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Title[原著]Proteomic Changes in a Squamous Cell CarcinomaCell Line Induced by an Effector Kinase of a Small G ProteinRap 2
Author(s) Kuninaka, Kouichi; Yamashiro, Yoshito; BayarJargal,Maitsetseg; Nishimaki, Tadashi; Kariva, Ken-ichi
Citation 琉球医学会誌 = Ryukyu Medical Journal, 26(3・4): 135-145
Issue Date 2007
URL http://hdl.handle.net/20.500.12001/1906
Rights 琉球医学会
Ryukyu MEd.J,26(3,4)135~145 2007 135
Proteomic Changesin a Squamous Cell Carcinoma Cell LineInduced
by an Effector Khase of a Small G Protein Rap2
KouichiKuninakal,2),Yoshito Yamashirol),Maitsetseg BayarJargall)
TadashiNishimaki2)and Ken-ichiKarlyal)
1)Division of CellBilobgy,Department of Molecuar and Cellutar Biology
GrαduαねSc/100叛 ル絶dic柁e
/小・/ヾ/・りJ可〃亘dJ・‥川′!=川′川!・ヾJ/J・ゞり:\・、ハり吊J両州り可=/二ごり/トJ年ん/J1日!.11・。/・イ肛
ハ′川互\・可l/・・′!J・・J仙・/’/J五日高車・可用・/抄/左\・J/バ、=/・///′川・日、・/′り〃りJ
(Received on December11,2007,aCCepted on December,17,2007)
ABSTRACT
TheTraf2-andNck-interactingkinase(TNIK)isadownstream effectorofaRas-1ike
SmallGproteinRap2identifiedbyus.VerylittleisknownaboutTNIK’srolesin physi0-
10glCalor pathologlCalcellular pr・OCeSSeS.ToexaminetheeffectofactivatedTNIKincan-
CerCells,OVer-eXPreSSionofTNIKwasinducedbytheuseofTetOffsysteminasquamous
cellcarcinomacellline.GlobalproteinexpressionprofilingwascarriedoutbyEttanTMtwo-
dimensionalfluorescencedifferencegelelectrophoresis(2TDDIGE).Proteinswereidenti-
fied by matrix-aSSistedlaser desorptionionization time-Of-flight(MALDI-TOF)massSPeCtrOmetry.Bycomparlngthecells with or without over-eXPreSSed TNIK,2-D DIGE
revealedup-regulationofthree proteins and down-regulation of six proteins byTNIK.
Peptidemass fingerprintingwith MALDI-TOFmass spectrometryidentified five out of
these nine proteins:isovaleryl coenzyme-A dehydrogenase(up-regulated 2.46-fold,
P<0.05);RhoGDPdissociationinhibitoralpha(down-regulated2.35-fold,Pく0.05);StOmatin-1ike
PrOtein 2(down-regulated1.57-fold,P<0.05);ribosomal protein,mitochondrial,S22
(down-regulated1.78-fold,P<0.05);Ornithineaminotransferase(down-regulated1.34-fold,
P<0.05).Three down-regulatedproteins,Rho GDPdissociationinhibitoralpha,StOmatin-
1ike protein2,and ornithine aminotr・ansferase were candidates for cancer prognostic
marker or target proteins for chemotheraputic drug development.旦yuhyuMbd.J,26(鋸の∬~145,2007
Keywords:Proteomics,Rap2,MAP4K,SquamOuS Cellcarcinoma
INTRODUCTION
Rap2isasmallGproteinhomologoustoRas,
aprototypicaloncogeneproductl).MembersofRas
familyplaycrucialrolesinregulationofvariousce1-
1ularprocesses such as cellgrowth,differentiation
anddeath.Thefamilyconsistsofsubfamiliessuch
asRas,Rap,Ral,etc.TheRapsubfamilyistheclos-
est relative of the Ras subfamily.Rap subfamily
containsRaplandRap2,Whichareabout60%iden-
ticaltoeachother・.Rapl’sfunctionisoftenanalo-
gous to that ofRas,butit sometimes counteracts
Ras function,because they shareidentical amino
acid sequenceintheireffector-bindingregl0n.For
instance,RaplinteractswithRaf-1,adownstream
effectorofRas.However,RapIcannotactivateRaf-
1forcomplexreasons,andasaresult,1eadstosup-
PreSSionofRas一mediatedactivationofextracellular・
Signal-aCtivated kinase(ERK),the growth-related
mitogen-aCtivatedproteinkinase(MAPK).Onthe
Other hand,Raplinteracts with and activates an-
OtherRaseffector,B-raf,mOreefficientlythanRas
andplaysamaJOrrOleinregulationofERKinsome
Celltypes.
