The kinase activity of the Ser/Thr kinase BUB1 promotes TGF

12 Pages·2014·1.45 MB·English

R E S E A R C H A R T I C L E
CANCER
The kinase activity of the Ser/Thr kinase BUB1
b
promotes TGF- signaling
Shyam Nyati,1,2 Katrina Schinske-Sebolt,2 Sethuramasundaram Pitchiaya,3,4
Katerina Chekhovskiy,2 Areeb Chator,2 Nauman Chaudhry,2 Joseph Dosch,5
Marcian E. Van Dort,5 Sooryanarayana Varambally,6 Chandan Kumar-Sinha,6,7
Mukesh Kumar Nyati,2 Dipankar Ray,2 Nils G. Walter,3,4 Hongtao Yu,8,9
Brian Dale Ross,1,5 Alnawaz Rehemtulla1,2*
Transforming growthfactor–b(TGF-b)signaling regulatescellproliferationanddifferentiation,which
contributestodevelopmentanddisease.UponbindingTGF-b,thetypeIreceptor(TGFBRI)bindsTGFBRII,
leadingtotheactivationofthetranscriptionfactorsSMAD2andSMAD3.UsinganRNAinterferencescreen
ofthehumankinomeandalive-cellreporterforTGFBRactivity,weidentifiedthekinaseBUB1(budding
uninhibitedbybenzimidazoles-1)asakeymediatorofTGF-bsignaling.BUB1interactedwithTGFBRIin D
thepresenceofTGF-bandpromotedtheheterodimerizationofTGFBRIandTGFBRII.Additionally,BUB1 o
w
interactedwithTGFBRII,suggestingtheformationofaternarycomplex.KnockingdownBUB1prevented n
therecruitmentofSMAD3tothereceptorcomplex,thephosphorylationofSMAD2andSMAD3andtheir lo
a
interactionwithSMAD4,SMAD-dependenttranscription,andTGF-b–mediatedchangesincellularpheno- de
d
typeincludingepithelial-mesenchymaltransition(EMT),migration,andinvasion.KnockdownofBUB1also f
impairednoncanonicalTGF-bsignalingmediatedbythekinasesAKTandp38MAPK(mitogen-activated ro
m
proteinkinase).TheabilityofBUB1topromoteTGF-bsignalingdependedonthekinaseactivityofBUB1. h
Asmall-moleculeinhibitorofthekinaseactivityofBUB1(2OH-BNPP1)andakinase-deficientmutantof ttp
BlinUeBs1.2sOuHpp-BreNsPsPe1daTdGmFi-nbisstigrantaiolinngtoamndicfeobrmeaartiinognloufntghecatercrninaorymcaoxmepnloegxrianftvsarreioduuscendorthmeaalmanoducnatnocfeprhcoesl-l ://stk
phorylatedSMAD2intumortissue.ThesefindingsindicatedthatBUB1functionsasakinaseintheTGF-b e
.s
pathwayinarolebeyonditsestablishedfunctionincellcycleregulationandchromosomecohesion. c
ie
n
c
INTRODUCTION inhumans.Duringmitosis,BUB1bindskinetochoresandplaysakeyrole em
Thetransforminggrowthfactor–b(TGF-b)familyofcytokinesregulates inestablishingthemitoticspindlecheckpointandaligningchromosomes ag
manyprocessessuchasimmunesuppression,angiogenesis,woundheal- (7),inadditiontoitscentralroleinensuringfidelityduringchromosomal .or
ing,andepithelial-mesenchymaltransition(EMT)(1,2).AbnormalTGF-b segregationintodaughtercells(8).Threemainregionshavebeenidentified og/
signalingislinkedtoautoimmuneandinflammatorydiseases,fibrosis, inBUB1:aconservedN-terminalregionthatcontainsthekinetochorelo- n
tumorformationandmetastasis,andvariousotherdisorders.Earlyin calizationdomain;anintermediate,nonconservedregionthatisrequiredas Ja
n
tumorigenesis,theproliferationofepithelialcellsretainsexquisitesen- ascaffoldfortherecruitmentofproteins;andaC-terminalregionthatcontains u
a
sitivitytoTGF-b,whereinTGF-belicitsatumor-suppressiveresponse. acatalyticserine/threoninekinasedomain(9).MutationsinBUB1areasso- ry
However,transformedcellsbecomerefractorytoTGF-b–mediatedgrowth ciatedwithaneuploidyandseveraltypesofcancer. 8
inhibitionandacquireaphenotypewhereintheintracellularsignaling Ligand-dependentactivationofTGF-breceptorsandregulationoftheir , 2
0
circuitryisaltered,leadingtotumorigenicandmetastaticeffectsinre- subsequentkinaseactivityisacomplexprocessthatcaninvolveseveral 1
5
sponsetoTGF-b(3).Theseresponsesarediverse,dependingonsignal- posttranslationalmodificationsofthereceptors[includingautophosphoryl-
ingfromgrowthfactorreceptorsactivatedinparallel(4),celldensityand ation(10),cross-phosphorylation(11),trans-phosphorylation(12),ubiqui-
cellcyclephase(5),andtheabundanceandactivityoftranscriptional tylation(13),sumoylation(14),anddephosphorylation(15)],aswellas
regulators(6). internalizationofthereceptor-ligandcomplex(16–19).Withsomuch
BUB1(buddinguninhibitedbybenzimidazoles-1)isaserine/threonine complexityinonepathway,wesetouttofindpotentiallynewregulators
kinasethatisencodedbytheBUB1gene[40kilo–basepairs(kbp),25exons] ofTGF-breceptoractivity.
1CenterforMolecularImaging,UniversityofMichigan,AnnArbor,MI48109, RESULTS
USA. 2DepartmentofRadiationOncology,UniversityofMichigan,AnnArbor,
MI48109,USA. 3SingleMoleculeAnalysisinReal-Time(SMART)Center,Univer- Kinase-targeted high-throughput small interfering RNA
sityofMichigan,AnnArbor,MI48109,USA. 4DepartmentofChemistry,University screen identifies regulators of TGF-b signaling
ofMichigan,AnnArbor,MI48109,USA. 5DepartmentofRadiology,Universityof
Michigan,AnnArbor,MI48109,USA. 6DepartmentofPathology,Universityof ToidentifyproteinsthatregulatesignalingbythetypeITGF-breceptor
Michigan,AnnArbor,MI48109,USA. 7MichiganCenterforTranslationalPathol- (TGFBRI), wetransfectedsmallinterferingRNAs(siRNAs)against
ogy,UniversityofMichigan,AnnArbor,MI48109,USA. 8HowardHughesMed- eachofthe720knownandpredictedkinasesintoA549lungadenocar-
icalInstitute,UniversityofTexasSouthwesternMedicalCenter,Dallas,TX75390,
cinomaandMDA-231-1833breastcancercellsthatstablyexpresseda
USA. 9DepartmentofPharmacology,UniversityofTexasSouthwesternMedical
reporterforTGFBRIkinaseactivity[(BTR(4)]andscreenedforanin-
Center,Dallas,TX75390,USA.
*Correspondingauthor.E-mail:[email protected] creaseinbioluminescenceaftertheadditionofTGF-bligandtotheculture
www.SCIENCESIGNALING.org 6January2015 Vol8Issue358ra1 1
R E S E A R C H A R T I C L E
medium(fig.S1A).BTRisareporterthatexhibitsincreasedlumines- precipitationanalysisrevealedthateitherTGFBRIorBUB1knockdown
cenceuponlossofTGFBRIkinaseactivity;thus,positivehitsfromthe impairedSMAD3bindingtoTGFBRIinthepresenceofligand(Fig.2,A
siRNAscreenidentifygenesthatencodeproteinsthatpromoteTGFBRI andB,andfig.S3,AtoC).
kinaseactivity.Weusedaquartile-basedmethodtoidentifyproteinsthat, Uponphosphorylation,receptor-regulatedSMADs(R-SMADs,includ-
whenknockeddown,inducedmorethan7.14-foldactivationofthereporter ingSMADs1,2,3,5,and8/9)translocatetothenucleus,whereinthey
inA549cellsandmorethan13.91-foldinMDA-231-1833cells(high- regulatetranscriptionofspecifictargetgenes.Weevaluatedtheeffectof
stringencyhits,targetederrorratea=0.0027),followedbyexperiment- BUB1knockdownonthenucleartranslocationofSMAD2asasurrogate
andplate-wiseanalysisofthenormalizedfoldinduction(Fig.1,AtoC, fortheactivationoftheTGF-bpathwaybyimmunofluorescencemicrosco-
andtableS1)aspreviouslydescribed(20).Eighthitswereidentifiedin py.A549andH358cellsweretransfectedwithcontrolorTGFBRI-or
A549-BTRcells(Fig.1A),whereas15wererecoveredinMDA-231-1833– BUB1-targetedsiRNAstimulatedwithTGF-bfor1hour.Incontrolcells,
BTRcells(Fig.1B).TwoofthesehitswerethekinasesBMPR2(inA549- SMAD2waspredominantlylocalizedtothenucleusuponTGF-bstimula-
BTRcells)andRPS6KB1(inMDA-231-1833–BTRcells),whichare tion;incontrast,incellsdepletedofeitherTGFBRIorBUB1,SMAD2
alreadyassociatedwiththeTGF-bpathway(tableS2).Analysisatalower translocationtothenucleuswassimilarlyimpaired(fig.S3D).Together,
stringency(5.4-foldinA549cells,10.29-foldinMDA-231-1833cells,tar- theseresultssuggestthatBUB1playsanimportantroleinpromotingcanon-
getederrorratea=0.046)revealedadditionalhits,severalofwhichalso icalandnoncanonicalTGF-bsignaling.
