THEJOURNALOFBIOLOGICALCHEMISTRY VOL.282,NO.30,pp.21598–21608,July27,2007 ©2007byTheAmericanSocietyforBiochemistryandMolecularBiology,Inc. PrintedintheU.S.A. Hsp27 Regulates Akt Activation and Polymorphonuclear Leukocyte Apoptosis by Scaffolding MK2 to Akt Signal Complex* Receivedforpublication,December11,2006,andinrevisedform,May15,2007 Published,JBCPapersinPress,May17,2007,DOI10.1074/jbc.M611316200 RuiWu‡1,HinaKausar‡1,PaulJohnson§,DiegoE.Montoya-Durango§,MichaelMerchant‡,andMadhaviJ.Rane‡§2 FromtheDepartmentsof ‡Medicineand§BiochemistryandMolecularBiology,UniversityofLouisville,Louisville,Kentucky40202 WehaveshownpreviouslythatAktexistsinasignalcom- cytes (PMN)3 take part in host defense mechanisms against plex with p38 MAPK, MAPK-activated protein kinase-2 infection and inflammatory diseases. Inappropriate termina- (MK2),andheatshockprotein27(Hsp27)andMK2phospho- tion of PMN activation or failure to remove apoptotic PMNs rylates Akt on Ser-473. Additionally, dissociation of Hsp27 resultsininflammation.Thisapoptoticprocesshasbeensug- from Akt, prior to Akt activation, induced polymorphonu- gestedtorepresentaninvivomechanismlimitingoxidant-in- clearleukocyte(PMN)apoptosis.However,theroleofHsp27 ducedtissueinjurycausedbyPMNsatthesitesofinflamma- in regulating Akt activation was not examined. This study tion. Although PMNs are constitutively committed to testedthehypothesisthatHsp27regulatesAktactivationand apoptosisfromthetimetheyentercirculation,therateofapo- promotes cell survival by scaffolding MK2 to the Akt signal ptosisisnotfixed.Wereportedthatinterleukin-8,granulocyte- complex.HereweshowthatlossofAkt/Hsp27interactionby macrophage colony-stimulating factor, LTB , and bacterial 4 anti-Hsp27 antibody treatment resulted in loss of Akt/MK2 lipopolysaccharide (LPS) delay constitutive PMN apoptosis interaction, loss of Akt-Ser-473 phosphorylation, and throughtheactivationoftheserine/threoninekinaseAkt(1,2). induced PMN apoptosis. Transfection of myristoylated Akt We demonstrated that p38 mitogen-activated protein kinase (AktCA) in HK-11 cells induced Akt-Ser-473 phosphoryl- (MAPK)activityisrequiredforAktphosphorylationandacti- ation, activation, and Hsp27-Ser-82 phosphorylation. Co- vation(3).Additionally,weshowedthatAktexistsinasignaling transfection of AktCA with Hsp27 short interfering RNA, module with p38 MAPK, MAPK-activated protein kinase-2 but not scrambled short interfering RNA, silenced Hsp27 (MK2),andheatshockprotein27(Hsp27)(3). expression, without altering Akt expression in HK-11 cells. Heatshockproteinsrepresentagroupofchaperoneproteins Silencing Hsp27 expression inhibited Akt/MK2 interaction, thatprotectthecellsagainstavarietyofstresses.Besidesbeing inhibitedAktphosphorylationandAktactivation,andinduced involved in functioning as a chaperone, Hsp27 has also been HK-11celldeath.Deletionmutagenesisstudiesidentifiedacidic shown to regulate stability of the cytoskeleton, cell motility linkerregion(aminoacids117–128)onAktasanHsp27binding (4–7), and apoptosis (8–13). When overexpressed in tumor region.Deletionofaminoacids117–128onAktresultedinloss cells, Hsp27 increases their tumorigenicity by overexpressing ofitsinteractionwithHsp27andMK2butnotwithHsp90as MMP-9 expression and down-regulating Src tyrosine kinase demonstrated by immunoprecipitation and glutathione Yes expression (14–16) and protects against apoptotic cell S-transferasepulldownstudies.Co-transfectionstudiesdemon- death triggered by various stimuli, including cytotoxic drugs stratedthatconstitutivelyactiveMK2(MK2EE)phosphorylated and ligation of the Fas/Apo-1/CD95 death receptor (17–19). Aktwt (wild type) on Ser-473 but failed to phosphorylate MiceoverexpressingHsp27wereprotectedfromlethalische- Akt mutantintransfixedcells.Thesestudiescollectively mia/reperfusion injury compared with their negative litter- (cid:1)117–128 define a novel role of Hsp27 in regulating Akt activation and mates(20).PossiblemechanismsofHsp27anti-apoptoticactiv- cellularapoptosisbymediatinginteractionbetweenAktandits ity are proposed to result from its activity as a molecular upstreamactivatorMK2. chaperone. Hsp27 binds to and inactivates the pro-apoptotic moleculesSmac,caspase3,caspase9,andcytochromec(21– 25). Hsp27-mediated suppression of Bid translocation to the mitochondria correlates with an inhibition of cytochrome c Apoptosisorprogrammedcelldeathisaseriesofeventsina release (25). Hsp27 has also been shown to promote survival cellthatleadstoitsdeath.Humanpolymorphonuclearleuko- 3Theabbreviationsusedare:PMN,polymorphonuclearleukocyte;Hsp27, *ThisworkwassupportedbyAmericanHeartAssociation-ScientistDevelop- heat shock protein 27; PDK1, phosphoinositide-dependent kinase-1; mentGrant0335278N(toM.J.R.)andNationalInstitutesofHealthGrant PDK2, phosphoinositide-dependent kinase-2; MAPK, mitogen-activated 1R56AI059165-01A2(toM.J.R.).Thecostsofpublicationofthisarticle proteinkinase;MK2,MAPK-activatedproteinkinase-2;HK-11cells,human weredefrayedinpartbythepaymentofpagecharges.Thisarticlemust renal proximal tubular cells; HEK-293, human embryonic kidney cells; thereforebeherebymarked“advertisement”inaccordancewith18U.S.C. AktCA,c-Myc-taggedmyristoylatedconstitutivelyactiveAkt;IEF,isoelec- Section1734solelytoindicatethisfact. tricfocusing;MES,4-morpholineethanesulfonicacid;PH,pleckstrinhomol- 1Bothauthorscontributedequallytothiswork. ogy;PMSF,phenylmethylsulfonylfluoride;fMLP,formylmethionylleucylphe- 2Towhomcorrespondenceshouldbeaddressed:UniversityofLouisville,570 nylalanine;MTT,3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium S.PrestonSt.,BaxterIBldg.South,102C,Louisville,KY40202.Tel.:502-852- bromide;FITC,fluoresceinisothiocyanate;siRNA,shortinterferingRNA;BisTris, 0014;Fax:502-852-4384;E-mail:[email protected]. 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol. This is an Open Access article under the CC BY license. 21598 JOURNALOFBIOLOGICALCHEMISTRY VOLUME282•NUMBER30•JULY27,2007 RegulationofAktActivationbyHsp27 pathways by modulating IKK complex stability and activity. latedconstitutivelyactiveAkt)constructswereobtainedfrom Parcellieretal.(26)demonstratedthatHsp27mediatesNF-KB UpstateBiotechnology,Inc.pUseAktwtwasshuttledintoGST activationandcellsurvivalbypromotingtheproteasomaldeg- vectorpGEX-4T-2asdescribedpreviously(8).pUseAkt (cid:1)117–128 radationofpolyubiquitinatedIKB.PhosphorylatedHsp27has wasshuttledintoGSTvectorpGEX-4T-2asdescribedunder been shown to bind an adaptor protein Daxx and to inhibit “Materials and Methods.” pcDNA3.1-Hsp27-wt was shut- Fas-mediatedapoptosis(27).Additionally,phosphorylationof tled into pGEX-5X-2 (GE Healthcare) vector as described Hsp27hasbeenshowntoberequiredforpropermaintenance previously (8). Constitutively active MK2EE cDNA con- ofcelladhesionandinhibitionofrenalepithelialcellapoptosis structwasobtainedfromtheGaestellaboratory(Hannover, (28).Furthermore,Shethetal.