDK2 with the lipoyl domains of E2 (L1, L2) and the E3-binding protein (L3) were characterized by AUC. PDK2 interacted very weakly with L2 (Kd approximately 175 microM for 2 L2/PDK2) but much tighter with dimeric glutathione S-transferase (GST)-L2 (Kd approximately 3 microM), supporting the importance of bifunctional binding. Reduction of lipoyl groups resulted in approximately 8-fold tighter binding of PDK2 to GST-L2red, which was approximately 300-fold tighter than binding of 2 L2red and also much tighter than binding by GST-L1red and GST-L3red. The E2 60-mer bound approximately 18 PDK2 dimers with a Kd similar to GST-L2. E2.E1 bound more PDK2 (approximately 27.6) than E2 with approximately 2-fold tighter affinity. Lipoate reduction fostered somewhat tighter binding at more sites by E2 and severalfold tighter binding at the majority of sites on E2.E1. ATP and ADP decreased the affinity of PDK2 for E2 by 3-5-fold and adenosine 5'-(beta,gamma-imino)triphosphate or phosphorylation of E1 similarly reduced PDK2 binding to E2.E1. Reversible bifunctional binding to L2 with the mandatory singly held transition fits the proposed "hand-over-hand" movement of a kinase dimer to access E1 without dissociating from the complex. The gain in binding interactions upon lipoate reduction likely aids reduction-engendered stimulation of PDK2 activity; loosening of binding as a result of adenine nucleotides and phosphorylation may instigate movement of lipoyl domain-held kinase to a new E1 substrate.
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PMID:Facilitated interaction between the pyruvate dehydrogenase kinase isoform 2 and the dihydrolipoyl acetyltransferase. 1281 49

The transacetylase component (E2) of PDC (pyruvate dehydrogenase complex) plays a critical role in the regulation of PDHK (pyruvate dehydrogenase kinase) activity. The present study was undertaken to investigate further the molecular mechanism by which E2 modulates the activity of PDHK. In agreement with the earlier results, it was found that the inner L2 (lipoyl-bearing domain 2) of E2 expressed with or without the C-terminal hinge region had little, if any, effect on the kinase activity, indicating a lack of direct allosteric effect of L2 on PDHK. In marked contrast, significant activation of PDHK was observed with the construct consisting of L2 and the E1BD (E1-binding domain) of E2 (L2-E1BD didomain) suggesting that co-localization and/or mutual orientation of PDHK and E1, facilitated by E2 binding, largely account for the activation of PDHK by the transacetylase component. Isothermal titration calorimetry and glutathione S-transferase pull-down assays established that binding of adenyl nucleotides to the PDHK molecule facilitated the release of L2 domain. In contrast, binding of the L2 domain caused a significant decrease in the affinity of PDHK for ATP. The cross-talk in binding of adenyl nucleotides and the L2 domain to PDHK may indicate the existence of a highly integrated mechanism whereby the exchange of lipoyl-bearing domains presented to PDHK by E2 is coupled with ADP/ATP exchange.
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PMID:Role of protein-protein interactions in the regulation of pyruvate dehydrogenase kinase activity. 1550 8

PKB/Akt is a protein involved in control of apoptosis, proliferation and cellular metabolism, and it has been found to be activated in many cancers. Activation of PKB involves recruitment of the enzyme by its PH domain to the cell membrane, and phosphorylation at two residues, T308 and S473. To produce active PKB kinase from Escherichia coli, we constructed a derivative of PKB lacking the PH domain and mutated to glutamate at residues S124, T450 and the activating residue S473 (DeltaPH-PKB-EEE). DeltaPH-PKB-EEE was expressed in E. coli together with PDK1, the kinase responsible for phosphorylating PKB at T308, which was expressed as a GST-fusion. Full-length DeltaPH-PKB-EEE was obtained by using a double tag strategy: His6 at the N-terminus and FLAG at the C-terminus. The protein was purified by nickel affinity chromatography, followed by passage over an anti-FLAG column. The final purification step, anion exchange over a monoQ column, separated phosphorylated from unphosphorylated protein. Active recombinant PKB kinase was thus produced from E. coli, by a simple, reproducible procedure.
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PMID:Expression and purification of active PKB kinase from Escherichia coli. 1580 34