haveknownrolesinregulatingTGF-bsignaling(tableS2),therebyfurther
validatingthescreen. BUB1promotesR-SMAD/SMAD4complexformationand
AlogicalrelationsanalysisoftheA549-BTRandMDA-231-1833– target gene transcription D
BTRscreensrevealedthatalargemajorityofthesignificanthitsweredis- ToextendourfindingthatdepletionofBUB1leadstoreducedR-SMAD o
w
tinctforeachcellline(Fig.1C);ofthese,sevenwerecommon(Fig.1C). recruitmenttothereceptorcomplexandhencereducedphosphorylation n
Signalingpathwayimpactanalysisdemonstratedthatsignalingnetworks andnucleartranslocation,weinvestigatedwhetherBUB1alsopromotes loa
previouslyimplicatedinpromotingthecellularresponsetoTGF-b[includ- SMAD2/3-SMAD4complexformationusingcoimmunoprecipitation de
d
ingthosemediatedbyreceptortyrosinekinases(RTKs),mitogen-activated assays. As expected, depletion of TGFBRI in A549 cells impaired f
proteinkinase(MAPK),andphosphoinositide3-kinase(PI3K)–AKT]were TGF-b–inducedformationofaSMAD2/3-SMAD4complex(Fig.2C ro
m
identifiedashitsinthescreen(tableS3andfig.S1,BandC)(4).Targets andfig.S4).ToevaluatearoleforBUB1inTGF-b–inducedgeneex- h
withintheprogesterone-mediatedoocytematurationandtheoocytemeiosis pression,weusedtheSBE4-Lucreporter(6).InA549cellstransfected ttp
ppearthtuwrbayatsi,oinnawchciucmhuBlaUtiBon1iisnaAk5e4y9c-BomTRpoannednt,McoDnAsi-s2t3en1t-l1y8e3x3h–iBbiTteRdcheigllhs wacittihvictyo,nbtruotltshiiRsNinAdu,cTtGioFn-wbainsdaubcreodgaatetdimbey-ddeeppelnetdioenntoinfcBreUaBse1i(nFriegp.o2r,teDr ://stk
(fig.S1,BandC). andE).Similarobservationswereseeninlung(NCI-H358),breast(MDA- e.s
BUB1 promotes canonical and noncanonical 231O-1n8e33o)f,tahnedecxeprrveiscsailo(nHseiLgan)atcuarnecseirncdeullcelidnebsy(FTiGg.F2-b,FsitgonHal)i.ngisthat cien
c
TGF-b signaling whichpromotesEMT,aprocessinepithelialcellsthatoccursduringdevel- e
m
Inanefforttobiochemicallyvalidatethesefindings,weknockeddown opmentanddiseaseprogressioninresponsetospecificstimuli(23).Addi- a
g
selecthitsinMRC5(humanfetallungfibroblast),Het1A(humanesoph- tion of TGF-b to epithelial cells in culture induces features of EMT, .o
agealepithelial),A549-BTR(lungadenocarcinoma),MDA-231-1833– includingtheadoptionofaspindle-likemesenchymalmorphologyand rg
BTR (breast cancer), MCF7 (breast cancer), and MCF10A (human invasivebehavior(24–26).Therefore,weinvestigatedtheroleofBUB1 o/
n
mammaryepithelial)cells.BUB1knockdowninducedthemostrobust inTGF-b–mediatedEMT.Asexpected,culturesofA549orNCI-H358cells J
a
decreaseinTGF-b–dependentSMAD2phosphorylation(fig.S2)and exhibitedatransitiontoamesenchymalphenotypeinresponsetoTGF-b; n
u
wasselectedforfurtherstudy.Similartoapositivecontrol,inwhich however,TGFBRIorBUB1knockdownpreventedthistransition(fig.S5, a
r
y
TGFBRI was silenced, knockdown of BUB1 in lung cancer (A549 AtoE).SimilartotheeffectofsilencingTGFBRI,knockingdownBUB1 8
andNCI-H358)andbreastcancer(MDA-231-1833)celllinesusingsiRNAs significantlyinhibitedTGF-b–mediatedinductionofcellmigrationandin- , 2
abrogatedtheactivationofSMAD2andSMAD3,aswellascomponents vasioninculture(fig.S6,AtoD).Theseresultsfurthersupportarolefor 01
ofthenoncanonicalsignalingcascade(c-JUN,p38MAPK,andAKT)in BUB1inmediatingTGF-bsignaling. 5
eachofthecelllines(Fig.1D),keydownstreameffectorsofTGF-bsignal-
ing(21,22).TransfectionwithBUB1siRNAcausedasimilardecreasein Catalytic activity is required for BUB1 to mediate
TGFBRIabundanceasdidTGFBRIsiRNA,butonlyinA549cells.Even TGF-b signaling
still,inthiscellline,theeffectsofBUB1siRNAdidnotmimictheeffectsof ToconfirmthatthekinaseactivityofBUB1mediatedTGF-bsignaling,we
TGFBRIsiRNA.Thus,theeffectsofBUB1knockdownondownstream conducted rescue experiments in A549 cells using siRNA-resistant
markersofTGF-bsignalingwerenotanartifactofoff-targetorindirect constructsofBUB1(27).IncellsdepletedofendogenousBUB1,restora-
knockdownofTGFBRI. tionwithwild-typeBUB1,butnotakinase-deficientBUB1mutant,res-
cuedSMAD2/3phosphorylationinaligand-dependentmanner(Fig.3A
BUB1 mediates TGF-b–dependent recruitment andfig.S7A).Inagreementwiththisobservation,reporteractivityin
of R-SMADs to the activated receptor BUB1-depletedA549cellsexpressingtheSBE4-Lucpromoterwasre-
BecausedepletionofBUB1reducedTGF-b–mediatedSMAD2/3phos- storeduponexpressionofwild-type,butnotakinase-deficient,BUB1
phorylation,wehypothesizedthatBUB1maymediatetheefficientre- (Fig.3B),demonstratingarequirementforBUB1’skinaseactivityinitsrole
cruitment of SMAD2/3 to the activated receptor in the presence of inTGF-bsignaling.Theconcentrationof2OH-BNPP,acatalyticBUB1
ligand. To test this hypothesis, A549 and human embryonic kidney inhibitor(9),negativelycorrelatedwiththephosphorylationofSMAD2
(HEK)293TcellsweretransfectedwithFlag-taggedSMAD3andHis- andSMAD3(fig.S7,BtoD).BlockingtheactivityofBUB1with2OH-
taggedTGFBRIinthepresenceofcontrolorTGFBRI-orBUB1-targeted BNPP1dose-dependentlyimpairedtheTGF-b–inducedphosphorylationof
siRNAandsubsequentlytreated withTGF-bfor 1hour.Coimmuno- proteinsthatmediate canonicalandnoncanonical TGF-bsignaling in
www.SCIENCESIGNALING.org 6January2015 Vol8Issue358ra1 2
R E S E A R C H A R T I C L E
A C A549 BTR 1833 BTR
20
a b c a b c
GUCY2C PANK1
e 16 JAK2 CSNK1G3
g
n GRK1
ha EPHB4 NME5
d c 12 PRKAA1
fol BUB1 BRDT BPIU4BK1II
TR 8 DGKQ BMPR2 PLK3 MAPK9
9 B FASTK
4 PRKAG1
A5 4 HSPB8
MAPK3
NEK6
0
MAP3K15
0 120 240 360 480 600 720
DYRK2
Target number NYD-SP25
B
50 BLK
GRK1 DGKQ
NYD-SP25 EPHB6 D
e 40 RPS6KB1 o
g w
han FRDA JKASRK22 nlo
d c 30 CIT ad
TR fol 20 FASKTPI4KII JRAPKS26KB1 BRD4 AATK TSK5 RSRRPAPSG6KEK1A6 ed fro
1833 B 10 BBULBK1 MAPN3EKK165 KSR2 BNLMRMBTKPP22R2 httpm
0 BFRRDDA4 ://stk
0 120 240 360 480 600 720 CDC2L5 e
ALK .s
Target number c
AATK ie
ERBB3 n
c
CRKL e
D A549 H358 1833 m
STK4
a
siTGFNBSRSI CPSIKN3KC1DA1 g.or
siBUB1 PFKFB2 g
(1 hour) BRDT o/
GUCY2C n
pSMAD3 ZAK Ja
PLK3 nu
SMAD3 XYLB a
r
TSKS y
pSMAD2 IETPPHKBB4 8, 2
0
SMAD2 1
5
0 12
BUB1
TGFBRI Fig.1.BUB1mediatesTGF-bligand–dependent
SMAD2/3 phosphorylation,aswellasMAPK
pc-JUN andAKTactivation.(AandB)Plotoftheinduc-
tionofaTGFBR1reporter(BTR)inA549cells
pP38MAPK (A) and MDA-231-1833–BTR (B) cells trans-
fectedwithsiRNAstargetinghumankinases.
pAKT
Red or blue triangles and circles indicate
AKT high-stringency (triangles) or low-stringency
(circles)hitseitherinplate-and experiment-
GAPDH wiseanalyses(red)orinexperiment-wiseanal-
ysis only (blue). Data are representative of
threeindependentexperiments.(C)Heatmapoflow-stringencyhitsinA549-BTRandMDA-231-1833–BTRcellsfromtriplicateexperiments.Genename
initalicsindicatescommonhits.(D)WesternblotanalysisofTGF-beffectormoleculesinA549,NCI-H358,andMDA-231-1833cellstransfectedwithsiRNA
againsteitherBUB1(siBUB1)orTGFBRI(siTGFBRI)orascrambledcontrolsiRNA[nonsilencingsiRNA(NSS)]inthepresenceofTGF-bligandfor1hour.
