Hsp27 Regulates Akt Activation and Polymorphonuclear Leukocyte Apoptosis by Scaffolding MK2 ...

12 Pages·2007·0.78 MB·English

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:[phone];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