The protein kinase AKT is a key regulator for cell growth, cell survival and metabolic insulin action. However, the mechanism of activation of AKT in vivo, which presumably involves membrane recruitment of the kinase, oligomerization, and multiple phosphorylation events, is not fully understood. In the present study, we have expressed and purified dimeric GST-fusion proteins of human protein kinase AKT2 (DeltaPH-AKT2) in milligram quantities via the baculovirus expression system. Treatment of virus-infected insect cells with the phosphatase inhibitor okadaic acid (OA) led to phosphorylation of the two regulatory phosphorylation sites, Thr309 and Ser474, and to activation of the kinase. Likewise, phosphorylation of Thr309 in vitro by recombinant PDK1 or mutation of Thr309 and Ser474 to acidic residues rendered the kinase constitutively active. However, even though the specific activity of our AKT2 was increased 15-fold compared to previous reports, GST-mediated dimerization alone did not lead to an activation of the kinase. Whereas both mutagenesis and phosphorylation led to an increase in the turnover number of the enzyme, only the latter resulted in a marked reduction (20-fold) of the apparent Km value for the exogenous substrate Crosstide, indicating that this widely used mutagenesis only partially mimics phosphorylation. Kinetic analysis of GST-AKT2 demonstrates that phosphorylation of Thr309 in the activation loop of the kinase is largely responsible for the observed reduction in Km and for a subsequent 150-fold increase in the catalytic efficiency (k(cat)/Km) of the enzyme. Highly active AKT2 constructs were used in autophosphorylation reactions in vitro, where inactive AKT2 kinases served as substrates. As a matter of fact, we found evidence for a minor autophosphorylation activity of AKT2 but no significant autophosphorylation of any of the two regulatory sites, Thr309 or Ser474.
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PMID:Activation of a GST-tagged AKT2/PKBbeta. 1589 Apr 50

To elucidate the role of 3-phosphoinositide-dependent protein kinase-1 (PDK1) in cellular signaling, we constructed and expressed a pseudosubstrate of PDK1, designated as deltaAL-PIF, and characterized its properties in cultured cells. deltaAL-PIF consists of two fused proteins of the protein kinase Cdelta (deltaPKC) activation loop (deltaAL) and PDK1-interacting fragment (PIF). The phosphorylation of deltaAL-PIF was detected with anti-deltaPKC phospho-Thr505-specific antibody and was increased in proportion to the expression level of co-expressed GST-PDK1, indicating that it acts as a pseudosubstrate of PDK1. In cells expressing deltaAL-PIF, basal phosphorylation level at the activation loop of PKBalpha, deltaPKC and gammaPKC was reduced, compared with that in control cells, suggesting that deltaAL-PIF functions as an inhibitory molecule for PDK1. deltaAL-PIF affected the stability, translocation and endogenous activity of PKCs. These effects of deltaAL-PIF on gammaPKC properties were confirmed by investigation using conditioned PDK1 knockout cells. Furthermore, apoptosis frequently occurred in cells expressing deltaAL-PIF for 3 days. These findings revealed that deltaAL-PIF served as an effective pseudosubstrate and an inhibitory molecule for PDK1, suggesting that this molecule can be used as a tool for investigating PDK-mediated cellular functions as well as being applicable for anti-cancer therapy.
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PMID:Fused protein of deltaPKC activation loop and PDK1-interacting fragment (deltaAL-PIF) functions as a pseudosubstrate and an inhibitory molecule for PDK1 when expressed in cells. 1692 25

