Proteomic analysis of novel targets associated with TrkA-mediated tyrosine phosphorylation signaling pathways in SK-N-MC neuroblastoma cells.

1.1 MB·English

by  Wu, Fang-Xiang

Proteomics2013,13,355–367 DOI10.1002/pmic.201200251 355
RESEARCHARTICLE
Proteomic analysis of novel targets associated with
TrkA-mediated tyrosine phosphorylation signaling
pathways in SK-N-MC neuroblastoma cells
Eun Joo Jung1, Sang-Yeul Lee2 and Choong Won Kim1
1DepartmentofBiochemistryandInstituteofHealthSciences,GyeongsangNationalUniversitySchool
ofMedicine,Jinju,SouthKorea
2DepartmentofPlasticandReconstructiveSurgery,SchoolofMedicine,KangwonNationalUniversity,
Chuncheon,SouthKorea
Tropomyosin-related kinase A (TrkA) is a receptor-type protein tyrosine kinase and exploits Received:June21,2012
pleiotypic roles via nerve growth factor (NGF)-dependent or NGF-independent mechanisms Revised:September28,2012
invariouscell types.Here,weshowedthattheinhibitionofTrkAactivity byGW441756 re- Accepted:October25,2012
sultedinthesuppressionoftyrosinephosphorylationofcellularproteinsincludingextracellular
signal-regulatedproteinkinase(ERK)andc-JunN-terminalkinase(JNK).Tofindnoveltargets
associatedwithTrkA-mediatedtyrosinephosphorylationsignalingpathways,weinvestigated
GW441756effectsonTrkA-dependenttargetsinSK-N-MCneuroblastomacellsbyproteomic
analysis.ThemajorTrkA-dependentproteinspotscontrolledbyGW441756weredetermined
by PDQuest image analysis, identified by MALDI-TOF MS and MALDI-TOF/TOF MS/MS,
andverifiedby2DE/Westernblotanalysis.Thus,wefoundthatmostoftheidentifiedprotein
spotsweremodifiedformsinanormalcondition,andtheirmodificationswereregulatedby
TrkAactivity.Especially,ourresultsdemonstratedthatthemodificationsof(cid:2)-tubulinandhet-
erogeneousnuclearribonucleoproteinsC1/C2(hnRNPC1/C2)weresignificantlyupregulated
byTrkA,whereas(cid:2)-enolasemodificationwasdownregulatedbyTrkA,anditwassuppressed
byGW441756,indicatingthatTrkAactivityisrequiredfortheirmodifications.Takentogether,
wesuggestherethatthemajornovelTrkA-dependenttargetssuchas(cid:2)-tubulin,hnRNPC1/C2,
and(cid:2)-enolasecouldplayanessentialroleinTrkA-mediatedtyrosinephosphorylationsignaling
pathwaysviaregulationoftheirposttranslationalmodifications.
