The 14-3-3 proteins in regulation of cellular metabolism

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ContentslistsavailableatSciVerseScienceDirect

Seminars in Cell & Developmental Biology

j our na l h o me p ag e :w w w . e l s e v i e r . c o m / l o c a t e / s e m c d b

Review

The 14-3-3 proteins in regulation of cellular metabolism

Rune Kleppe

^a

, Aurora Martinez

^a,b

, Stein Ove Døskeland

^a

, Jan Haavik

^a,b,c,∗

aDepartmentofBiomedicine,UniversityofBergen,JonasLiesvei91,5009Bergen,Norway

bK.G.JebsenCentreforResearchonNeuropsychiatricDisorders,UniversityofBergen,JonasLiesvei91,5009Bergen,Norway

cHaukelandUniversityhospital,Bergen,Norway

a r t i c l e i n f o

Articlehistory:

Available online 22 August 2011

Keywords:

14-3-3Proteins Metabolism Tyrosinehydroxylase Membraneassociation Cellsignaling

a b s t r a c t

Thirtyyearsago,itwasdiscoveredthat14-3-3proteinscouldactivateenzymesinvolvedinaminoacid metabolism.Inthefollowingdecades,14-3-3shavebeenshowntobeinvolvedinmanydifferentsignaling pathwaysthatmodulatecellularandwholebodyenergyandnutrienthomeostasis.Largescalescreening forcellularbindingpartnersof14-3-3hasidentiﬁednumerousproteinsthatparticipateinregulationof metabolicpathways,althoughonlyaminorityofthesetargetshaveyetbeensubjecttodetailedstudies.

Becauseofthewidedistributionofpotential14-3-3targetsandtheresurginginterestinmetabolic pathwaycontrolindiseaseslikecancer,diabetes,obesityandcardiovasculardisease,wereviewthe roleof14-3-3proteinsintheregulationofcoreandspecializedcellularmetabolicfunctions.Wecite illustrativeexamplesof14-3-3actionthroughtheirdirectmodulationofindividualenzymesandthrough regulationofmasterswitchesincellularpathways,suchasinsulinsignaling,mTOR-andAMPdependent kinasesignalingpathways,aswellasregulationofautophagy.Wefurtherillustratethequantitative impactof14-3-3associationonsignalresponseatthetargetproteinlevelandwediscussimplications ofrecentﬁndingsshowing14-3-3proteinmembranebindingoftargetproteins.

Contents

1. Introduction. . ... 714

2. Directmodulationofmetabolicenzymefunctionby14-3-3proteins... 714

3. Regulationofmetabolicprocessesbysignalingpathways– impactof14-3-3. ... 716

4. Effectormechanismsof14-3-3proteins... 717

4.1. Localizationtomembranes ... 717

4.2. Effectsontargetproteinphosphorylation... 717

5. Concludingremarks... 717

Acknowledgements... 718

References... 718

Abbreviations:4E-BP1,eIF4E-bindingprotein1;AANAT,arylalkylamineN-acetyltransferase;ACC,acetyl-CoAcarboxylase;AKT,proteinkinaseB;AMPK, AMPdependentproteinkinase;AS,ATPsynthase;AS160,AKTsubstrate160;ATG,autophagyrelated;BAD,Bcl-2antagonistofdeath;

BAX,Bcl-2associatedXprotein;BIM,Bcl-2-interactingmediatorofdeath;CRTC,cAMP-regulatedtranscriptionalcoactivator;FAS,fattyacidsynthase;

GAPDH,glyceraldehyde-3-phosphasedehydrogenase;GLUT,glucosetransporter;GSK3,glycogensynthasekinase3;

HIOMIT,hydroxyindole-O-methyltransferase;LKB1,liverkinaseB1/STK11;MARK1–4,MAP/microtubule-regulatingkinase1–4;

mTOR,mammaliantargetofrapamycin;PFK-2,6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase;PK,pyruvatekinaseM;

PKA,cAMPdependentproteinkinase;PRAS40,prolinerichAKTsubstrate40;RAPTOR,regulatory-associatedproteinofmTOR;

RICTOR,rapamycin-insensitivecompanionofTOR;SK61,p70ribosomalS6kinase1;SIK1–3,salt-induciblekinase1–3;

TH,tyrosinehydroxylase;TPH,tryptophanhydroxylase;TSC1/2,Tuberoussclerosisprotein1/2;ULK1,unc-51-likekinase1.

