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The
International
Journal
of
Biochemistry
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Cell
Biology
j ou rn a l h o m ep a g e :w w w . e l s e v i e r . c o m / l o c a t e / b i o c e l
CK2
phosphorylation
of
C/EBP
␦
regulates
its
transcription
factor
activity
Lisa
Schwind,
Andreas
D.
Zimmer,
Claudia
Götz,
Mathias
Montenarh
∗MedicalBiochemistryandMolecularBiology,SaarlandUniversity,Building44,D-66424Homburg,Germany
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t
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c
l
e
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n
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o
Articlehistory:
Received29September2014
Receivedinrevisedform26January2015 Accepted3February2015
Availableonline11February2015 Keywords: Phosphorylation Transcription Transcriptionfactor ProteinkinaseCK2 Protein–proteininteraction
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ProteinkinaseCK2playsanessentialroleincellviabilityinlowerandhighereukaryotes.Asaglobal regu-latoritphosphorylatesandtherebyregulatesabroadarrayofcellulartargetsincludingalargenumberof transcriptionfactors.Here,wehaveidentifiedtheCCAAT/enhancerbindingprotein␦(C/EBP␦)asanew substrateforCK2.UsingpointmutantsofC/EBP␦themajorphosphorylationsiteforCK2wasmappedto serine57,whichislocatedwithinthetransactivationdomainofC/EBP␦.Forproperfunctioningasa tran-scriptionfactorC/EBP␦hastobetranslocatedintothenucleuswhereitformsheterodimerswithother membersoftheC/EBPfamilyofproteinsandATF4.Here,wefoundthatCK2phosphorylationdoes nei-therinfluencethesubcellularlocalizationofC/EBP␦noritsinteractionwithC/EBP,butratherdoesCK2 phosphorylationmodulatethetranscriptionalactivityofC/EBP␦.Moreover,wefoundthatCK2bound toC/EBP␦,whichmighthelptotargetCK2tothetranscriptionalmachinerywhereitcanphosphorylate othertranscriptionfactorsorco-activators.
©2015ElsevierLtd.Allrightsreserved.
1. Introduction
Agreaterunderstandingoftheregulationofgeneexpression is achievedbyanalysingtheregulation of transcriptionfactors. Transcriptionfactorsareabsolutelyrequired foraccurate trans-criptionalresponseineverycelltype.Thistranscriptionalresponse enablesgeneexpressioninaspecificcelltypeoratdistinct devel-opmentstagesaswellasduringdifferentiation(Struhl,1989;Abel andManiatis,1989).Onekeystepintheregulationoftranscription factorsseemstobethetranslocationoftheproteinfromthe cyto-plasmintothenucleusorback.Anotherprocess,thatseemstobe implicatedintheregulationoftranscriptionalactivity,isthe inter-actionoftranscriptionfactorswithothercellularproteins,which inmanycasesenablesthecorrectinteractionofthetranscription factorswithspecificDNAsequences.Inaddition,post-translational modificationsof transcriptionfactorsrepresent anotherlevelof regulation.Amongthepost-translationalmodificationsreversible phosphorylationplaysa prominentrole.Accordingtothemode of action, transcription factors can be classified as zinc finger proteins,basichelixloophelixcontainingproteinsorbasic-leucine zipper(bZIP)transcriptionfactorstomentionjustbutafew.The CCAAT/enhancerbindingproteinfamily(C/EBPs)isafamilyof tran-scriptionfactorswithahighlyconservedbZIPdomain.Sixisoforms
∗ Correspondingauthor.Tel.:+4968411626502;fax:+4968411626027. E-mailaddress:Mathias.Montenarh@uks.eu(M.Montenarh).
namelyC/EBP␣toC/EBPwereidentifiedtodate(Williamsetal., 1991;Lekstrom-HimesandXanthopoulos,1998).Numerous stud-ieshaveattributedaroleofC/EBPsincontrollingandregulation of cellular proliferation, differentiation and metabolism (Ramji andFoka,2002;Rosen,2005).Ithasbeendemonstratedthat iso-formsofC/EBPscanregulatethetranscriptionofgeneswhichare importantforfatmetabolismoradipocytedifferentiation.C/EBP␦ isanimportantmemberoftheC/EBPfamilywhichisexpressed duringadipogenesisandcontributestoitbyregulatingthe expres-sion of other key transcription factors suchas the peroxisome proliferator-activatedreceptor␥(PPAR␥)andC/EBP␣(Caoetal., 1991;Clarkeetal.,1997).Inaddition,C/EBP␦interactswithC/EBP toinducePPAR␥expressionduringadipogenesis(Wuetal.,1996). Oneof theprotein kinasesthatphosphorylates transcription factors is protein kinase CK2formerly known as casein kinase II.Amongthedifferentclassesoftranscriptionfactorsanumber oftranscriptionfactorswerealreadyidentifiedasCK2substrates (Meggio and Pinna, 2003)including the CCAAT/enhancer bind-ing protein (C/EBP)family of proteins. A known CK2substrate withinthis family is C/EBP alsoknownasCHOP,GADD153 or DDIT3(Ubeda andHabener,2003).PhosphorylationofCHOPby CK2inhibitsitstranscriptionactivationfunction,whichhas impli-cations for ER stress induced apoptosis (Zinszner et al., 1998; Yamaguchiand Wang,2004)or differentiation(Tangand Lane, 2000).CHOPandothermembersoftheC/EBPfamilyofproteinscan formheterodimerswithmembersoftheactivatingtranscription factors(ATFs).Recently,weidentifiedATF4asanewsubstrateand
bindingpartnerofCK2(Schneideretal.,2012;Ampofoetal.,2013). WehaveshownthatanATF4phosphorylationmutant,whichcan nolongerbephosphorylatedbyCK2,exhibitsareduced transcrip-tionfactoractivity(Ampofoetal.,2013).Thus,wehypothesizethat probablyothermembersoftheC/EBPproteinsmightbesubstrates forCK2andthatCK2mightregulatetheseproteinsby phosphor-ylation.Here,wedescribeC/EBP␦asanewsubstrateforCK2and analysetheimpactofthisphosphorylationonC/EBP␦functions.