Keywords:
(cid:2)-Enolase/(cid:2)-Tubulin/CellBiology/GW441756/hnRNPC1/C2/TrkA
1 Introduction
In response to nerve growth factor (NGF), tropomyosin-
related kinase A (TrkA) is dimerized and phosphorylated
Correspondence:ProfessorChoongWonKim,DepartmentofBio- on several tyrosine residues in the catalytic domain (Y-670,
chemistry,GyeongsangNationalUniversitySchoolofMedicine, Y-674,andY-675)andnoncatalyticintracellulardomain(Y-
92Chilam-dong,Jinju660-751,SouthKorea
490 and Y-785) [1,2]. It has been known that NGF-induced
E-mail:[email protected]
Fax:[phone] autophosphorylationofY-670,Y-674,andY-675isessential
forTrkAactivityandcausestrans-phosphorylationofTrkAat
Abbreviations:ERK,extracellularsignal-regulatedproteinkinase; Y-490andY-785,resultingintheactivationofNGF-mediated
hnRNPC1/C2,heterogeneousnuclearribonucleoproteinsC1/C2; TrkAsignaling[3].TheactivatedTrkArecruitsandphospho-
IEF, isoelectric focusing; JNK, c-Jun N-terminal kinase; NGF,
rylates the adaptor protein Shc via Y-490 phosphorylation,
nerve growth factor; PLC-(cid:2), phospholipase C-(cid:3); PMF, peptide
andthusthephosphorylatedShcassociateswiththeadaptor
massfingerprinting;Tet,tetracycline;TrkA,tropomyosin-related
proteinGrb2andtheRasexchangefactorSos,leadingtothe
kinaseA
(cid:2)C 2013WILEY-VCHVerlagGmbH&Co.KGaA,Weinheim www.proteomics-journal.com
356 E.J.Jungetal. Proteomics2013,13,355–367
activationofRassignaling[4].NGF-inducedY-490phospho- that(cid:2)-and(cid:4)-tubulin,heterogeneousnuclearribonucleopro-
rylationofTrkAisupregulatedbyconstitutivelyactiveSrcin teinsC1/C2(hnRNPC1/C2),peroxiredoxin-6,and(cid:2)-enolase
SK-N-MCcells,causingenhancementofextracellularsignal- are novel TrkA-dependent targets and could play an im-
regulatedproteinkinase(ERK)signaling[5].However,inter- portant role in TrkA-mediated tyrosine phosphorylation
actionofTrkAwithSHP-1,aphosphotyrosinephosphatase, signaling pathways via regulation of their posttranslational
via Y-490 phosphorylation induces dephosphorylation at Y- modifications.
674 and Y675, leading to the inhibition of NGF-mediated
cellular processes including phosphorylation of ERK, phos-
pholipase C-(cid:3) (PLC-(cid:3)), and Akt [6]. These results suggest 2 Materials and methods
that the cellular effects of TrkA through Y-490 phospho-
rylation are dependent on the downstream signaling path- 2.1 Reagents
ways. Y-785 phosphorylation is required for the interaction
ofTrkAwithPLC-(cid:3),resultingintheactivationofRassignal- DMEM, tetracycline-screened FBS, and penicillin/
ingfollowedbyNGF-inducedapoptosisinmedulloblastoma streptomycin were from Gibco-BRL. Tetracycline (Tet)
cells[7].MutationofTrkAatY-785(Y785F)protectsfromthe was from Duchefa. Blasticidine and zeocin were from
apoptosisinducedbyeitherc-MycorUVtreatment,whereas Invitrogen. Aqueous mounting media was from Biomeda.
mutation of TrkA at Y-490 (Y490F) inhibits c-Myc-induced Twenty percent formaldehyde was from Tousimis. Super
apoptosisbutnotUV-,showingadifferentialapoptoticsig- signal west pico stable peroxide solution was from Pierce.
naling pathways on the tyrosine phosphorylation resides of Protran nitrocellulose (NC) membrane (BA83) was from
TrkA [8]. Paradoxically, it has been well known that TrkA Whatman. GW441756, iodoacetamide, trichloroacetic acid,
plays an important role in the signal transduction of cell Bradford reagent, Commassie Brilliant Blue G, octyl (cid:4)-
survival in response to NGF [1,2,9,10]. Until now, differ- D-1-thioglucopyranoside, and (cid:2)-cyano-4-hydroxycinnamic
ential mechanisms of cell survival and death by TrkA are acid (CHCA) were from Sigma-Aldrich. Forty percent
still unclear. Nonetheless, Y-490 phosphorylation, at least, acrylamide/bis-acrylamide 37.5:1 solution, urea, thiourea,
seemstobeessentialforbothTrkA-inducedcellsurvivaland CHAPS,Tris,Tween-20,ammoniumsulfate,andDTTwere
death. fromAmresco.Bio-Lyte3/10ampholyte,IPGstrip(pH4–7,
Intriguingly,NGF-independentactivationofTrkAcanbe 7cm),IPGstrip(pH4–7,17cm),mineraloil,andprotean
induced by various stimuli including amyloid-beta [11,12], plusoverlayagarosewerefromBio-Rad.IPGbuffer(pH4–7)
proteosome inhibitors such as MG-132 and lactacystin was from Amersham Biosciences. Bromophenol blue was
[13,14], and dorsomorphin, a selective inhibitor of bone fromPharmaciaBiotech.RecombinantDNaseI(RNase-free)
morphogenetic protein signaling [15]. Inhibition of protea- wasfromTakara.ProteaseinhibitorcocktailsetIwasfrom
someactivitycausesintracellularproteinaggregation,which Calbiochem.Ortho-phosphoricacid(85%)wasfromMerck.