∗Correspondingauthorat:DepartmentofBiomedicine,UniversityofBergen,JonasLiesvei91,5009Bergen,Norway.Tel.:+4755586432;fax:+4755586360.

E-mailaddress:[email protected](J.Haavik).

doi:10.1016/j.semcdb.2011.08.008

Open access under CC BY-NC-ND license.

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714 R.Kleppeetal./SeminarsinCell&DevelopmentalBiology22 (2011) 713–719 1. Introduction

Inunicellularaswellasmulticellularorganisms,metabolism needs to be tightly regulated according to demand of cellu- larprocessessuchascellgrowth,proliferation,heat production andmechanicaltransformations,e.g.musclecellcontractionand migration. At the organism level, different tissues are formed tocopewithspecializedfunctionsensuringoptimalenergycon- servationandnutrientavailabilityforlifeandreproduction.Key signalingpathwayselicittheco-regulationofmetabolismatthe cellularand tissue level and during thepast decades,evidence has accumulated about the involvement of 14-3-3 proteins in this regulation. The 14-3-3 proteins are being implicated in a growingnumberofcellbiologyprocesses,attestingtothemulti- functionalityofthisubiquitouseukaryoticadaptorproteinfamily.

Herewereviewexamplesof14-3-3involvementinsomemetabolic processes,emphasizingtheirregulatoryrolesinenergymetabolism andbiosyntheticreactionpathways.

Affinitypurificationofcellular14-3-3bindingproteinsinpro- teomic studies provide evidence for several hundred different bindingpartners(possibly>500)associatedwithmostcellularpro- cesses[1–8].Althoughtheassociationswithmanyofthesebinding partnershavenotbeencompletelyverifiedyet,theseproteomic and interactomic studies clearly illustrate the diverse biologi- calfunctionsassociated withthis proteinfamily. Theextensive interactome of the 14-3-3 proteins and its regulation by protein phosphorylationevents suggest a fundamental function of theseproteinsinsignalingrelatedtocellularmetabolicstates.The archetypicalpeptidesequencerequirementsforbindingto14-3- 3havebeenknownfor alongtime [9]and haverecentlybeen reviewed[10],thoughstructuralfeaturesoutsidethebindingmotif canalsocontribute[11].

The14-3-3proteinsseemtobeexpressedinalleukaryoticcell typesandarehighlyabundantinthemammaliannervoussystem [12].Amongthesevenmammalian14-3-3isoforms(␣/␤,␧,␩,␥,

␶/␪,␦/␨,␴)therearereporteddifferencesintheisoformexpres- sionpatternbetweencell-types,tissuesandvariouslaboratorycell lines[13,14].Theunderlyingregulatorymechanismsresponsible forcontrollingthecellularlevelsofdifferent14-3-3isoformsare stillpoorlyunderstood.Still,evidencepointstoarichanddynamic regulationof14-3-3expression,exempliﬁedbyepigeneticregula- tionof14-3-3␴incancer[15],transcriptionalregulation[16–18], andregulationbymicro-RNA[19,20].

Thetwo14-3-3isoformsinyeastseemtoprovideoverlapping functionalityas suggested byviable singleversus lethaldouble knockout.Similargrossredundancyislikelytoexistforthemam- malian14-3-3proteins,butwithmoregeneticallymodifiedmodels becomingavailableand theuseofrefinedwholeanimal analy- sis,one willexpect more isoform-specific functionstoemerge.

Thus,disruptionof the14-3-3␧ isoform(YWHAE geneproduct) hasledtoitsfunctionalassociationwithbraindevelopmentand neuronal migration and it is founddeleted in individuals with Miller–Diekersyndrome[21].IthasbeenreportedthataYWHAE polymorphismisassociatedwithschizophrenia[22],however,in othersamplesthisassociationhasnotbeenreplicated[23].Further- more,14-3-3␩(YWHAHgeneproduct)hasbeenassociatedwith psychoticbipolardisorder[24,25].Lessfunctionalspecialization seemstobeassociatedwith14-3-3␥basedonitsdisruptionin mouse[26],yetalinktobrainandheartdevelopmenthasbeen foundby similarstudiesin zebraﬁsh[27].The14-3-3␴isoform hasbeenmainly associatedwithregulationof cellproliferation andappears tobethemost specializedof themammalian isoforms,whichmayexplainwhyitpreferentiallyformshomodimers.