2. Materialsandmethods
2.1. Reagentsandantibodies
Protease inhibitor cocktail CompleteTM was obtained from
RocheDiagnostics(Mannheim,Germany).DMSOwaspurchased from Merck (Darmstadt, Germany). CX-4945 was bought from Selleckchem(Munich, Germany)and quinalizarinfromLabotest (Niederschöna,Germany). Glutathione agarosewas fromPierce (Rockford,USA). Anti-␣-tubulinand Anti-FLAG antibodieswere obtainedfrom Sigma–Aldrich(Munich,Germany). Theantibody againstC/EBP␦waspurchasedfromSantaCruzBiotechnology (Hei-delberg,Germany). Anti-GST antibody and proteinA sepharose wereobtainedfromAmershamBiosciences(Freiburg,Germany). DetectionofCK2wasperformedbyusingtherabbitantibody#26 againstCK2␣(Faustetal.,1999)andthemousemonoclonal anti-body 6D5 (Nastainczyk et al., 1995) wasused to detect CK2. Goat,mouseandrabbitsecondaryantibodieswereallboughtfrom Dianova(Hamburg,Germany).
2.2. Cellcultureanddrugtreatment
HCT116 cells (ATCC Number: CCL-247) were cultured at 37◦C and 5% CO2 in McCoy’s 5A medium (PromoCell,
Heidel-berg,Germany)containing10%foetalcalfserum(FCS).TheCK2 inhibitorsCX-4945and quinalizarinweredissolvedindimethyl sulfoxide (DMSO) to a 10mM stock solution. Six hours after transfectioncellsweretreatedwiththeCK2inhibitorsatfinal con-centrationsof10M(CX-4945),50M(quinalizarin)orthesame volumeofDMSOascontrolforanoverallperiodof24h.
2.3. Plasmidsandcloningofpromoterconstructs
ThecDNAofhumanC/EBP␦waskindlyprovidedbyDr.Esta Ster-neck(CentreforCancerResearchNCI-Frederick,USA).ThecDNA wasamplifiedbyPCRwiththespecificprimershC/EBP␦-for:5 -AGAGAGGGATCCATGAGCGCCGCG-3;hC/EBP␦-rev:5-AGAGAG GAATTCTTACCGGCAGTCTGCTGT-3andclonedinto pGEX4T-1vector(BamHI/EcoRI),whichwasfromGEHealthcare(Munich, Germany).ThecreationoftheGST-C/EBP␦mutantswasperformed by “splicing by overlap extension”-PCR (SOE-PCR). Aside from primershC/EBP␦-forwardand–reversemutationspecificprimers C/EBP␦-S57A-SOE-for:5-TGTACG ACG ACGAGGCCGCCA TCG ACTTCA-3;C/EBP␦-S57A-SOE-rev:5-TGAAGTCGATGGCGGCCT CGTCGTCGTACA-3;C/EBP␦-S57D-SOE-for:5-TGTACGACGACG AGGACGCCATCGACTTCA-3;C/EBP␦-S57D-SOE-rev:5-TGAAGT CGATGGCGTCCTCGTCGTCGTACA-3;C/EBP␦-T159A-SOE-for:5 -ACCCCGCCCGCGTCGCCGGAG-3;C/EBP␦FLAG-T159A-SOE-rev: 5-CTCCGGCGACGCGGGCGGGGT-3wereused.
Thep3xFLAG-CMV-7.1vectorwasfromSigma–Aldrich(Munich, Germany).TogeneratetheexpressionplasmidsFLAG-C/EBP␦WT,
FLAG-C/EBP␦S57AandFLAG-C/EBP␦S57D,therespectivecDNAwas
subclonedfromthepGEX4T-1 constructintop3xFLAG-CMV-7.1 vectorusingEcoRIandBamHI.
The human cDNA of C/EBP LAP2 was from Addgene Inc. (Cambridge,U.S.A).UsingPCR,itwasamplifiedwiththeprimers hC/EBP-for:5-AGAGAGGGATCCATGGAA GTGGCCAAC-3;
hC/EBP-rev:5-AGAGAGGAATTCCTAGCAGTGGCCGGA-3and clonedintopRSETAvector(BamHI/EcoRI),whichwasfrom Invitro-gen(Carlsbad,USA).
