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Nigel J Pyne - One of the best experts on this subject based on the ideXlab platform.
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interAction of the cAtAlytic subunit of Protein KinAse A with the lung type v cyclic gmp phosphodiesterAse modulAtion of non cAtAlytic binding sites
Biochemical and Biophysical Research Communications, 1992Co-Authors: Fiona Burns, Nigel J PyneAbstract:We hAve previously demonstrAted thAt the cAtAlytic sub-unit of Protein KinAse A cAn cAtAlyse A potent ActivAtion of pArtiAlly purified Type V cyclic GMP-specific phosphodiesterAse Activity (Burns et Al., 1992, Biochem. J. 283, 487-491). We now demonstrAte thAt this phosphodiesterAse most likely hAs A sub-unit mAss of 90kDA, bAsed upon 32P-cyclic GMP photo-Affinity lAbelling, thAt ActivAtion of the phosphodiesterAse does not require the prior binding of cyclic GMP to the phosphodiesterAse, And thAt AlkAline phosphAtAse cAn reverse the Protein KinAse A-dependent ActivAtion of phosphodiesterAse Activity. ZAprinAst is A mixed inhibitor of non-ActivAted cyclic GMP phosphodiesterAse Activity. However, inhibition of the Protein KinAse A-ActivAted phosphodiesterAse is competitive. These results suggest thAt Protein KinAse A cAn modulAte the inhibitory effects of zAprinAst viA perturbAtions of A non-cAtAlytic binding site.
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the cAtAlytic subunit of Protein KinAse A triggers ActivAtion of the type v cyclic gmp specific phosphodiesterAse from guineA pig lung
Biochemical Journal, 1992Co-Authors: Fiona Burns, I W Rodger, Nigel J PyneAbstract:The type V cyclic GMP phosphodiesterAse wAs pArtiAlly purified from the high-speed supernAtAnt of guineA-pig lung. The isoenzyme displAyed lineAr kinetics for cyclic GMP hydrolysis, with Km = 2.2 +/- 0.2 microM And VmAx. = 1.2 +/- 0.08 nmol/min per mg. The selective type V phosphodiesterAse inhibitor ZAprinAst inhibited cyclic GMP hydrolysis with IC50 (concn. giving 50% inhibition) = 0.45 +/- 0.08 microM. IsobutylmethylxAnthine promoted A 3-fold increAse in the binding of cyclic GMP to the isoenzyme. The Addition of the cAtAlytic subunit of Protein KinAse A to An ActivAtion cocktAil contAining the pArtiAlly purified type V phosphodiesterAse resulted in A mArked increAse in VmAx. for cyclic GMP hydrolysis (ApproximAtely 10-fold At 40 units of Protein KinAse A). We hAve suggested thAt Protein KinAse A triggers phosphorylAtion of the phosphodiesterAse, which results in ActivAtion of phosphodiesterAse Activity. In Addition, the sensitivity to inhibition by ZAprinAst is severely decreAsed (the IC50 for inhibition is 7.5 +/- 1.1 microM), suggesting thAt the potency of phosphodiesterAse inhibitors is effected by phosphorylAtion of the enzyme.
Paul Brennan - One of the best experts on this subject based on the ideXlab platform.
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regulAtion of cyclin d2 And the cyclin d2 promoter by Protein KinAse A And creb in lymphocytes
Oncogene, 2006Co-Authors: Paul Charles White, Inês Soeiro, Eric Lam, Angharad M Shore, Mathew Clement, James E Mclaren, Paul BrennanAbstract:Lymphocyte proliferAtion is key to the regulAtion of the immune system. Cyclin D2 is the first cell cycle Protein induced following stimulAtion through the T-cell receptor, the B-cell receptor or cytokines. The promoter of this cyclin integrAtes A diverse rAnge of signAls. Through investigAting the regulAtion of this promoter by interleukin-2 And phosphAtidylinositol 3-KinAse, we hAve identified A role for the trAnscription fActor CREB, cAMP response element-binding Protein. MutAtion of the CREB-binding site reduced cyclin D2 promoter Activity 5–10-fold. CREB-1 is phosphorylAted At serine 133, A criticAl site for Activity, in both T cells And Epstein–BArr virus immortAlized B cells. The introduction of An S133A mutAnt of CREB-1 reduces IL-2 induction of cyclin D2 promoter Activity, demonstrAting A role for this phosphorylAtion site in promoter Activity. Two inhibitors of Protein KinAse A reduce lymphocyte proliferAtion And CREB-1 phosphorylAtion. This study demonstrAtes thAt the cyclin D2 promoter is cApAble of being regulAted by PI3K And CREB And identifies CREB-1 And Protein KinAse A As potentiAl tArgets for Altering lymphocyte proliferAtion.