Thisbrings up a second unresolvedissue of 14-3-3 function – homodimersversusheterodimers,andthesevereeffectsof14-3-3␧ disruptionmightberelatedtoitsabilitytoformheterodimerswith

mostoftheotherisoforms[28–30].Interestingly,somecellular functionshavebeenassociatedwithspeciﬁc14-3-3heterodimers suchasaldosteronestimulatedsodiumchanneltranslocation(14- 3-3␤/␧)andkeratinocytemigrationbythemoreunusual14-3-3␨/␶ [28,31].

Themainfocusof14-3-3biologyinmammaliansystemshas beenontheirmodulationofcellularsignalingpathways,celldeath, cell-cycleandcytoskeletaldynamics.However,theﬁrstfunctional targetsfor14-3-3proteinswereenzymesofspecializedaminoacid metabolism,i.e.tyrosine-andtryptophanhydroxylase[32](THand TPH,respectively),givingrisetotheirnametyrosine-andtrypto- phanhydroxylaseactivators(YWHAs)andputtingtheminacentral positionas modulatorsof catecholamineand serotoninbiosyn- thesis.Inplants,14-3-3functionhasbeenmainlyassociatedwith metabolicregulation(reviewedin[33]),thoughseveralexamples oftargetsinsignaltransductionhavebeenreportedandcellsig- nalingisanincreasinglyestablishedfunctionof14-3-3sinplants [33–35].

2. Directmodulationofmetabolicenzymefunctionby 14-3-3proteins

Historically,thefirstreportedtargetsofproteinSer/Thrkinases wereenzymesinvolvedincentralmetabolicpathways[36],dis- coveries which contributed to the understanding of hormonal control of cellularmetabolism. In fact, metabolicregulation by Ser/Thr phosphorylation is crucial to cellular function. How- ever,the impactof 14-3-3 proteinson regulationof metabolic enzyme activities is still poorly understood in mammals. In a largescaleaffinitypurificationstudyof14-3-3bindingpartners from HeLa cell lysates, using yeast 14-3-3(BMH1) as bait and elution with the 14-3-3 binding peptide ARAApSAPA, MacKin- tosh and colleagues identified several key enzymes in central metabolism as potential binding partners of 14-3-3 [2]. These included enzymes involved in glycolysis, pentose phosphate pathway, fattyacidsynthesis, nucleotide synthesis, methionine metabolism,andreductivemetabolism.Interestingly,inasimilar studyusingmammalian14-3-3␨asbait,severalofthesemetabolic enzymes were confirmed, including pyruvate kinase M (PK), ATP-synthase (AS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH),fattyacidsynthase(FAS)andthebifunctionalenzyme6- phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2) [1]

(Fig. 1). Many of these enzymes and additional proteins have beenfoundtointeractwith14-3-3insubsequentinvestigations [7,37]. Interestingly, the heart isoform of PFK-2 was identi- ﬁed as a 14-3-3 target upon phosphorylation by AKT but not 5AMP-activated protein kinase (AMPK), locking the enzyme in its glycolysis stimulating state in response to insulin [38]

(Fig.1).

Together, these ﬁndings indicate that 14-3-3 proteins may directlyinteractwithandmodifythefunctionsofenzymesthat areofcentralimportance inmetabolicregulation.However,for mostoftheproteintargetsthatwereidentiﬁedintheabovescreen- ingstudies,thebindingkineticsandbiologicalimplicationsofthe interactionsarenotyetfullyelucidated.Instead,mostofourknowl- edgeof14-3-3actiononmetabolicregulationcomesfromdetailed studiesonalimited numberof regulatoryenzymesinvolved in specialized biological pathways. The regulation of monoamine biosynthesisbythearomaticaminoacidhydroxylasesisofpartic- ularimportancebothforhistoricalreasonsandbecausethedetails ofinteractionshavebeenstudiedinquantitativetermsbymany differentgroups.