IncontrasttoRas/Rap1,1ittlehadbeenknown
aboutRap2’sroleincellularslgnaling.Theeffector-
binding reglOn Of Rap2differs from thosein Ras
andRaplbyasingleaminoacid:Rap2hasPheat
136 Effect of a MAP4K on cancer proteome
amino acid position 39,but Ras/Rapl have Ser.
ThisdifferencemightsuggestthatRap2hasitsspe-Cific downstream effectors.In our previous study,
therefore,Rap2TSPeCificinteracting proteins were
SearChedfromratbrainbyafocusedproteomic ap-
PrOaCh:the combination of Rap2-affinity column
Chromatographyandproteinidentificationbymass
SPeCtrOmetry.A protein of155kDa wasisolated
andidentifiedasTraf2-andNck-interactingkinase
(TNIK)2).TNIK belongs to the Ste20group of
kinases that act upstream of c-Jun N-terminal
kinase(JNK),aStreSS-relatedMAPK3).Inaddition,
WehaveisolatedanotherSte20groupkinase,HPK/
GCK-11ike kinase(HGK),also known as MAPK
kinasekinasekinase4(MAP4K4),aSanOtherRap2-
SPeCificinteracting pr・Otein by the yeast two-hybrid
screening4).Thus,itturnedoutthatRas/Rapland
Rap2,Withdifferenteffector-bindingreglOnatPOSi-
tion39,aCtuPStreamOfdifferenttypesofMAPKs,
namely,ERKandJNK,reSPeCtively.
Ste20group members share similar kinase
domainstothatofSTE20,ayeaStMAP4K3).Thegroup consists of two families.The first family,
p21-aCtivated kinase(PAK)family contains C-
terminal catalytic and N-terminal regulatory do一
mains,Whilethesecondfamily,thegerminalcenter
kinase(GCK)family contains N-terminal kinase
and C-terminal regulatory domains.TNIK and
HGK/MAP4K4belong to thelatter,and their C-
terminalregulatorydomainisthebindingsitefor
Rap2.TNIK and HGK/MAP4K4are about90%
identicalin their・kinase and r・egulatory domains,
butidentitydropstoabout50%inintermediated0-
main between the kinase and the regulatory d0-
mains.Onereasonforthisdropisthepresenceof
thr・ee alternatively spliced modules(amino acids
447-475,537-591and795-8020fhumanTNIK),and
thelargest human TNIKisoform consists of1360
aminoacids.TNIKisnamedafteritsabilitytoin-
teractwithadaptorproteinsTraf2andNckatthis
intermediate domain5).However,nO eVidence has
been reported so far that Traf20r Nck activates
TNIK,thusRap2istheonlyknownupstreamacti-
vatorofTNIK.
RegardingJNKactivationbyTNIK,SOmeCOn-
troversyexistsastowhetherGCKfamilymembers
Shouldbetermed“MAP4K”ornot,Sincetheyappear
toactivateMAP3Kswithoutphosphorylatingthem.
Rather,the members help formation of MAP3K
Oligomers:the C-terminal regulatory domains of
the GCK family members have an ability to
Oligomerize and at thesametimeserveasbinding
SitesforMAP3Ks.Thus,formationofGCKfamily
memberoligomersthroughtheC-terminalregulaTtorydomainsinturnleadstoformationofMAP3K
Oligomers,Withinwhichtrans-Phorphorylationand
activationofMAP3Ktakeplace:kinasedomainof
the family members are notnecessary3).ConsisT
tent with this,Simple artificialover-eXPreSSionof
theGCKfamilymemberscanactivateJNK3):thisis
also the case for TNIK,and over-eXPreSSion of a
kinase-deficientmutantTNIKcanleadtoactivation
ofJNK5).
Verylittleis known about TNIK’s rolesin
PhysiologlCal or pathological cellular processes.
Oneobviousapproachtoaddressthisissue,Which
isnotinthescopeofthisstudy,istosubjectcells
WithactivatedorinactivatedTNIKtoavarietyofcell
biologlCalassays.However,this kind ofhypothesis-
drivenapproachcouldoverlooksomethingthatiscom
pletelyunexpected.Onenon-hypothesis-drivenapproach
istheproteomicappr・OaCh6).Recently,thestrategyof
COmbining EttanTM two-dimensional fluorescence
difference gel electrophoresis(2-D DIGE)system
(GEHealthcare)withmatrix-aSSistedlaserdesorption/
ionizationtime-Of-flight(MALDI-TOF)massspec-trometryhasbecomeoneofstandardstrategiesfor
proteomicanalysis7).The2-DDIGEsystemhascir-
CumVented thelimitations of previous stain-based
two-dimensional electrophoresis system.In this
SyStem,PrOteinsinthecontrolandtestsamplesare
labeledwithdiffer・entfluor・esCentCyDyesTMsuchas
Cy3andCy5.Thisallowstw0-dimensionalsepara-
tionofthecontrolandtestsamplepalrOnthesame
gel,enablingperfectprotein spot matching.AIso,
1ineardynamicrangesforfluorescentdyesar・efive
Ordersofmagnitude,allowlngVeryaCCuratequanti-
tativecomparisons.Proteinspotsshowingquanti-
tative differences are examined by MALDI-TOF
mass spectrometry,and pr・Oteins containedin the
SPOtSareidentified.