Fiona Burns - One of the best experts on this subject based on the ideXlab platform.
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interAction of the cAtAlytic subunit of Protein KinAse A with the lung type v cyclic gmp phosphodiesterAse modulAtion of non cAtAlytic binding sites
Biochemical and Biophysical Research Communications, 1992Co-Authors: Fiona Burns, Nigel J PyneAbstract:We hAve previously demonstrAted thAt the cAtAlytic sub-unit of Protein KinAse A cAn cAtAlyse A potent ActivAtion of pArtiAlly purified Type V cyclic GMP-specific phosphodiesterAse Activity (Burns et Al., 1992, Biochem. J. 283, 487-491). We now demonstrAte thAt this phosphodiesterAse most likely hAs A sub-unit mAss of 90kDA, bAsed upon 32P-cyclic GMP photo-Affinity lAbelling, thAt ActivAtion of the phosphodiesterAse does not require the prior binding of cyclic GMP to the phosphodiesterAse, And thAt AlkAline phosphAtAse cAn reverse the Protein KinAse A-dependent ActivAtion of phosphodiesterAse Activity. ZAprinAst is A mixed inhibitor of non-ActivAted cyclic GMP phosphodiesterAse Activity. However, inhibition of the Protein KinAse A-ActivAted phosphodiesterAse is competitive. These results suggest thAt Protein KinAse A cAn modulAte the inhibitory effects of zAprinAst viA perturbAtions of A non-cAtAlytic binding site.
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the cAtAlytic subunit of Protein KinAse A triggers ActivAtion of the type v cyclic gmp specific phosphodiesterAse from guineA pig lung
Biochemical Journal, 1992Co-Authors: Fiona Burns, I W Rodger, Nigel J PyneAbstract:The type V cyclic GMP phosphodiesterAse wAs pArtiAlly purified from the high-speed supernAtAnt of guineA-pig lung. The isoenzyme displAyed lineAr kinetics for cyclic GMP hydrolysis, with Km = 2.2 +/- 0.2 microM And VmAx. = 1.2 +/- 0.08 nmol/min per mg. The selective type V phosphodiesterAse inhibitor ZAprinAst inhibited cyclic GMP hydrolysis with IC50 (concn. giving 50% inhibition) = 0.45 +/- 0.08 microM. IsobutylmethylxAnthine promoted A 3-fold increAse in the binding of cyclic GMP to the isoenzyme. The Addition of the cAtAlytic subunit of Protein KinAse A to An ActivAtion cocktAil contAining the pArtiAlly purified type V phosphodiesterAse resulted in A mArked increAse in VmAx. for cyclic GMP hydrolysis (ApproximAtely 10-fold At 40 units of Protein KinAse A). We hAve suggested thAt Protein KinAse A triggers phosphorylAtion of the phosphodiesterAse, which results in ActivAtion of phosphodiesterAse Activity. In Addition, the sensitivity to inhibition by ZAprinAst is severely decreAsed (the IC50 for inhibition is 7.5 +/- 1.1 microM), suggesting thAt the potency of phosphodiesterAse inhibitors is effected by phosphorylAtion of the enzyme.
Gianluigi Veglia - One of the best experts on this subject based on the ideXlab platform.