Recent work has highlighted a role for PDK1 in adaptive immunity, however its contribution to innate immunity has not been addressed. We have investigated the role of PKB and PDK1 in IL-1beta-induced NF-kappaB activation. Over-expression of either in HCT 116 and HEK 293T cells, effected a reproducible NF-kappaB activation. This was validated in a one-hybrid assay utilizing Gal4-RelA and Gal4-luciferase assay. N-tosyl phenylalanyl chloromethyl ketone (TPCK), wortmannin and Ly294002 inhibited IL-1beta-induced NF-kappaB activation in both systems indicating involvement of the PI3K axis in this response. p65 (Rel A) Ser536 phosphorylation was not affected by the PI3K inhibitors but was dose-dependently attenuated by TPCK. Evaluation of IKK-associated activity using GST-p65 substrate phosphorylation in immune complex assays, revealed that whilst TPCK attenuated this, neither of the PI3K inhibitors had any effect. Furthermore whilst TPCK inhibited IL-1beta-induced p65 DNA binding, this was not apparent with either of wortmannin or Ly294002. Similarly, over-expression of PDK1 but not PKB resulted in promotion of p65 DNA binding. Using a p65-S536A reporter construct, we found inhibition of only PDK1 over-expression-induced, but not PKB over-expression-induced NF-kappaB activation. This was supported using biochemical analysis in which immunoprecipitated IKKgamma from IL-1beta-activated cells was unable to phosphorylate a p65-S536A substrate, confirming this as the dominant IKK-dependent site. In further support of a dissociated response, we observed an attenuation of the Ser177/181 IKK phosphorylation by TPCK but not in response to PI3K inhibition. Our data reveals for the first time that PDK1 and PKB may differentially activate NF-kappaB, and that TPCK may subserve a useful anti-inflammatory function by inhibiting IKKbeta.
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PMID:Investigation of interleukin 1beta-mediated regulation of NF-kappaB activation in colonic cells reveals divergence between PKB and PDK-transduced events. 1713 79

The serine-threonine protein kinases PDK1 and PKB each contain a pleckstrin homology (PH) domain that binds the membrane-bound phosphatidylinositol 3,4,5-triphosphate [PI(3,4,5)P3] second messenger and is required for PDK1-catalyzed phosphorylation and activation of PKB. While X-ray structures have been reported for the individual regulatory PH and catalytic kinase domain constructs of both PDK1 and PKB, diffraction quality crystals of full length constructs have yet to be obtained, likely due to conformational heterogeneity. In developing alternative approaches to understanding the potential role of conformational dynamics in regulating PKB phosphorylation by PDK1, an efficient in vitro method for protein trans-splicing was developed, which utilizes the N- and C-terminal split inteins of the gene dnaE from Nostoc punctiforme [(N)NpuDnaE] and Synechocystis sp. strain PCC6803 [(C)SspDnaE], respectively. For conjugating the regulatory PH domain to the catalytic kinase domain of PDK1, the recombinant trans-splicing fusion constructs KINASE(AEY)-(N)NpuDnaE-His6 and GST-His6-(C)SspDnaE-(CMN)PH were designed, PCR assembled, overexpressed, and affinity purified. The cross-reacting (N)NpuDnaE and (C)SspDnaE inteins generated full length spliced-PDK1 with kobs = (2.8 +/- 0.3) x 10(-5) s(-1) and with < or =5% of any competing trans-cleavage reactions. Spliced-PDK1 was efficiently purified to > or =95% homogeneity from the reaction mixture by subsequent His6 affinity and ion exchange chromatography steps. In vitro kinase assays and phosphopeptide mapping studies confirmed that spliced-PDK1 retained the ability to colocalize and selectively phosphorylate Thr-309 of PKBbeta in a PI(3,4,5)P3-dependent manner. The high-level production and reconstitution of functional spliced-PDK1 establishes the feasibility of incorporating domain-specific biophysical probes for spectroscopic studies of regulatory PH domain mediated catalytic specificity.
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PMID:Reconstitution of modular PDK1 functions on trans-splicing of the regulatory PH and catalytic kinase domains. 1750 May 9


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