Brassinosteroid signal transduction from cell-surf

LETTERSBrassinosteroidsignaltransductionfromcell-surfacereceptorkinasestonucleartranscriptionfactorsTae-WukKim1,ShenhengGuan2,YuSun1,ZhipingDeng1,WenqiangTang1,Jian-XiuShang3,YingSun3,AlmaL.Burlingame2andZhi-YongWang1,4Brassinosteroid(BR)regulatesgeneexpressionandplantdevelopmentthroughareceptorkinase-mediatedsignaltransductionpathway1.Despitetheidentificationofmanycomponentsofthispathway,itremainsunclearhowtheBRsignalistransducedfromthecellsurfacetothenucleus2.HerewedescribeacompleteBRsignallingpathwaybyelucidatingkeymissingsteps.WeshowthatphosphorylationofBSK1(BR-signallingkinase1)bytheBRreceptorkinaseBRI1(BR-insensitive1)promotesBSK1bindingtotheBSU1(BRI1suppressor1)phosphatase,andBSU1inactivatestheGSK3-likekinaseBIN2(BR-insensitive2)bydephosphorylatingaconservedphospho-tyrosineresidue(pTyr200).Mutationsthataffectphosphorylation/dephosphorylationofBIN2pTyr200(bin2‑1,bin2‑Y200Fandquadrupleloss-of-functionofBSU1-relatedphosphatases)supportanessentialroleforBSU1-mediatedBIN2dephosphorylationinBR-dependentplantgrowth.TheseresultsdemonstratedirectsequentialBRactivationofBRI1,BSK1andBSU1,andinactivationofBIN2,leadingtoaccumulationofunphosphorylatedBZR(brassinazoleresistant)transcriptionfactorsinthenucleus.ThisstudyestablishesafullyconnectedBRsignallingpathwayandprovidesnewinsightsintothemechanismofGSK3regulation.SteroidhormonesarecriticalfordevelopmentofallmulticellularorganismsandinplantsBRshaveessentialrolesinawiderangeofdevelopmentalandphysiologicalprocesses3.Unlikeanimalsteroidhormones,whichfunctionthroughnuclearreceptors,BRsbindtoareceptorkinase(BRI1)atthecellsurfacetoactivatetheBRresponsetranscriptionfactorsBZR1andBZR2(alsoknownasBES1)throughasignaltransductionpathway1,4.Althoughmanycomponentshavebeenidentifiedandstudiedindetail,ourunder‑standingoftheBRsignallingpathwayremainsincomplete,withmajorgapsbetweenthereceptorkinasesatthecellsurfaceanddownstreamcomponentsinthecytoplasmandnucleus(SupplementaryInformation,Fig.S1a)1,2.TheupstreamBR‑signallingcomponentsattheplasmamembraneincludeBRI1(refs5,6)andBAK1(BR‑insensitive1‑associatedreceptorkinase1;refs7,8)receptorkinases,anewprotein,BKI1,thatinhibitsBRI1(ref.9)andtheplasmamembrane‑associatedBR‑signallingkinases(BSKs)10.BRbindingtotheextracellulardomainofBRI1causesdisas‑sociationofBKI1fromBRI1(ref.9,11)andinducesassociationandtrans‑phosphorylationbetweenBRI1anditsco‑receptorBAK1(ref.12),leadingtoactivationofBRI1kinaseandphosphorylationofitsBSKsub‑strates10.ProteomicandgeneticstudieshavedemonstratedanessentialroleforBSKsintransducingthesignaltothedownstreamcomponents,buttheirdirecttargetremainsunknown10.DownstreamBRsignallinginvolvestheGSK3‑likekinaseBIN2(ref.13),theKelch‑repeats‑containingphosphataseBSU1(ref.14),the14‑3‑3fam‑ilyofphosphopeptide‑bindingproteins15,andBZR1andBZR2,whichdirectlybindtoDNAandregulateBR‑responsivegeneexpression16–19.AsanegativeregulatorofBRsignalling,BIN2phosphorylatesBZR1andBZR2atnumeroussitestoinhibittheiractivitiesthroughseveralmecha‑nisms2.Theseincludeacceleratingproteasome‑mediateddegradation20,promotingnuclearexportandcytoplasmicretentionbythe14‑3‑3pro‑teins15,21,andinhibitingDNAbindingandtranscriptionalactivity2,15,22.Bycontrast,theBSU1phosphataseisapositiveregulatorofBRsignalling14.OverexpressionofBSU1increasesdephosphorylatedBZR2andactivatesBRresponses4,14.However,BSU1doesnotinteractwithoreffectivelydephosphorylateBZR2invitroandthebiochemicalfunctionofBSU1remainsunknown4,14.ItisbelievedthatBRinducesrapiddephosphor‑ylationofBZR1andBZR2byinhibitingBIN2and/oractivatingBSU1.However,themechanismsbywhichupstreamBRsignallingregulatesBIN2andBSU1remainunclear(SupplementaryInformation,Fig.S1a)2,23.TounderstandhowBRsignallingregulatesBIN2,weanalysedBR‑inducedchangesinBIN2phosphorylationusingimmunoblottingoftwo‑dimensionalgelelectrophoresisandfoundthattreatmentwithbrassi‑nolide(themostactiveformofBR)causeddisappearanceoftheacidicformsandanincreaseinthebasicformsofepitope‑taggedBIN2(Fig.1a),suggestingthatBRinduceddephosphorylationofBIN2.ThisresultledustoinvestigatetheroleofBSU1phosphataseinBRregulationofBIN2.BothBSU1anditsclosesthomologueBSL1promoteBRsignallinginvivo(SupplementaryInformation,Fig.S2a)14andshowmanganese‑dependent1DepartmentofPlantBiology,CarnegieInstitutionforScience,Stanford,CA94305,USA.2DepartmentofPharmaceuticalChemistry,UniversityofCalifornia,SanFrancisco,CA94143,USA.3InstituteofMolecularCellBiology,HebeiNormalUniversity,Shijiazhuang,Hebei,050016,China.4CorrespondenceshouldbeaddressedtoZ.-Y.W.(e-mail:[e-mail]).Received24April2009;accepted4June2009;publishedonline6September2009;DOI:10.1038/ncb19701254naturecellbiologyVOLUME11|NUMBER10|OCTOBER2009©2009MacmillanPublishersLimited.Allrightsreserved.LETTERSphosphataseactivity(SupplementaryInformation,Fig.S2b,c).BSU1onlypartiallyreducedthephosphorylationofBZR1orBZR2whenco‑incubatedwithBIN2andBZR1orBZR2(SupplementaryInformation,Fig.S3a,b)14,andfailedtodephosphorylateBZR1andBZR2whenaddedafterBIN2andATPhadbeenremovedfromthekinasereaction(Fig.1b;SupplementaryInformati