蛋白質和蛋白質組學課件：4.Protein Phosphorylation3

ProteinPhosphorylation

AGlobalRegulatonofCellularProteinActivityHistory

Doesphosphorusonlyfunctionasanutrient?Asearlyasthe19thcenturyitwasknownthatphosphatescouldbeboundtoproteins.Mostexamplesofthese‘phosphoproteins’werefoundinmilk(caseins)andeggyolk(phosvitin卵黃高磷蛋白)andweresimplyconsideredabiologicalmethodofprovidingphosphorusasanutrient.

FindingofproteinkinaseandphosphorylationIn1954,anenzymeactivitywasobservedthattransferredaphosphateontoanotherprotein—abiologicalreactioncalledphosphorylation.Theproteinresponsiblewasaliverenzymethatcatalyzedthephosphorylationofcaseinandbecameknownasaproteinkinase,thefirstofitskindtobediscovered. GlycogenmetabolismwasregulatedbyphosphorylationAyearlater,theroleofphosphorylationbecamemoreinterestingasFischerandKrebs(receivedtheNobelPrizeinmedicinefortheirpioneeringefforts1992),andWosilaitandSutherland,showedthatanenzymeinvolvedinglycogenmetabolismwasregulatedbytheadditionorremovalofaphosphate,suggestingthatreversiblephosphorylationcouldcontrolenzymeactivity.Today,itisthoughtthatonethirdoftheproteinspresentinatypicalmammaliancellarecovalentlyboundtophosphatePhosphorylationanddephosphorylationPhosphorylationreferstotheadditionofaphosphatetooneoftheaminoacidsidechainsofaprotein.Dephosphorylation？？Increasingordecreasingthebiologicalactivityofanenzyme.Helpingmoveproteinsbetweensubcellularcompartments.Allowinginteractionsbetweenproteinstooccur.Labelingproteinsfordegradation.RegulationbyphosphorylationHowcanphosphorylationcontrolenzymeactivity?Phosphatesarenegativelycharged(witheachphosphategroupcarryingtwonegativecharges)sothattheiradditiontoaproteinwillchangethecharacteristicsoftheprotein.Thischangeisoftenaconformationalone,causingtheproteintochangehowitisstructured.Molecularswitchphosphorylationreactionisreversiblebyaprocesscalleddephosphorylation.Theproteinswitchesbacktoitsoriginalconformationwhenthephosphorusisremoved.Ifthesetwoconformationsprovidetheproteinwithdifferentactivities(i.e.beingenzymaticallyactiveinoneconformationbutnottheother),phosphorylationoftheproteinwillactasamolecularswitch,turningtheactivityonoroff.ProteinkinasesandphosphatasesThetransferofphosphatesontoproteinsiscatalyzedbyavarietyofenzymesinthecell.Althoughthevarietyislarge,alloftheseenzymessharecertaincharacteristicsandfallintooneclassofproteins,calledproteinkinases.Theirsimilaritiesstemfromthegroup'sabilitytotakeaphosphateoffthechemicalenergy-carryingmoleculeATPandplaceitontoanaminoacidsidechainofaprotein.Thehydroxylgroups(-OH)ofSer,Thr,TyrorHissidechainsarethemostcommontarget.Asecondclassofenzymesisresponsibleforthereversereaction,inwhichphosphatesareremovedfromaprotein.Thesearetermedproteinphosphatases.Specific?Someoftheseenzymesareextremelyspecific,potentiallyphosphorylatingordephosphorylatingonlyafewtargetproteins,whileothersareabletoactbroadlyonmanyproteins.TargetsofphosphorylationTargetsofphosphorylationincludemostproteincomponentsofthecell,includingenzymes,structuralproteins,cellreceptors,ionchannelsandsignalingmolecules.Theuseofthephosphorylation/dephosphorylationofaproteinasacontrolmechanismhasmanyadvantagesItisrapid,takingaslittleasafewseconds.Itdoesnotrequirenewproteinstobemadeordegraded.Itiseasilyreversible.ExternalSignalscanactivateproteinkinasesandphosphatasesphosphorylationcascade

Inanimalcells,thesecascadesaremediatedbytwotypesofkinases:Serine/threoninekinases(whichphosphorylateserineandthreonineaminoacidsidechains)Tyrosinekinases(whichphosphorylatetyrosineaminoacidsidechains).PhosphorylationdoesnotalwayschangetheactivityofaproteinPhosphorylationinresponsetoasignalproducesasecondoutcomeapartfromtheactivationofkinasesandphosphatases,whichinvolvestheproductionofbindingsitesforproteinstointeract.Thisprocessisdifferentfromtheactivationofaproteinbyphosphorylation,sinceitdoesnotnecessarilychangetheinherentactivityofthemoleculethathasbeenphosphorylated.Instead,itcreatesaphosphorylatedaminoacidonthemoleculethatanotherproteincanbindto.Uponreceptionofasignal,somemembrane-boundreceptorswillbecometyrosinephosphorylated.Free-floatingproteinsthenbindtothesephosphotyrosinesitesandarethusconcentratednearthereceptor.Thisconcentrationoftenleadstotheactivationofadditionalproteinsbybringingtogethermoleculesthatnormallywouldnotbeincloseproximity.Throughtheuseofphosphorylationcyclesandcascades,thecellisabletoregulateadiversesetofprocesses,includingcellularmovement,reproductionandmetabolism.Itisthesimplicity,reversibilityandflexibilityofphosphorylationthatexplainswhyithasbeenadoptedasthemostgeneralcontrolmechanismofthecell.ProteinkinaseAproteinkinaseisanenzymethatcantransferaphosphategroupfromadonormolecule(usuallyATP)toanaminoacidresidueofaprotein.Theproteinkinasemechanismisusedinsignaltransductionfortheregulationofenzymes:phosphorylationcanactivate(orinhibit)theactivityofanenzyme.Althoughmostproteinkinasesarespecializedforasinglekindofaminoacidresidue,someexhibitdualkinaseactivity(theycanphosphorylatetwodifferentkindsofaminoacid).Classificationofkinases