Thebiosynthesis ofcatecholamineandserotoninneurotrans- mitters and hormones is tightly regulated at the level of the rate-limiting enzymesin these pathways(tyrosinehydroxylase

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Fig.1.Involvementof14-3-3proteinsinregulationofcellularenergymetabolism.Theﬁguresummarizesbindingof14-3-3(blue)toimportanttargetsandtheirplacement incoremetabolicpathwaysandregulatorysignalingpathways.Themetabolicpathwaysareillustratedwithbroadarrowsorspirals(fattyacidsynthesisoroxidation)and onlyselectedintermediatesandenzymes,whicharereportedas14-3-3bindingpartners,areshown.Interactionswithmetabolicenzymesthatarereportedininteractomics studiesareshownwithdottedlines.Phosphorylationeventsareshownas(P)witharrowsfromthekinaseinvolved,wherestimulatingphosphorylation(and14-3-3binding) areshownonwhitebackgroundandinhibitoryphosphorylationonblack.MetabolicintermediatesareabbreviatedGluc(glucose),Gluc-6P(glucose-6-phosphate),Fruc- 6P(fructose-6-phosphate),PEP(phosphoenolpyruvate),Pyr(pyruvate),AcCoA(acetylcoenzymeA),Glyc-3-P(glycerol-3-phosphate),Rib-5P(ribose-5-phosphate)andthe enzymesCL(citratelyase),ACC(acetyl-CoAcarboxylase)andPKC(proteinkinaseC).Forotherabbreviationswerefertothetextandabbreviationssection.

(TH)andtryptophanhydroxylase(TPH),respectively).Beforeany otherfunctionalrolesof14-3-3proteinshadbeendiscovered,ina seriesofpioneeringstudiesitwasshownthatactivationofTHand TPHbyphosphorylationwasdependentonanadditionalprotein factorthatcouldbeseparatedfromthehydroxylases[32].Upon subsequentidentificationoftheactivatorproteinasamixtureof 14-3-3proteins[39],accesstopurifiedenzymesandspecificanti- bodies,ithasbeenshownthatallsevenhuman14-3-3isoforms, aswellastheproteinsBMH1andBMH2bindtophosphorylated THandTPH,albeitwithdifferentaffinitiesandbindingspecificities [40–43].

InratsandhumansTHisencodedbyasinglegenethatissubject toalternativesplicing,resultinginmultipleisoformswithslightly different N-terminal sequences, also affectingtheir phosphorylation and 14-3-3 binding sites [41,44]. The 14-3-3-dependent THactivation/stabilizationrequiresthephosphorylationatSer19 [41,44].Phosphorylation atthis positionalsoincreases therate ofSer40phosphorylationbyPKA,amodiﬁcationthatreleasesthe feedbackinhibitionbycatecholamines[44–47].Bindingto14-3-3 preserves thestability of thehighlyactive and polyphosphory- latedTH,anditalsoappearstobeimportantforthesubcellular enzyme localization and coordinated dopa/dopamine synthesis

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716 R.Kleppeetal./SeminarsinCell&DevelopmentalBiology22 (2011) 713–719

andrelease(seebelow).TPHis encodedbytwoseparategenes, resultinginthehomologousenzymesTPH1andTPH2,withdif- ferenttissuedistributions,regulatorypropertiesandphysiological functions [48]. As for TH, 14-3-3 binding to TPH1 and TPH2 consolidatestheenzymeactivationthatisinducedbyphospho- rylation,protectsthephosphoproteinsagainstdephosphorylation, andincreasestheirthermalstability[40,43].Anotherexampleof ﬁneregulationofametabolicpathwaybyregulationofenzyme activityand stabilitythroughcomplexformationwith14-3-3is foundformelatoninsynthesisinthepinealgland.Melatoninsyn- thesisconstitutestheﬁnalstageinthisbiosyntheticpathwayfrom tryptophan (serotonin→N-acetylserotonin→melatonin), where arylalkylamine N-acetyltransferase (AANAT) is the penultimate enzyme.Boththeintrinsiccircadianclockandlightexposureregu- lateAANATatthetranscriptionalandtranslationallevelinconcert withTPHand hydroxyindole-O-methyltransferase (HIOMT)that operateinthepreceedingandsucceedingstepsinthepathway.