(13)showedthatintroductionof Germany). recombinantHsp27causeddelayofPMNapoptosis;however, IsolationofPMNsandCultureConditions—PMNswereiso- mechanismsregulatingthisdelayofPMNapoptosiswerenot lated from venous blood obtained from healthy volunteers as determined. described previously (3, 8). PMN preparations routinely con- We recently demonstrated direct protein/protein interac- tained(cid:2)95%PMNs,asdeterminedbymorphology,andwere tion between Akt/Hsp27 (3, 8). The physical association of (cid:2)99% viable by trypan blue dye exclusion. PMNs were sus- Hsp27 with Akt is a critical determinant of PMN survival, as pended in RPMI 1640 medium supplemented with 10% fetal removalofHsp27fromtheAktsignalmodulepreventedAkt calfserum,L-glutamine,penicillin,andstreptomycinandincu- phosphorylation and activation and resulted in accelerated batedfortheindicatedtimesat37°Cin5%CO . 2 PMNapoptosissuggestinganimportantroleforHsp27inreg- Generation of pUseAkt Mutant by Site-directed (cid:1)117–128 ulatingAktactivity(8).MK2hasbeenshowntobindandphos- Mutagenesis—In-framedeletionofaminoacids117–128from phorylate Hsp27 (29), and MK2 is PDK2 for Akt in human pUseAktwtcDNAconstructwascarriedoutusingtheTrans- PMNs(3).Recently,Zhengetal.(30)demonstratedthatMK2is former site-directed mutagenesis kit from BD Biosciences requiredforp38MAPKandHsp27interaction;however,asso- according to the manufacturer’s instructions. The in-frame ciationofHsp27andAktwasnotdependentonMK2.Hencewe deletionprimerwas5(cid:3)-AGGCAGGAAGAAGAGTCAGGGG- hypothesizedthatHsp27regulatesAktactivationandapopto- CTGAAGAG-3(cid:3), and the selection primer for pUseAktwt sisbyscaffoldingMK2totheAktsignalcomplex. (mutatingtheKpnIsite)was5(cid:3)-GTTAAGCTTGAATCCGA- Akt contains an N-terminal pleckstrin homology (PH) GCTCG-3(cid:3). Cloning and mutation were confirmed by DNA domain and a catalytic kinase domain (residues 1–116 and sequencing. 148–411 respectively) linked by a highly acidic linker region SubcloningandPurificationofGSTBeadsandRecombinant (residues 117–147). The C-terminal tail region lies between Proteins—Akt wasexcisedfrompUSEAkt with (cid:1)117–128 (cid:1)117–128 residues412and480.Phosphoinositidesareknowntobindthe restrictionenzymesBamHI/PmeIandligatedintoBamHI/SmaI PHdomainofAktandrecruitittotheplasmamembranefor sitesofpGEX-4T-2(GEHealthcare)vector.GenerationofGST- fullactivationbyPDK1andPDK2(28,31–33).Inthepresent Hsp27pGEX-5X-2, GST-AktpGEX-4T-2, was described previ- study we show that amino acids 117–128 within the acidic ously (8). GST-pGEX-4T-2, GST-Hsp27pGEX-5X-2, GST-Akt- linkerregionofAktarerequiredforinteractionwithHsp27.An pGEX-4T-2, and GST-Akt pGEX-4T-2 cDNAs were (cid:1)117–128 in-frame deletion mutant of Akt (Akt ), lacking the transformed into Escherichia coli BL21(DE3)PlysS, and the (cid:1)117–128 Hsp27bindingregion,interactswithHsp90butnotwithHsp27 expressionandpurificationofGST,GST-Hsp27,GST-Aktwt,and andMK2.DisruptionofAkt/Hsp27interactionpreventsMK2 GST-Akt fusion proteins were performed as described (cid:1)117–128 association with Akt, resulting in loss of MK2-mediated Akt previously (8). pRSETA vector was digested with restriction Ser-473phosphorylation,activation,andinductionofapopto- enzymeEcoRIfollowedbygenerationofabluntendbytreat- sis. These studies demonstrate for the first time that Hsp27 mentwithKlenowenzymefollowedbydigestionwithBamHI regulates Akt activation and cellular apoptosis by scaffolding restrictionenzyme.ThisvectorwasthenligatedtoeitherAktwt MK2totheAktsignalcomplex. or Akt , which were excised from pUSEAktwt or (cid:1)117–128 pUSEAkt with restriction enzymes BamHI/PmeI. All MATERIALSANDMETHODS (cid:1)117–128 positive clones were confirmed by DNA sequencing. Expres- Anti-PH domain Akt, anti-Akt, and anti-p38 antisera were sion of pRSET-Aktwt and pRSET-Akt plasmids was (cid:1)117–128 obtained from Cell Signaling Inc. (Beverly, MA). Anti-phos- carried out in BL21(DE3)pLysS chemically competent E.coli pho-Ser-473-AktwasobtainedfromSantaCruzBiotechnology cells,andproteinwaspurifiedusingtheProBondpurification (Santa Cruz, CA). Anti-MK2 antibody was obtained from system(Invitrogen). Sigma. Mouse isotype control antibody was obtained from Immunoblot Analysis—Immunoblotting procedures were Santa Cruz Biotechnology (Santa Cruz, CA). Recombinant performedasdescribedpreviously(3,8).Fifty(cid:1)gofproteinwas Hsp27 and anti-mouse Hsp27 were obtained from StressGen subjected to 10% SDS-PAGE and immunoblot analysis with Biotechnologies Corp. (Victoria, British Columbia, Canada). anti-pAktSer-473(1:1000,SantaCruzBiotechnology),anti-Akt ProteinA-Sepharoseandglutathione-Sepharosewereobtained (1:1000, Santa Cruz Biotechnology), anti-Hsp27 (StressGen), from Pharmingen. Histone H2B was obtained from Roche anti-Ser(P)-82Hsp27(CellSignaling),anti-c-Myc(CellSignal- Applied Science. Recombinant GST-Akt-(1–149), recombi- ing),anti-MK2(Sigma),andanti-Hsp90(SantaCruzBiotech- nant active MK2, and recombinant active catalytic protein nology)antisera(3,8). kinaseAwereobtainedfromUpstateBiotechnologyInc.(Lake Gel Filtration Chromatography—The system used for the Placid,NY).pUseAktwt(wildtype)andpUseAktCA(myristoy- size exclusion chromatography experiments consisted of a JULY27,2007•VOLUME282•NUMBER30 JOURNALOFBIOLOGICALCHEMISTRY 21599 RegulationofAktActivationbyHsp27 HiPrep26/60SephacrylS-300HRprepackedchromatography mMphenylmethylsulfonylfluoride,20mMNaF,1mMsodium columnconnectedtoanAKTApurifier10liquidchromatogra- pyrophosphate,1mMsodiumorthovanadate,and1%(v/v)Tri- physystem(AmershamBiosciences),equippedwithaFrac-900 tonX-100.AppropriateGSTbeads(10(cid:1)l)wereaddedtothe automated fraction collector, and controlled by the UNICORN lysatesandincubatedat4°Cfor1hwithshaking.Thebeads version4.00software(AmershamBiosciences).Priortochro- were washed three times with Krebs buffer, and 15 (cid:1)l of 2(cid:4) matography,thecolumnwasequilibratedin50mMTris-Cl,pH Laemmli buffer was added to each tube. The samples were 7.4, 1 mM EDTA, 150 mM NaCl, 1.5 mM MgCl , 5% glycerol, boiled for 3 min and then subjected to 10% SDS-PAGE. Pro- 2 0.5%TritonX-100.Forthechromatographicseparationofthe