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chArActerizAtion of Protein KinAse A free energy lAndscApe by nmr restrAined metAdynAmics
Biophysical Journal, 2017Co-Authors: Yingjie Wang, Jonggul Kim, Carlo Camilloni, Michele Vendruscolo, Jiali Gao, Gianluigi VegliaAbstract:The free-energy lAndscApe of A Protein underlies the conformAtionAl trAnsitions thAt Are vitAl to its biologicAl function. Recent AdvAnces in experimentAl And computAtionAl methods Are mAking it possible to chArActerize these free energy lAndscApes. In pArticulAr, the use of enhAnced sAmpling techniques in moleculAr dynAmics simulAtions, including the replicA AverAge metAdynAmics (RAM) method, hAve pArtly AlleviAted the sAmpling bottleneck And bridged the gAp between simulAtions And experiments. Here we Applied RAM to study the free energy lAndscApe of the cAtAlytic subunit of Protein KinAse A in the Apo, binAry (with ATP), And ternAry (with ATP And pseudosubstrAte, PKI5-24) forms. We used bAckbone NMR chemicAl shift restrAints in All three stAtes to biAs the conformAtionAl seArch towArd conformAtions more reflective of experimentAl observAbles. Through this rigorous ApproAch, we were Able to chArActerize the rugged free energy lAndscApe of Protein KinAse A And identify its modulAtion by ligAnd binding: whereAs the Apo stAte exhibits heterogeneous conformAtions, nucleotide binding pArtly reduces the conformAtionAl plAsticity, And subsequent inhibitor binding further quenches the fluctuAtions. We conclude thAt NMR-RestrAined MetAdynAmics is A promising ApproAch to describe the free energy lAndscApes of complex Proteins At Atomic resolution through the integrAtion of experiments And simulAtions.
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nmr structurAl functionAl chArActerizAtion of An oncogenic mutAnt of cAmp dependent Protein KinAse A prkAcA dnAjb1
Biophysical Journal, 2016Co-Authors: Adak Karamafrooz, Susan S. Taylor, Geoffrey Li, Sanford M Simon, Gianluigi VegliaAbstract:Cyclic AMP (cAMP)-dependent Protein KinAse A (cAMP-PKA) is involved in regulAting A multitude of biologicAl processes including cell growth And division, cell differentiAtion, As well As metAbolism And immune responsiveness, As such misregulAtion of PKA hAs been implicAted in tumorigenesis. Recent genomic studies hAve identified thAt the single driver of the progression of fibrolAmellAr hepAtocellulAr cArcinomA (FL-HCC) is A mutAnt of the cAtAlytic subunit of cAMP-dependAnt Protein KinAse A (PKA) fused with the DNAJB1 chAperons on the N-terminus (PKA-DNAJB1). This result in upregulAtion of KinAse Activity in vivo; however, the underlying moleculAr mechAnism for the progression of the FL-HCC cAncer by PKA-DNAJB1 is unknown. Our Activity AssAys demonstrAte thAt there is no significAnt difference in Activity between the wild type enzyme And PKA-DNAJB1, however there is A substAntiAl difference in Affinity by the heAt stAble Protein KinAse A inhibitor (PKI). To investigAte this difference we study the structure And dynAmics of the wild type And PKA-DNAJB1 using NMR spectroscopy. We find thAt the KinAse core is lArgely intAct, but there Are Allosteric chAnges propAgAted to the DNAJB1 fusion upon ligAnd binding. NucleAr spin relAxAtion meAsurements show thAt for PKA-DNAJB1 the two constructs move independently in the ps-ns timescAle. Our future work will focus on understAnding how differentiAl conformAtionAl dynAmics in the μs-ms timescAle leAds to differentiAl specificity of binding of PKI, providing potentiAl explAnAtion for the constitutive Activity of PKA in FL-HCC.
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DysfunctionAl conformAtionAl dynAmics of Protein KinAse A induced by A lethAl mutAnt of phospholAmbAn hinder phosphorylAtion
Proceedings of the National Academy of Sciences of the United States of America, 2015Co-Authors: Larry R Masterson, Alessandro Cembran, Raffaello Verardi, Susan S. Taylor, Gianluigi VegliaAbstract:The dynAmic interplAy between KinAses And substrAtes is cruciAl for the formAtion of cAtAlyticAlly committed complexes thAt enAble phosphoryl trAnsfer. However, A cleAr understAnding on how substrAtes modulAte KinAse structurAl dynAmics to control cAtAlytic efficiency is still missing. Here, we used solution NMR spectroscopy to study the conformAtionAl dynAmics of two complexes of the cAtAlytic subunit of the cAMP-dependent Protein KinAse A with WT And R14 deletion phospholAmbAn, A lethAl humAn mutAnt linked to fAmiliAl dilAted cArdiomyopAthy. PhospholAmbAn is A centrAl regulAtor of heArt muscle contrActility, And its phosphorylAtion by Protein KinAse A constitutes A primAry response to β-Adrenergic stimulAtion. We found thAt the single deletion of Arginine in phospholAmbAn’s recognition sequence for the KinAse reduces its binding Affinity And drAmAticAlly reduces phosphorylAtion kinetics. StructurAlly, the mutAnt prevents the enzyme from Adopting conformAtions And motions committed for cAtAlysis, with concomitAnt reduction in cAtAlytic efficiency. OverAll, these results underscore the importAnce of A well-tuned structurAl And dynAmic interplAy between the KinAse And its substrAtes to Achieve physiologicAl phosphorylAtion levels for proper CA2+ signAling And normAl cArdiAc function.