The firefly luciferase reporter vector (pGL4.10) was from Promega (Mannheim, Germany). The mouse PPAR␥2 promoter fragment,containing1302nucleotidesupstreamofthestartcodon, wasobtainedbyPCRamplificationfrompurifiedmousegenomic DNAwiththeprimersmPPAR␥2-prom-for:5-AGAGAGCTCGAG GGGGAGTCATTAAATGATG-3;mPPAR␥2-prom-rev:5-AGAGAG CCATGGACAGCATAAAACAGAGATTTG-3andcloned(XhoI/NcoI) intopGL4.10.AllDNAconstructswereverifiedbysequencing. 2.4. Expressionandpurificationofbacteriallyexpressedproteins
C/EBP␦wild-typeand mutantswereexpressedin Escherichia coli BL21(DE3) after induction with 0.5mM isopropyl- -d-thiogalactopyranoside (IPTG) for 3h at 37◦C. Proteins were extractedwithGSTextractionbuffer(2mMMgCl2,500mMKCl,5%
(v/v)glycerol,0.65%(v/v)Chapsin1×PBS(pH7.4))and sonifica-tion,purifiedbyaffinitychromatographywithglutathioneagarose andelutedwith10mMglutathioneinGSTextractionbuffer.
Recombinant␣-and-subunitsoftheCK2holoenzymecloned inapT7-7vectorwereexpressedinE.coliBL21(DE3),andthe pro-teinswerepurifiedaccordingtoGrankowskietal.(1991). 2.5. InvitrophosphorylationwithproteinkinaseCK2
RecombinantGST-taggedC/EBP␦proteins(1g)weremixed withCK2holoenzymeinavolumeof20lofkinasebuffer(50mM Tris–HCl,pH7.5,150mMNaCl,5mMMgCl2,50M ATP,1mM
DTT).Tostartthereaction,5lofkinasebufferincluding2Ci [32P]␥ATPwereaddedandthesampleswereincubatedfor15min
at37◦C.Thereactionwasstoppedbyadding10lof3×SDSsample buffer(195mMTris–HCl,pH6.8,0.03%(w/v)bromophenolblue, 15%(v/v)-mercaptoethanol,30%(v/v)glycerol,6%(w/v)SDS). Sampleswereseparatedthrougha12.5%SDSpolyacrylamidegel. ThegelwasstainedwithCoomassieblue(0.2%CoomassieR250, 0.01%CoomassieG250,50%methanol,10%aceticacid)toverify equalamountsofproteinandvacuum-dried.Phosphorylationwas detectedbyautoradiography.
Forthecompetitionexperimentsthepeptidewiththesequence AMTDDESAIDFSACcomprisingtheCK2phosphorylationsitewas pre-incubatedwith100ngofCK2holoenzymeat37◦Cfor10min. Subsequently,1gofC/EBP␦proteinwasaddedandthemixture wasincubatedforadditional5minat37◦C.Sampleswereanalysed asdescribedabove.
2.6. Invitrotranscription/translation
InvitrotranscriptionandtranslationofHis-C/EBPwere per-formedwiththeonestepkitTNT®T7CoupledReticulocyteLysate System(Promega,Mannheim,Germany)accordingtothe manu-facturer’sinstructions.
Briefly,thepRSETA-C/EBP-LAP2plasmidwasincubatedinthe presenceof[35S]methioninefor90minat30◦Cinatotalreaction
volume of 50l. Thereafter, 7l translation mixture per GST-C/EBP␦proteinwasusedforpull-downassayorthewholereaction mixturewasfrozenat−80◦C.
2.7. Pull-downassay
TheassaywasperformedasdescribedbyHagemeieretal.(1993)
buffer(50mMTris–HCl,pH8.0,140mMNaCl,100mMNaF,200M sodiumorthovanadate,0.5%(v/v)NP40).AfterwashingwithNETN buffer(20mMTris,pH8.0,100mMNaCl,1mMEDTA,0.5%(v/v) NP40),boundproteinswereelutedand separatedbySDS poly-acrylamidegelelectrophoresis.Detectionofboundproteinswas accomplishedeitherbyWesternblot(CK2)orbystainingthegel withCoomassieblue,dryingandautoradiography(C/EBP). 2.8. Transienttransfection,celllysisandluciferaseassay
TransfectionofcellswasperformedbyusingtheFuGENE®HD transfectionreagent(Promega,Mannheim,Germany)accordingto themanufacturer’sinstructions.
For the luciferasereporter assay,HCT116 cells wereseeded intoa 24-wellplate(62,500cells perwell) ina total volumeof 0.5ml/wellofcellculturemediumandculturedovernight.Cells werethentransfectedwithFuGENE®HDtransfectionreagentusing atotalof0.5gofplasmidDNA(transfectionmixtureperwell: 0.05gofpromoterreporterplasmid,0.45gofexpression plas-mid(s),1.25lFuGENE® HD transfectionreagentand25lcell culturemediumwithoutFCS).
To measure luciferase activity,cells werecollected at given time points after transfection by lysing in cell culture lysis reagent(Promega)andmeasuredwiththeLuciferaseAssaySystem (Promega)followingthemanufacturer’srecommendations.
For Western blot analysis, cell lysates were centrifuged at 13,000×gtoremovecelldebris.Theproteincontentwas deter-minedwiththeBradfordmethodusingtheBioRadproteinassay reagent(BioRad,Munich,Germany).Proteinextractswere imme-diatelyusedforWesternblotanalysisorstoredat−20◦C.
