文档介绍：该【autophagic degradation determines the fate of t315i-mutated bcr-abl protein haruka shinohara参考-匠人 】是由【抱琴】上传分享，文档一共【4】页，该文档可以免费在线阅读，需要了解更多关于【autophagic degradation determines the fate of t315i-mutated bcr-abl protein haruka shinohara参考-匠人 】的内容，可以使用淘豆网的站内搜索功能，选择自己适合的文档，以下文字是截取该文章内的部分文字，如需要获得完整电子版，请下载此文档到您的设备，方便您编辑和打印。LETTERSTO?THEEDITORdifferentfromthatofothertypesofBCR--T315I-mutatedBCR-ABLproteinBCR--47sup-pressestranscriptionofBCR--MarkedclinicalimprovementhasbeenachievedbythesionofBCR-ABLmRNAwasdecreasedaftertreatmentuseofBCR-ABL-tyrosinekinaseinhibitors;however,withAIC-47inbothWT-andT315I-BCR-ABL-harboringmutationsinBCR-moncells(OnlineSupplementaryFigureS3A).AIC--ABLmRNAlevelsinprimarycellsfromWepreviouslyfoundthatAIC-47,anovelautophagypatients(OnlineSupplementaryFigureS3B).Atsteadyinducer,suppressestheexpressionofBCR-ABL,1,2sug-state,thedecayofBCR-ABLmRNAwasnotsignificantlygestingthatAIC-47couldaffecttheexpressionofmutat-differentbetweenWT-andT315I-BCR-ABLcells(OnlineedBCR-,inthepresentstudyweestimatedtheSupplementaryFigureS3C).Moreover,inhibitionoftrans-effectsofAIC-47onBCR-ABLmutantcellsbyusinglationofBCR-ABLmRNAbycycloheximideorBCR-ABL--47puromycinresultedinadelayintheeliminationofwasfoundtosuppressthephosphorylationandexpres-T315I-BCR-paredwiththatofWT-BCR-ABLsionofwildtype(WT)-BCR-ABL;however,T315I-BCR-(Figure1B),suggestingthatdegradationcontributedtoABLexpressionwashardlysuppressedbyAIC-47(FigurethelevelofBCR-,the1AandOnlineSupplementaryFigureS2A).InBaf3p210majorityofintracellularproteinsaredegradedbythecellsharboringotherBCR-ABLmutations(M351Torubiquitin-),thesuppressionwasobserved(hattheautophagyinhibitor3-methyladenine(3-MA)SupplementaryFigureS2B),suggestingthattheregulationsuppressedthedegradationofbothtypesofBCR-ABL;ofT315I-BCR-ABLgenerationand/ordegradationwaswhereastheproteasomeinhibitorMG-132didnotsup--BCR-ABLprotein.(A)EffectsofAIC-47orimatinibonphosphorylationandexpres-sionofWT-andT315I-BCR-(DMSO;Control:C),AIC-47(47;10μM)orimatinib(IM;)for48h,followedbywesternblottinganalysis.(B)Time-dependentexpressionofBCR--ABLproteins,Ycellsweretreatedwithcycloheximide(CHX;)orpuromycin(Puro;1pg/mL).ToinhibitdegradationofBCR-ABLproteins,anautophagyinhibitor3-methyladenine(3-MA;100μM)andaproteasomeinhibitorMG-132(10μM)-ABL,p62,andLC3Bproteinsweredeterminedbywesternblottinganalysis.(C)EffectofAIC-47onautophagyfluxandBCR--ABLandp62,andconversionofLC3B,YcellstreatedwithAIC-47(5or10μM)(takenas“1”),whosevaluesweredeterminedbydensitometry.(D)DistributionofABLandtheautophagymarkerLC3inAIC-47--47(5μM)for12handthenco-immunostainedwithc-;.(E)Effectoftheautophagyinhibitors3-MAandchloroquine(CQ)onBCR--BCR-ABL-harboringcellswerepretreatedwith3-MA(100μM)orCQ(1μM)for4h,andthentreatedwithDMSOorAIC-47(5μM)-;104:e191LETTERSTO?THEEDITORpresstheirdegradation(Figure1B).Thesefindingsindi-BCR-ABLcells(OnlineSupplementaryFigureS1C).ThecatethatautophagicdegradationwascloselyassociatedimpairedautophagyintheT315I-mutatedcellswasalsowiththefateofBCR--treatedcells(Figure1CandOnlineNext,weinvestigatedtheinductionofautophagyinSupplementaryFigureS5).AfterAIC-47treatment,LC3AIC-47-,AIC-47treatmentwaspunctateandco-localizedwithBCR-ABLinWT-resultedinseveralfindingsofautophagy,including:(i)BCR-ABLcells,indicatingthatBCR-ABLhadbeenLC3lipidation,asindicatedbytheconversionofLC3BIdegradedinautophagosomes,whereasLC3punctaweretoII;(ii)inductionofAtgproteins;and(iii)degradationofbarelyobservedinT315I-BCR-ABLcells(Figure1D).Top62inthecellswithWT-BCR-ABL,butnotinthosewithfurtherinvestigatetheAIC-47-inducedautophagicdegra-T315I-BCR-ABL(Figure1CandOnlineSupplementarydationofBCR-ABL,weinhibitedautophagyfluxinWT-FigureS4).urspreferen-BCR-ABLcellsbyusing3---treatmentwiththeseautophagyinhibitorspartlyabol-berofWT-BCR-ABLcellsintheG0/G1phaseincreasedishedthedecreasedexpressionofBCR-ABLinducedbyaftertreatmentwithAIC-47,whereasachangeintheAIC-47(Figure1E).Basedontheseresults,wespeculatedprofileofthecell-cyclewasbarelyobservedinT315I--2fromBeclin-1wasimpairedinT315I-BCR-ABL-harboringcells.(A)-47(5μM)-2,Beclin-1orBcl--BikantibodycouldnotdetectBikofmouseorigin.(B)EffectsofsilencingBcl--2(5or10nM)for72h;andthenexpressionlevelsofBCR-ABLandp62,andtheconversionofLC3B,wereexaminedbywesternblottinganalysis.(C)EffectsofoverexpressionofBcl--fectedwithpIRESneo-Bcl-2vectorsfor72h,andthenexpressionlevelsofBCR-ABLandp62,andconversionofLC3B,wereexaminedbywesternblottinganaly-sis.(D)EffectsofAIC-47onthephosphorylationandexpressionofBcl-2,JNK,(DMSO;Control,C),orAIC-47(5or10μM)for24h,followedbywesternblottinganalysis(leftpanel).Anti-phospho-Bcl-2antibodycouldnotdetectphosphory-latedBcl--2wasquantifiedbydensitometryscanningandnormalizedtotheexpressionlevelofBcl-2(rightpanel).Dataareexpressedasmeans±SDofthreedifferentexperiments.*P<,***P<(Studentt-test).(E)TheheatmapshowsallgenesthatweredifferentiallyexpressedbetweenWT-BCR-ABLcells(WT)andT315I-BCR-ABLcells(MT).Genesetenrichmentanalysis(GSEA)resultsofc2ref-erencegenesetsrevealedthatRas-relatedsignatureswereenrichedinsteady-stateWT-BCR-ABLcells(falsediscoveryrate=,P<).“NRASsignaling”signatureswerealsoenrichedinAIC-47-treatedWT-BCR-ABLcells(falsediscoveryrate=,P<)..(F)--GTPwasquantifiedbydensitometryscanningandnormalizedtothetotallevelofRas.(G)SchematicdiagramofthedifferenceinautophagicdegradationbetweenWT-andT315I-BCR--BCR-ABL-harboringcells,theactivationofRas/MAPKsignalingislessthanthatinWT-BCR-ABL-harboringcells,whichreducesthephosphorylationofBcl--2fromBeclin-1isthusimpairedinT315I-BCR-ABLcells,resultinginimpairedautophagicdegradationofT315I-BCR-;104:e192LETTERSTO?THEEDITORtheincreasedstabilityofT315I-BCR-ABLandmightbedemonstratedthatRas/MAPKsignalingpromoteselldeaththroughtheNOXA/Beclin-1path-,Bcl-2interactswithBeclin-betweenanactiveGTP-,7GDP-,theamountofGTP-TreatmentwithZ36,anotherinducerofautophagy,boundRaswaslessinT315I-BCR-ABLcellsthaninWT-whichdissociatesBcl-2/Bcl-xLandBeclin-1,8resultedinBCR-ABLcells(Figure2F),indicatingthattheactivationinductionofLC3lipidationinWT-andT315I-BCR-ABLofRaswasassociatedwiththedifferenceinactivationofcells(OnlineSupplementaryFigureS6).TheexpressionMAPK/,furtherinvestigationislevelofBCR--andT315I-BCR-ABLcells(heincreasedstabilityofT315I-BCR-ABLproteinwasSupplementaryFigureS6).