Blotsarerepresentativeofthreeindependentexperiments.
www.SCIENCESIGNALING.org 6January2015 Vol8Issue358ra1 3
R E S E A R C H A R T I C L E
multiplecancercelllines(Fig.3,CtoE)andnormalcelllines(fig.S7,E SMAD2(fig.S7,GandH),SMAD2/3-SMAD4complexformation(Fig.3,
andF).SimilartotheeffectsofapharmacologicalTGFBRIinhibitor FandG),andSBE4-Lucreporteractivity(Fig.3,HandI,andfig.S7,I
[SB431542(28)], 2OH-BNPP impaired the nuclear translocationof andJ)inresponsetoTGF-binvariouscelllines.
A NSS B NSS C NSS BUB1 colocalizes and interacts
siTGFBRI siTGFBRI siTGFBRI with TGFBRI in response to
siBUB1 siBUB1 siBUB1 TGF-b stimulation
FL-SMAD3 FL-SMAD3 3 - OnthebasisoftheobservedroleofBUB1
His-TGFB-RI His-TGFB-RI IP:MAD2/ IB: SMAD4 iTnGpFroBmRoIti(nFgigt.h2e,reAcraunitdmBen,taonfdSfMigA.SD33,tAo
FBRI IB: FL FBRI IB: FL S SMAD4 andB)andpreviousevidenceofascaffold-
TG TG ingfunctionforBUB1(7–9,29),weinves-
P:IB: His P:IB: His pSMAD3 tigatedwhetherBUB1physicallyinteracts
I I
withTGFBRItomediateitsactivityusing
BUB1 BUB1
SMAD3 totalinternalreflectionfluorescence(TIRF)
Input His Input His ut pSMAD2 mthiecproressceonpceyo(f3B0U).BT1IRanFdiTmGaFgBeRsIreinvepaulnecd-
FL FL Inp SMAD2 tpaltaensetr(uFcitgu.re4sAw).itShpinecthtrealplaansamlyasmisedmetbercatnede Do
w
negligiblecolocalizationofTGFBRIand n
TGFBRI BUB1at1and24hoursafterligandstim- loa
uction)D108 247482 hhhooouuurrrsss ****** E NSS GABPUDBH1 7utdh2aleanhtcsiooieugnornsfa(wcflyiifgttrho.opTSmlaG8stAFmh-)eib,cr,pBetohlUaestBsipiv1rbeo.llyIpynobhcrteoiigconhatnruaaossbfet,ucbonoyf-- hded from
SBE4-Luc (fold ind 246 LucisfiBeBrUUaBBs-1e1 lbecoxolecpndaWrsleti(zreFsuesidncgiote.Tnx4wtG,oaeAFfsxBaBstmouRUfIiCfnBia)ec1.ndiedunwsBthinUfeogtBhrea1irtsMsfioignyrnctceae-rldtsaacgodtvgoieoeurnd-- ttp://stke.scie
withTGFBRI,andifthiswouldaffectthe n
c
0 GAPDH phosphorylationofSMAD2/3.Heterologous em
NSS siBUB1 expressionofBUB1inA549cellsresulted a
g
inthecoimmunoprecipitationofBUB1and .o
F 20 ** G 4 H 2.0 TGFBRIregardlessofTGF-b ligand(Fig. rg
4Dandfig.S8B),butthiswasnotsufficient o/
uction)16 uction) 3 ** uction) 1.6 ** taobsinenitciaeteoSfMligAanDd2,/o3nplyhomspohdoersytllyatiionncrienastehde n Janu
old ind12 old ind 2 old ind 1.2 pinhothspehporreysleatniocenooffSTMGAFD-b3((Fbiugt.n4oDtSaMnAdDfi2g). ary 8
BE4-Luc (f 48 BE4-Luc (f 1 BE4-Luc (f 00..48 STb–8GsCFti)Bm.ROuIlvaietnerdeduxcpinerdetesarsaiomcntaioroknfedbbloeytthwgreBeeaUnteBrB1TUGaBnFd1- , 2015
S S S
and TGFBRI and further increased the
0 0 0.0 phosphorylationofSMAD3(Fig.4Dand
NSS siBUB1 NSS siBUB1 NSS siBUB1
- - - - - - fig.S8,BandC).Theseresultsdemonstrate
thatoverexpressionofTGFBRIand/orBUB1
Fig. 2. BUB1promotestherecruitmentofSMAD3toTGFBRI,SMAD2/3-SMAD4complexformation,and enhancestheactivationofthesignalingpath-
transcriptionalresponse.(AandB)Immunoprecipitation(IP)fortheTGFBRIantibodyandthenimmuno- wayonlyinthepresenceoftheTGF-bligand.
blotting(IB)fortheFlagorHistagsinHEK293T(A)andA549(B)cellstransfectedwithcontrolsiRNA Myc-taggedBUB1coimmunoprecipitated
(NSS),TGFBRIsiRNA,orBUB1siRNAalongwithFlag-taggedSMAD3(FL-SMAD3)and6XHis-tagged withaHis-taggedcytoplasmictailofTGFBRI
TGFBRI(His-TGFBRI)andtreatedwithTGF-b(1hour).(C)IPforSMAD2/3followedbyblottingforSMAD4 fromA549celllysates(Fig.4E),demon-
inlysatesfromA549cellstransfectedwithcontrolsiRNA,TGFBR1siRNA,orBUB1siRNAandtreatedwith stratingthatBUB1mayinteractwiththe
TGF-b(1hour).(D)Relativefireflyluciferaseactivity(normalizedtoGaussialuciferase)afteradditionofTGF-b cytoplasmictailofTGFBRI.
(10ng/ml)andtransfectionwithmock(NSS)orBUB1siRNAfor24to72hoursinA549cellstransientlytrans-
fectedwithanSBE4-LucreporterandGLucplasmids.(E)ImmunoblottingforluciferaseandBUB1inlysates BUB1mediatesTGF-b–dependent
fromA549cellstransfectedandtreatedasin(D)andharvested24hoursafterTGF-btreatment.(FtoH) TGFBRI-TGFBRIIheteromeric
RelativeluciferaseactivityinNCI-H358(F),MDA-231-1833(G),andHeLa(H)transfectedasin(D) complexformation
andtreatedwithTGF-b(10ng/ml)for24hours.Blotsarerepresentativeofthreeindependentexperiments. Inthepresenceofligand,TGFBRIisre-
Dataaremeans±SEMofthreeindependentexperiments.**P<0.001,two-sidedStudent’sttest. cruitedtothetypeIIreceptor(TGFBRII)
www.SCIENCESIGNALING.org 6January2015 Vol8Issue358ra1 4
R E S E A R C H A R T I C L E
AsiBNubS1S B 6 ** andformsastablereceptorcomplexthrough
MMyycc--BBUUVBBe11c W tKoDTr nduction)4 0.02 trroalnesf-oprhoBsUphBo1ryinlattihoenf(o3r1m).aTtioonexoafmthiinsehtehte-
d i eromericreceptorcomplex,A549cellswere
ol
pSSMMAADD33 SBE4-Luc (f2 itsmriaRmnNsufAencoatpnerddectwripeiaittthaetdecsowfnirttohrmoTlGtohFre-Bbre,UsauBnltd1in-TgtaGrlygFseBattReedsI
pSMAD2 0
NSS wereprobedforTGFBRII.BUB1depletion
SMAD2 siBUB1 abrogated the formation of the TGFBRI-
Vector
BUB1 Myc-BUB1 WT TGFBRIIcomplex(Fig.4F).Theabundance
Myc Myc-BUB1 KD ofSARA[SMADanchorforreceptoractiva-
- (1 hour)
GAPDH tion(32)]wasdetectedintheimmunoprecipi-
C D E tatedfractionirrespectiveofBUB1expression
(Fig.4F).TGF-b signalingisknowntode-
pendonthecellcycle(33),andBUB1isa
kcoM kcoM kcoM knownregulatorofthecellcycle(8);however,
pSMAD2 wedidnotobservecellcyclearrestinBUB1-
depletedcells(fig.S8D). D
SMAD2 TotestwhetherBUB1kinaseactivity o
w
playedaroleintheformationofthehetero- n
pSMAD3 meric receptor complex, we transfected loa
d
SMAD3 A549cellswithHis-taggedTGFBRIfol- e
d
lowedbytreatmentwith2OH-BNPP1for f
pP38MAPK 1hourandstimulationwithTGF-bforan ro
m
P38MAPK additional hour. The TGFBRI inhibitor h
SB431542wasusedinparallelexperiments ttp
pc-JUN asacontrol.InhibitionofBUB1kinaseac- ://s
tivitysignificantlyreducedtheformationof tk
pAKT theTGFBRI-TGFBRIIcomplex(Fig.4G e.s
AKT andfig.S9).InhibitionofBUB1kinaseac- cie
tivityalsodecreasedthecoimmunoprecipi- n
c
TGFBRI tationofMyc-taggedBUB1withHis-tagged em
TGFBRI(Fig.4H).Becausethekinaseac- a
GAPDH g
tivity of BUB1 promoted the TGFBRI- .o
r
IP:InputSMAD2/3FIBpp: SSSSSSMMMMMMAAAAAADDDDDD443223 kcoM kcoM 245134BS- 1PPNB-HO2 IP:nputSMAD2/3GIBpp: SSSSSMMMMMAAAAADDDDD42324 kcoM kcoM 245134BS- 1PPNB-HO2 HSBE4-Luc (fold induction)02468 Mock ** ISBE4-Luc (fold induction)1122050505 Mock ** TiwTB(Ft(nafflGGUihittaggeeeFgFB.dr.taBh-BS1StcewR1aR1trwig1hI0oIB.)gaIe)n,IsUe,nncMdwanoBevnoymi1SxitdttchprMp-caorBaloTeeAldkuUxtsGiihlsDrndBeoFfeaoc2d1uBdstregimdRrcikheanioiIadcsn,iMsttHmalianwyosyEoymenesptcKfhur-piaoenB2onnhrvvs9dooUepeT3spahsBipGTlrttoesihe1FgdrcocyoaBaiertlwthpaynRletalildndes--It on January 8, 2015g/
BUB1 I SMAD3 ate purified SMAD3 invitro (fig. S12).