2.9. SDSpolyacrylamidegelelectrophoresisandWesternblot analysis
Proteins were separated on a 12.5% sodium dodecylsulfate-polyacrylamidegelandtransferredontoapolyvinylidene difluo-ridemembrane (PVDF) bytankblotting using a transfer buffer containing20mMTris–HCl,pH8.3and150mMglycine.The mem-brane was blocked with 5% dry milk in PBS containing 0.05% Tween-20for1hatroomtemperatureandthenincubatedwith thespecificantibody,whichwasdilutedinPBSwith0.05% Tween-20containing1%drymilkpowderfor1hatroomtemperatureor overnightat4◦C.ThemembranewaswashedwithPBSTween-20 containing 1% skimmed milk (2× 10min), before being incu-batedwithaperoxidase-coupledsecondaryantibody(anti-rabbit 1:30,000,anti-mouse1:10,000oranti-goat1:5000)for1hatroom temperature.ThemembranewaswashedagaininPBSTween-20 (2×10min).Signalsweredevelopedandvisualizedbythe Lumi-lightsystemfromRocheDiagnostic(Mannheim,Germany). 2.10. Immunofluorescenceanalysis
ForimmunofluorescenceanalysisHCT116cellswereseededin 6-wellplatesoncoverslips(300,000cellsperwell)andcultured overnight.CellswerethentransfectedwithFuGENE® HD trans-fectionreagentusingatotalof2.5gofplasmidDNAperwellas indicated.Afterwards,cellswereincubatedfor24h. Immunofluo-rescenceanalysiswasperformedasdescribed(Faustetal.,2002). FortheidentificationoftransfectedFLAG-C/EBP␦,themouse mono-clonalantibodycloneM2(1:600)againsttheFLAG-tagwasused. 2.11. Co-immunoprecipitation
Forco-immunoprecipitationexperiments,HCT116 cellswere seededin100mmcellculturedishesandculturedovernight.Cells were then transfected with FuGENE® HD transfection reagent
Fig.1.C/EBP␦containstwoputativeCK2phosphorylationsiteswithhigh conserva-tion.(A)Aminoacid(aa)sequenceofhumanC/EBP␦proteinandlocalizationofthe twoputativephosphorylationsitesforCK2(boxes,arrows).Transactivationdomain: aa41–108(Jietal.,2003;Svotelisetal.,2005),basicregion:aa195–222andleucine zipper:aa226–254[www.uniprot.org].(B)Analysisofconservationamongdifferent specieswithrespecttotheputativephosphoacceptorsites(boxes).
using a total of 8g of plasmid DNA (4g FLAG-C/EBP␦ and 4gFLAG-C/EBP)andculturedforadditional20h.Proteinswere extractedwithRIPAbuffer(50mMTris–HCl,pH8.0,150mMNaCl, 0.5%sodiumdesoxycholate,1%TritonX-100,0.1%sodium dode-cylsulfate (SDS)) and 2mg of total protein were subjected to immunoprecipitation.
For co-immunoprecipitation of CK2, nuclear proteins were extracted.Therefore,cellswereresuspendedinbufferA(10mM HEPES, pH 7.9, 1.5mM MgCl2, 10mM KCl, 0.5mM DTT,
CompleteTM)andincubatedonicefor20min.Aftercentrifugation
thesupernatantcontainingcytoplasmicproteinswasremovedand thepelletwasresolvedinbufferC(20mMHEPES,pH7.9,1.5mM MgCl2,0.2mMEDTA,0.5mMDTT,CompleteTM).Nuclearproteins
2.12. Stainingofphosphoproteins
Detection of phosphoproteins was accomplished with the Pro-Q® Diamond Phosphoprotein Blot Stain Kit (P33356) from MolecularProbes(ThermoFisherScientific,USA)accordingtothe manufacturer’sinstructions. Briefly, proteins werefixed onthe PVDFmembrane,washed,stainedwithPro-Q®DiamondBlotStain Reagent and destained.After drying theblot, fluorescencewas detectedwiththeTyphoon-Trioimagingsystem(GE-Healthcare) usingthe580-nmband-passfilter(580BP30)incombinationwith theImageQuantTLsoftware7.0(GE-Healthcare).
Afterstainingofphosphoproteins,totalproteinswerestained withPonceauSsolution(0.2%(w/v)PonceauS, 15%(v/v)acetic acid,40%(v/v)methanol).Afterwashing,theblotwasblockedand incubatedwithantibodiesasdescribedin2.9.
2.13. Statistics
MicrosoftExcel2010softwarewasusedtoanalysethedata. Results were expressed as arithmetic mean±SEM. Differences betweentheexperimentalgroupswereanalysedusingStudent’s
t-test(two-tail,unpaired),statisticalsignificantdifferenceswere shownasfollows:***p<0.001,**p<0.01or*p<0.05.