teinsweretransferredontonitrocelluloseandimmunoblotted samples, control and fMLP (0.3 (cid:1)M)-stimulated PMNs were withappropriateantibodies. harvestedandresuspendedinAktlysisbuffercontaining20mM GSTPulldownAssaywithRecombinantProteins—Appropri- Tris-HCl,pH7.4,150mMNaCl,1%(v/v)TritonX-100,0.5% ateGSTbeads(10(cid:1)l)wereaddedto50(cid:1)lofkinasebuffer(20 (v/v)NonidetP-40,1mMEDTA,1mMEGTA,20mMsodium mMHEPES,10mMMgCl ,10mMMnCl )containing50ngof orthovanadate,10(cid:1)Mp-nitrophenolphosphate,20mMNaF,5 appropriaterecombinant2protein.Thesam2 pleswereincubated mMPMSF,21(cid:1)g/mlaprotinin,and5(cid:1)g/mlleupeptin.Thecell at4°Cfor1hwithshaking.Thebeadswerewashedthreetimes lysatewasclearedbycentrifugationat14,000rpmfor15min. withKrebsbuffer,and15(cid:1)lof2(cid:4)Laemmlibufferwasaddedto Next,3–5mloftotalclearedcelllysatewasinjectedontothe each tube. The samples were boiled for 3 min and then sub- column with a manual injection through a 50-ml capacity jected to 10% SDS-PAGE and immunoblotting. Recombinant superloop system (Amersham Biosciences). Isocratic elution GST-Akt-PHdomain(1–149aminoacids)wasfirstconjugated with 2.5 column volumes of the same buffer was performed. withglutathione-Sepharosebeadsbyincubatingthetwoat4°C Both a constant flow rate of 1.0 ml/min and the absorbance for1hwithshaking.Thesebeadswerethenincubatedwith50 profilesatA andA weremonitoredduringtheentire 230nm 280nm ngofrecombinantHsp27asdescribedabove. chromatographicprocedure.Fractionsof1.0mlwerecollected, Cell Culture—HK-11 cells (human renal tubular epithelial and 100 (cid:1)l of every fifth fraction was used for Western blot cells)immortalizedbytransductionwithadenovirus12-SV40 analysis.Allprocedureswereperformedat4°C. were obtained from Dr. Racusen (34). Cells were cultured in Isoelectric Focusing (IEF) Electrophoresis—PMN lysates (25 Dulbecco’smodifiedEagle’smedium/Ham’sF-12(Invitrogen) (cid:1)g)weresubjectedtoIEFelectrophoresis.Proteinsweresepa- supplementedwith5%fetalcalfserum(Sigma)andpenicillin/ rated based on their isoelectric point. Precast IEF (NOVEX) streptomycin (100 units/ml) (Invitrogen). Fresh growth gels(Invitrogen)wererunaccordingtomanufacturer’sinstruc- medium was added to cells every 3–4 days until confluent. tions.Gelswerefixedin100mlof20%methanolsolutionfor20 HEK-293 cells were cultured in Dulbecco’s modified Eagle’s min.Gelwasrinsedin1(cid:4)SDS-runningbufferfor20minand mediumsupplementedwith10%fetalbovineserumandpeni- transferredontonitrocellulosemembraneusingsemidrytrans- cillin/streptomycin(100units/ml)(Invitrogen). fer apparatus (Invitrogen) for 20 min at 20 V. Nitrocellulose membraneswerewashedwithKrebs(cid:5)bufferandthenimmu- cDNA Transfections—HK-11or HEK-293 cells were plated on 6-well trays a day prior to performing transfections, to noblottedwithanti-Hsp27andanti-Ser(P)-82Hsp27antisera. Two-dimensional PAGE—Protein lysates (50 (cid:1)g) were achieve60%confluence.Onthedayoftransfectionappropriate cellswerewashedwithserum-freeRPMI1640medium.One(cid:1)g dilutedintourea/thiourearehydrationbuffer(GenomicSolu- of appropriate cDNA was transfected into these cells using tions),andproteinswereseparatedbytwo-dimensionalPAGE. GenePORTERreagentaccordingtothemanufacturer’sproto- IPG strips, pH 3–10 (Invitrogen), were rehydrated overnight col(GeneTherapySystems).Twentyfourhoursaftertransfec- withproteinsamples.Proteinswereseparatedonthebasisof tioncellswerelysedin100(cid:1)lofAktlysisbuffercontaining20 their isoelectric point by IEF using the ZOOM IPG Runner (Invitrogen)withamaximalvoltageof2000Vand50(cid:1)Aper mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% (v/v) Triton X-100, gel.FollowingIEF,IPGstripswereincubatedtwiceinequilibra- 0.5% (v/v) Nonidet P-40, 1 mM EDTA, 1 mM EGTA, 20 mM tionbufferI(6Murea,130mMdithiothreitol,30%glycerol,45 sodiumorthovanadate,10(cid:1)Mp-nitrophenolphosphate,20mM mM Tris base, 1.6% SDS, 0.002% bromphenol blue; Genomic NaF,5mMPMSF,21(cid:1)g/mlaprotinin,and5(cid:1)g/mlleupeptin, Solutions)andonceinequilibrationbufferII(6Murea,135mM andproteinsconcentrationsweredetermined.Proteinlysates iodoacetamide, 30% glycerol, 45 mM Tris base, 1.6% SDS, (50(cid:1)g)weresubjectedtoSDS-PAGEandimmunoblotanalysis 0.002% bromphenol blue; Genomic Solutions) for 10 min. ortoimmunoprecipitationstudies. EquilibratedIPGstripswereappliedto4–12%BisTrisgradient Construction of Hsp27 siRNA—Construction of siRNA gels (Invitrogen), and proteins were separated in the second was performed as described previously (35). Hsp27 siRNA dimensionbasedontheirmolecularsizeusingNuPAGEMES/ was generated using Silencer(cid:1) siRNA construction kit SDSbuffer(Invitrogen)at200Vfor40min.Followingelectro- (Ambion, Austin, TX). Hsp27 sequence 5(cid:3)-AAGACCAAG- phoresis,gelsweretransferredontonitrocelluloseandimmu- GATGGCGTGGTG-3(cid:3) was targeted to generate Hsp27 noblottedwithanti-Hsp27antibody. siRNA.ThesenseandantisensesiRNAoligonucleotidesused GST Pulldown Assay—PMNs (2 (cid:4) 107) were untreated or togenerateHsp27siRNAwere5(cid:3)-AACACCACGCCATCCT- treatedwithisotypecontrolantibodyorHsp27antibodyfor2h TGGTCCCTGTCTC-3(cid:3) (sense) and 5(cid:3)-AAGACCAAGGAT- at37°C.Thecellswerelysedwith200(cid:1)loflysisbuffercontain- GGCGTGGTGCCTGTCTC-3(cid:3)(antisense).TheHsp27siRNA ing1%(v/v)NonidetP-40,10%(v/v)glycerol,137mMNaCl,20 and a scrambled siRNA were transfected by using Gene- mMTris-HCl,pH7.4,1(cid:1)g/mlaprotinin,1(cid:1)g/mlleupeptin,5 PORTERreagentasoutlinedabove. 21600 JOURNALOFBIOLOGICALCHEMISTRY VOLUME282•NUMBER30•JULY27,2007 RegulationofAktActivationbyHsp27 AssessmentofCellViability—Cellviabilitywasmeasuredby assessment of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H- tetrazolium bromide (MTT) reduction (Sigma) as described previously(36,37).ThesolubleformofMTTwasreducedby mitochondria of live cells, resulting in a water-insoluble salt. Product formation was monitored by reading absorbance at 540nmusingamicroplatereader. Akt Immunoprecipitation Assays—Appropriate cells were lysedinAktlysisbuffercontaining20mMTris-HCl,pH7.4,150 mMNaCl,1%(v/v)TritonX-100,0.5%(v/v)NonidetP-40,1mM EDTA,1mMEGTA,20mMsodiumorthovanadate,10(cid:1)Mp-ni- trophenolphosphate,20mMNaF,5mMPMSF,21(cid:1)g/mlapro- tinin, and 5 (cid:1)g/ml leupeptin. Following centrifugation at 15,000 (cid:4) g for 15 min at 4°C, cleared lysates were incubated with20(cid:1)lofanti-AktPHdomainagarosebeadsorwithmouse FIGURE1.EnhancedAkt/Hsp27interactionduringLPSdelayofapo- ptosis.LysatesfromhumanPMNsfreshlyisolatedorinculturefor24hin isotype control antibody beads as described