In the present study,We have attempted to
examine TNIK’s rolein cancer cells by the above
PrOteOmicapproach:Wehaveexaminedtheeffectof
OVer-eXPreSSed TNIK on globalprotein expression
profileinacancercelllineinuitro.Wehavechosen
amousesquamouscellcarcinomacelllinePam212,
Whichfor・mStumOrSinmiceaftersimplesubcutane-
ousimplantation9),allowinglnUiuotestofinuitro
findings.
Kuninaka K.et al.
MATERIALSANDMETHODS
Generation of Pam212cell clones for induced expres-sion of TNIK.
Unlessotherwiseindicated,Pam212cellswere
maintainedinastandardgrowthmedium,Dulbecco’s
modifiedEagle’s medium(DMEM)containinglO%
fetalbovine serum(FBS)supplemented withpeniT
Cillinandstreptomycln.Toestablishcellsthatcan
beinducedtoover-eXPreSSTNIK,WeuSedtheRev-
Tet-Off System(BD Clontech).According to the
manufacturer’sprotocol,tWODNAconstructswere
transducedintoPam212cellsusingretroviruses:[1]a
COnStruCtforexpressionofatranscriptionalactiva-
torprotein,tTA,thatallowsinductionofageneof
interestonlyintheabsenceoftetracycline(i.e.,坦二
racyclineshuts!些theinduction);[2]aconstruct
that expresses a cDNA for thelongestisoform of
human TNIK2)fused to the HA-ePitope tag(HA-
TNIK)under・the controlof tTA.The neomycin-
and hygromycln-reSistant genes containedin the
tTAandHA-TNIKconstructs,reSPeCtively,allowed
Selection of doubly transduced cells.The selected
Cells over-eXPreSSed HA-TNIK only when cultur・ed
intheabsenceoftetracyclinederivative,doxycycline
(Dox).Forcloningofhigh1yover-eXPreSSingcellsusing96-Wellculture plates,Selected cells were di-
1utedtothepointofonecellper200〃lofmedium,
andlOO FLIwere added to each well.Cloned cells
thatformedsinglecoloniesweretransferredto60-
mm dishes,PrOPagated,and subjected to Tet-
r・egulatedinductionofHA-TNIK.
Until the time ofinduction,Cloned cells were
PrOPagatedin the standard growth medium con-
tainingl〃g/mlofDoxtosuppressexpressionof
HA-TNIK.Forinduction,Cells were trypslnized,
and trypsln WaSinactivated with DMEM supple-
mented withlO%”Tet System-Approved FBS”
(Clontech)to avoid any contamination oftetracyT
ClinederivativesfromordinaryFBS.Thecellswere
fed with the same Dox-free medium next day and
fur・therincubatedfortwomoredaysbeforeexperi一
ments(72hinduction,“TNIKcells”).Ontheother
hand,tO prePare Cells that are notinduced,Cells
Wer・egrOWnfor3daysinthepresenceofDox.In-
duction of HA-TNIK was examined with Western
immunoblottingwithamousemonoclonalanti-HA
antibodyasdescribedpreviously2).Twoclones(1ines
137
AandB)thatexhibited stronginductionwere se-
1ectedandmaintained.Thesame72hinductionpr0-
tocoIwasusedthroughoutthestudy.
Proteinextraction and fluorescent dye labeling
Prot,ein extraction from Pam212 cells and
fluorescentdyelabelingwere carriedoutsimilarly
as described previously for oculartissues except
that cells wereharvested by scraplng,COllected by
centrifugation,andhomogenizedbysonicationonlylO,11)
Briefly,COnfluentcontroIcellsandTNIKcellsin60-
mm dishes were washedinice-COld phosphate-
buffered saline,and collectedin150 ml of
homogenizationbuffer(2Mthiourea,7Murea,4%
W/v3-[(3-Cholamidopropyl)dimethylammonio]-1-
propane-Sulfonate(CHAPS),30mMTris-HCl,PH
8.5),SuPPlemented with Complete EDTA-free
ProteaseInhibitors(Roche).Afterhomogenization
Withabriefsonication,thecrudehomogenatewas
Centrifugedat16,000gfor・30minat40C.Proteins
inthesupernatantwerepurifiedwiththe2-DClean
UpKit(GE)andre-dissoIvedinthehomogenization
buffer.Proteinconcentrationwasdeterminedusing
the2-DQuantKit(GE)andadjustedt05mg/ml,
beforestorlngat-800C.Afterthawingoniceand
Centrifugation at16,000g forlO min at40C,PrO-
teins werelabeled with CyDye DIGE fluorescent
dyesaccordingtomanufacturer’sinstr・uCtions(“m
inimallabeling”protocol,GE).ControlandTNIK
CellsamplesweredifferentiallylabeledwithCy3and
Cy5,reSPeCtively,OrViceversa.