GAPDH BUB1 Thus,albeitimportant,thetargetofBUB1’s
GAPDH kinaseactivity in the TGFBRIsignaling
complexwaselusive.
Fig. 3.BUB1inhibitor2OH-BNPP1 abrogates TGF-b signalingin adose-dependent manner.(A)Im-
munoblotting for total and phosphorylated(p) proteinsas indicatedinlysates from A549 cells tran- BUB1 interacts with TGFBRII in
sientlytransfectedwithcontrol siRNA(NSS) orBUB1siRNA alongwithwild-typeBUB1(Myc-BUB1 response to TGF-b stimulation
WT)orakinase-deficientmutant(Myc-BUB1KD),serum-starved,andthentreatedwithTGF-b (10ng/ml, WenextevaluatedwhetherBUB1alsoin-
1hour).(B)RelativeluciferaseactivityafteradditionofTGF-binA549cellculturesdescribedin(A)expressing teractedwithTGFBRII,whichwouldbe
theSBE4-LucandGLucplasmids.Dataaremeans±SEMofthreeindependentexperiments.(CtoE) indicativeoftheformationofaternarycom-
ImmunoblotsoflysatesfromA549(C),NCI-H358(D),andMDA-231-1833(E)cellstreatedwithvehicle, plexbetweenBUB1,TGFBRI,andTGFBRII.
vehicleandTGF-b(10ng/ml),orTGF-bandtheindicatedconcentrationof2OH-BNPP1for1hour.(Fand HEK293TcellsweretransfectedwithHis-
G)IPforSMAD2/3andthenblottingforSMAD4inlysatesfromA549(F)andNCI-H358(G)cellstreated TGFBRIandwild-typeMyc-BUB1andtreated
withvehicle,vehicleandTGF-b,orTGF-bandeitherSB431542or2OH-BNPP1(10mM)for1hour.(HandI) with2OH-BNPP1for1hour,followedby
RelativeluciferaseactivityinA549(H)andNCI-H358(I)cellstreatedasin(F)and(G)for24hours.Dataare stimulationwithTGF-bforanadditional
means±SEMofthreeindependentexperiments.**P<0.001,two-sidedStudent’sttest.Blotsarerepresent- hour.Coimmunoprecipitationassaysrevealed
ativeofthreeindependentexperiments;blotsfrom(C)to(E)arequantifiedinfig.S7. anincreaseinTGF-b–stimulatedinteraction
www.SCIENCESIGNALING.org 6January2015 Vol8Issue358ra1 5
R E S E A R C H A R T I C L E
betweenTGFBRIIandBUB1inHEK293Tcellsinakinase-dependent treatedmice(Fig.5,AandB),supportingaroleforBUB1inSMAD
manner(Fig.4I).Inparallelstudies,treatmentofcellswiththeTGFBRI- activation.
specificinhibitorSB431542didnotaffectthisinteraction(Fig.4I). Together,ourdataindicatetheformationofaternarycomplexbetween
TGFBRI,TGFBRII,andBUB1,andthatthekinaseactivityofBUB1may
BUB1 inhibitor 2OH-BNPP1 abrogates SMAD2 promotecanonicalandnoncanonicalTGF-bsignaling(Fig.6).
phosphorylation in vivo
Wefolloweduponourcellularstudiesbyevaluatingtheabundanceof
DISCUSSION
phosphorylated SMAD2 in subcutaneous A549 tumor xenografts
harvested4hoursaftermiceweretreatedwith2OH-BNPP1.Similar ReversegeneticscreensinmammaliancellsusingsiRNAhavebeenused
totumorsfrommicetreatedwiththeTGFBRIinhibitor,tumorsfrom forassessing gene function, target validation, pathwayanalysis,gene
2OH-BNPP1–treated mice had a significantly decreased abundance redundancy,andtherapeutictargetingofseveraldisorders.Inaneffortto
of phosphorylated SMAD2 compared with tumors from vehicle- elucidatenovelregulatorsoftheTGF-bpathway,siRNAlibraryscreens
BUB1 TGFBRI Merge
Fig.4.BUB1colocalizeswithTGFBRI,coim- A D Vector
munoprecipitateswithTGFBRIandTGFBRII, Myc-BUB1
andpromotesheteromericTGFBRI/IIcom- His-TGFBRI
palnedxTfoGrmFBatRioInc.o(lAo)caTlIiRzFatiaonnaliynsiAs5o4f9BcUeBll1s Mock IP:TGFBRITGF-βIB (:1 Mhoyucr) Dow
treatedwithTGF-b(10ng/ml)for72hours. n
Scalebar,10mm.(B)LinescanacrossBUB1 Myc loa
d
and TGFBRI particles within inset of (A). His e
d
(C)Extent of colocalizationnormalizedto f
themocksample.Dataaremeans±SEM - TGFBRI ro
m
of three independent experiments (1000 put h
particles,>20cells).(D)IPforTGFBR1and In pSMAD3 ttp
thenblotting forMyc inlysatesfrom A549 B C SMAD3 ://s
cells transfected with Myc-BUB1 and/or Mock tk
His-TGFBRI,andtreatedwithTGF-b.1hour: pSMAD2 e.s
10ng/mlfor1hour.(E)IPforHisandthen SMAD2 cie
blottingforMycinlysatesfromA549cells n
transfectedwithMyc-BUB1andHis-tagged GAPDH cem
cytosolicdomainofTGFBRI.(F)IPforTGFBRI E Vector F NSS 1 2 3 4 5 6 7 8 ag
andthenblottingforTGFBRIIandSARAin His-TGFBRI cytopls siBUB1 .o
lctyreosnaatttreeodlsowfrriotBhmUTBAG15Fs4-bi9R.N(cGAe),llssIPetrrfuaomnrs-TsfGteacFrvtBeeRddI,waannitddh IP: HisMyIcBI-BB: :uM Hbyi1sc IP:TGFBRITIGBF:I- BβT :G( 1SF AhBoRRuAIrI) on Jrg/
thenblottingforHisandTGFBRIIinlysates BUB1 an
fsreormumA-5s4ta9rvceedlls,atranndsfterecateteddwwithithHSisB-T4G3F1B5R42I, nput MHyisc Input TGFSBARRIAI uary 8
or2OH-BNPP1(1hour)andthenTGF-b.(H) IGAPDH TGFBRI , 2
ITwa(P1GnithdfhFooBHrturRiTesrI-G)aITtiGaFnenBFdlydBRswRIathiIatteehansnndSdfrTBothMGm4ey3FncA1-b-5b5B.44lUo(92ItB)tic1noIeP,grlsl2sfefooOrturrraHmMMn--sBysyftcceNarcPvaatePnendddd1, HiGsT2-GTOFSG-HB Fβ-4BB V3(RN1e1I cPh5 WtoP4ou12Trr) +–––– +–––– ++––– +++–– +++–– GAHPMiDsy2TH-cTGO-SGF HBB-F -U4βHBB3B RN(111IP5 WhWP4o12TTuIrg)G++––– IP ++–––TG+++––FBRI++++– IP ++++– I MycHMiTsy2G-IcTBOF-SG- H:BB βF T-u4B BG(3bR1NF11 IhBP5 WWoRP4uI12TTrI) ––––– ++––– +++–– ++++– ++++– 015
tHahnEedKn2Hb9isl3o-TTttGicnFegBllsRfoIt,rrasTenGrsufFemBc-tsRetaIdIrviwneidthl,yasMnaydtecst-rBefUarotBemd1 IP: TGFBRIIB: TGIBF:B HRiIsI P:TGFBRI IBIB: :M Hyics IP: TIGBF: BMRyIcI
I
with 2OH-BNPP1 or SB431542 (10 mM, Myc
1hour)andthenTGF-b.Blotsarerepresent- TGFBRII Myc
ativeoftwo(I)oratleastthree(DtoH)in- TGFBRI His
dependentexperiments. His
Input pSSMMAADD33 nput pSMAD3 Input pSSMMAADD33
I
pSMAD2 SMAD3 pSMAD2
SMAD2 pSMAD2 SMAD2
SMAD2 GAPDH
www.SCIENCESIGNALING.org 6January2015 Vol8Issue358ra1 6
R E S E A R C H A R T I C L E
haveidentifiedkeyregulatorsofTGF-bsignaling(34–40),althoughscreens Het1A)aswellastumorcelllinesofvariousorigins(A549,NCI-H358,
usingTGFBRIkinaseactivityasareadouthavenotbeenconducted.The MCF7,andMDA-231-1833)demonstratedarequirementforBUB1ex-