3. Results
TheC/EBPtranscriptionfactorfamilyofproteinsisknowntobe modifiedbypost-translationalmodificationswhichareimportant mechanismstoregulatethem.C/EBP␣,and␦arephosphorylated bydifferentkinasessuchasp38kinaseorGSK3-kinase(Terragni etal., 2011;Galibert et al.,2001).In1996 Osadareportedthat C/EBP␦wasphosphorylatedbyproteinkinaseCK2.A phosphor-ylationsitewas,however,notidentifiedinthisstudy(Osadaetal., 1996).WestartedaninsilicoanalysisofC/EBP␦forthepresence ofCK2phosphorylationsitesonthepolypeptidechainofhuman C/EBP␦. Asshown inFig.1Awe foundtwoputativeCK2 phos-phorylationsitesonthepolypeptidechainofC/EBP␦(twoboxes). Bothsites fulfiltherequirementsfor a minimalCK2consensus sequence,whichrequiresanacidicresidueatposition+3 down-streamfromthephosphoacceptorsite(S/T-x-x-D/E/pS/pY)(Salvi etal.,2009).Furtheranalysisrevealedthatbothsitesarehighly conservedamongdifferentspecies(Fig.1B).Inthenextstepwe
Fig.2.C/EBP␦isasubstrateofCK2andisphosphorylatedatpositionserine57onthepolypeptidechain.(A)PhosphorylationofGST-C/EBP␦byCK2invitrokinaseassay.The recombinantGST-C/EBP␦wasincubatedwithorwithoutrecombinantCK2holoenzymeinthepresenceofkinasebufferand2Ciof[32P]␥ATPfor15minat37◦C.Thesamples
Fig.3. SubcellularlocalizationofC/EBP␦isunaffectedafterCK2inhibitionormutationofSer57.(AandB)ImmunofluorescenceanalysisofHCT116cellstransfectedwith FLAG-C/EBP␦WT.SixhoursaftertransfectioncellsweretreatedwithDMSO,10MCX-4945or50Mquinalizarinandincubatedforadditional18h.(CandD)Immunofluorescence
analysisofHCT116cellstransfectedfor24hwithFLAG-C/EBP␦WT,-S57Aor-S57D.FLAG-C/EBP␦wasdetectedwiththeFLAGantibodyM2andlabelledbyAlexaFluor
488-coupledsecondaryantibody(green).ThenucleuswasstainedwithDAPI.Onerepresentativeofatleast3experimentsisshownhere.BandDarehighermagnificationsofA andB.(Forinterpretationofthereferencestocolourinthisfigurelegend,thereaderisreferredtothewebversionofthisarticle.)
attemptedtoprovethatC/EBP␦isasubstrateforproteinkinase CK2.Therefore,weexpressedaGST-taggedC/EBP␦proteinin bacte-ria,purifiedtheproteinandincubateditwithCK2and[32P]␥ATP.
AscontrolsweusedtheGST-tagalonewith[32P]␥ATP,CK2with
[32P]␥ATPandC/EBP␦withoutCK2butwith[32P]␥ATP.Samples
wereanalysedonanSDSpolyacrylamidegel.AsshowninFig.2A lowerpanel,wefoundastronglyphosphorylatedproteinbandfor GST-taggedC/EBP␦whichcannotbeobservedintheabsenceofCK2 andalsointheCK2control.TheGST-tagitselfshowsno phosphor-ylation.Theotherstrongphosphorylatedprotein-bandbelongsto CK2which isautophosphorylated.Theupperpanelshowsthe Coomassieblue stainedgelwhich belongs tothe autoradiogra-physhowninthelowerpanel.Thestainedgelshowstheproteins presentinthedifferentsamplesanddepictsthatsimilaramounts ofproteinwereusedfortheassay.Thus,weshowthatC/EBP␦is indeedasubstrateforproteinkinaseCK2.
Inorder todefinewhich ofthetwo putativesitesidentified bytheinsilicoanalysisorwhetherbothsitesarephosphorylated byCK2wegeneratedalaninemutantsatposition57or159and a mutantwhere bothsites weremutatedtoalanine.Wild-type C/EBP␦ and thephosphorylation mutantswere phosphorylated byCK2 and [32P]␥ATP and analysed onan SDSpolyacrylamide
gel.AsshowninFig.2Bwild-typeC/EBP␦(WT)and themutant T159AwerephosphorylatedbyCK2whereastherewasnoband forthephosphorylatedS57AmutantofC/EBP␦.Therewasalsono phosphorylatedproteinbandforthedoublemutant(DM).Since wild-typeandmutantsofC/EBP␦containaGST-tagandthereisno phosphorylatedproteinband,theseresultsalsoexcludethatthe GST-tagisphosphorylatedbyCK2.WealsoconstructedaC/EBP␦
mutantwhereserine57hadbeenreplacedbyglutamicacidinorder tomimicanegativechargeatposition57.Thereisafaintbandfor thismutantproteinafterphosphorylationwith[32P]␥ATPwhich
mightindicatethegenerationofanewweakphosphorylationsite (Fig.2B).TheupperpanelshowsthecorrespondingCoomassieblue stainedgel,demonstratingthatweusedequalamountsofC/EBP␦ protein for the phosphorylationreactions. These data provided strongevidencethatserine57isthemajorCK2phosphorylation site.
InordertosupportthisconclusionwephosphorylatedC/EBP␦ withCK2intheabsenceorpresenceofincreasingconcentrations ofapeptidewiththesequenceAMTDDESAIDFSACrepresentingthe sequencearoundserine57.AsshowninFig.2Cinthepresenceof increasingamountsofthispeptidewefoundareduction inthe phosphorylationofC/EBP␦comparedtothecontrolwhichfurther supportstheideathatserine57isthemajorCK2phosphorylation site.TheupperpartofFig.2CshowstheCoomassiebluestained gelcorrespondingtotheautoradiograminthelowerpartofFig.2C showingthatweusedthesameamountofGST-C/EBP␦proteinfor thephosphorylationexperiments.