But ﬁne regulation of AANAT activity occurs through light- dependentphosphorylationattwositesintheenzyme,i.e.Thr31 andSer205,thatdifferentlyaffecttheafﬁnityoftheenzymefor 14-3-3␨[49].

3. Regulationofmetabolicprocessesbysignalingpathways –impactof14-3-3

Cellularfunctions,growthandproliferationarecontrolledby signalingpathwaysthat sensetheenergyandnutrientstatusof thecell.TheAMPKs(AMPK1–3)areimportantcellularsensorsof theAMP:ATPratioand arekey regulatorsofenergy consuming andenergy producing pathways.These heterotrimeric(␣,␤,␥) enzymesareactivatedbyAMPbindingtotheir␥-subunit,making theactivationloopofthecatalytic␣-subunitavailableforactivation byThr172phosphorylationbyanupstreamAMPKKinase[50].In mostcellsthetumorsuppressorliverkinaseB1(LKB1/STK11)acts asthemasterregulator,notonlyofAMPK,butalsoofotherkinases intheAMPKfamily,includingsalt-induciblekinase1–3(SIK1–3) andtheMAP/microtubule-regulatingkinase1–4(MARK1–4)[51]

(Fig.1).

ControloflocalizationandactivityofAMPKsby14-3-3proteins wasﬁrstdescribedfortheMARKs(alsocalledPartitioningdefective (Par)1a–d),whichareinvolvedinregulatingcellularpolarity[52]

(Par-5isalsoa14-3-3proteininCaenorhabditiselegans),butisalso reportedfortheSIKs[53].InparticularSIK2,butalsoMARK2and AMPKplayanimportantroleinmetabolicregulationthroughthe repressionoftheCREBcoactivator,cAMP-regulatedtranscriptional coactivator(CRTC)andtherebysuppresshepaticgluconeogenesis [54].Phosphorylation-dependentbindingof14-3-3toCRTCwith resultinglocalizationtothecytosolseemstobeanimportantreg- ulatorymechanism [55], suggesting that14-3-3 proteinsactto consolidatesuppressionofgluconeogenesisthroughSIK2andCRTC aswellasinsulinsignaling(below)(Fig.1).

Thereis considerablecross-talk betweentheAMPKpathway andotherkeyenergyregulatorypathwayssuchastheTargetof rapamycin(TOR) signalingcomplex 1 (TORC1) and insulin signaling.TheTORC1 playsa key role inadapting cellulargrowth tonutrientavailability[56].Theregulatory-associatedproteinof TOR(RAPTOR)containingcomplexstimulatesribosomebiogenesis, proteintranslationthroughphosphorylationofS6kinase1(S6K1) andeIF4E-bindingprotein1(4E-BP1)andinhibitsautophagyby phosphorylationof unc-51-like kinase1 (ULK1)and autophagy related 13 (ATG13) [56,57] (Fig. 1). The mammalian TORC1 (mTORC1)also depends on stimulatory inputfrom the GTPase RHEB(Rashomologueenrichedinbrain),whichreceivesinhibitory inputfromtheGAPcontainingproteincomplexofTuberoussclero- sisprotein1and2(TSC1/HamartinandTSC2/Tuberin)[56].Much

ofthemodulationofmTORC1ismediatedthroughTSC1/2,suchas growthfactorsignaling,AMPKandhypoxia.Inhibitoryphosphory- lationofTSC1/2byAKTandMAPKAPK2facilitatesbindingof14-3-3 [58,59],andaprevailinghypothesisofhypoxia-inducedinhibition ofmTORC1isbyRebb1inductionandsequestrationof14-3-3away fromTSC2[60](Fig.1).