-2/Bcl-xLandBeclin-1isassoci-()significantlyinhibitedcellgrowthinWT-BCR-atedwiththeimpairedautophagyinT315I-BCR-ABLABLcells,,weexaminedtheinteractionofBcl-2/Bcl-xLT315I-BCR-ABLcells(OnlineSupplementaryFigureS9).withBeclin-1inAIC-47--47treat-ThedissociationofBcl-2/Beclin-1bytreatmentwithZ36ment,Bcl-2clearlydissociatedfromBeclin-1inWT-orsilencingofBcl-2inducedgrowthinhibitioninbothBCR-ABLcells;whereasbindingremainedintheT315I-WT-andT315I-BCR-ABLcells(OnlineSupplementaryBCR-ABLcells(Figure2A).Bcl-xLslightlydissociatedFigureS9),butthiseffectwasmoremarkedintheT315I-fromBeclin-1(Figure2A).ThereareseveraldifferentBCR--meanstoregulatethedissociationofBcl-2fromBeclin-tentionthattheautophagicdegradationofBCR-ABL1,petitivedisplacementoftheBeclin-,thisstudyhasBH3domainbyotherBcl-2familyproteins,orbyMAPK6,7limitationstoelucidatetheassociationbetween(JNKandErk)-mediatedphosphorylationofBcl--47couldinteractionwithBH3-onlyproteinsBikorNOXAandhavemultipletargetsotherthanautophagicsignalingandBcl-2wasincreasedaftertreatmentwithAIC-47inWT-exhibitanti-leukemiceffectseveninT315I-BCR-ABLBCR-ABLcells(Figure2A).BindingwithBcl-,wasnotobserved(Figure2A),suggestingthatBikandasthesameregulators,suchasBcl-2,cancontrolbothNOXAboundtotheBH3-bindinggrooveofBcl-(1nM)couldalsodisruptedtheinteractionbetweenBeclin-1andBcl-(OnlineBcl-xLboundBad;however,theinteractionwasslightlySupplementaryFigureS9).Furtherstudiesareneededtodecreased(Figure2A).AninteractionbetweenBikorrevealtheassociationbetweenautophagicdegradationofNOXAandBcl-xLwasnotobserved(Figure2A).BasedBCR-,wespeculatedthatBcl-2,ratherthanBcl-AutophagyisessentialforthemaintenanceofcellularxL,contributedpredominantlytotheinductionofAIC--,autophagycaneitherleukemiacells,dissociationofBcl-2andBeclin--BCR-ABLcells,whereasbinding12thatautophagyregulatesleukemogenesisandthatphar-remainedinT315I-BCR-ABLcells(OnlineSupplementarymacologicalinhibitionofautophagyenhancestheeffectsFigureS7A).TheknockdownofBcl--anddecreasedBCR-ABLexpressioneveninT315I-BCR-dencewithrespecttothetumor-inhibitingeffectsofABLcells(Figure2B).OverexpressionofBcl-2byusinga9autophagy,whichshowedthatBCR-ABLactsasasup-pIRESneo-Bcl-2vectorreversedtheexpressionofBCR-14ABL(Figure2C).ABcl-2inhibitor,ABT-737,induceddis--leukemiceffectsofarsenictrioxidearealsocausedbyautophagicdegrada-sociationofBcl-2/Beclin-1andautophagicdegradation15ofbothWT-andT315I-BCR-ABL;theseeffectsweretionofWT-BCR--binationwithAIC-47(OnlineciationofBcl-2fromBeclin-1wasimpairedinT315I-SupplementaryFigureS7B).TheseresultssuggestedthatBCR-ABLcells,resultingintheimpairedautophagicBcl-2wasoneoftheessentialmoleculesforautophagicdegradationthatcontributedtotheextensionofthehalf-degradationofBCR--BCR-ABL(Figure2G).AlthoughinhibitionMAPK-mediatedphosphorylationofBcl--47ofautophagyiseffectiveatenhancingthesensitivityofdecreasedBcl-2expression;whereastheratioofphos-tyrosinekinaseinhibitorstoBCR-ABL,itcouldalsocon-phorylatedBcl-2wasincreasedinWT-BCR-ABLcellstributetoincreasingthestabilityofBCR--(Figure2D).AftertreatmentwithAIC-47,thelevelofingssuggestthatthereisstillroomforargumentaboutphosphorylationofJNKwasdecreasedinWT-BCR-ABLautophagy-;however,thelevelofphosphorylationofErkwasHarukaShinohara,1YosukeMinami,2,3TomokiNaoe4andincreasedinthesec