(lowerpanel).TheseresultsdemonstratedthatC/EBP␦isa phos-phoproteindetectablewiththePhosphostain.Inthenextstepwe repeatedtheexperimentbutcellswereadditionallyincubatedwith theCK2inhibitors quinalizarin(Cozza et al.,2009)or CX-4945 (Siddiqui-Jainetal.,2010).Fig.2Eshowsareductionoftheintensity oftheC/EBP␦proteinbandlabelledwithPhosphostaininthe pres-enceofeitherquinalizarinorCX-4945(middlepanel)supporting theideathatitisphosphorylatedbyendogenousCK2.After normal-izationofthephosphorylatedbands(Phosphostain,middlepanel) fromthreeindependentexperimentstotherespectivetotalC/EBP␦ (lowerpanel,WB)thephosphorylationwasreducedto84%bythe quinalizarintreatmentandto90%bytheCX-4945treatment.
ItisknownforseveralCK2substratesthattheirsub-cellular localizationisregulatedbyCK2phosphorylation(Hübner etal., 1997;JansandJans,1994;Miyataetal.,2011;Wilsonetal.,2002). Therefore,wewantedtoknowwhetherCK2phosphorylationof C/EBP␦ would influence the subcellular localization of C/EBP␦. CellsweretransfectedwithFLAG-C/EBP␦WTandthentreatedwith
quinalizarin(Q)orCX-4945orthesolventcontrolDMSOfor18h. TheimmunofluorescenceanalysisrevealedthatC/EBP␦wasfound exclusivelyinthenucleus(Fig.3AandB).Asafurtherprovewe transfectedeither FLAG-C/EBP␦WT orFLAG-C/EBP␦S57A or
FLAG-C/EBP␦557D into HCT116 cells and analysed the localization of
C/EBP␦byimmunofluorescence.AsshowninFig.3CandD, wild-typeandthetwomutantC/EBP␦swerefoundexclusivelyinthe nucleus. C/EBP␦ is evenly distributed over the nucleus with a slightlymoreperinuclearintensity.Fromthesetwoexperiments weconcludethatCK2phosphorylationofC/EBP␦hasnoinfluence onthenuclearlocalizationofC/EBP␦.
C/EBP␦forms stablecomplexeswithanothermemberofthe C/EBPfamilynamelyC/EBP(NewmanandKeating,2003).Inorder toanalysewhetherphosphorylationofC/EBP␦byCK2might influ-encethisinteractionweincubatedhighlypurifiedGST-C/EBP␦WTor
thetwomutantsC/EBP␦S57AandC/EBP␦S57Dwith[35S]methionine
labelledinvitrotranslatedC/EBP.Complexeswereprecipitated withGSH-sepharoseandthen analysedonanSDS polyacrylam-idegel.Fig.4A upperpartshows a Coomassieblue stainedgel demonstratingthatweusedequalamountsofGST-C/EBP␦forthe pull-downassay.ThelowerpartshowstheboundC/EBP demon-strating that equal amounts of C/EBPwere bound toC/EBP␦. Thus,CK2phosphorylationofC/EBP␦doesnotinfluencebinding toC/EBP.
In order to confirm these results under in vivo conditions we transfected FLAG-C/EBP␦WT or FLAG-C/EBP␦S57A or
FLAG-C/EBP␦S57DtogetherwithFLAG-C/EBPintoHCT116cells.C/EBP␦
wasprecipitatedwithaspecificantibodyandcomplexeswere ana-lysedonanSDSpolyacrylamidegel.Proteinbandswerevisualized withanantibodyagainsttheFLAG-tag. Asshown inFig.4Bwe foundequalamountsofC/EBPintheco-immunoprecipitatesfor C/EBP␦WT aswellasfortheCK2phosphorylationmutants.This
resultconfirmstheobservationshowninFig.4AthatCK2 phos-phorylationdoesnotinfluencecomplexformationbetweenC/EBP␦ andC/EBP.
An increasing number of proteins bind to CK2 and these protein–proteininteractions provideanotherlevel ofregulation forCK2inthecell(MontenarhandGötz,2013).Therefore,inthe nextstepweaskedwhetherC/EBP␦mightbindtoCK2.We incu-batedGST-C/EBP␦ortheGST-tagalonewiththeCK2holoenzyme. As shown in Fig. 5A GST-C/EBP␦ co-precipitated withthe CK2 holoenzyme.TherewasnobindingofCK2totheGST-tagorto GSH-agarose.InordertosupportthefindingaboutbindingofCK2 toC/EBP␦weattemptedtoco-immunoprecipitateCK2andC/EBP␦ fromHCT116cells.CellsweretransfectedwiththeFLAG-tagged C/EBP␦construct.24haftertransfectioncellswerelysedand FLAG-C/EBP␦wasprecipitatedfromthecellextractwithaC/EBP␦-specific antibody.TheprecipitatewasanalysedonanSDS-polyacrylamide
Fig.4.BindingofC/EBP␦toC/EBPisnotaffectedbyphosphorylationofC/EBP␦ byCK2.(A)GSTpull-downassay.BacteriallyexpressedandpurifiedGST-tagged C/EBP␦WT andmutantproteinswerebound toglutathione(GSH)agaroseand
thenincubatedwithinvitrotranslatedHis-C/EBPfor1h.Proteinswere sepa-ratedona12.5%SDS-polyacrylamidegelfollowedbystainingwithCoomassie blue(upperpanel).BindingofHis-tagged-C/EBPwasdetectedby autoradiogra-phy(lowerpanel).Onerepresentativeofatleast3experimentsisshownhere. (B)Co-immunoprecipitationofFLAG-C/EBP␦andFLAG-C/EBP.HCT116cellswere transfectedwith FLAG-C/EBP␦andFLAG-C/EBP. Lysateswereincubatedwith C/EBP␦specificantibodyM-17.Elutedproteinsfromimmunoprecipitateswere detectedwithananti-FLAGantibodyM2.Input:1%ofcellextractusedinthe experiment;CE:cellextractafterIP;C:pre-precipitate;IP:immunoprecipitate;S: sepharosecontrol;AB:antibodycontrol.
