PROJECTSUMMARY Overthepastdecades,theHippopathwayhasbeenrecognizedasacrucialsignalingpathwaythat controlsorganandtissuesize,byrestrictingcellproliferationandanti-apoptosis.TheHippopathwaycanbe regulatedbyawiderangeofextracellularsignaling,includingperceivedphysicalsignalsfromcell microenvironment(i.e.contactinhibition,cellpolarity,cytoskeleton-basedmechanicalcues),growth factors/mitogenichormones(i.e.LPAandS1PregulatedGPCRsignaling),andrecentlydiscoveredmetabolic status(i.e.energystress,hypoxia).Mechanistically,almostallthesestimuliorconditionsoperateYAPactivity throughLATS1/2kinases.ThemajorknowledgegapforcurrentHipposignalingcomesfromthe uncharacterizedmechanismforLATS1/2regulation.Theoverallobjectiveofthisproposalistoelucidatethe regulatorymechanismforLATS1/2kinasesinresponsetoHippoupstreamsignalingevents. ThedetailedregulatorymechanismforLATS1/2kinasescouldbecomplex,sincemultipleupstream kinasesincludingMST1/2,MAP4Ks,TAO1-3,areabletophosphorylateLATS1/2andrequiredforLATS1/2 activation.Itisstillunclearhowtheseidentifiedkinasesarecoordinatedtotransduceupstreamsignalingto activateLATS1/2.Besides,smallRhoGTPasetogetherwithF-actincansenseupstreamsignalingtomodulate LATS1/2activities,however,theunderlyingmechanismisalsolargelyunknown.Therefore,overthepastfew years,wehaveconductedaproteomicanalysisofthemajorcomponentsandregulatorsintheHippopathway topursuetheanswertothesequestions.Unfortunately,ourfindingsandfindingsfromotherlabsfailed identifyingsuchaputative?mediator?tofillthecurrentknowledgegapintheHippopathway.Unexpectedlyand excitingly,ourpreliminarystudieshavediscoveredphosphatidicacid(PA)anditrelatedlipidsignalingasa criticalsignalingaxisinvolvedintheHippopathwayregulation.PAcouldfunctionasasecondmessengerto directlyassociatewithLATS1/2kinasesandregulateLATS1/2activities.Remarkably,PA?slevelaswellasthe activityofPLD1,akeyenzymethatcatalyzesPAproduction,arebothdecreasedinresponsetotheHippo- activatingstimuliorconditions.Onthebasisoftheseobservations,wehypothesizedthatPLD1-PAaxiscould playacrucialroleinregulationofLATS1/2activities.Specifically,weproposeto1)determinetheroleofPAin YAPregulation,particularlyfocusingontheindependentroleofPAfromLPAinYAPactivation;?2)elucidate themechanismbywhichPAactivatesYAP.WewilldissecttheroleofPAinLATS1/2suppressionthroughits associationwithLATS1/2andNF2;?and3)investigatetheroleofPLD1-PAaxisinLATS1/2regulationin responsetoHippoupstreamsignaling.Collectively,ourproposedstudywillrevealacrucialregulatory mechanismtocontrolLATS1/2activationinresponsetotheHippoupstreamsignalingandconceptually advanceourunderstandingoftheHippopathwaybyfillingthisknowledgegap.