Forthefirstdimensionofisoelectricfocuslng
(IEF),animmobilizedpHgr・adient(IPG)drystrip
WaS uSed(pH4-7,18-cmlinear,Immobiline Dry
Strips,GE).ApairoflabeledcontrolandTNIKcell
SamPles(50mgproteinseach)weremixed(tota124
ml),COmbinedwith320mlofrehydr・ationbuffer[8
Murea,2%CHAPS,0.002%W/v bromophenolblue
(BPB),0.5%V/vIPG buffer(GE)and1.2%V/v
Destreak Reagent(GE)]and applied to the dry
strip.Afterovernight“rehydr・ation」oading”(also
Called“in-gelrehydration”)ofsampleproteins,the
StripwassubjectedtoIEFwithLKB2117Multiphor
II(GE)and aprogrammedvoltage gradient(O to
500Vr・amPforlmin,500to3,500Vrampfor1.5h,
and3,500Vfor6h).For the second dimension of
SOdiumdodecylsulfate-pOlyacrylamidegelelectroporesis
138 Effect of a MAP4K on cancer proteome
(SDS-PAGE),the strip afterIEF was equilibrated
for・15mininequilibr・ationbuffer(50mMTris-HCl,
PH8.8,6Murea,30%V/v glycerol,2%W/v SDS,
0.002%BPBand1%W/vdithiothreitol).Thestrip
WaS then mounted on top of a12.5%gel(1-mm
thick,24Ⅹ24cm)thatwascastusinglow fluores-
CenCeglassplates(NihonEIDO,Japan)andfixedin
PlacewithasharkTtOOthcomb.SDS-PAGEwascar-
riedoutataconstanttemperature(200C)andcon-
StantPOWer(60mA)untiltheBPBdyefrontwas
approximatelyl mm from the bottom of the gel.
The gelwas scanned uslng the Typhoon Variable
ModeImager(GE)toacquireCy3andCy5images.
Protein spots from eachimage were outlined and
quantifiedusingImageQuantToolandImageMas-
terPlatinumsoftwareprograms(GE).Thequan-
tity of anindividual spotin eachimage was
expressedas%volume(%Ⅴ),relativetothesumof
alldetectedspotsfromeachimage12).
・ヾりいぬJ与・りJJ・/ん/川/小・り//=〃 可′1招iJl〃由り/年り、I
pressedproお花S
OnlyproteinspotsthatexhibitedsimilarTNIK-
inducedalteration(up-Ordown-regulation)ina112-D
DIGEanalyseSwereselected.Usingthe%Vvalue,a ratio of TNIK-induced alteratJion(%Vin TNIK
Cells/%Vin controIcells)was calculated for each
analysIS.Subsequently,theaverageratiofromall
analyses was calculated.Only protein spots that
exhibited statistically slgnificant alterations were
definedas alteredbyTNIKandsubjectedtoprotein
identification.For proteinidentification,aSamPle
Of500mg protein,Of whichlOO mg waslabeled
WithCyDyes,WaSSubjectedtotwo-dimensionalsepa-
ration.Proteinsin the gels wer・e tranSfer・red to
POlyvinylidenedifluoride(PVDF)membrane(ProBlott
PVDF,AppliedBiosystems)followed by Colloidal
Gold Total Protein staining(Bio-Rad).Protein
SPOtS defined as altered by TNIK were excised(1
mmxlmmsquare)fromthemembrane.Immobi-
1ized proteins were reduced,S-Carboxymethylated,
anddigestedinsituwithAchroTTWbacterproteaseI
(a Lys-C),Which cleaves after Lysl3).Molecular
massanalysesofLys-Cfragmentswereperformed
byMALDI-TOFmassspectrometry,uSlngApplied
Biosystems Voyager-DE/STR14).Identification of
PrOteinswascarriedoutbythepeptidemassfinger-
Printing technique and screenlng the NCBI non-
redundant protein database uslng the MASCOT
SOftwarewithamasstolerancesettingof±0.03Da.