BTRreporterisuniqueinthatlossofTGFBRIkinaseactivityresultsina pression in the cellular response to TGF-bstimulation, and thus was
gainoffunction(increaseinbioluminescencesignal),andthusislesslikely followedupformechanisticstudies.AdditionalconfidenceforBUB1as
toresultinnonspecificoroff-targetbiologicaleffectsthanarecommonly atruehitwasderivedfromstudiesdemonstratingthatdepletionofBUB1
observedintraditionalluciferase-basedscreens(4).Recentstudiesusinga inthreeindependentcelllinesresultedinabrogationofcanonical(phos-
GFP(greenfluorescentprotein)–fusedSMAD2reporterinalarge-scale phorylatedSMAD2andphosphorylatedSMAD3)aswellasnoncanonical
RNAi(RNAinterference)screenobservedthatabout1%ofhitsexhibited (phosphorylatedp38MAPK,AKT,andc-JUN)downstreameffectormol-
off-targeteffects(41).Incontrast,depletionoftargetgenesthatleadsto ecules.BecauseTGF-bsignalinghasbeenshowntobecellcycle–dependent
cellulartoxicityoroff-targeteffectswouldbescoredasfalsenegativebut (49,50),weaddressedthepossibilitythatBUB1(aregulatorofmitosis)
notfalsepositivewhenusingtheBTRreporter.Thisprovideduswithcon- knockdownresultsinadecreaseinTGF-b–mediatedsignalingduetothearrest
fidencethatsiRNAsthatresultedinanincreaseinreporteractivityrepre- ofcellsinmitosis.ExistingevidenceindicatesthatsiRNAmediatingcom-
sentedtruehits.Alackofoverlapbetweenhitsderivedfromthetwocell pletedepletionofBUB1doesnotresultinacellcyclearrest(51),afinding
linesisnotsurprisingconsideringthatthephysiologicresponseofcellsto confirmedinourstudies(fig.S9).Inaddition,cellcycledependenceof
TGF-bstimulationisoftencelltype–andcontext-dependent(42,43).We TGF-b–mediatedcellularresponsesisunderstoodtobeadownstreamevent
focusedonsixhitsthatwerecommonbetweenthetwocelllines,assuming whereinchangesinR-SMADphosphorylationarenotalteredinacell
thatthesemayrepresentimportantbiologicalregulators.Ofthese,BMPR2, cycle–dependentmannerbutareratherregulatedthroughinteractionwith
RPS6KB1,NEK6,andJAK2havebeendescribedtobeassociatedwiththe SKIandSNON(5).Last,theabilityofaBUB1kinaseinhibitortoabrogate D
TGF-bpathway(44–48).However,theidentificationofBUB1inoursiRNA TGF-b–dependentsignalingwithinanhouroftreatment,aperiodduring o
w
screenusingtheTGFBRIkinaseassaywassurprisingandunexpecteddue whichacellcyclearrestwouldnothaveoccurredfurther,supportsadirect n
tothepreponderanceofliteraturedescribingBUB1asacomponentofthe roleforBUB1inreceptor-mediatedsignaling. loa
d
mitoticmachinery,whereinitensuresbipolarattachmenttospindlemicro- ThefunctionalinsightsobtainedinthecurrentstudysuggestthatBUB1 e
tubulessothatchromatidsegregationcanoccurwithhighfidelity(7,8). isrecruitedtoTGFBRIinaligand-dependentmanner.Coimmunoprecipi- d f
r
Confirmatorystudiesconductedinnormalcells(MCF10A,MRC5,and tationstudiesdemonstratedrecruitmentofBUB1toTGFBRIwithinan o
m
hourofTGF-btreatment,whereasTIRFmicroscopydemonstratedcoloca- h
A MMMoooccckkk (((DDDMMMSSSOO)) B lizationonlyatthe72-hour,butnotearlier,timepoint.Superiorsensitivity ttp://s
tk
e
.s
c
ie
n
60 ** ** ce
m
a
g
50 .o
r
g
SSSBBB444333111555444222 (((111000 mmmggg///kkggg))) 40 nn == 24 SMBo4c3k1 542 MEMBRANE on/
n = 5 2OH-BNPP1 Ja
n
u
30 BU B 1 ary
R-SMAD 8
20 , 2
0
1
5
P
10 -
R-SMAD
222OOOHHH---BBBNNNPPPPPP111 (((555000 mmmggg///kkggg)))
0
Mock SB431542 2OH-BNPP1 -
Treatments -
Fig. 5. BUB1 inhibitor blocks TGF-b
signaling in vivo. (A) Representative
EMT/migration/invasion
immunohistochemistry staining for
phosphorylated SMAD2 in A549 xe-
nografts harvested from SCID mice
4hoursaftertreatmentwith2OH-BNPP1 Fig.6. AmodelforaroleofBUB1inTGF-bsignaling.Onthebasisofour
(50mg/kg),SB431542(10mg/kg),orvehicle[dimethylsulfoxide(DMSO)]. findings,weproposethatBUB1formsaternarycomplexwithTGFBRIand
Scalebar,200mm.(B)Numberofcellsstainingpositivefornuclearphos- TGFBRII.OurdatasuggestthattheinteractionofBUB1withTGF-breceptors
phorylatedSMAD2incontrol(n=4),SB431542-treated(n=2),or2OH- isenhanceduponTGF-bstimulation,requiresthekinaseactivityofBUB1,
BNPP1–treated (n =5) mice bearingtumors. Data are means ±SEM, andpromotesstabilizationoftheheteromericcomplexbetweenTGFBRI-
cellcountsfromthreerandomfieldsforeachtumor.**P<0.001,two-sided TGFBRII,R-SMADrecruitment,andsubsequentcanonicalandnoncanonical
Student’sttest. TGF-bsignalingcascades.
www.SCIENCESIGNALING.org 6January2015 Vol8Issue358ra1 7
R E S E A R C H A R T I C L E
andsignaltonoiseofthecoimmunoprecipitationexperimentsmayex- gle’smedium(DMEM)inthesameconditionsmentionedabove.NCI-
plainthisdiscordantobservation.Alternatively,BUB1-TGFBRIinter- H358lungcancercelllinewasprovidedbyD.Beer(UniversityofMichigan,
actions potentially occur at membrane-proximal regions, which are AnnArbor).NormallungfibroblastcelllineMRC5,esophagealcellline
opticallyinaccessiblebyTIRFmicroscopy,atearliertimepoints,and Het1A,breastepitheliallineMCF7,cervicalcancercelllineHeLa,andnormal
thesecomplexesareenrichedattheplasmamembraneonly72hoursafter kidneycelllineHEK293TweremaintainedinDMEM,whereasnormal
ligandtreatment—ahypothesisthatisstillconsistentwithcoimmuno- breastepithelialcelllineMCF10Awasmaintainedinspecialmedium.
precipitationexperimentsthatsamplewhole-celllysates.Additionally, AllcelllineswereobtainedfromATCC.Cellculturesweregrowninahu-
arequirementforBUB1inthestabilizationofthetypeIIandIreceptor midifiedincubatorat37°Cand5%CO.A549andMDA-231-1833cell
2
complex,R-SMADrecruitmenttothereceptor,aswellasco-SMAD/R-SMAD linesstablyexpressingwild-typeBTRandmutantreporterweregenerated
complexformation,andthereforeinthedownstreamtranscriptionalre- andmaintainedasdescribed(4).