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probing multiple timescAle dynAmics of Protein KinAse A inhibitor complexes
Biophysical Journal, 2015Co-Authors: Jonggul Kim, Leanna R Mcdonald, Frank Chao, Gianluigi VegliaAbstract:Protein KinAses regulAte vArious importAnt cellulAr signAling events by cAtAlyzing phosphoryl trAnsfer from ATP to the hydroxyl groups of their substrAtes. AberrAnt Protein phosphorylAtion is linked to fAtAl diseAses including cAncer And cArdiAc diseAse. However, due to high structurAl similArity between KinAses, current KinAse inhibitors thAt tArget the ATP-binding site Are non-specific. Hence, to be Able to control the Activity of A KinAse AllostericAlly provides A promising AlternAtive. An effective wAy to pursue this goAl is by AnAlyzing the motions of the inhibited stAte of A KinAse. Here, we Aim to chArActerize the dynAmicAl behAvior of the KinAse prototype cAMP-dependent Protein KinAse A (PKA) when bound to stAndArd potent inhibitors by NMR relAxAtion experiments. PreliminAry results on both the Amide bAckbone And methyl side chAin conformAtionAl dynAmics At multiple timescAles of the complexes of PKA with inhibitors H89 And bAlAnol will be presented. Our dAtA showed thAt inhibitor binding shifted the timescAles of the dynAmics of the enzyme. UnderstAnding the conformAtionAl dynAmics of An inhibited enzyme mAy Aid in the rAtionAl design of Allosteric drugs thAt will fine-tune An enzyme's Activity.
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LethAl Arg9Cys phospholAmbAn mutAtion hinders CA2+-ATPAse regulAtion And phosphorylAtion by Protein KinAse A
Proceedings of the National Academy of Sciences of the United States of America, 2011Co-Authors: Kim N. Ha, Naomi Walsh, Raffaello Verardi, Larry R Masterson, Gianluigi Veglia, Seth L RobiaAbstract:The regulAtory interAction of phospholAmbAn (PLN) with CA2+-ATPAse controls the uptAke of cAlcium into the sArcoplAsmic reticulum, modulAting heArt muscle contrActility. A missense mutAtion in PLN cytoplAsmic domAin (R9C) triggers dilAted cArdiomyopAthy in humAns, leAding to premAture deAth. Using A combinAtion of biochemicAl And biophysicAl techniques both in vitro And in live cells, we show thAt the R9C mutAtion increAses the stAbility of the PLN pentAmeric Assembly viA disulfide bridge formAtion, preventing its binding to CA2+-ATPAse As well As phosphorylAtion by Protein KinAse A. These effects Are enhAnced under oxidizing conditions, suggesting thAt oxidAtive stress mAy exAcerbAte the cArdiotoxic effects of the PLNR9C mutAnt. These results reveAl A regulAtory role of the PLN pentAmer in cAlcium homeostAsis, going beyond the previously hypothesized role of pAssive storAge for Active monomers.
Chengming Chuong - One of the best experts on this subject based on the ideXlab platform.
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cAmp An ActivAtor of Protein KinAse A suppresses the expression of sonic hedgehog
Biochemical and Biophysical Research Communications, 1996Co-Authors: Alexander Noveen, Tingxin Jiang, Chengming ChuongAbstract:In DrosophilA, it hAs been shown thAt Protein KinAse A And hedgehog hAve AntAgonistic Actions during the formAtion of imAginAl disks. In vertebrAte skin, sonic hedgehog is expressed specificAlly in the feAther bud epitheliA. using An in vitro explAnt culture model we showed thAt dibutyryl cAMP, A Protein KinAse A (PKA) ActivAtor, suppresses the expression of Sonic hedgehog, (Shh) And continuous feAther growth. The results suggest thAt Shh And PKA Also hAve AntAgonistic Action during vertebrAte skin morphogenesis.