Bulk CloneA
DoxT Dox+ Dox- Dox+
_二二司 〃m±二
1 2 3 4
Fig.1StrongbutleakyexpressionofHA-TNIKin clonedcells
Strong butleaky expression of HA-TNIKin cloned
cells.Bulkand cloned cells were cultured eitherin the
absence(-)orpresence(+)ofDox(1pg/ml)for72
hours.After cellharvesting,10FLgOftotalcellpro-
teins weresubjectedtoWesternimmunoblottingusing
mouse monoclonalanti-HA antibody.ArTOWSindicate
POSitions of HA-TNIK.
RESULTS
As one way to analyze the role of signaling
pathways mediated by TNIKin a context of
SquamOuS Cell carCinoma,We SOught toidentifyPrOteins whose expressionlevels were altered by
OVer-eXPreSSion of HA-TNIKin a squamous cell
CarCinoma cell1ine,Pam212,uSing the2-D DIGE/
MALDI-TOF/MSproteomicapproach.Tothisend,
WeutilizedtheRev-Tet一〇ffsystemtoenglneerthe
CellssothattheyexpressHA-TNIKwhencultured
intheabsenceofatetracyclinederivativeDox.Two
Celllines(AandB)thatexpresshighlevelofHA-
TNIK were established by clonlng from bulk cul-
ture.However,during the cloning procedure,We
noticed that cells expressinglarge amOnt Of HA-TNIK when culturedin the absence ofDox also ex-
PreSSSOmeamOuntOfHA-TNIKevenwhencultured
inthepresenceofDox(socalled“1eakyexpression”).
InFig.1.,HA-TNIKexpressionofbulkandcloneA
Cells are compared.Bulk cells expressed modest
amountofHA-TNIKintheabsenceofDox(1anel)
butnegligibleamountofHA-TNIKinthepresence
OfDox(1ane2).Ont,heotherhand,CloneAcel1sex-
PreSSedlargeamOuntOfHA-TNIKintheabsenceof
Dox(1ane3).However,they also expressed some
amount ofHA-TNIKin thepresence ofDox(lane
2).Toavoidpotentialeffectofthisleakyexpression
On PrOtein expression profile,We uSed parental
Pam212cells as“controI cells”and cloned cells ex-
PreSSinginducedHA-TNIKas“TNIKcells’’forcom-
parison.PotentialeffectofclonlngWaSaVOidedby
Kuninaka K.et al.
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Fig.2Representative2-D DIGEimages
Representative2rDDIGEimages.Proteinsamplesfromcontroland TNIK cells(50mgprotein each)
Werelabeled with Cy3and Cy5fluorescent dyes,reSPeCtively,Or Vice versa.Samples were mixed
and separatedin the same gelon the basis ofpI(x-aXis)andmolecularmass(yraxis).Fouranaly-
SeS uSingindependent palrS Ofcontroland TNIK cells gave essentially the same results.Shown are
representative fluorescentimages from one ofthe fouranalyses(grayscaleimages foreachindivid-
ualchannelforCy3andCy5).BothCy3(control)and Cy5(TNIK)images are shown.Arrowswithnumbersindicate protein spots whoseintensities were consistently altered by TNIKin all four
analyses(spots#1,#2and#4,uP-regulated;SPOtS#3,#5,#6,#7,#8and#9,down-regulated).
excludingfindingsnotcommontobothclones.
For screening of proteins thatare differen-
tiallyexpr・eSSedbetweenthecontr・01andTNIKcells,
we have carried out the Dox-COntrOlled over-
expression experiments of HA-TNIK four times:
twiceforeachoftheclonedcelllines,AandB.Ex-
Periments wer・e Car・ried out on differ・ent OCCaSions,
generating TNIK-A and TNIK-B cells twice each
(TNIK-A#1,TNIK-A#2,TNIK-B#1andTNIK-B#2).
ControIcells were also prepared each at the same
time(control#1to#4).Weextr・aCtedproteinsfrom
COntrOlandTNIKcellsandexaminedglobalprotein
expressionprofilesusing2-DDIGE,apOWerfultwo-
dimensionaldifferentialgelelectrophoresis system
(Fig.2).A pair・Of protein samples wer・elabeled
With different fluorescent CyDyes and mixed.
Mixedlabeledproteins were separatedinthe same
gel,aCCOrdingtotheirisoelectricpoint(pI)inthe
first dimension and to their molecularmaSSin the
SeCOnd dimension.Separated proteins were repreT
Sentedbyfluorescentspots onthegel,Whichwere
visualized by obtain1ng fluorescenceimages ofthe
gel for each CyDye.Usingthis system,We ana-
1yzed protein expression profilesin four palrS Of
COntrOl and TNIK cell samples.We found nine
PrOteinspotswhoseintensitieswereconsistentlyal-
teredbyover-eXPreSSionofHA-TNIKinallanalyses
(designatedspot#1tospot#9inFig.2).