previously (8). thepresenceandabsenceofLPS(100ng/ml)weresubjectedtoanti-Akt Immunoprecipitated proteins were eluted with 40 (cid:1)l of 2(cid:4) immunoprecipitation(IP)andimmunoblotted(IB)withanti-Hsp27anti- body.LysatesfromfreshlyisolatedPMNswerealsosubjectedtoisotype Laemmlidye.Sampleswereboiledfor2min;beadswerepre- controlimmunoprecipitationascontrol.Asacontrol,immunoprecipitates cipitatedbyaquickspininapicofuge,and40(cid:1)lsupernatant were immunoblotted with anti-Akt antibody to demonstrate equal amountsofimmunoprecipitationsineachcondition. containing eluted proteins was subjected to SDS-PAGE and immunoblotanalysis. AktKinaseAssay—HK-11cellsweretransfectedwithpUSE control,zerotimePMNlysatesweresubjectedtoisotypecon- vectororpUSEAktCA(c-Myc-taggedmyristoylatedconstitu- trol antibody immunoprecipitation and immunoblotted with tivelyactiveAkt;UpstateBiotechnology,Inc.)orpUSEAktCA anti-Hsp27andanti-Aktantisera(Fig.1,4thlane).Asexpected, alongwithHsp27siRNA.Transfectedlysatesweresubjectedto noAktorHsp27bindingwasdetectedinPMNlysatesimmu- anti-Aktimmunoprecipitation(asdescribedabove).Immuno- noprecipitatedwithisotypecontrolantibody(Fig.1,4thlane). precipitatedAktwassubjectedtoaninvitroAktkinaseassay Akt/Hsp27interactionwasdetectedinzerotimePMNlysates usinghistoneH2Bassubstrateasdescribedpreviously(8). subjected to anti-Akt immunoprecipitation (Fig. 1, 3rd lane). Annexin V binding was performed as described previously Akt-Hsp27 association was markedly reduced in PMNs cul- (8).Briefly,1(cid:4)106PMNswerewashedandresuspendedin100 turedfor24hintheabsenceofLPS(Fig.1,2ndlane);however, (cid:1)l of RPMI 1640 medium without fetal calf serum. The cells thisassociationwasmaintainedinPMNsculturedfor24hin were prewarmed for 5 min at 37°C; anti-Hsp27 antibody (10 thepresenceofLPS(Fig.1,1stlane).Theseresultssuggestthere (cid:1)g)orisotypecontrolantibody(10(cid:1)g)wasthenadded,andthe isaneedtodeterminethecauseandeffectrelationshipbetween cellswerefurtherincubatedfor2hat37°C.PMNswerewashed Akt/Hsp27 interaction and PMN viability, suggesting the once with RPMI 1640 medium without fetal calf serum and importanceofAkt/Hsp27interactionintheregulationofPMN centrifuged at 400 (cid:4) g for 2 min. Cells were resuspended in apoptosisduringinflammation. ice-cold1(cid:4)BindingBuffer(10mMHepes/NaOH,pH7.4,150 SeparationofAktSignalComponentsinControlandfMLP- mMNaCl,5mMKCl,1mMMgCl ,2.5mMCaCl )ataconcen- stimulated PMNs—We have previously shown in PMNs that 2 2 tration of 106 cells/0.3 ml. Next 3 (cid:1)l of annexin conjugate AktexistsinacomplexwithHsp27,MK2,andp38MAPKand ApopNexinFITCwasaddedtothecellsuspensionandincu- thatp38MAPK-dependentkinaseMK2regulatesAktactiva- bated at room temperature for 15 min in the dark. The cells tion by phosphorylating Ser-473 on Akt (8). In addition, we were immediately examined using Zeiss Axiovert 100 micro- demonstratedthatuponfMLPstimulation,Hsp27dissociates scopeandLSM510software. from the Akt signal complex, whereas MK2 and p38 MAPK continuetoassociatewithAkt(3,8).Toestimatepercentageof RESULTS Hsp27orMK2orp38MAPKfromPMNsthatassociatewith EnhancedAkt/Hsp27InteractionduringLPSDelayofPMN Akt,wesubjectedcontrolandfMLP-stimulatedPMNlysatesto Apoptosis—Cultured PMNs undergo constitutive apoptosis gelfiltrationchromatography.Fractionscollectedwereimmu- suchthatonlyabout30%areviableafter24h.However,treat- noblotted with anti-Hsp27, anti-MK2, anti-p38 MAPK, and mentofPMNswithlipopolysaccharide(LPS)delaysconstitu- anti-Akt(Fig.2A).TheresultsdemonstratedthatHsp27eluted tivePMNapoptosis,suchthatabout70%areviableafter24h inthesamefractionsasAktincontrolPMNlysates.Inaddition, (2).WedeterminedwhetherincreasedPMNviabilityat24hin MK2andp38MAPKelutedinthesamefractionsasHsp27and thepresenceofLPScouldbeattributedtoincreasedAkt/Hsp27 Akt;however,bothMK2andp38weredetectedinadditional interaction. Freshly isolated PMNs (zero time control) and fractionsintheabsenceofAktandHsp27.Furthermore,p38 PMNs in culture for 24 h with or without LPS were lysed. MAPK was detected in some fractions in the absence of Akt, Lysates were subjected to anti-Akt immunoprecipitation and Hsp27,andMK2.Theseresultssuggestthatundercontrolcon- immunoblotted with anti-Hsp27 antibody. Immunoprecipi- ditions, almost 100% of Hsp27 and Akt associate with MK2, tates were also immunoblotted with anti-Akt to determine whereasasmallpercentageofMK2existsinacomplexwithp38 equalimmunoprecipitationineverycondition.Additionally,as MAPKintheabsenceofAktandHsp27(Fig.2A).Theseresults JULY27,2007•VOLUME282•NUMBER30 JOURNALOFBIOLOGICALCHEMISTRY 21601 RegulationofAktActivationbyHsp27 FIGURE2.SeparationofAktsignalcomponentsincontrolandfMLP-stim- ulatedPMNs.HumanPMNlysatesgeneratedfromcontrol(A)and0.3(cid:1)M fMLP-treated (B) PMNs were subjected to gel filtration chromatography. Elutedproteinfractionswerecollectedasdescribedunder“Materialsand Methods.”ProteinfractionsweresubjectedtoSDS-PAGEandimmunoblot- tingwithanti-Akt,anti-Hsp27,anti-MK2,andanti-p38MAPKantisera.Results showthatAktandHsp27eluteoutinthesamefractionsincontrolPMN lysates,althoughamajorityofHsp27elutesoutpriortoAktinfMLP-treated PMNlysates,suggestingitsdissociationfromAkt.MK2andp38MAPKasso- ciatewithAktinthepresenceandabsenceoffMLP. wereconfirmedbyperformingAktimmunoprecipitationsand immunoblottingwithanti-Hsp27andanti-MK2antisera(data notshown).InfMLP-stimulatedPMNs,wedemonstratedthat Hsp27 eluted out prior to Akt (Fig. 2B). Thus, only a small FIGURE3.PhosphorylationinducesanacidicshiftinHsp27pIwithout fractionofHsp27associateswithAkt,whereasalargeamount alteringitsmolecularsizeinhumanPMNs.A,humanPMNlysatesgener- of Hsp27 was not in a complex with Akt in fMLP-stimulated atedfromcontrol(C)and5-minfMLP(F5)(0.3(cid:1)M)-treatedPMNsweresub- PMNs. MK2 and p38 MAPK continue to associate with Akt jectedtoIEFelectrophoresisandimmunoblotted(IB)withanti-Hsp27and anti-Ser(P)-82Hsp27antiseraasdescribedunder“MaterialsandMethods.”IEF afterfMLPstimulation. immunoblottingdemonstratedtwospeciesofHsp27withdifferentisoelec- PhosphorylationInducesanAcidicShiftinHsp27pIwithout tricpointsinbothcontrolandfMLP-stimulatedPMNlysates.Increasedabun- danceofthemoreacidicspeciesofHsp27wasseeninfMLP-treatedlysates. AlteringItsMolecularSizeinHumanPMNs—Theoligomeriza- Immunoblotanalysiswithanti-Ser(P)-82Hsp27antibodyconfirmedthatthe tion status of Hsp27 in human PMNs has not been demon- increaseintheacidicformofHsp27correlatedwithenhancedHsp27-Ser82 stratedtodate.TodeterminewhetherHsp27existedatdiffer- phosphorylation.B,controland5-minfMLP(0.3(cid:1)M)-treatedPMNlysates