has been quite observed in a neurodegenerative disorder Sequencing grade-modified trypsin was from Promega.
such as Alzheimer’s disease, Parkinson’s disease and amy- Antibodiesusedinthestudywere:TrkA(763,SantaCruz),
otrophiclateralsclerosis[16,17].Themicrotubule-associated phospho-TrkA(E-6,SantaCruz),phospho-Tyr(Transduction
protein tau is one of the major aggregating proteins, and Laboratories), phospho-Y204-ERK (Bioworld Technology),
its aggregation is predominantly associated with phospho- phospho-T183/Y185-JNK (Bioworld Technology), (cid:4)-actin
rylation by the protein kinases such as tau-tubulin kinase (Sigma-Aldrich), (cid:2)-tubulin (B-5–1-2, Sigma-Aldrich), acety-
1, glycogen synthase kinase 3(cid:4), and cyclin-dependent pro- lated (cid:2)-tubulin (6–11B-1, Santa Cruz), (cid:4)-tubulin (H-235,
teinkinaseCDK5/p25complexes[18,19].Thephosphoryla- Santa Cruz), hnRNP C1/C2 (4F4, Santa Cruz), (cid:2)-enolase
tiondownregulatestauinteractionswithphosphatidylinosi- (L-27,SantaCruz),secondarygoatanti-rabbitandanti-mouse
tol3-kinase,PLC-(cid:3),Grb2,andSrcfamilykinases,resulting HRPconjugates(Bio-Rad),andanti-rabbitTRITCconjugate
in the disruption of the intracellular networks of tau [20]. (Sigma-Aldrich).
Moreover, Tau is associated with A(cid:4)-induced axonal trans-
portdefects[21],andA(cid:4)regulatesposttranslationalmodifi-
cationoftauandtubulinfollowedbymicrotubuledisruption 2.2 Cellculture
[22].
We have previously showed that TrkA overexpression We have previously established TrkA-inducible stable cells,
predominantly chose cell death rather than cell survival SK-N-MC-TrkA#9and#21,whichcaninduceTrkAoverex-
in both neuronal SK-N-MC cells and non-neuronal U2OS pressioninthepresenceoftetracyclinebutnotintheabsence
cells in an NGF-independent mechanism [23]. To better [23]. The cells were maintained with medium A [DMEM,
understand TrkA-mediated intracellular pathways, we have tetracycline-screened10%FBS,1%penicillin/streptomycin]
investigated novel TrkA-dependent targets associated with containing1.25(cid:5)g/mLblasticidineand25(cid:5)g/mLzeocinin
tyrosine phosphorylation signaling pathways using the ahumidified5%CO incubatorat37(cid:6)C.TrkAoverexpression
2
inhibitioneffectofTrkAactivitybyGW441756inSK-N-MC inSK-N-MC-TrkAcellswasinducedbyaddingof2(cid:5)g/mL
neuroblastoma cells by proteomic analysis. We show here tetracyclineinthemediumAfortheindicatedtimes.