sponse,wasalsodemonstrated.Furthermore,ourfindingsusingakinase
dead(KD)mutantofBUB1aswellasasmall-moleculeinhibitordemon- Antibodies, reagents, and siRNA library
stratedthatthekinaseactivityofBUB1promotestherecruitmentofBUB1 Antibodiestophosphorylated(pSer465/467)ortotalSMAD2,TGFBRI,
toTGFBRIandTGFBRII,aswellasforTGFBRI-TGFBRIIcomplexfor- TGFBRII,pSer63c-JUN,pSer473ortotalAKT,pThr180/Tyr182ortotal
mationuponligandstimulation,leadingtothepropagationofthecellular p38MAPK,andGAPDH(glyceraldehyde-3-phosphatedehydrogenase)
responsetoTGF-b.ThebiologicalsignificanceofBUB1’sroleinTGF-b wereallfromCellSignalingTechnology.AntibodyagainstSMAD3was
signalingwasemphasizedbythefindingthattreatmentoftumor-bearing fromInvitrogen;antibodiestoTGFBR1(H100)andTGFBRII(L21)were
animalswith2OH-BNPP1resultedinalossofTGF-b–mediatedSMAD2 fromSantaCruz;antibodiestoMyc-tagandfireflyluciferasewerefrom D
phosphorylationtoanextentsimilartoSB431542,aninhibitorofTGFBRI Millipore;antibodiestoNterminusofBUB1werefromAbcam(rabbit) o
w
kinaseactivity.OurinvitrokinaseassaysfailedtodemonstrateTGFBRIor orSanta Cruz(goat);antibodytoFlag–horseradishperoxidase (HRP) n
SMAD3asdirectsubstratesofBUB1,althoughweobservedaninteraction wasfromSigma;andantibodiestoHis-HRPwerefromInvitrogen(clones loa
d
betweenBUB1andSMAD2inHEK293Tcells.Futurestudieswillfocus H3andC-term)orMillipore(H8clone).HRP-andfluorophore-conjugated e
onidentificationofsubstratesofBUB1kinaseactivitywithintheTGF-b secondaryantibodieswerefromJacksonImmunoResearch.Recombi- d f
signalingpathway.AlthoughweshowthatBUB1formsaternarycomplex nanthumanTGF-b1wasobtainedfromHumanZyme.TGFBRIinhibi- ro
m
byinteractingwithTGFBRI,aswellasTGFBRII,therequirementfor torSB431542wasobtainedfromCaymanChemical.D-Luciferinwas h
TGFBRIIintherecruitmentofBUB1toTGFBRIwasnotdirectlyinvesti- fromXenogenCorp.ThesiGENOMESMARTpoolsiRNAlibrarytargeted ttp
gated.OurstudiesinH358cells,whichlackTGFBRII(52–54),demonstrated againstallhumankinases,NSS,andindividualsiRNAfromthesiGENOME ://s
arequirementofBUB1expressionandkinaseactivityinthepropagationof SMARTpool,andcustomsiRNAsagainstBUB1wereobtainedfromDharmacon. tk
theTGF-bsignalingpathway,suggestingthatTGFBRIImaybedispensable Aspecific,small-moleculeinhibitorofBUB1kinaseactivity(2-[(4-amino- e.s
forIthnesuinmtemraacrtyi,owneofprBoUviBde1cwoimthpTelGlinFgBeRvIi.dencethatBUB1anditskinase 1(9-)(tewrta-sbusytynl)t-h1eHsi-zpeydraizno-lhoo[3u,s4e-.d]pyrimidin-3-yl)methyl]phenol;2OH-BNPP1) cien
c
activity are an integral component of TGF-b signaling and promote e
m
TGFBRI/II receptor complex formation, thus regulating downstream High-throughput siRNA screening against a
g
signalingcascades,includingtheSMAD,MAPK,andPI3K/AKTpath- human kinases .o
r
ways. We further show that BUB1 interacts with both TGFBRI and Forthehigh-throughputscreen,A549(5000cellsperwell)andMDA-231- g
TGFBRIIandmediateTGF-b–dependentEMT,cellmigration,andinva- 1833cells(6000cellsperwell)stablyexpressingthewild-typeBTRrepor- o/
n
sion.Weanticipatethatourresultswilllaythefoundationforfuturestudies terwereplatedinclear-bottomwhite-walled96-wellplates(CorningInc.), J
a
thatwillprovidenewavenuesfortherapeutictargetingofTGF-bsignaling 1daybeforetransfection.Twentymicrolitersof2mMofeachsiRNAfrom n
u
indisease. thesiRNAkinaselibrarywasaddedtoaV-bottomintermediateplate.Simi- a
r
y
laramountofcontrolsiRNAwasalsoaddedincolumns1and12inthe 8
intermediateplate(fig.S1A).ThesiRNAsweredilutedbyadding40ml , 2
MATERIALSANDMETHODS ofOpti-MEMandincubatedfor5minatroomtemperature.Eightymicro- 01
litersofDharmaFECT1andOpti-MEMmixwasaddedtotheintermediate 5
Plasmid DNA plateandincubatedforanadditional30min.Allliquidhandlingwas
Wild-typeBTRreporterhasbeendescribedearlier(4).SBE4-Luc(6) doneusingBiomekNXPLaboratoryAutomationWorkstation(Beckman
reporterplasmidwasprovidedbyB.Vogelstein(Addgeneplasmid#16527). CoulterInc.).ThefinalassayconcentrationofeachsiRNAwas50nM,
pCMV5-TGFBRI-His (#19161), pCS2-Flag-SMAD2 (#14042), and whereas0.2mlofDF-1wasusedforeachwell.Cellswereincubatedfor
pCS2-Flag-SMAD3(#14052)wereprovidedbyJ.Massague(55),and 72hoursinStoreXSTX44ICprecisionincubator(LiCONiCInstruments),
pCMV5B-TGFBRI-K232R(#11763)wasagiftbyJ.Wrana(32).siRNA- whichwasconnectedtoPlateHandlerIIrobot(PerkinElmer)andEnVision
resistantwild-typeMyc-BUB1andMyc-BUB1KDhavebeenpreviously 2104PlateReader(PerkinElmer)withluciferininjector.TGF-b(10ng/ml)
described(27). wasaddedtocells1hourbeforeaddingD-luciferin(50mg/ml)andmeasuring
bioluminescence.Alltheprocesseswereautomated.Foldchangeinreporter
Cell culture and transfection activitywascalculatedoverchangeinactivityinnonsilencingcontrolsiRNA
ThehumanlungcarcinomacelllineA549[AmericanTypeCulturecollec- (NSS)–transfectedcells.
tion(ATCC)]wasmaintainedinRPMI1620mediumsupplementedwith Tovalidatehits,100nMsiRNAtoselectedgenes,alongwithTGFBRI
10%heat-inactivatedfetalbovineserum,1%glutamine,and0.1%penicillin/ siRNAandNSS,wastransfectedinA549,NCI-H358,andMDA-231-1833cells
streptomycin/gentamicin(Gibco-Invitrogen).Cellculturesweregrownina usingDharmaFECT1.Thetransfectedcellswereincubatedfor60hours
humidifiedincubatorat37°Cand5%CO.Breastcancercellline1833(56) andserum-starvedovernight,andTGF-b(10ng/ml)wasadded1hourbe-
2
derivedfromMDA-MB-231wasprovidedbyJ.Massague(MemorialSlo- foreharvestingcellsforWesternblottingforphosphorylatedSMAD2/3,
anKetteringInstitute,NY)andmaintainedinDulbecco’smodifiedEa- p38MAPK,c-JUN,andAKT.Additionally,MRC5,Het1A,MCF7,and
www.SCIENCESIGNALING.org 6January2015 Vol8Issue358ra1 8
R E S E A R C H A R T I C L E
MCF10Acelllinesweretransfectedinparallelexperiments,andWestern Healthcare)for2to3hoursandthenwashedfourtimeswithlysisbuffer.
blottingwasdonetoprobeforphosphorylatedSMAD2. TheresultingpelletwasresolvedbySDS-PAGEandtransferredtoPVDF
membraneforWesternanalysis.
Analysis of the siRNA screen
DatafromthesiRNAscreenwereinitiallyanalyzedonMSExcel.A SBE4-Luc reporter assay
quartile-basedmethodwasusedforHTS(high-throughputscreening)hit ForSBE4-Lucreporterstudies,1.25×105cells(A549,NCI-H358,MDA-231-
selection(20).NormalizationwasdonewithacontrolnontargetingsiRNA 1833,andHeLa)wereplated1daybeforetransfection.Cellsweretransfected
(NSS)placedoneachindividualassayplate,andmedian(Q2),first(Q1), thenextdaywith100ngofSBE4-Lucplasmid,100nMsiRNA,500ngof
andthird(Q3)quartilevalueswerecalculatedforallnormalizedvalues variousDNA,and20ngofGaussialuciferaseforeachwellwithLipofectamine
andsubjectedtoplate-by-plateanalysis.Tofacilitateexperiment-wide 2000.Cellswereserum-starved24hoursaftertransfectionfor6hoursand
analysis,Q1,Q2,andQ3valueswerecalculatedasdescribedabove. treatedwithTGF-b(10ng/ml)for24hours(orupto72hoursforA549)before
AveragesandSEcalculationsweredonefortriplicatesoflog(x/median) measuringSBE4-Lucbioluminescenceactivity.Alternately,cellsweretreated
values.UpperandlowerboundarieswerecalculatedasQ3+2c(Q3−Q2) with10mMSB431542or2OH-BNPP1inthepresenceofTGF-b.Cellswere
andQ1−2c(Q2−Q1),respectively,forc=1.7239,correspondingtoahigh- washedtwicewithphosphate-bufferedsaline(PBS)andkeptinfreshme-
stringencytargetederrorrate(a=0.0027;morethan7.14-foldactivationin diumfor8to24hoursbeforemeasuringGaussialuciferaseactivity.Relative
A549cellsandmorethan13.91-foldinMDA-231-1833cells),andc= fireflyluciferaseactivitywasnormalizedtoGaussialuciferaseactivity.