weresubjectedtotwo-dimensionalPAGE(2D-PAGE)immunoblotanalysis ent isoelectric points by the virtue of its phosphorylation, withanti-Hsp27antibody.ResultsdemonstratedtwospeciesofHsp27as control and fMLP-stimulated PMN lysates were subjected to seeninIEFgels;however,boththesespecieswereofidenticalsize.Collec- IEF gel electrophoresis and immunoblotting with anti-Hsp27 tively,theseresultsdemonstratethatphosphorylationofHsp27inducedan acidicpIshiftofHsp27withoutalteringitssizeinhumanPMNs. antibody.TheresultsdemonstratedtwospeciesofHsp27with different isoelectric points in both control and fMLP-stimu- lated PMN lysates. However, the intensity of the more acidic two-dimensional polyacrylamide gels were subsequently sub- formofHsp27wasenhancedinfMLP-stimulatedlysatecom- jected to immunoblotting with anti-Hsp27 antibody. Results paredwithcontrollysate(Fig.3A,leftpanel).Immunoblotanal- demonstratedtwospeciesofHsp27;however,boththesespe- ysis with anti-Ser(P)-82Hsp27 antibody confirmed that the cieswereidenticalinsize(Fig.3B).Collectively,IEFandtwo- increaseintheacidicformofHsp27correlatedwithenhanced dimensionalPAGEHsp27immunoblotanalysisofcontroland Hsp27 Ser-82 phosphorylation (Fig. 3A, right panel). Having fMLP-stimulated PMNs lysates demonstrated that phospho- demonstrated that two forms of Hsp27 exist at different iso- rylationofHsp27inPMNsinducedashiftinpIwithoutaltering electric points, we next sought to determine the size of these sizeofHsp27.Thus,underthesecurrentexperimentalcondi- twoformsofHsp27.Toaccomplishthisgoal,PMNlysateswere tions,Hsp27doesnotexistasanoligomericproteininPMNs. subjectedtotwo-dimensionalPAGE.PMNproteinsweresep- Hsp27 Depletion Induces PMN Apoptosis by Inhibiting Akt aratedinthefirstdimensiononpIandseparatedonthesecond Association with Its Upstream Kinase MK2—We have previ- dimensionbythevirtueoftheirmolecularweightorsize.These ously shown that loss of Akt/Hsp27 association in human 21602 JOURNALOFBIOLOGICALCHEMISTRY VOLUME282•NUMBER30•JULY27,2007 RegulationofAktActivationbyHsp27 tion with the Hsp27-interacting proteinMK2.Asdemonstratedpre- viously, incubation of PMNs with anti-Hsp27 but not isotype control antibody induced apoptosis as shown by enhanced annexin V binding in anti-Hsp27-treated PMNs(Fig.4C).Theseresultssug- gest that disruption of Akt/Hsp27 association results in loss of Akt/ MK2 and induction of PMN apoptosis. Silencing Hsp27 Expression Dis- rupts Akt/MK2 Interaction—We further validated the role of Hsp27 inscaffoldingMK2toAktandregu- latingcellularapoptosisbyanHsp27 FIGURE4.Hsp27depletioninducesPMNapoptosisbyinhibitingAktassociationwithitsupstream kinaseMK2.A,humanPMNsweretreatedwithisotypecontrol(IC)antibodyorHsp27antibodyfor4hat37°C knockdown approach. Effects of asdescribedpreviously(8).PMNlysatesweresubjectedtoanti-Aktorisotypecontrolimmunoprecipitation(IP) silencingHsp27onAkt/MK2inter- followedbyimmunoblot(IB)analysiswithanti-Hsp27antibody.ResultsdemonstrateAkt/Hsp27interactionin isotypecontrol-treatedPMNsbutnotinanti-Hsp27treatedPMNs(n(cid:6)3).PMNlysatesobtainedabovewere actionweredetermined.Celllysates incubatedwithGSTorGST-Akt-Sepharose.B,proteinswereseparatedbySDS-PAGEandimmunoblottedwith from HK-11 cells transfected with anti-MK2antibody(n(cid:6)3).Isotypecontrol-treatedPMNlysatewasincludedasapositivecontrolforMK2(5th scrambled or Hsp27 siRNA were lane).ResultsshowthatdepletionofHsp27inhibitsAkt/MK2interaction.C,PMNsweretreatedwithmouse isotypecontrolantibodyormonoclonalanti-Hsp27antibodyfor4hat37°C.Isotypecontrolandanti-Hsp27 immunoblotted with anti-Hsp27 antibody-treatedcellswerestainedwithFITC-conjugatedannexinVandviewedbyconfocalmicroscopy. and anti-Akt antisera to demon- AnnexinV-positivestainingwasobservedinanti-Hsp27antibody-treatedcells. strate specific silencing of Hsp27 expression. Fig. 5A shows that PMNsresultsinlossofAktactivationandPMNapoptosis(8). Hsp27siRNAbutnotscrambledsiRNAsignificantlyinhibited TodeterminewhetherlossofAktphosphorylationandactiva- Hsp27expressionwithoutalteringAktexpression.GSTpull- tion in the absence of Hsp27 results from loss of interaction downstudiesperformedwithabovelysatesdemonstratedAkt/ between Akt and its upstream activator kinase MK2, PMNs MK2 interaction in scrambled siRNA-treated cells but not in wereincubatedinthepresenceorabsenceofanti-Hsp27anti- Hsp27 siRNA-treated cells (Fig. 5B). As expected no binding bodyorisotypecontrolantibodiesasdescribedpreviously(8). wasdetectedinGST-Sepharosecontrol(Fig.5B,lane3).These SuccessfulintroductionofFITC-taggedantibodiesintoPMNs resultswereconfirmedbyAktimmunoprecipitationstudiesin was confirmed by confocal microscopy and trypan blue scrambled siRNA and Hsp27 siRNA-transfected HK-11 cells quenchingasdescribedpreviouslybyourgroup(8).After4hof (Fig.5C).Collectivelytheseresultsdemonstrateanimportant appropriate antibody incubation cell lysates generated were role of Hsp27 expression in regulating Akt/MK2 interaction subjectedtoisotypecontroloranti-Aktimmunoprecipitation (Fig.5BandFig.3C). tovalidatedisruptionofAkt/Hsp27interactioninthepresence Disruption of Akt/MK2 Interaction by Silencing Hsp27 of anti-Hsp27 antibody. Fig. 4A demonstrates association of Expression Inhibits Akt Phosphorylation and Activation—Be- Akt/Hsp27 in isotype control-treated cells but not in cells causeMK2actsasPDK2forAkt,wenextdeterminedwhether treated with anti-Hsp27 antibody (2nd and 3rd lanes, top loss of Akt/MK2 interaction would regulate Akt activation. panel). In addition, as expected nonspecific binding was not HK-11cellsweretransfectedwithvectororc-Myc-taggedcon- detectedinisotypecontrolimmunoprecipitation(Fig.4A,1st stitutivelyactivemyristoylatedAkt(AktCA)inthepresenceof lane, top panel). Equal Akt immunoprecipitation was docu- scrambledsiRNAorHsp27siRNA.AktCAcDNAconstructis mented by immunoblotting with anti-Akt antibody (Fig. 4A, myristoylated and hence targeted to the plasma membrane 2ndand3rdlanes,bottompanel).Havingdemonstratedlossof allowing constitutively activated PDK2 to phosphorylate Akt Akt/Hsp27 interaction in anti-Hsp27-treated cells, we next on Ser-473. Fig. 6A shows that Hsp27 siRNA selectively sought to determine association of Akt/MK2 in the presence silencedHsp27expressionwithoutalteringc-Myc-taggedAkt andabsenceofAkt/Hsp27interaction.PMNlysatesgenerated expression(Fig.6A,2ndlane,panels1and2),whereasscram- above were subjected to GST or GST-Aktwt pulldown assay. bledsiRNAhadnoeffectonHsp27orAktexpression(Fig.6A, ProteinswereresolvedbySDS-PAGEfollowedbyimmunoblot 