Reactiveoxygenspecies(ROS)areemergingascriticalsecondmessengersinmanysignalingpathways relatedtohealthanddisease.Whilemuchprogresshasbeenmadeinunderstandingthemechanismsby whichROSlevelsareregulatedinsidecells,lessisknownaboutthemolecularsignalingeventsthatoccur downstreamofROSgeneration.Agrowingbodyofevidencesuggeststhatproteinkinasesaredirectly regulatedbyROSmodification.Forinstance,thereversibleoxidationofspecificCysresiduesinredox- sensitivekinaseshasbeenshowntoinfluencetheiractivity(eitherpositivelyornegatively),subcellular localization,andprotein-proteininteractions.Inmanycases,themodifiedCysintheaffectedkinaseis conservedamongothermembersinthesamekinasefamily.Thisraisesthepossibilitythatreversibleoxidation maybeageneralmeansofregulatingkinasefunctioninsidecells.Toexplorethispossibilityfurther,we recentlyusedfunctionalproteinmicroarraystoexaminetheimpactofoxidationontheglobalsubstrate selectionofaseriesofAGCandCMCGkinasefamilymembers.ThesestudiessuggestthatH2O2-dependent oxidationshiftsthesubstratepreferenceofmanykinases,leadingtodistinctsubstrateprofilesintheoxidized andreducedstates.Interestingly,inmostcases,bothincreasesanddecreasesinsubstratephosphorylation wereobserved.Asaconsequence,reversibleoxidationmayplayanimportantroleincontrollingthesignaling specificityofredox-sensitivekinasesincells.Toinvestigatethesequestionsfurther,wepropose1)toexamine themolecularmechanismsunderlyingtheH2O2-inducedshiftsinkinasesubstrateselection(Aims1&2)and 2)tobegintoexplorethefunctionalconsequencesofredoxmodificationonkinase-dependentsignaling processesinsidethecell(Aim3).Duringthesestudies,wewillfocusontworepresentativeAGCandCMGC familymembers,namelyPKAandERK2.Notonlywillthisprovidenewinsightsintotheredoxregulationof theseimportantkinases,butitwillalsolayafoundationfortheanalysisofotherredoxsensitivekinases identifiedinthemicroarrayexperiments.Together,thesestudieswillofferuniqueinsightsintoROS-mediated regulationofkinasefunctionandprovidethefoundationforfuturestudiesintocrosstalkbetweenROS-and phosphorylation-dependentsignalingpathwaysinphysiologicalandpathologicalstates.
Intheproposedproject,weplanto1)investigatethebiochemicalmechanismsunderlyingoxidation- inducedshiftsinproteinkinasesubstrateselectionand2)begintoexplorethefunctionalconsequencesof redoxmodificationonkinase-dependentsignalingprocessesinsidecells.Duringthesestudies,wewillfocus onPKAa?andERK2asrepresentativemembersoftheAGCandCMGCkinasefamilies.Notonlywillthe proposedstudiesprovidenewinsightsintotheredoxregulationoftheseimportantkinases,buttheywillalso layafoundationfortheanalysisofotherredox-sensitivekinases.Together,thesestudieswillhelpidentify pointsofsignalintegrationbetweenROS-andphosphorylation-dependentpathways.Theywillalso complementexistingmodelsofpathologicaloxidativestressandprovidenewopportunitiesfortargeted antioxidanttherapeuticsformanydiseases(e.g.,cancer,diabetesandcardiovasculardisease).