(cid:2)C 2013WILEY-VCHVerlagGmbH&Co.KGaA,Weinheim www.proteomics-journal.com
Proteomics2013,13,355–367 357
2.3 Westernblotanalysis solutionandtheheavy14%SDS-PAGEsolutioncontaining
15% sucrose; at 20 mA for 1 h and subsequently maintain
Whole cells were extracted with SDS sample buffer and with 30 mA during electrophoresis. The gels were fixed in
boiledfor5–10minat95(cid:6)C.ProteinswereseparatedonSDS fixingsolution(40%methanol,5%ortho-phosphoricacid)at
polyacrylamide gel and transferred to NC membrane. The roomtemperaturefor3handstainedwithCommassieBlue
blot was blocked for 1 h at room temperature in blocking staining solution (17% ammonium sulfate, 3% phosphoric
buffer(3%skimmilk/0.1%Tween-20/PBS)andthenincu- acid,34%methanol,0.1%CommassieBrilliantBlueG)for
batedwithprimaryantibodyat4(cid:6)Covernight.Theblotwas 24h. After destaining withH O,gel images were obtained
2
washed with PBST (0.1% Tween-20/PBS) for 15 min three usingaUMAXscanner(PowerLook2100XL).
times and incubated with HRP-conjugated secondary anti-
bodyinblockingbufferfor2h.AfterwashingwithPBST,the
blotwasanalyzedbythesupersignalECLdetectionsystem. 2.7 Imageanalysisandstatisticalsignificance
Quantitative analysis of Commassie Blue stained images
2.4 Confocalimmunofluorescencemicroscopy
wascarriedoutusingPDQuestsoftware(Bio-Rad)according
to the manufacturer’s instructions. Quantity of each spot
SK-N-MC-TrkA cells (#9 and #21) werecultured in the ab- was normalized by total valid spot intensity. Protein spots
sence(–Tet)orpresence(+Tet)oftetracyclineon6-welldish associated with TrkA-mediated tyrosine phosphorylation
with a cover slide for 20 h. The cells were washed with signaling pathways were determined by the inhibitory ef-
PBS, fixed with 3% formaldehyde/PBS for 45 min, perme- fectsofGW441756onupregulatedordownregulatedTrkA-
atedwith0.5%TritonX-100/PBSfor3–5min,blockedwith dependent targets in SK-N-MC cells. The statistical signif-
1% BSA/PBS for 1 h, and then incubated with anti-TrkA icance of image analysis was determined by the Student’s
antibodyfor2h.AfterwashingwithPBS,thecellswerein- t-test(statisticallevelofp<0.05issignificant).
cubatedwithanti-rabbitTRITCconjugatedantibodyfor1h,
washedwithPBSandmountedwithaqueousmountingme-
2.8 In-geltrypticdigestion
dia.Cellmorphologyandintracellularlocalizationofprotein
wereanalyzedbyconfocalmicroscopy(OlympusFV-500).