1st lane, panels 1 and 2). Hsp27 siRNA but not scrambled analysiswithanti-MK2antibody(Fig.4B).Fig.4Bdemonstrates siRNA inhibited AktSer-473 phosphorylation of transfected associationofAkt/MK2inisotypecontrolantibody-treatedbut myristoylated Akt and also inhibited AktCA-induced Hsp27 not in anti-Hsp27 antibody-treated PMN lysates. No binding Ser-82 phosphorylation (Fig. 6A, 2nd lane, panels 3 and 4). wasdetectedincontrolGSTbeads(Fig.4B,2ndand4thlanes). Additionally,Hsp27siRNAbutnotscrambledsiRNAinhibited Isotypecontrolantibody-treatedPMNlysatewasrunasapos- theabilityofimmunoprecipitatedAkttophosphorylatehistone itivecontrol(Fig.4B,5thlane).Theseresultssuggestthatdis- H2B from AktCA transfected HK-11 cells (Fig. 6B). Loss of ruptionofAkt-Hsp27associationresultsinlossofAktinterac- Akt/MK2interactionandsubsequentlossofAktphosphoryla- JULY27,2007•VOLUME282•NUMBER30 JOURNALOFBIOLOGICALCHEMISTRY 21603 RegulationofAktActivationbyHsp27 FIGURE6.DisruptionofAkt/MK2interactionbysilencingHsp27expres- sioninhibitsAktphosphorylationandactivation.Humanrenaltubular FIGURE5.SilencingHsp27expressiondisruptsAkt/MK2interaction. cells(HK-11)weretransfectedwithc-Myc-taggedmyristoylatedAkt(AktCA) HK-11cellsweretransfectedwithscrambledorHsp27siRNA.A,celllysates constructwithscrambledorHsp27siRNA.A,transfectedcelllysateswere fromtransfectedcellswereimmunoblotted(IB)withanti-Hsp27andanti- subjected to immunoblot analysis with anti-Hsp27 (panel 1), anti-c-Myc AktantiseratodemonstratespecificsilencingofHsp27expression(n(cid:6)3). (panel2),anti-phosphoAktSer-473(panel3),andanti-phosphoHsp27Ser-82 B,abovelysatesweresubjectedtoGSTorGST-Aktwtpulldownassayto (panel4)antisera(n(cid:6)3).ResultsshowthattransfectionofHsp27inhibits determineAkt/MK2interactioninthepresenceofscrambledorHsp27 Hsp27expressionwithoutalteringexpressionofc-Myc-taggedAkt.Addition- siRNA (n (cid:6) 3). C, above lysates were also subjected to mouse anti-Akt ally, AktSer-473 phosphorylation and Hsp27Ser-82 phosphorylation were immunoprecipitation(IP)andimmunoblottedwithrabbitpolyclonalanti- inhibitedinthepresenceofHsp27siRNAbutnotinthepresenceofscram- MK2andrabbitpolyclonalanti-Aktantisera(n(cid:6)3).Theresultsdemon- bledsiRNA.B,HK-11cellsweretransfectedwithvector(1stlane)orAktCA stratelossofHsp27expressionabrogatesAkt/MK2interaction. alongwithscrambledsiRNA(2ndlane)orAktCAalongwithHsp27siRNA(3rd lane). Lysates were immunoprecipitated with anti-Akt antibody and sub- tionandactivationinthepresenceofHsp27siRNAsuggesta jectedtoaninvitroAktkinaseassayusinghistoneH2Bassubstrate(n(cid:6)3). AutoradiographdemonstratesthattransfectionofAktCAinducesincreased criticalroleforHsp27intheregulationofAktactivation. Akt-mediatedhistoneH2Bphosphorylationinthepresenceofscrambled Disruption of Akt/MK2 Interaction by Silencing Hsp27 siRNAbutnotinthepresenceofHsp27siRNA.Theseresultsdemonstratethat Expression Induces HK-11 Cell Death—Silencing Hsp27 silencingHsp27expressioninhibitsAktphosphorylationandactivation. expression inhibits Akt/MK2 interaction and Akt activation. Thus,wedeterminedeffectsofsilencingHsp27expressionon containstheAktPHdomain(1–116aminoacids)andtheacidic HK-11cellviability. linker region (117–149) to interact with recombinant Hsp27. HK-11 cells alone or HK-11 cells in the presence of Gene- Recombinant GST-Akt-(1–149) was conjugated to glutathi- PORTER reagent, scrambled siRNA, or Hsp27 siRNA were one-Sepharose as described under “Materials and Methods.” incubatedat37°Cin5%CO for24h,andcellsweresubjected Fig.8AdemonstratesthatGST-Akt-(1–149)(2ndlane),butnot 2 to MTT cell viability assay. Hsp27 siRNA but not scrambled glutathione-Sepharose (1st lane), specifically interacts with siRNA significantly inhibited Hsp27 without altering Akt recombinantHsp27suggestingaroleforthePHdomainand/or expression (Fig. 7, bottom panel). Additionally, Hsp27 siRNA the acidic linker region to interact with Hsp27. Recombinant but not scrambled siRNA significantly decreased HK-11 cell GST-Akt-(1–149)andrecombinantHsp27wererunaspositive viability.GenePORTERreagentbyitselfhadnoeffectoncell controls(Fig.8A,3rdand4thlanes).Todeterminetheroleof viability(Fig.7,toppanel).TheseresultssuggestthatHsp27isa Akt PH domain in interacting with Hsp27, recombinant survival protein, and its regulation of HK-11 survival may be Akt (mutant lacking N-terminal PH domain or 1–116 (cid:1)PH mediatedbyscaffoldingMK2toAkt,allowingAktactivationto aminoacids)wasprecipitatedwithGSTorGST-Hsp27.Fig.8B occur,therebypromotingcellsurvival. demonstrates interaction of recombinant Akt with GST- (cid:1)PH AktPHDomainIsNotRequiredforInteractionwithHsp27— Hsp27butnotwithGST-Sepharose.RecombinantAkt was (cid:1)PH WehavedemonstratedpreviouslythatHsp27directlyinteracts runaspositivecontrol(Fig.8B,lane3).Theseresultssuggest with Akt (8). To identify Hsp27-binding site(s) on Akt, we that Akt-PH domain is not required for its interaction with determinedtheabilityofGST-Akt-(1–149)-Sepharose,which Hsp27. 21604 JOURNALOFBIOLOGICALCHEMISTRY VOLUME282•NUMBER30•JULY27,2007 RegulationofAktActivationbyHsp27 Hsp27DirectlyInteractswiththeAcidicLinkerRegionofAkt— In-framedeletionmutantofAktwasgeneratedsuchthatamino acids117–128fromtheacidiclinkerregionofAktweredeleted. InaGSTpulldownassayrecombinantHsp27wasprecipitated byGST-AktwtbutnotbyGSTorGST-Akt -Sepharose (cid:1)117–128 (Fig.9A,bottompanel).TransferredgelwasstainedwithCoo- massieBluetoserveasaloadingcontrolforGSTbeadsutilized in the assay. Additionally, HEK-293 cells were co-transfected withc-Myc-taggedpUseAktwtorpUseAkt alongwith (cid:1)117–128 Hsp27 cDNA. After transfection cells were lysed, and lysates weresubjectedtoanti-c-Mycimmunoprecipitationandimmu- noblotted with anti-Hsp27 antibody. Fig. 9B (bottom panel) demonstratesinteractionbetweenAktwtandHsp27,whereas nointeractionisdetectedbetweenAkt andHsp27.To (cid:1)117–128 demonstrate overexpression of c-Myc-tagged Akt constructs andHsp27,totallysateswereimmunoblottedwithanti-c-Myc andanti-Hsp27antisera(Fig.9B,topandmiddlepanel).Fur- thermore, these results were confirmed in human PMNs by performing a GST pulldown assay. GST-Akt but not GST or FIGURE 7. Disruption of Akt/MK2 interaction by silencing Hsp27 GST-Akt(cid:1)117–128 interacted with Hsp27 from PMN lysates expression induces HK-11 cell death. HK-11 cells were treated with (Fig.9C).TransferredgelswerestainedwithCoomassieBlueto GenePORTER(transfectionreagent)ortransfectedwithscrambledsiRNA demonstrateequalloadingofGST-AktandGST-Akt - or Hsp27 siRNA. Transfected cells were subjected to MTT cell viability (cid:1)117–128 assay.MTTreductionwasdeterminedasameasureofHK-11cellviability Sepharose.Theseresultscollectivelyindicatethataminoacids inthepresenceofGenePORTER,scrambledsiRNA,orHsp27siRNA.Results 117–128intheacidiclinkerregionofAktarerequiredforinter- wereexpressedas%viabilityrelativetocellalone(100%).SilencingHsp27 expressioninducedsignificantcelldeath(*,p(cid:7)0.05versuscellalone,n(cid:6) actionwithHsp27. 