0.9826,correspondingtoalower-stringencytargetederrorrate(a=0.046;
morethan5.4-foldinA549andmorethan10.29-foldinMDA-231-1833cells). Cell migration and invasion assay D
High-stringencyhitswerechosenasthosetargetsthatscoreda≤0.0027in Multiwellcellcultureinserts(8.0-mmporesize,BDBioscience)wereused o
w
bothplate-by-plateandexperiment-wideanalyses.Selectedtargetsdistrib- forinvitromigrationassays,whereasMatrigelprecoatedinserts(8.0-mm n
utedequallyamongalltheplates,whichindicatesthatourdatadidnotshow poresize,BDBioscience)wereusedforinvasionassays.A549cellswere loa
any“alphabeticalclustering,”ratheragreeingonthesparse-hithypothesis, transfectedwith100nMsiRNA,serum-starvedfor6hourswith0.5%fetal de
whichassumesthathits arerandomlyspacedthroughout thedataset. bovineserum–containingmedium,treatedwithTGF-b(10ng/ml)ormock d f
r
SelectedtophitsweresubjectedtoPathwayAnalysisusingPathwayGuide for72hours,trypsinized,washed,andplatedinserum-freemediuminthe o
m
(AdvaitaCorp.)toidentifythepathwaysthataresignificantlyaffectedin inserts(25Kformigration,50Kforinvasion).Mediumwith5%serumwas h
theBTRassay.Thissoftwareusesanimpactanalysisthatincludestheclas- addedinthebottomchamber,providingachemotacticgradient.Plateswere ttp
sicalstatisticsandalsoconsidersthemagnitudeofeachgene’sexpression incubatedfor8hours(migrationassay)orfor24hours(invasionassay).At ://s
change,theirtypeandpositioninthegivenpathways,theirinteraction,etc. theendoftheassay,cellswereremovedfromthetopsideoftheinsertusing tk
(57,58).Two-wayevidenceplot,totalperturbationaccumulation(TA) acottonswab.Cellsthatpenetratedtotheundersidesurfacewerefixed e.s
plot,andpathwaymapsweregeneratedbyPathwayGuide.Functional usingabsolutemethanol,stainedwith0.5%crystalviolet(Sigma),and cie
proteinassociationnetworkanalysisofthehitswascarriedoutusingString countedunderthemicroscope.Theassaywasrepeatedthreetimes.The n
c
(http://string-db.org/).Heatmapsoflow-andhigh-stringencyhitsweregen- meanofthreerepeatsrunintriplicatesisplotted. e
m
eratedusingtMeV(59)toshowdatareproducibility. a
g
Immunofluorescence .o
r
Western blot analysis A549cellswereplatedonglasscoverslipsinsix-wellplatesandtransfected g
Westernanalysiswascarriedoutusingstandardprotocols.Cellsweregrown with100nMsiRNA,serum-starvedovernight,treatedwithTGF-b(10ng/ml) o/
n
inculturedishes,transfectedwithspecificsiRNA,ortreatedwithselect for1hour,andprocessedforimmunofluorescence.Fortheinhibitorstudies, J
a
compoundsfordesignatedtimeperiods,andcelllysateswereresolvedon cellswereplatedonglasscoverslips,serum-starvedfor24hours,andtreated n
u
SDS–polyacrylamidegelelectrophoresis(SDS-PAGE)gelsandtransferred withTGF-b(10ng/ml)inthepresenceof10mMinhibitors(SB431542or a
r
y
topolyvinylidenedifluoride(PVDF)membranes.Membraneswereprobed 2OH-BNPP1)for1hour.CellsoncoverslipswerewashedwithPBSand 8
againstspecificprimaryantibodiesfollowedbyHRP-conjugatedsecondary fixedin10%(v/v)bufferedformalin(5minatroomtemperature),followed , 2
antibodiesandthenvisualizedusingtheenhancedchemiluminescence(ECL) bymethanolfixationat−20°Cfor20min.Coverslipswererehydratedby 01
WesternBlottingSystem(GEHealthcare).Signalintensitywasmeasured incubatingthreetimesinPBSfor5minandpermeabilizedin0.2%(v/v)Tri- 5
usinganimageprocessingandanalysisprogram(ImageJ,v1.45)(60). tonX-100–PBScontaining1%(w/v)bovineserumalbumin(BSA)onicefor
5min.Coverslipswereblockedby5%(w/v)BSA,5%(v/v)goatserum,and
Coimmunoprecipitation 10%(v/v)donkeyseruminPBSfor1hour.Anti-SMAD2antibodyat1:200
Forcoimmunoprecipitationstudies,HEK293TorA549cellsweretrans- dilutionwasaddedinPBScontaining0.5%(w/v)BSAandincubatedfor
fectedwithvariousplasmids.Lysatesweremade48hoursaftertransfection 1hourat37°Cinahumidifiedchamber.Coverglasseswerewashedthree
innativelysisbuffer[50mMtris(pH7.4),1%NP-40,0.25%deoxycholate timesinPBScontaining0.025%(v/v)TritonX-100andincubatedwithanti-
sodiumsalt,150mMNaCl,10%glycerol,and1mMEDTA]supplemented rabbitAlexaFluor488secondaryantibodyfor1hour.Afterwashing,
with1×PhosSTOP(Roche),1×ProteaseInhibitorCocktail(Roche),sodium coverslipsweremountedinProLongGold(Invitrogen)containingDAPI
orthovanadate,sodiumfluoride,phenylmethylsulfonylfluoride(PMSF), (4′,6-diamidino-2-phenylindole),driedovernightatroomtemperature,and
andb-glycerolphosphate(2mMeach).Cellpelletwassolubilizedin600ml storedat−20°Cuntilmicroscopy.MicrographsweretakenusingOlympus
oflysisbuffer,rockedfor60minat4°C,andcentrifugedat14,000rpmfor BX51uprightfluorescencemicroscopefittedwithanOlympusDP70high-
15min.Proteinestimationwasperformedwithdetergent-compatibleDC resolutiondigitalcolorcamera.
AssayKit(Pierce).Lysateswerepreclearedbyincubatingwith50mlof
agarosebeadsfor1hourat4°Candcentrifuged.Coimmunoprecipitation Tumor xenograft experiments
wascarriedoutbyincubatingpreclearedcelllysate(400mgofprotein)with and immunohistochemistry
1to1.5mgofspecificantibodyovernightat4°C.Theimmunecomplexwas Cellline–derivedxenograftswereestablishedbyimplanting2.5×106A549
capturedusing30mlofslurryofproteinA/G–coupledSepharosebeads(GE cellssubcutaneouslyintoeachflankof4-to6-week-oldmaleSCIDmice.
www.SCIENCESIGNALING.org 6January2015 Vol8Issue358ra1 9
R E S E A R C H A R T I C L E
Whentumorsreachedavolumebetween40and60mm3,micewereinjected 5′-triphosphate)and300mMcoldATP.Reactionswererunat30°Cfor
withasingleintraperitonealdoseofSB431542(10mg/kgbodyweight),a 0.5to1hour,quenchedusingLaemmlibuffer,andresolvedusinga4to
dosepreviouslyreportedtoinhibitTGF-bsignalinginmousemodels(61), 12%bis-trisgel.QuantitativeautoradiographywasperformedwithaTy-
2OH-BNPP1(50mg/kg),orvehicle(DMSO).Tumorswereexcised4hours phoonFLA9000scanner(GEHealthcare).
aftertreatmentandfixedinformalin.Paraffin-embeddedsectionswere
stainedusinganantibodyforphosphorylatedSMAD2,andmicrographswere SUPPLEMENTARYMATERIALS
takenwithanOlympusmicroscopefittedwithanOlympusDP70high-
www.sciencesignaling.org/cgi/content/full/8/358/ra1/DC1
resolutiondigitalcamera.Theproportionofnucleithatstainedpositivefor Fig.S1.siRNAscreeningandpathwayanalysis.
phosphorylatedSMAD2werecountedinthreerandomfieldspertumor Fig.S2.BUB1mediatesTGF-b–dependentSMAD2/3phosphorylation.
pertreatmentcondition[vehicle(n=4),SB431542(n=2),and2OH-BNPP1 Fig.S3.DepletionofBUB1leadstoreducedSMAD3recruitmenttoTGFBRIandTGF-b–
dependentaccumulationofSMAD2inthenucleus.
(n=5)].Atwo-sidedStudent’sttestwasperformedtoassessstatisticalsig- Fig.S4.DepletionofBUB1attenuatesTGF-b–dependentSMAD2/3-SMAD4complexformation
nificance.SlideswereadjustedforbrightnessandcontrastwithAdobePhoto- inA549cells.
shop CS2 (Adobe Inc.), but the micrographs underwent no further Fig.S5.DepletionofBUB1inhibitsTGF-b–mediatedEMTinA549andNCI-H358cells.
manipulations.AllmouseexperimentswereapprovedbytheUniversity Fig.S6.DepletionofBUB1inhibitsTGF-b–mediatedmigrationandinvasionofA549cells.
Fig.S7.BUB1kinaseactivitymediatesTGF-b–dependentphosphorylationandnuclear
CommitteeontheUseandCareofAnimalsoftheUniversityofMichigan.
activityofR-SMAD.