AMPKisreportedtoinhibitmTORC1byactivationofTSC1/2and byinhibitionofRAPTORbyphosphorylation-inducedbindingof14- 3-3[61],bothofwhichwillstimulateautophagy.Recently,adirect stimulatorypathfromAMPKtoautophagywasdescribedthrough phosphorylationofULK1 [62], andcomplex formation between ULK1,mTORC1andAMPKhasbeenfoundtocoincidewithRAPTOR phosphorylationandbindingof14-3-3[63](Fig.1).Judgingfrom the14-3-3:RAPTORinteraction,14-3-3seemstobeonbothsides oftheequationforautophagy.Interestingly,14-3-3␨hasrecently beenreportedtoinhibitautophagythroughinhibitionoftheclass IIIphosphatidylinositol-3-kinase(PI3K-III)[64],whichisnecessary for autophagosomevesicle nucleation.14-3-3␨is alsoan inter- actionpartnerofBH3-onlyfamilymembersoftheBcl-2protein family,suchasBcl-2antagonistofdeath(BAD)andBCL-2inter- actingmediatorofcelldeath(BIM),aswellastheporeforming Bcl-2associatedXprotein(BAX)[65–67].Hence,increased14-3- 3␨shouldshiftthebalanceofBCL-2proteinstowardssurvival,but alsoreleasemoreBCL-2/BCL-XLforbindingtoBECLIN-1(ATG6)and therebysuppressautophagyofmitochondriaandER[68](Fig.1).

Downstreamofinsulinandgrowthfactorstimulation,AKTis activatedbyPDK1 andtheRICTOR(rapamycin-insensitive com- panionofTOR)containingTORcomplex2(TORC2),leadingtoAKT andERKmediatedinhibitionofTSC1/TSC2andstimulationofthe mTORC1pathway.AsecondpathofinsulintomTORC1activation ismediatedbyAKTphosphorylationandinhibitionoftheTORC1 inhibitoryproteinProline-richAKTsubstrate40(PRAS40),which associateswithRAPTOR[69].Bindingof14-3-3tophosphorylated PRAS40isimportanttoreleaseitssuppressionofTORC1[70],but theexactmechanismisstillnotsettled.ThepresenceofPRAS40 seemstobeimportantforTORC1activity,whereasTORC1mediated phosphorylationofPRAS40isimplicatedinthereleaseofPRAS40 suppression[71](Fig.1).

Amajoreffectofinsulininskeletalmuscleandadiposetissue isincreaseduptake ofglucosefromtheblood. Thisismediated byincreasedtranslocationofglucosetransporter4(GLUT4)tothe plasmamembranebyRab-mediatedvesiculartransport.Inresting cellsthistransportisinhibitedbyRab-GAPssuchasAktSubstrate 160 (AS160/TBC1D4) and TBC1D1, which are both phosphory- latedbyAKTinresponsetoinsulin[72,73].AKTphosphorylation facilitatesbindingof14-3-3andcytosolictranslocationofAS160 and TBC1D1withsubsequentrelease ofits suppressiveactivity [74] (Fig. 1). Interestingly, a knock-in mouse of AS160 with a disrupted phosphorylation- and binding site of 14-3-3 showed decreasedinsulin-stimulatedGLUT4-translocationinmuscleand insulininsensitivitywithrespecttoglucosetolerance[75].Paral- leltoincreasedglucoseuptake,insulinstimulatesglucosestorage asglycogeninliverandmusclebyAKT-mediatedinactivationof glycogensynthasekinase3(GSK3),whichsuppressesglycogensyn- thesis.The␤-isoformofGSK3isaknownbindingpartnerfor14-3-3 uponSer9phosphorylation,butthisinteractionhasreceivedmore attentionforitsroleinGSK3␤mediatedphosphorylationofTau [76](Fig.1).TherelativerolesofGSK3␣and␤inregulationofglyco- gensynthesisindifferenttissuesareyetnotresolved.However,a recentlydiscoveredlinkbetweenGSK3␤andinactivationofRICTOR ofTORC2[77],importantforinsulininduceduptakeofglucosein muscle[78],suggestsapossibleroleof14-3-3inGSK3␤-mediated insulinresistance.

Theapparentinvolvementof14-3-3proteinsintheregulation ofenergymetabolism,mTORsignalingandautophagyinaddition totheirwell-known roles in apoptotic and cell-cycle signaling,

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alsomakestheminterestingforunderstandingcarcinogenesis(see reviewbyYaffeetal.inthisissue)[93].