4).IB,immunoblot. We next determined the ability of GST, GST-Aktwt, and GST-Akt tointeractwithHsp90fromPMNlysates. (cid:1)117–128 Hsp90 has been shown previously to directly interact with Aktviaaminoacids229–309(38).Toconfirmthatdeletion of117–128aminoacidsfromAktdidnotalterproteinconfor- mationanditsabilitytointeractwithHsp90,wesubjectedGST, GST-Akt,andGST-Akt toaGSTpulldownassaywith (cid:1)117–128 PMNlysate.Fig.9DdemonstratesthatasexpectedbothGST- Aktwt and GST-Akt but not GST interacted with (cid:1)117–128 Hsp90fromPMNlysates.PMNlysaterunaspositivecontrol immunoreacted with anti-Hsp90 antibody (Fig. 9D, lane 1). TransferredgelswerestainedwithCoomassieBluetodemon- strateequalloadingofGST-AktandGST-Akt -Sepha- (cid:1)117–128 rose. These results collectively indicate that Hsp27 interacts with Akt via amino acids 117–128, and the deletion of this regiondoesnotseverelyalteritsconformationandbindingto itsknownbindingproteinHsp90. MK2FailstoInteractwithAkt andPhosphorylateIt (cid:1)117–128 onSer-473exVivo—HavingdisruptedAkt/Hsp27interaction bydeletingHsp27bindingregiononAkt,wenextdetermined theabilityofGST,GST-Aktwt,andGST-Akt toprecip- (cid:1)117–128 itateMK2fromPMNlysates.Fig.10AshowsthatGST-Aktwt, but not GST or GST-Akt , interacted with MK2 from (cid:1)117–128 PMN lysate. Transferred gels were stained with Coomassie FIGURE8.AktPHdomainisnotrequiredforinteractionwithHsp27. Blue to demonstrate equal loading of GST-Akt and GST- A,recombinantHsp27wasincubatedwithglutathione-Sepharose(Seph,1st Akt -Sepharose.HavingshownlossofMK2bindingto lane),glutathione-SepharosecoupledtorecombinantGST-Akt-(1–149)(2nd (cid:1)117–128 Akt , we next determined the ability of constitutively lane),andsubjectedtoGST-pulldownassayandimmunoblotted(IB)with (cid:1)117–128 anti-Hsp27andanti-Aktantisera(n(cid:6)3).RecombinantGST-Akt-(1–149)and activatedMK2(MK2EE),aknownupstreamactivatorofAkt,to recombinantHsp27alonewererunaspositivecontrols.ResultsshowthatAkt phosphorylateAktwtandAkt intransfectedHEK-293 (1–149aminoacids)mediatesinteractionwithrecombinantHsp27.B,recom- (cid:1)117–128 binant Akt (PH domain 1–116 amino acids deleted from N terminus) cells.Fig.10BshowsthatMK2EEphosphorylatesAktwtonSer- (cid:1)PH mutantwasincubatedwithGST(lane1)orGST-Hsp27-Sepharose(lane2)and 473butfailstophosphorylateAkt .Theseresultsestab- immunoblottedwithanti-Aktantibody(n(cid:6)3).RecombinantAkt wasrun (cid:1)117–128 (cid:1)PH lishanovelroleforHsp27inregulatingAktactivationbyscaf- aspositivecontrol(lane3).ResultsshowthatPHdomainofAktisnotrequired forinteractionwithHsp27. foldingMK2toAktsignalcomplex. JULY27,2007•VOLUME282•NUMBER30 JOURNALOFBIOLOGICALCHEMISTRY 21605 RegulationofAktActivationbyHsp27 Based on these studies, we postu- lated that Hsp27 regulates PMN apoptosisbyactingasascaffolding protein in the Akt signaling com- plex. Hsp27 binds to p38 MAPK, MK2, and other proteins required for activation of Akt. In this study weshowforthefirsttimerelevance ofAkt/Hsp27interactiontoconsti- tutive apoptosis and LPS delay of apoptosis. Akt/Hsp27 interaction wasdocumentedinfreshlyisolated PMNs and was markedly reduced inPMNsculturedfor24h,atime point at which only about 30% of PMNs are viable in culture (2). In contrast, Akt-Hsp27 association was markedly enhanced in PMNs culturedfor24hinthepresenceof LPS.Wehaveshownpreviouslythat LPS delays constitutive PMN apo- ptosiswith70%viablePMNsat24h (2).Inaddition,wehaveshownthat Hsp27isamemberoftheAktsignal complex, and removal of Hsp27 from the complex results in loss of Akt activation and induction of PMN apoptosis. These findings indicatearoleforHsp27inthereg- FIGURE9.Hsp27directlyinteractswiththeacidiclinkerregionofAkt.A,GST,GST-Aktwt,orGST-Akt (117–128aminoacidsdeleted)wassubjectedtoapulldownassaywithrecombinantHsp27(50ng)(cid:1).1T1r7a–n12s8- ulationofinflammationbymodula- ferredgelwasstainedwithCoomassieBluetoserveasaloadingcontrolofGSTbeadsutilizedintheassay. tion of Akt activation and PMN RecombinantHsp27associatedwithGST-AktwtbutfailedtobindGSTorGST-Akt -Sepharose.B,HEK- (cid:1)117–128 apoptosis. 293cellsweretransfectedwithHsp27alongwitheitherc-Myc-taggedAktwtorin-frameAktdeletionmutant Akt (117–128aminoacidsdeleted).Transfectedlysateswereimmunoprecipitated(IP)withanti-c-Myc IsolationofAktsignalcomplexin (cid:1)117–128 antibodyandimmunoblotted(IB)withanti-Hsp27antibody(middlepanel).Transfectedlysateswerealso the presence and absence of fMLP immunoblottedwithanti-Hsp27andanti-c-MycantibodiestodemonstrateexpressionofHsp27andc-Myc- taggedAktconstructs(topandbottompanels).C,PMNlysatewasincubatedwithGST,GST-Akt,andGST- by gel filtration chromatography Akt(cid:1)117–128andsubjectedtoaGSTpulldownassayandimmunoblottedwithanti-Hsp27antibody(n(cid:6)3). demonstrated that Hsp27 dissoci- TransferredgelswerestainedwithCoomassieBluetodemonstrateequalloadingofGST-AktandGST-Akt - (cid:1)117–128 atedfromAktuponfMLPstimula- Sepharose.D,PMNlysatewasincubatedwithGST,GST-Akt,andGST-Akt andsubjectedtoaGSTpulldown assayandimmunoblottedwithanti-Hsp90antibody(n(cid:6)3).Transferred(cid:1)g1e1l7s–w12e8restainedwithCoomassieBlueto tionasdemonstratedpreviouslyby demonstrateequalloadingofGST-AktandGST-Akt(cid:1)117–128-Sepharose.ResultsshowthatAkt(cid:1)117–128mutantbinds our group (3). In addition, IEF gel Hsp90butfailstointeractwithHsp27. electrophoresis/immunoblotanaly- sisdemonstratedanacidicpIshiftin DISCUSSION Hsp27 in fMLP-stimulated PMNs compared with control, The first indication that Hsp27 might regulate Akt activity coincidingwithHsp27Ser-82phosphorylation.Moreover,two- wasreportedbyKonishietal.