BasedonaminoacidstobephosphorylatedSerine/threonine-specificproteinkinasesTyrosine-specificproteinkinasessubstratespecificityMitogen-activatedproteinkinases(MAPkinases)

regulatorsProteinkinasescanberegulatedbyActivatorproteinsInhibitorproteinsPseudosubstratesAutoinhibition(apartoftheproteinkinasemimicsapseudosubstrate)LigandbindingtoregulatorysubunitsCofactors/secondmessengerPhosphorylationintheactivecenter(intrastericalregulation)by:otherproteinkinases(trans-phosphorylation)itself(cis-phosphorylation/autophosphorylation)LocationwithinthecellPKAPKB/AKTPKCPKDPKRReceptortyrosinekinasesPKASerine/threonineproteinkinase

cAMP-dependentproteinkinase,proteinkinaseA.ProteinkinaseAhasseveralfunctionsinthecell,includingregulationofglycogen,sugar,andlipidmetabolism.ItiscontrolledbycAMP:intheabsenceofcAMP,thekinaseisatetrameroftworegulatoryandtwocatalyticsubunits(R2C2),withtheregulatorysubunitsblockingthecatalyticcenterofthecatalyticsubunits.BindingofcAMPtotheregulatorysubunitleadstodissociationofactiveRCdimers.Also,thecatalyticsubunititselfcanberegulatedbyphosphorylation.DownregulationofproteinkinaseAoccursbyafeedbackmechanism:oneofthesubstratesthatisactivatedbythekinaseisaphosphodiestrase,whichconvertscAMPtoAMP,thusreducingtheamountofcAMPthatcanactivateproteinkinaseA.PKB/Akt

Serine/threonineproteinkinaseBCurrentmodelofserine/threonineproteinkinaseB(PKB/Akt)regulationbyreceptortyrosinekinases.Activationofphosphatidylinositol3-kinase(PI3K)bygrowthfactorsleadstogenerationofphosphatidylinositol(3,4,5)trisphosphate(PtdIns(3,4,5)P3),whichrecruitsinactivePKB/Akt(yellow)totheplasmamembrane.ResiduesThr308inthecatalyticsiteandSer473inthehydrophobicmotifaresubsequentlyphosphorylatedbyphosphoinositide-dependentproteinkinase-1(PDK1)andanunidentified‘Ser473’kinase,respectively.ActivatedPKB/Aktcanthenphosphorylatesubstratesattheplasmamembrane,inthecytosol,andinthenucleus.ActivationofPKB/AktisinhibitedbyPTEN(phosphatasesandtensinhomologuedeletedonchromosome10)andpresumablyalsobyCTMP(carboxyl-terminalmodulatorprotein)andTRB3(tribblehomologue3),whichinhibitsphosphorylationofPKBonSer473.Thekinaseisinactivatedbyproteinphosphatase2A(PP2A).

PKCSerine/threonineproteinkinaseProteinkinaseC(PKC)isacyclicnucleotide-independentenzymethatphosphorylatesserineandthreonineresiduesinmanytargetproteins.ManydifferentisoformsofproteinkinaseC(PKC)existincellsandtheyarecentraltomanysignaltransductionmechanisms.

TherearethreeclassesofPKC;theconventionalPKCsareassociatedwithandactivatedbyanionicphospholipids.

TheconventionalPKCmembersare:PKCα,PKCβIandβII,PKCγ.

NovelPKCsinclude:PKCδ,PKCε,PKCηandPKCθ.

AtypicalPKCsinclude:

PKCλandPKCζ.

PKDSerine/threonineproteinkinaseProteinkinaseD(PKD,alsocalledPKCmu)isaserine/threoninekinasewithpotentiallyimportantrolesingrowthfactorsignalingaswellasinstress-inducedsignaling.ReceptortyrosinekinasesThesekinasesconsistofatransmembranereceptorwithatyrosinekinasedomainprotrudingintothecytoplasm.Theyplayanimportantroleinregulatingcelldivision,cellulardifferentiation,andmorphogenesis.Morethan50knownreceptortyrosinekinasesareknowninmammals.StructureofReceptortyrosinekinases

Theextracellulardomainservesastheligandreceptor.Itcanbeaseparateunitthatisattachedtotherestofthereceptorbyadisulfidebond.Thesamemechanismcanbeusedtobindtworeceptorstogethertoformahomo-orheterodimer.Thetransmembraneelementisasingleαhelix.Theintracellularorcytoplasmicdomainisresponsibleforthe(highlyconserved)kinaseactivity,aswellasseveralregulatoryfunctions.RegulationofReceptortyrosinekinases

Ligandbindingcausestworeactions:Dimerizationoftwomonomericreceptorkinasesorstabilizationofaloosedimer.Manyligandsofreceptortyrosinekinasesaremultivalent.Sometyrosinereceptorkinases(e.g.,theplateletderivedgrowthfactorreceptor)canformheterodimerswithothersimilarbutnotidenticalkinasesofthesamesubfamily,allowingahighlyvariedresponsetotheextracellularsignal.Trans-autophosphorylation(phosphorylationbytheotherkinaseinthedimer)ofthekinase.ExamplesfortheregulationbyphosphorylationTheeffectsofphosphorylationonadaptorproteinfunctionAdaptorprote