4. Effectormechanismsof14-3-3proteins 4.1. Localizationtomembranes

Theregulatoryfunctionsof14-3-3proteinsextendbeyondacti- vation,inhibition, stabilizationor orientationof itspartners, as 14-3-3alsomodulatethespecificsubcellularlocalizationoftheir cargos[79].Itiswellestablishedthat14-3-3sequestersproteins inthecytoplasm,inhibitingnuclearormitochondriallocalization ofspecificpartners,thoughlessisknownaboutisoform-specific differences[80,81].However,thereisalsoincreasingevidenceon theroleof14-3-3inthetransferofbindingpartnerstomembranes [82,83].

Inadditiontotheuniqueroleofbiologicalmembranesincell compartmentalization,membranesallowtheformationandfunc- tion of specialized, multi-enzyme units. Several of the protein componentsoftheseunitsinteractwithmembranesperipheral- andreversiblyandthisinteractioncanbemediatedinseveralways, including phosphorylation and modulations of charge–charge relationships [84]. The lowered dielectric constant and lower pH in the vicinity of a biological negatively charged membrane are often the driving forces for peripheral interactions.

We haverecentlyshown that14-3-3␥interactsreversibly with negatively charged membranes [84,85]. The membrane afﬁnity of 14-3-3␥ is high for phospholipid compositions that mimic that of synaptic vesicles, with a high content of 1-stearoyl-2- docosahexaenoyl-phosphatidylserine(SDPS)andincreaseswhen aSer19-phosphorylatedTH-derivedpeptideisboundto14-3-3␥ [84],suggestingaroleofthis14-3-3isoforminmembranelocal- izationofTH.Thoughmainlycytoplasmatic,afractionofTHisalso foundasmembrane-boundbothinbrain,notablyatnerve end- ingsandsynapticvesicles,andincatecholaminesecretorygranules inadrenalmedulla[86].ThesigniﬁcanceofthebindingofTHto membranesisnotclearbutitisassumedtohavearoleinthecoordi- nationofdopaminesynthesisandreleasesincedopadecarboxylase alsoformsfunctionalcomplexeswithTHatmembranes[87].

4.2. Effectsontargetproteinphosphorylation

Byspecificallyassociatingwiththephosphorylatedstateofthe targetproteins, the14-3-3 proteinswillshield against dephos- phorylation.Theefficiencyoftheprotectionwilldependonthe dissociationconstant(K_d)ofthecomplexandtheeffectivecon- centrationsofthebindingpartners.For14-3-3targetprotein(TP) association,K_d-valuesinthesub-nanomolartomicromolarrange aregenerallyreported[41,80,88].Theinfluenceof14-3-3binding onthesteadystateleveloftargetphophorylationcanthereforebe predicted,asexemplifiedbythesimplecaseofmonovalentphos- phorylationandTPbindingto14-3-3(Fig.2AandB).Inthismodel, evenformoderatebindingaffinities(K_d=0.5␮M),a∼6-foldampli- ficationofthesignal(phospho-TP)responseisobserved,andfor higheraffinitiessignificantsensitizationispredictedinadditionto prominentsignalamplification.

Bindingofdimeric14-3-3coversaconsiderablesurfaceareaof theTPandthismayaffectitslocalization,oligomerization/complex formation,or furtherproteinmodifications. 14-3-3 Bindingcan beconsideredefficientanddynamicmeansofinducingsecondary effects of phosphorylationonprotein function, ascompared to otherproteinsthatobtainsimilarresponsesthroughconforma- tionalchanges.The14-3-3proteinsareabletosimultaneouslybind twophospho-targetsites,eithertoobtainhighaffinityinteraction (coincidencedetector)ortoelicitstructuraleffectsuponbinding