Fig.S8.TGFBRIandBUB1colocalizationbyTIRFmicroscopyat1and24hoursand
Cell cycle analysis coimmunoprecipitationofHis-TGFBRIandMyc-BUB1.
A549cellswereplatedinsix-wellplatesandtransfectedwith100nMsiRNA. Fig.S9.BUB1kinaseactivitymediatesTGFBRI-TGFBRIIinteraction. D
Cellsweretrypsinized72hoursaftertransfection,counted,resuspendedin Fig.S10.TGFBRIisnotadirectsubstrateofBUB1kinaseactivity. o
Fig.S11.Wild-typeMyc-BUB1interactswithFL-SMAD2inHEK293Tcells. w
PBS(50,000cells/ml),fixedusing50%ethanol,stainedwithpropidiumio- Fig.S12.SMAD3isnotadirectsubstrateofBUB1kinaseactivity. n
dide(50mg/ml)supplementedwithribonucleaseA(100mg/ml),andreadona TableS1.HumankinomesiRNAscreeninA549-BTRandMDA-231-1833–BTRcells. loa
flowcytometer.Themeanofatleastthreeexperimentsisplotted. TableS2.HitsobtainedinA549-BTRandMDA-231-1833–BTRhumankinomescreen. de
TableS3.PathwayimpactanalysisbasedonthefoldinductionoftheBTRreporter. d
f
r
TIRF microscopy o
REFERENCESANDNOTES m
A549cellswereplatedonglasscoverslipbottom35-mmdishes(MatTek)and 1. Y.Drabsch,P.tenDijke,TGF-bsignallinganditsroleincancerprogressionandme- h
serum-starvedthenextdayfor24hours.CellsweretreatedwithTGF-b(10 tastasis.CancerMetastasisRev.31,553–568(2012). ttp
n(fvigx//vam)tilbo)unffofaretr1e−,d22f04o°,rComraf7oli2rnh2foo0urmr5simann.indCawetlarlssohowemderwteemirthephPeyrBdatSrua.rtCee,defloblslylowiwenrecedufbbiaxyteimdngeintht1ha0nre%oel 23.. CJRc.ae.XnlDlcuRee,rreSysp.n.rLco1kag9,mr,eRo1s.u5siJi6lol.e–n,A1.R7kN2h.auD(tr2.es0Grty,0en9Anc).e.ktB,.Ta2Gl9m,Fa1-ibn1-,7in–Td1Gu2cF9e-bd(2es0ip0gi1nth)a.elilniagltionmtuemseonrcshuypmparelstrsaionnsitaionnd. ://stke.s
tXim-1e0s0–inPBPSBcSofnotarin5inmgi1n%anBdSAweorneipceerfmore5abmiilniz.eCdelilnsw0.e2re%bl(ovc/kve)dTursitinogn 4. Sof.NTGyaFtib,-Kin.dSuccheidnsSkem,aDd.2R/3ayp,hMo.sNphyoartiy,lBat.ioDn.Rreovsesa,lAs.aRerohleemfoturllare,cMeoplteocrutlyarroisminaeginkig- cien
c
5%(w/v)BSA,5%(v/v)goatserum,and10%(v/v)donkeyseruminPBS nasesinmodulatingTGFbsignaling.Clin.CancerRes.17,7424–7439(2011). e
m
for1hour.Primaryantibodiesatappropriatedilution[anti-TGFBRI,1:500; 5. A.Zieba,K.Pardali,O.Söderberg,L.Lindbom,E.Nyström,A.Moustakas,C.H.Heldin, a
U.Landegren,IntercellularvariationinsignalingthroughtheTGF-bpathwayandits g
anti-BUB1(goat),1:35]wereaddedinPBScontaining0.5%(w/v)BSA relationtocelldensityandcellcyclephase.Mol.Cell.Proteomics11,M111.013482(2012). .or
awnedreinwcuasbhaeteddthforree1thimouersaitnroPoBmStceomnptaeirnaitnugre0i.n02a5h%um(vid/vif)ieTdricthoanmXb-e1r.0P0laatneds 6. LH.uZmaawneSl,mJ.aLd.3Daani,dPS.mBuacdk4haarueltsse,qSu.eBn.cZeh-ospue,Kci.fiWct.raKninszclreipr,tiBon.Vaocgtievalsttoerisn.,MS.oEl..CKeellrn1,, og/
incubatedwithfluorophore-labeledsecondaryantibodiesfor1hourinthedark. 611–617(1998). n J
7. V.L.Johnson,M.I.F.Scott,S.V.Holt,D.Hussein,S.S.Taylor,Bub1isrequiredfor a
Afterwashing,freshPBSwasaddedintotheplates,andTIRFmicroscopywas n
kinetochorelocalizationofBubR1,Cenp-E,Cenp-FandMad2,andchromosomecon- u
performedattheSingleMoleculeAnalysisinReal-Time(SMART)Center gression.J.CellSci.117,1577–1589(2004). ar
usingacell-TIRFsystembasedonanOlympusIX81microscope,equipped 8. H.Yu,Z.Tang,Bub1multitaskinginmitosis.CellCycle4,262–265(2005). y 8
witha60×1.49NA(numericalaperture)oilimmersionobjective(Olympus). 9. J.S.Kang,M.J.Yang,B.Li,W.Qi,C.Zhang,K.M.Shokat,D.R.Tomchick,M.Machius, , 2
SInasmtrupmlesenwtse)rweipthlaxcyezdpoonsitaionnapnioemzoecteorn-ptrroelc.iIsmioangemsowteorreizaecdqustiaregdeo(nPrainnciXetoonn 10. HBSu..bLT1a..wYMlueo,rl,.SXCtr.euHlclt.3uF2re,en3a9gn4,d–R4s.0uH5b.s(tC2ra0ht0ee8n)r,.eEcr.uMitm.Menatruoofkthae,Ch.umWa.nTusrpcink,dlIe.Gchrieswckoplodi-nPtrekinnnaesre, 015
UltraEMCCD(electronmultiplyingcharge-coupleddevice)camera(512× R.Derynck,ThetypeIItransforminggrowthfactor-breceptorautophosphorylatesnot
512pixels;Andor).AlexaFluor488andAlexaFluor647dyesconjugated onlyonserineandthreoninebutalsoontyrosineresidues.J.Biol.Chem.272,
tothesecondaryantibodieswereexcited,oneafteranother,bythe488-and 14850–14859(1997).
11. S.Souchelnytskyi,P.tenDijke,K.Miyazono,C.H.Heldin,PhosphorylationofSer165
640-nmlasers,respectively.Theirfluorescencewasfilteredthroughacus-
inTGF-betatypeIreceptormodulatesTGF-beta1-inducedcellularresponses.EMBO
tomizedquad-bandfiltercubeconsistingofaZT405/488/561/640rpcdi- J.15,6231–6240(1996).
chroic filter (Chroma) and a ZET405/488/561/640m emission filter 12. A.J.Galliher,W.P.Schiemann,SrcphosphorylatesTyr284inTGF-btypeIIreceptor
(Chroma).ImagesofBUB1(AlexaFluor488)andTGFBRI(AlexaFluor andregulatesTGF-bstimulationofp38MAPKduringbreastcancercellproliferation
andinvasion.CancerRes.67,3752–3758(2007).
647)wereprocessedusingacustom-compiledImageJmacrotoidentifyspa-
13. P.Kavsak,R.K.Rasmussen,C.G.Causing,S.Bonni,H.Zhu,G.H.Thomsen,
tiallydistinctparticlesandcalculateextentofcolocalization. J.L.Wrana,Smad7bindstoSmurf2toformanE3ubiquitinligasethattargetstheTGFb
receptorfordegradation.Mol.Cell6,1365–1375(2000).
In vitro kinase assay 14. J.S.Kang,E.F.Saunier,R.J.Akhurst,R.Derynck,ThetypeITGF-breceptoris
covalentlymodifiedandregulatedbysumoylation.Nat.CellBiol.10,654–664(2008).
Invitrokinaseassayswereperformedwith200ngofprotein(wild-typeBUB1or
15. W.Shi,C.Sun,B.He,W.Xiong,X.Shi,D.Yao,X.Cao,GADD34–PP1crecruitedby
akinaseinactivemutantBUB1KD)and2mgofsubstrate(wild-typeTGFBRI, Smad7dephosphorylatesTGFbtypeIreceptor.J.CellBiol.164,291–300(2004).
TGFBRIKD,SMAD3,orH2A)in1×kinasebuffer[50mMtris-HCl,150mM 16. F.Huang,Y.G.Chen,RegulationofTGF-breceptoractivity.CellBiosci.2,9(2012).
NaCl,10mMMgCl ,1%(v/v)glycerol,0.1%(v/v)TritonX-100,DTT 17. J.S.Kang,C.Liu,R.Derynck,NewregulatorymechanismsofTGF-breceptor
(dithiothreitol),PMSF2,Na VO (1mMeach),2mMNaF,andb-glycerol function.TrendsCellBiol.19,385–394(2009).
3 4 18. C.LeRoy,J.L.Wrana,Clathrin-andnon-clathrin-mediatedendocyticregulationof
phosphate]in 20-ml volume containing 10mCi [32P]ATP (adenosine cellsignalling.Nat.Rev.Mol.CellBiol.6,112–126(2005).
www.SCIENCESIGNALING.org 6January2015 Vol8Issue358ra1 10