AsshowninTablel,these proteinspots ex-
hibitedvariousextentsofHA-TNIK-inducedquan-
titative alterations as revealed by“average ratio”
Calculationasfollows:[1]Theintensityofeachspot
ineachanalysis wasexpressedas%V,Wherevol-
ume(integratedareaandfluorescencestrength)of
eachspotisnormalizedtothesumofvolumeOfall
the spotsin the gel.[2]In each analysis,%V of
eachspotinHA-TNIK-0Ver-eXPreSSingsampleswas
nomalizedtothatofcontroIsamplestoglVearatio
OfHA-TNIK-inducedchange.[3]Theratiosinall
four analyses wer・e aVeraged to glVe the“aver・age
ratio”:nOte that“average%Ⅴ”valuesgivenin
Tablelareaveragesofal1fouranalyses,and“aver
age ratio”Calculated as above([1]-[3])is not the
ratio of“average%Ⅴ”for TNIK cells to“aver・age
%Ⅴ”forcontroIcells(seenotesonTablelformore
detai1edproceduresandtheirreasons).Amongthe
nineproteinspots,the“averageratio”Variedfrom
O.23to4.09,and allalterations wer・e statistical1y
Significant(p<0.01forspots#2,#4and#8,P<0.05
for spots#1,#3,#5,#6,#7,#9by paired t-teSt;
041
Effect of a MAP4K on cancer proteome
Tablel Altered proteins
% vol
tOpS ControI TNIK Averageratio p-Value
1 2 3 4 5 6 7 00 9
0.0876 0.1213
0.0168 0.0355
0.0945 0.0265
0.3174 0.5803
0.1726 0.0685
0.3392 0.2322
0.0783 0.0459
0.3377 0.1392
0.2594 0.1943
2.4644
4.0949
0.2261
1.8540
0.4255
0.6489
0.5615
0.3585
0.7468
0.0124
0.0063
0.0197
0.0036
0.0261
0.0132
0.0366
0.0074
0.0244
Fourpairedcomparisons between controland eitherTNIKrA orTNIK-B cells were
Carried out[(1)-(4)].However,aCtually6experiments were performed as follows:
(1a)control#1-Cy5vs TNIK-A#1-Cy3
(1b)control#1rCy3vs TNIK-A#1-Cy5
(2)control#2-Cy5vs TNIKrA#2-Cy3
(3a)control#3-Cy5vs TNIK-B#1-Cy3
(3b)control#3rCy3vs TNIK-B#1rCy5
(4)control#4-Cy5vs TNIKrB#2-Cy3
In the fisrt pairing of controlvs TNIK-A cells[(1)]or controIvs TNIK-B cells
[(3)],eXPeriments were performedin duplicate by reversing CyDyes[(1a)and(1b);
(3a)and(3b)].Thus,%V of each spotsin experiments(1a)and(1b)were averaged
to give the%V of comparison(1),Which were used for subsequent calculations.Simir
larly,%Vofeach spotsinexperiments(3a)and(3b)wereaveraged to give the%V for
the comparison(3).On the other hand,int,he second pairing[(2)and(4)],Only sinr
gle experiment each was performed.In these cases,%V of each spot,from each experi-
ments were used directly for subsequent calculations.
The reason why two experiments were performed for comparison(1)and(3)
eachby switchingCyDyesisbecausewehadfoundpreviouslythatafewproteins were
labeled by Cy5more efficiently than Cy3,Or Vice versa.Thus,any PrOtein spot show-
inginconsistent behaviorin different CyDye combinations,e.g.,uP-regulatedin(1a)
but down-regulatedin(1b),Wereneglectedinallcomparisonsas apotentially misleadr
ing spots.In addition,any SPOt that show different behaviorin any comparison,e.g.,
up-regulatedin allcomparison except forin(4),WaS also neglected.
Tablel).Thus,these nine spots were defined as
beingalteredby over-eXPr・ession ofHA-TNIK.As
expressedbythe“averageratio”Values,threeoutof
theninespotswereup-regulatedbyTNIK(spots#1,
#2and#4),While the other six spots were down-
regulated(spots#3,#5,#6,#7,#8and#9).
Wesucceededtoidentifyproteinscontainedin
five out ofthese nine spots.Proteinidentification
WaS Carried outbyanalyzingpeptides obtainedby
PrOteaSedigestion ofeachproteinwithinthespot.
Wehavechosenourmethodforcollectingpeptides
Of highest concentration based on our own
experiences12・13),Which we also foundin theliter・a-
ture:[1]In2-DDIGEgels,Pr・Oteinspotscaptured
byfluorescenceCyDyescannlngarenOtalwaysthe
pointofhighestproteinconcentration7).Mostpro-teinspotscontainlabeledaswellasunlabeledpr・0-
tein:Only a smallfraction ofproteinsare fluor・es-
CenCe-1abeledwith ourminimallabeling procedure.