ThetargetspotsstainedwithCommassieBluewereexcised
andpreparedforMALDI-TOFMSanalysisinamannersimi-
2.5 Samplepreparationfor2DE lartothatpreviouslydescribed[24]withaslightmodification.
Briefly, excised spots were destained with 50% acetonitrile
WholecellswerewashedwithPBS,suspendedin2DElysis solution, reduced in 10 mM DTT/100 mM NH HCO so-
4 3
buffer (8 M urea, 4% CHAPS, 40 mM Tris, 100 mM DTT, lution for 45 min at 56(cid:6)C, and alkylated in 55 mM iodoac-
0.5%Bio-Lyte3/10ampholyte,1Xproteaseinhibitorcocktail) etamide/100 mM NH HCO solution for 30 min in the
4 3
using22-gaugesyringeneedle,andthenincubatedonicefor dark. The gel pieces were incubated for 5 min in 100 mM
30min.Thecelllysatesweretreatedwith20U/mLofDNase NH HCO andthenfor15minafteraddinganequalvolume
4 3
Ionicefor1handprecipitatedwithtrichloroaceticacid(15– ofacetonitrile.Aftervacuumdry,thegelpiecesweretreated
20%).Afterwashingwithacetone,thepelletwasdissolvedin with20(cid:5)Loftrypsindigestionsolution(50mMNH HCO ,
4 3
rehydration solution (7 M urea, 2 M thiourea, 2% CHAPS, 0.01%octyl(cid:4)-D-1-thioglucopyranoside,12.5(cid:5)g/mLtrypsin)
100mMDTT,0.0002%bromophenolblue)followedbypro- for13–15hat37(cid:6)C.Thetryptic-digestedpeptides(2(cid:5)L)were
teinquantificationusingBradfordreagent. mixedwith2(cid:5)Lofsaturatedmatrixsolution(15mgCHCA,
15 mg NC membrane, 75% acetone, 25% 2-propanol) and
1 (cid:5)L of mixed calibrants (bradykinin (0.28 pmole/(cid:5)L) and
2.6 2DEandCommassieBluestain
neurotensin (0.15 pmole/(cid:5)L)). The mixed tryptic peptides
(1.5 (cid:5)L) were immediately spotted onto stainless steel
Protein samples (500 (cid:5)g each) were added with 0.5% IPG
MALDI-TOFsampletargetplate.
buffer (pH 4–7) and rehydrated with 17 cm IPG strip
(pH 4–7) for 14 h at 20(cid:6)C in the isoelectric focusing (IEF)
cellfollowedby1Disoelectricfocusinginamaximumcur- 2.9 MALDI-TOFMSanddatabasesearching
rent of 50 (cid:5)A/IPG strip; at 250 V for 15 min, at 10 000 V
for3h,andthenat10000Vfor65000V-h.Thestripswere Mass measurement of tryptic peptides was performed with
equilibratedinbufferI(50mMTris(pH8.8),6Murea,30% MALDI-TOF MS (Voyager-DE-STR; Applied Biosystems),
glycerol,2%SDS,10mg/mLDTT)for15minandinbuffer andmassspectrawereacquiredforthemassrangeof800–
II(50mMTris(pH8.8),6Murea,30%glycerol,2%SDS, 3500DaandcalibratedwithBradikininm/z(904.4681)and
25mg/mLiodoacetamide)for15min.The2Delectrophore- Neurotensinm/z(1672.9175)asstandardpeaks.Theproteins
sisofthestripswasperformedon8.5–14%sucrosegradient wereidentifiedbypeptidemassfingerprinting(PMF)onthe
polyacrylamidegelpreparedusingthelight8.5%SDS-PAGE basis of the Swiss-Prot database using the search program
(cid:2)C 2013WILEY-VCHVerlagGmbH&Co.KGaA,Weinheim www.proteomics-journal.com
358 E.J.Jungetal. Proteomics2013,13,355–367
MASCOT(http://www.matrixscience.com),allowingpeptide at 20(cid:6)C for 14 h in the isoelectric focusing cell followed by
mass tolerance of 1.2 Da and one missed cleavage. Signifi- 1Disoelectricfocusinginamaximumcurrentof50(cid:5)A/IPG
canceofthesearcheddatawasjudgedfromthescoresmore strip; at 250 V for 15 min, at 4000 V for 2 h, and then at
than56(p<0.05)andatleastsevenmatchedpeptidemasses. 4000Vfor30000V-h.Thestripswereequilibratedinbuffer
Ifor15minandsubsequentlyinbufferIIfor15min,and
thenperformedby10%SDS-PAGE.Theproteinspotswere
2.10 MALDI-TOF/TOFMS/MSanddatabase
transferredtoNCmembraneandanalyzedbyWesternblot.
searching
Proteinsamples(50(cid:5)geach)for17cmIPGstrip(pH4–7)
wereseparatedby2DEusing8.5–14%sucrosegradientpoly-
Protein spots were excised from Commassie Blue stained
acrylamidegelsasdescribedabove.Theareasoftheidentified
2DE gels and performed in-gel digestion using modified
proteinspotswereexcisedandtransferredtoNCmembrane
porcinetrypsinasdescribed[25].Peptidemassspectrawere
followedbyWesternblotanalysis.