Fig.5. ProteinkinaseCK2bindstoC/EBP␦invitroandinvivo.(A)GST-C/EBP␦WT
Fig. 6.Mutation of CK2 phosphorylation site Ser57 reduces transactivation capabilityofC/EBP␦.(A)Schematicrepresentationofthereportergeneconstruct forthemurinePPAR␥2promoterwithdepictedC/EBPbindingsiteandTATA-Box. LuciferasereportergeneassayswerecarriedoutinHCT116cellsaftertransient co-transfectionofexpressionplasmidsFLAG-C/EBP␦WTalone(B)orFLAG-C/EBP␦WT,
FLAG-CK2␣andFLAG-CK2(C)orFLAG-C/EBP␦WT,S57AandS57D(D)togetherwith
gelfollowedby WesternblotwithCK2␣and CKspecific anti-bodies.Asacontrolprecipitate FLAG-C/EBP␦wasdetectedwith aFLAG-antibody.AsshowninFig.5BbothCK2␣andCK2were co-immunoprecipitatedwithC/EBP␦confirmingthedataobtained withthepull-downexperimentshowninFig.5A.Fromtheseresults weconcludethatC/EBP␦bindstoCK2.
Itiswellknownthatmanytranscriptionfactorsareregulated by phosphorylation(Bohmann, 1990).Moreover,protein kinase CK2phosphorylationof transcription factors isknown to stim-ulateor toabrogate transcriptionalactivity(Meggioand Pinna, 2003).Therefore, weanalysed whetherCK2phosphorylationof C/EBP␦ mightalsoinfluence itstransactivationfunctions.It has been shown that thePPAR␥2 promoter is regulatedby C/EBP␦ (Mooreet al.,2003)and thereforethis promoter wasclonedin frontofaluciferasereporterconstruct(pGL4-PPAR␥2,Fig.6A).This reporterconstructwastransfectedintoHCT116cellstogetherwith theFLAG-C/EBP␦WTconstruct.Asacontrolweusedthe
correspond-ingreporterconstructwithoutthePPAR␥2promoter(pGL4basic vector).Fig.6BshowsthatthePPAR␥2promoterbutnotthebasic vectorconstructwasactivatedbyC/EBP␦.
Next,wetestedwhetherCK2mightmodulateC/EBP␦ transcrip-tionalactivity.Therefore,FLAG-C/EBP␦wastransfectedeitheralone ortogetherwithCK2␣andCK2andthePPAR␥2reporter con-struct.TheresultofthisreporterassayisshowninFig.6C.C/EBP␦ stimulatedtranscriptionofthePPAR␥2promoter.Inthepresence ofCK2therewasanevenhigherleveloftheluciferaseactivity. TheCK2holoenzymealonehadnoeffectonthereporterconstruct. Thus,theseresultsindicatethat CK2phosphorylationofC/EBP␦ increasesthetranscriptionalactivityofC/EBP␦.
Tofurthervalidatethisfindingweusedthephosphorylation mutantsC/EBP␦S57AandC/EBP␦S57DtoanalyseaninfluenceofCK2
phosphorylationontheC/EBP␦transactivation.AsshowninFig.6D wefoundanincreaseintheluciferaseactivityforFLAG-C/EBP␦WT
inthepresenceofthePPAR␥2promoter comparedtothebasic vectorconstruct.ThephosphorylationmutantC/EBP␦S57Ashowed
areducedtranscriptionalactivitycomparedtowild-typeC/EBP␦. ThephosphorylationmutantC/EBP␦S57DwhichmimicsaCK2
phos-phorylationbyproviding anegativechargeshowedanelevated luciferaseactivitycomparedtothealaninemutantandtowild-type C/EBP␦.Theseresultsconfirmthedataobtainedwithan overex-pressionoftheCK2holoenzyme(Fig.6C).