[2]Fluorescence-1abeling causes retarded migra-
tion ofproteinsinthe second dimension,aneffect
moremar・kedforsmallproteins7).To subjectthe
areaofhighestproteinconcentration totheproteinidentification procedures based on these
Kuninaka K.et al.
Table2Identified proteins
141
%
Coverage
32
Totalamino MatchedPept,idesacidrecidues (residueNo.)
424 57-65
Spot ProteinName MolecularNo. (accessionNo.) Mass(Da)
1 isovalerylco-Adehydrogenase 46296(NP_062800)
5 Rho GDPdissociationinhibior・ 23393
dpha(NP_598577)
6 stomatin-1ikeprotein2 38361
(NP_075720)
7
9
ribosomalprotein,mitJOChondria1, 41167S22
(NP_079761)
0rnithineaminotransferase
(NP_058674)
48324
204
77-83
154-178
195-211
212-230
244-260
327-346
400-424
34-43
100-105
106-113
114-127
128-135
142-167
168-178
187-199
353 115-140
359
439
141-145
179-187
188-200
201-221
255-288
292-321
322-351
66-80
89-103
258-277
333-359
33-49
50-66
103-107
108-129
136-151
363-374
375-386
422-434
435-439
47
47
21
27
information,We electro-blotted protein spots to
PVDFmembranes,Visualized them by colloidalgold
Stainlng,and excisedthem.Additionally,tranSfer・
Ofprotein spots from the gel to a membrane a1-
10wed efficient reductionandS-alkylationofdisu1-
fidebonds and digestion withLys-C protease(on-
membrane digestion).We obtained peptide mass
SPeCtra by MALDI-TOF mass spectrometry,and
identified proteins uslng bioinformatics database
SearChing(Mascot SearCh,PePtide mass finger-
Printing).Foridentified proteins,database acces-
Sionnumber,theoreticalmolecularmass,tOtalamino
acid number,matChed peptides(amino acid residue
numbers),andpercentageofcoverageofproteinse-
quence(%coverage,matChed amino acid/total
aminoacidxlOO)areShowninTable2.
By combining the datain Tablel and Table
2,theidentified proteinsincluded(spot number,
andnatureofalterationinducedbyTNIK,inparen-
theses):isovalerylcoenzyme-Adehydrogenase15)(spot
l,up-regulated2.46-fold);Rho GDP dissociation
inhibitoralpha16)(spot5,down-regulated2.35-fold);
stomatin-1ike protein21n(spot6,down-regulated
1.57-fold);ribosomalprotein,mitochondrial,S2218)
(spot 7,down-regulated l.78-fold);0mithineaminotransferase19)(spot9,down-regulated1.34-
fold).
DISCUSSION
Proteomic consequence of TNIK activationin
thecontextofsquamouscellCarCinomawasexam-
inedinthepresentstudybyuslngPam212cells.By
COmparlngglobalproteinexpressionprofilebetween
142 Effect of a MAP4K on cancer proteome
the controIPam212and HA-TNIK-OVer-eXPreSSing
Celllines,Wefoundthatexpressionofnineproteins
WereCOnSistentlyalteredby TNIK over-eXPreSSion
(activation).Outofthem,fiveproteinswereidenti-
fied:[1]isovalerylcoenzyme-Adehydrogenase;[2]
RhoGDPdissociationinhibitoralpha;[3]stomatin-
1ikeprotein2;[4]ribosomalprotein,mitochondrial,
S22;[5]ornithine aminotransferase.Strikingly,
down-regulationofRhoGDPdissociationinhibitor
alpha([2]),down-regulationofstomatin-1ikepro-
tein 2([3]),and down-regulation of ornithine
aminotransferase([5])observedinHA-TNIK-0Ver-
expresslngCelllinesallmadeustOSPeCulatethatac-
tivation of TNIK would make Pam212 cell tumor
morebenignandsensitivetochemotherapy.