(39),whodemonstratedanasso- dimensional PAGE immunoblotting demonstrated that this ciation of Hsp27 and Akt in COS-7 cells. Subsequently, we acidicpIshiftinHsp27occurredwithoutalteringthemolecular demonstrated for the first time that Akt exists in a signaling sizeofHsp27.Thus,phosphorylationofHsp27doesnotalter complexwithp38MAPK,MK2,andHsp27inhumanPMNs themolecularsizeofHsp27inPMNs.Therefore,Hsp27disso- (3).Morerecentlyp38MAPK-dependentactivationofAkthas ciationfromAktuponPMNactivationisdependentonHsp27 beendemonstratedinavarietyofcells,includingcardiomyo- phosphorylationandnotonthemolecularsizeofHsp27. cytes,keratinocytes,andendothelialcells(40–47).Thus,p38 Heatshockproteinsareinducedundervariousstresscondi- MAPKregulationofAktactivationisnotuniquetoPMNs.In tionstocombatstress-inducedapoptosiscausedbyaggregation this study we show that Hsp27 directly interacts with Akt ofdenaturedproteins.Heatshockproteinsbindtoandrefold through its acidic linker region. The physical association of denaturedproteinsandchaperonethemtoappropriatecellular Hsp27 with Akt is a critical determinant of PMN survival, as compartmentsortargetthemtotheproteasomefordegrada- removal of Hsp27 from the Akt signal module prevents Akt tion.Hsp27bindsactinandstabilizesthecellulararchitecture activationandresultsinacceleratedPMNapoptosis(8).More- (48). The protective effect of Hsp27 on the cytoskeleton is over, MK2 in addition to phosphorylating Hsp27 (29) acts as mediatedbytheabilityofHsp27tobinddenaturedactinand PDK2 for Akt in human PMNs, phosphorylating Ser-473 (3). prevent its aggregation (49). Ariggo et al. (50) demonstrated 21606 JOURNALOFBIOLOGICALCHEMISTRY VOLUME282•NUMBER30•JULY27,2007 RegulationofAktActivationbyHsp27 Akt, we hypothesized that Hsp27 modulated interactions within the Akt-Hsp27-MK2 signal complex by acting as a scaffolding protein. This study defined a novel scaffolding roleofHsp27intheregulationofAktactivationandcellular apoptosis. Disruption of Akt/Hsp27 interaction by depleting Hsp27 fromhumanPMNsresultedinlossofAktinteractionwithboth Hsp27 and MK2 and induction of PMN apoptosis. Because MK2isanHsp27-bindingprotein,wenextdeterminedwhether depletionofHsp27nonspecificallyremovedMK2fromtheAkt signal complex. To address this question, siRNA technology was utilized to specifically silence Hsp27 expression from HK-11 cells to determine effects of silencing Hsp27 on Akt/ MK2interactionandHK-11cellviability.Hsp27siRNAbutnot scrambled siRNA-transfected HK-11 cells demonstrated a markeddecreaseinHsp27expression,lossofAkt/MK2inter- action,andinductionofHK-11celldeath.Theseresultssug- gestedaroleforHsp27inregulatingAkt/MK2interactionand Aktactivation.CelldeathinducedbysilencingHsp27wasnot entirely surprising as Schepers et al. (58) have shown that FIGURE10.MK2failstointeractwithAkt andphosphorylateiton silencing Hsp27 expression resulted in a 2-fold increase in (cid:1)117–128 Ser-473exvivo.A,PMNlysatewasincubatedwithGST,GST-Akt,andGST- VP-17-induced apoptosis in acute myeloid leukemia cells. Akt andsubjectedtoaGSTpulldownassayandimmunoblottedwith anti(cid:1)-M117K–212a8ntibody(n(cid:6)3).TransferredgelswerestainedwithCoomassieBlue However, induction of cell death by modulating Akt/MK2 todemonstrateequalloadingofGST-AktandGST-Akt(cid:1)117–128-Sepharose. interaction was a novel observation. To determine more ResultsshowthatAkt mutantfailstointeractwithMK2fromPMN lysates.B,HEK-293cell(cid:1)s1w17e–1re28transfectedwithvectororco-transfectedwith directlytheroleofHsp27inregulatingAktactivation,HK-11 Hsp27 and MK2EE (constitutively active MK2) along with either Aktwt or cells were transfected with myristoylated-c-Myc-tagged Akt Akt .Transfectedlysateswereimmunoblottedwithanti-Hsp27,anti- (cid:1)117–128 (AktCA)inthepresenceandabsenceofHsp27siRNA.Immu- MK2,anti-c-Myc,andanti-pAktS473antisera.ResultsshowthatactiveMK2 failstophosphorylateAkt exvivoinHEK-293cells. noblottingstudiesdemonstratedthatlossofHsp27expression (cid:1)117–128 inhibited AktSer-473 phosphorylation and activation. These thatHsp27inhibitedapoptosisbymaintainingtheredoxequi- studiesclearlyidentifiedaroleforHsp27inregulatingAktacti- libriumofthecell.Inaddition,overexpressionofHsp27inhib- vation. Hsp27 negatively regulates protein kinase C(cid:2)activity itedoxidantstress-inducedcellulardamagebyincreasinglevels andinhibitscellapoptosis(59);however,weshowforthefirst of cellular glutathione (51). Overexpression of various heat time that Hsp27 positively regulates Akt activation and pro- shockproteinshasbeenshowntoinduceasurvivalresponse. motescellsurvival. Contrarytomostheatshockproteins,Hsp60(52,53)hasbeen We have demonstrated direct protein/protein interaction shown to induce apoptosis by interaction with caspase-3, betweenHsp27andAkt(3,8).Thisstudyidentifiedaminoacids whereasHsp27(21–25),Hsp70(54–56),andHsp90(57)have 117–128intheacidiclinkerregionofAkttomediateinterac- beenshowntoinhibitapoptosisbybindingtoandinactivating tionwithHsp27.Anin-frameAktdeletionmutant(Akt ) specific components of the apoptosis machinery. In general interactedwithHsp90(cid:3)butfailedtointeractwithHs(cid:1)p12177–a1n28d heatshockprotein-inducedprotectionfromstresswasshown MK2.Thisfindingisinaccordancewiththeliterature,which toactattheleveloftheapoptosomebypreventingdownstream demonstratedthatAktinteractedwithHsp90(cid:3)viaaminoacids activationofcaspases.ThisstudydefinesanovelroleofHsp27 229–309(38). intheregulationofcellularapoptosisbyactingasascaffolding ThesestudiessuggestedthatlossofAkt interaction protein. (cid:1)117–128 withMK2andHsp27wasnotbecauseofalteredconformation WehaverecentlydemonstratedthatHsp27existsinacom- of the mutant. The importance of Hsp27 in regulating Akt/ plexwithAkt,p38MAPK,MK2,andHsp27,andMK2actsas MK2 interaction and AktSer473 phosphorylation was docu- PDK2 for Akt (3). In addition, we demonstrated that MK2- mented by co-transfecting HEK-293 cells with constitutively binding protein Hsp27 dissociated from Akt signal complex activeMK2(MK2EE)andHsp27alongwithAktwtorAkt . after Akt activation (3). However, disruption of Akt/Hsp27 (cid:1)117–128 interaction prior to Akt activation resulted in loss of fMLP- MK2EEphosphorylatedAktwtonSer473asexpected;however, stimulated Akt activation and induced PMN apoptosis (8). itfailedtophosphorylateAkt(cid:1)117–128onSer473,acriticalsite ThesestudiesindicatedtheimportanceofAkt/Hsp27interac- knowntoregulateAktactivation(60).Furthermore,thephys- tiontoAktactivationandPMNsurvival.RecentlyZhengetal. iologicconsequenceoflossofAkt/MK2interactionandlossof (30) demonstrated that MK2 was required for p38 MAPK/ Ser473 phosphorylation resulted in cellular apoptosis. These Hsp27interaction.However,MK2wasnotrequiredforasso- studies collectively define a novel role of Hsp27 in regulating ciationofHsp27withAkt.GiventhatAkt/Hsp27interaction Aktactivationandapoptosisbymediatinginteractionbetween wasnotregulatedbyMK2andthatMK2servesasPDK2for AktanditsupstreamactivatorMK2. JULY27,2007•VOLUME282•NUMBER30 JOURNALOFBIOLOGICALCHEMISTRY 21607