4. Discussion
The transcription factor C/EBP␦ is a member of the family of CCAAT/enhancer binding protein transcription factors which modulatemanybiological processessuchascelldifferentiation, proliferation, mobility, cell death, inflammationand metastasis (BalamuruganandSterneck,2013).Likeallothermembersofthis family of transcription factors C/EBP␦ contains a basic region-leucine zipper DNA binding domain in the C-terminus and an N-terminaltransactivationdomain.C/EBPtranscriptionfactorscan bindtoDNAonlyasdimersbyvirtueoftheleucinezipperdomain. Bindingpartners areallmembersoftheC/EBPfamily including C/EBP whichis alsoknownasCHOPand alsoATF4(Grigoryan
pGL4-PPAR␥2promoterconstruct(B-D)oremptyreporterplasmid(pGL4basic vector)asnegativecontrol(B).Theemptyexpressionplasmidp3xFLAG-CMV-7.1 servedasvectorcontrolineachcase.Relativepromoteractivitiesmeasured24h aftertransienttransfectionarepresentedhere.PPAR␥2promoteractivitymeasured afterco-transfectionwithC/EBP␦WTwassetto100%.Meansandstandard
devia-tionsofthreeindependentexperimentsareshown.*p<0.05;**p<0.01;***p<0.001. Westernblotanalysesofco-transfectedHCT116cellswereanalysedinparallel andillustrateexpressionofFLAG-C/EBP␦WT(B),FLAG-C/EBP␦WT,FLAG-CK2␣and
FLAG-CK2(C)andFLAG-C/EBP␦WT,S57AandS57D(D).Anti-FLAGantibodywasused
etal.,2009).Both,CHOPand ATF4werefoundtobesubstrates forproteinkinaseCK2(UbedaandHabener,2003;Ampofoetal., 2013)andtheinteractionbetweenCK2andbothtranscription fac-torsplaysarole intheERstressresponseregulation(Schneider etal.,2012).InthepresentstudyweidentifiedC/EBP␦asasubstrate forproteinkinaseCK2byaninsilicoanalysiswiththeCK2 phos-phoacceptorsitewiththeconsensussequenceST-x-x-D/E/pS/pY (Salvietal.,2009).Theresultofthisinsilicoanalysiswasconfirmed byaninvitrophosphorylationofC/EBP␦withCK2.Since thein silicoanalysisrevealedtwoputativeCK2phosphorylationsiteswe createdpointmutationstomapdownthemajorphosphorylation sitetoserine57.Serine57islocatedwithinanacidicenvironment whichfitsperfectlytotherequirementsforCK2phosphorylation. TheotherputativeCK2phosphorylationsiteissurroundedbya proline-richregionwhichisanegativeselectionforCK2 phosphor-ylationsites(Marinetal.,1992).Thesequencearoundserine57is highlyconservedamongdifferentspecieswhichhintstoa func-tionalrelevantregion.BystainingC/EBP␦fromHCT116cellswith Phosphostainwehaveshownthatitisaphosphoprotein.The inhi-bitionofCK2withquinalizarinorwithCX-4945resultedinslightly butreproduciblereducedstaining,implyingthattheCK2isone amongdifferentotherkinasesthatphosphorylateC/EBP␦.
SinceC/EBPs formheterodimers withothermembers ofthe C/EBPfamilyofproteinsitwasanobviousquestionwhetherthe CK2phosphorylationmightinterferewiththeheterodimerization. However,aspresentedhere,CK2phosphorylationdoesnot influ-enceheterodimerizationwithC/EBP.Theleucinezipperregion, whichisimplicatedinbindingoftheotherC/EBPs,islocatedin theC-terminusofC/EBPs.TheCK2phosphorylationsiteislocated intheN-terminuswhichmightsomehowexcludeadirect influ-enceoftheCK2phosphorylationontheheterodimerizationandis consistentwiththepresentresults.
Thenuclearlocalizationofmanyproteinsisregulatedby phos-phorylation (Anet al., 2010; Lam et al., 1999; Li et al., 1997; Krempleretal.,2005)andinparticularbyCK2phosphorylation (Kawamura et al.,1981; Xiaoet al.,1998; Hübner etal., 1997; JansandJans,1994;Wilsonetal.,2002).InsomecasestheCK2 phosphorylationsitesareinclosevicinityofthenuclear localiza-tionsignal(NLS).Asshownherebyusinginhibitorsoftheprotein kinaseactivityofCK2orbyusingCK2phosphomutantsofC/EBP␦ itturnedoutthatCK2phosphorylationofC/EBP␦doesnot influ-enceitssubcellularlocalization. Wefoundstrongevidencethat besidesbeingasubstrateforCK2,C/EBP␦alsobindstoCK2.This isaninterestingobservationbecauseinadditiontothe transcrip-tionalfunctionC/EBP␦hasatransportfunctionfortheDNArepair proteinFANCD2intothenucleus(Wangetal.,2010).Thus,itmight bepossiblethatC/EBP␦targetsCK2inthenucleusto phosphory-lateotherC/EBPfamilymemberssuchasCHOPorthetranscription factorandbindingpartnerofC/EBP␦ATF4.
Proteinphosphorylationisaversatiletoolfortheregulationof thetranscription factoractivity.It hasbeenshown thatprotein phosphorylationoftranscriptionfactorscaneitherenhanceDNA bindingactivityandtranscriptionalefficacy(Molkentinetal.,1996) ordisruptDNAbindingandtranscriptionfactoractivity(Ramsay etal.,1995;Grigoryanetal.,2009).ThemajorCK2phosphorylation siteserine57islocatedinthetransactivationdomainofC/EBP␦. Here,weshowthatco-expressionofC/EBP␦withCK2enhances thetranscriptionfactoractivityofC/EBP␦,concerningthePPAR ␥2-promoter.AnelevatedphosphorylationofC/EBP␦mightalterthe conformationofthepolypeptidechaininordertoenhanceDNA bindingortherecruitmentofotherproteinsofthetranscriptional machinery.AlternativelyphosphorylationofC/EBP␦mightdirectly helptorecruitothercellularco-activatorsorco-factorstoenhance transcription.
Insummary,wehaveidentifiedC/EBP␦asanewsubstratefor CK2;we localized themajorphosphorylation site toserine 57.
CK2phosphorylationofC/EBP␦didneitherinfluenceits subcellu-larlocalizationnortheinteractionwithC/EBP.However,weshow thatthetranscriptionfactoractivityofC/EBP␦isregulatedbyCK2 phosphorylation.Inadditionwe demonstratethatCK2bindsto C/EBP␦whichmayreflectasubstrate/enzymeinteractionorwhich mightindicatetargetingofCK2toothermembersofthe transcrip-tionmachinery.
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