Rho family small G proteins,Rho,Rac and
Cdc42,Wereinitiallylinked to regulation of actin
cytoskeleton20).However,nOWitiswidelyaccepted
thattheir roles extend beyond theseinitialobser-
Vations and cover diverse aspects of cellular proc-
essesincluding gene transcription and cell cycle
progression21).smallGproteinsincludingRhofam-
ilyactasmolecularSWitchescyclingbetweenGDP-
boundinactive and GTP-bound active forms.Rho
GDPdissociationinhibitoralphawasidentifiedasa
negativeregulatorofRhofamilymemberstypified
byitsabilityto prevent dissociation ofGDP from
them,therebykeeplngtheminGDP-boundinactive
forms16).Despite such negative rolesinitially at-
tributed to Rho GDP dissociationinhibitor alpha,
accumulated evidence has come to suggest thatit
mayalsoactasapositiver・egulator・forRhofamily
membersby regulatingtheirspatialdistribution22,23)
For an example relevant to tumor biology,the
transformlngaCtivityofconstitutivelyactiveCdc42
(F28L)mutantin NIH3T3 fibroblasts was sup-
PreSSed by down-regulation of Rho GDP dissocia-
tioninhibitor throughRNAinterference23).ThetransformlngaCtivity was also suppressed by ad-
ditional R66A mutationin Cdc42 that abolishes
interaction with Rho GDP dissociationinhibitor,
but the R66A mutJation also abolished plasma
membrane distribution of the Cdc42(F28L,R66A)
mutant.Infurthersupportoftumorlgenicroleof
Rho GDP dissociationinhibitor,itis high1y exTpressedinmultipletypesofhumancancersinclud-
1ng OVarian cancer24)and breast cancer25).More
importantly,reCent Studies have shown that Rho
GDP dissociationinhibitoris highly expressedinchemoresistant cancer celllines,SuCh as melanoma26)
andbreastcancercells27).Down-regulationofRho
GDP dissociationinhibitor by RNAinterference
(RNAi)sensitizedthechemoresistantcellstodrugS27).
Thus,Rho GDP dissociationinhibitorlikely plays
SOmerOleinthedevelopmentofchemoresistance.
Humanstomatinisanerythrocytemembrane
PrOteinofunknownfunction.Stomatin-1ikeprotein
2is unlqueinits structure amOng mammalian
StOmatin-1ikeproteinsandcouldplaysome rolein
thecontrolofionchannelconductanceormembrane
lipid domain organization17).zhang et al.first
reported that stomatin-like protein 2is highlyexpressedin human esophageal squamous Cell
CarCinoma,1ung cancer,1aryngeal cancer,and
endometrialadenocarcinoma28).Further,theyshowed
thatesophagealsquamous cellcarcinomacelllines
transducedwithastomatin-likeprotein2antisense
COnStruCt PrOduced slower一grOWing tumorin nude
mice.Elevated expression ofstomatin-1ike protein2
wasalsofoundinhumanosteosarcoma29),inlaryngeal
SquamOuSCellcarcinomaandbreastcancerasprog-
nosticfactorswithhigherexpressionbeingfoundin
patients with metastasis30・31),andin paclitexel-
resistanthumanbreastcancercellline32).Finally,
Cui et al.reported thatin human endometrial
adenocarcinoma,SenSe and antisense constJruCt,S Of
StOmatin-1ikeprotein2acceleratedanddelayedcell
gr・OWth,r・eSPeCtively,inuitro33).Thus,StOmatin-like
protein2isalsoinvoIvedintumorlgeneSisandma-
1ignantprogression.
0rnithineaminotransferasehas recentlybeen
found to be necessary for mitotic cell division of
human cancer cells but not of normal cells34).
Down-regulation ofornithineaminotransferaseby
RNAiin human cervical carCinoma HeLa cells and
osteosarcoma U20S cellsled to accumulation of cell
arrestedattheG2/Mphaseofthecellcycleandan
increasein mitotic spindle defects,Subsequently
CauSlngmaSSivecelldeath.This“secondfunction”of
ornithineaminotransferaseis unrelated toits role
inaminoacidmetabolismandis blockedbyanatu-
rally occurrlnganti-CanCer COmPOund diazonamide
AanditssyntheticanalogAB-534,35).Inuitro,AB-5
inhibited growth ofhuman colon,breast,0Varian,
PanCreatic,lung and prostate cancer celllines and
melanomacelllineswithnannomolargrowthinhibi-
tory50(GI50)values similar to two widely used
antimitotics,Vinblastine and paclitaxel35).In uiuo,
AB-5inhibited growth ofimplanted tumors of
HCT116colon cancercellline,PC3prostate cancer
Kuninaka K.et al.
cel11ine and MDA一MB-435 breast cancer cellline
With efficacies comparable to those of vinblastine
andpaclitaxelwithoutanyovertsideeffectssuchas
weightlossandneutropeniainnudemice35).Inconclusion,OurPrOteOmicanalysisofTNIK-
OVereXPreSSlng Pam212 celllines revealed down-
regulationofthreeproteinsthatarerecentlyunder
extensive study as candidate proteins for cancer
prognositc marker and target proteins for
Chemotheraputic drug development.Although we
need to be cautiousin examinlng reSults of such
Studies,itwouldbeinterestingtoexaminewhether
implantedtumorofTNIK-OVereXPreSSlngPam212
Celllines and parentalPam212cells behave differ-
entlylnmiceinL,iuo,Sincethereexistsaprecedent
inthatTetOffsystem(feedingwithDox+OrDox-
Water)wasusefulforinductionofgeneexpression
inimplantedtumor36).
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