Fig.2. Theconsolidatingactionof14-3-3ontargetphosphorylation.Panel(A) showspartofasignalingpathwaywhereaproteinkinaseisactivated(modeledby fractionalactivation)byasignalinput(e.g.externalsignalorinternalsecondmes- senger)accordingtoasigmoidactivationfunction,whereSisthesignalinput,S0.5is thesignalinputthatgiveshalffractionalactivationofthekinase(here10nM)andh istheHillcoefficientofthekinasesignalresponse(here1.5).Panel(B)showsadown- streamtargetprotein(TP)([TP]=0.5␮M)oftheactivatedkinase(Kinase*),whichis dephosphorylatedbyaproteinphosphatase(PP)andwhichmayinteractwith14- 3-3proteins(setto5␮M)inphosphorylationdependentmanner.Thesteadystate amountoftotalTPphosphorylationinresponsetothesignalinputiscalculatedfor TPwithdifferentaffinitiesto14-3-3(Kd=0.5,50,500and∞nM).Thus,fromamaxi- malTPphosphorylationresponseof9%intheabsenceof14-3-3binding,decreasing theKd(500nM,;50nM,;0.5nM,)bothamplifies(5.8-,10-and11-fold,respectively)andsensitize(about2-,5-and10-fold,respectively)theTPphosphorylation responsetoS.ThemodelingwasperformedinCopasi(v4.5)[90]usingrateof phosphorylationVK^∗=kphos(S^h/(S^h_0.5+S^h))·[Kinasetot]·[TP],where[Kinasetot]=1, kphos=0.5␮M⁻¹s⁻¹andrateofdephosphorylationVPP=5␮M⁻¹s⁻¹·[PPtot]·[pTP]

([PPtot]=1),whereastheassociationwith14-3-3,kawassetto0.5␮M⁻¹s⁻¹andKd

tofulﬁlltheKd[39].

(gatekeeperphosphorylation,molecularanvil[89]).Theseissues arediscussedin[10],andthecoincidencedetectorpropertyof14- 3-3caneasilybevisualizedfromFig.2B,where twoversusone phosphorylationeventsdramaticallyincreasethebindingafﬁnity, resultinginbothsignalampliﬁcationandsensitization.

5. Concludingremarks

Theregulationofcellularmetabolismby14-3-3proteinsisan arearipeforfurtherinvestigations.The14-3-3interactomicstudies suggestthatkeymetabolicenzymesarewellrepresentedamong 14-3-3’scellularbindingpartners.Thecurrentunderstandingof how 14-3-3 proteinsregulate theirtargetsis likely tobevalid forothermetabolicenzymesaswell.Thiswillalsoprovideaddi- tionalmechanisticinsightintohowsuchenzymesareregulatedby phosphorylation.Potentialavenuesforfutureresearchincludehow 14-3-3proteinscontrolenzymeactivitiesandturnover,modulate single-ormultiplephosphorylationevents,andregulatemetabolic activitiesatdifferentcellularlocations.

Thepresenceofmanyalternativebindingpartnersfor14-3-3s inthecellposesseveralimportantandunresolvedissuesrelatedto ourunderstandingoftheirabilitytomodulatecellularprocesses.

Howdodifferent14-3-3ligandscompeteforcomplexformation?

Thetotalcellularlevelof14-3-3proteinsisquitehigh,butthemany interactionpartnersmeansthatonlyaproportionofthe14-3-3

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718 R.Kleppeetal./SeminarsinCell&DevelopmentalBiology22 (2011) 713–719

proteinswillbefreetoengagepartnersinthecell.Unfortunately, theconcentrationoffree14-3-3forthevarious14-3-3isoforms remainsunknown.

The14-3-3proteinsarecertainlyinvolvedinmanycellularpro- cessesthatalreadyareinfocusforpharmacologicalintervention, buttheirbroadinvolvementmakesonewonderwhether14-3-3 proteinsarerealisticastargetsfordrugseitherinhibitingorstabi- lizingtheirinteractionsingeneral.Afeasibleapproachmaybeto targettheuniqueprotein–proteininterfacepresentforeach14-3- 3:targetproteincomplex.Itremainstobeseenifthisspeciﬁcitycan beachievedattheorganismlevel(seereviewsbyFuetal.fordis- cussion)[12,91].Recently,thefungalphytotoxinfusicoccin,which stabilizestheinteractionbetweenplant14-3-3andH⁺-ATPase,was showntopromoteplateletaggregation,presumablyviastabiliza- tionofthe14-3-3-glycoprotein Ib␣complex[92].Clearly,more basicknowledgeand improvedmodelsanddrugcandidatesare requiredtoexploitthepotentialof the14-3-3-proteinsasdrug targets.

Acknowledgements

ThisworkwassupportedbytheResearchCouncilofNorway, TheKristianGerhard Jebsen Foundation,theNorwegian Cancer SocietyandtheWesternNorwayHealthAuthorities.

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