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1、TECHNICALREPORTSMolecularimagingofAktkinaseactivityLiminZhang1,KueiCLee2,MahaveerSBhojani1,AmjadPKhan1,AllaShilman1,EricCHolland3,BrianDRoss2,4&AlnawazRehemtulla1,4Theserine/threoninekinaseAktmediatesmitogenicandanti-RESULTSapoptoticresponsesthatresultfromactivationofmult
2、ipleConstructionofabioluminescentAktreportersignalingcascades.ItisconsideredakeydeterminantofThehybridpolypeptidebioluminescenceAktreporter(BAR;Fig.1a)tumoraggressivenessandisamajortargetforanticancerdrugcontainsanAktconsensussubstratepeptide21,consistingofadomaindevelopm
3、ent.Here,wedescribeanewreportermoleculethatbindsphosphorylatedaminoacidresidues(FHA2)22flankedbywhosebioluminescenceactivitywithinlivecellsandinmicetheN-terminal(N-Luc)andC-terminal(C-Luc)domainsofthefireflycanbeusedtomeasureAktactivity.Aktactivityinculturedluciferasereporte
4、rmolecule23.Inadditiontothiswild-typereporterhttp://www.nature.com/naturemedicinecellsandtumorxenograftswasmonitoredquantitativelyand(BAR(WT)),wealsoconstructedtwoadditionalreporters(Fig.1a),dynamicallyinresponsetoactivationorinhibitionofreceptoroneinwhichThr596wasmutated
5、toalanine(BARmut1)andasecondtyrosinekinase,inhibitionofphosphoinositide3-kinase,orinwhichallthreonineandserineresidues(Ser590,Ser594andThr596)directinhibitionofAkt.Theresultsprovideuniqueinsightswithinthesubstratepeptideweremutatedtoalanine(BARmut2).Theintothepharmacokine
6、ticsandpharmacodynamicsofagentsfunctionalbasisofthereporterisshownschematicallyinFigure1b.InthatmodulateAktactivity,revealingtheusefulnessofthisthepresenceofAktkinaseactivity,phosphorylationoftheAktreporterforrapiddoseandscheduleoptimizationinthedrugconsensussubstratesequ
7、encewithinthereporterwouldresultinitsdevelopmentprocess.interactionwiththeFHA2domain,thusstericallypreventingrecon-stitutionofafunctionalluciferasereportermolecule.IntheabsenceofTheserine/threoninekinaseAkt(alsocalledPKB)functionsAktkinaseactivity,lossofthisstericconstrai
8、ntwouldallowrecon-asasignalinghubwheremanyupstreamsignalingpathwaysstitutionoftheluciferaserepor