obtainedbyMALDI-TOF/TOFMS/MS(ABI4800plus;Ap-
pliedBiosystems),andinternalcalibrationwasaccomplished
byanalysisofautolytictrypsincleavageproducts.Searchfor
3 Results
proteinidentitywascarriedoutusingtheNCBInrdatabase
byProteinPilotv.3.0(withMASCOTasthedatabasesearch
3.1 TrkAoverexpressioncausesmorphological
engine) with peptide and fragment ion mass tolerance of
changeofSK-N-MCneuroblastomacells
50 ppm [26]. The other parameters for searching were one
missed trypsin cleavage, carbamidomethylation of cysteine, It has been reported that ectopic expression of TrkA in-
oxidationofmethionine,andmonoisotopic.Significanceof duces apoptosis of neuroblastoma cells in a p53-dependent
theidentifiedproteinswasbasedonthenumberofmatching mechanism [27]. We have previously showed that TrkA
peptidemassesandcomparisonofexperimentalandtheoreti- overexpressionresultedinasignificantcelldeathandmor-
calpropertiesoftheproteins,inadditiontodatabasesearched phological change of U2OS cells to a neuron-like pheno-
proteinscores;greaterthan84wereconsideredasastatisti- type [28]. Here, we investigated the effects of TrkA over-
callysignificant(p<0.05). expression on the morphology of SK-N-MC neuroblastoma
cells.AsshowninFig.1A,TrkA-induciblestablecelllines,
2.11 Confirmationoftheidentifiedproteinsby #9 and #21, were changed to a considerable long cell body
2DE/Westernblotanalysis or rounded phenotype in the presence of TrkA expression
(+Tet panel) but not in the absence (–Tet panel). TrkA ex-
Protein samples (10 (cid:5)g each) for 7 cm IPG strip (pH 4–7) pression was predominantly located in the cytoplasm with
wereaddedwith0.5%IPGbuffer(pH4–7)andrehydrated alargeaggresomearoundnucleusinboth#9and#21cells
Figure1. MorphologicalchangeofSK-N-MCneuroblastomacellsbyTrkAoverexpression.SK-N-MC-TrkAcells(stableclones#9and#21)
wereculturedintheabsence(–Tet)orpresence(+Tet)oftetracyclinefor20h.ThecellswerefixedandthenstainedwithTRITC-labeledred
againstanti-TrkAantibody.Differentialinterferencecontrast(DIC)imageandtheintracellularlocalizationofectopicTrkAwereanalyzedby
confocalmicroscopy.MergepanelshowscombinedimageofDICandTrkA.
(cid:2)C 2013WILEY-VCHVerlagGmbH&Co.KGaA,Weinheim www.proteomics-journal.com
Proteomics2013,13,355–367 359
Figure 2. GW441756 effects on the
cellular processes by TrkA overex-
pression.(A)SK-N-MC-TrkAcells(#9)
wereuntreatedortreatedwith2(cid:5)M
of GW441756 for 12 h in the ab-
sence or presence of tetracycline.
(B) SK-N-MC-TrkA cells (#9) were
culturedintheabsenceorpresence
of tetracycline for 12 h and then
untreated or treated with 2 (cid:5)M of
GW441756for 24 h,leading to 36 h
Tet-Oninduction.Wholecellextracts
wereanalyzedbyWesternblotusing
antibodies against TrkA, phospho-
Tyr, phospho-Y204-ERK, phospho-
T183/Y185-JNK,and(cid:4)-actin.
(see yellow-colored arrows in Fig. 1B). Unexpectedly, TrkA Fig.3.PDQuestimageanalysiswasperformedwithtwosets
expression was also detected in the nucleus of certain cells ofwell-separated2DEimages,andthentenproteinspotswere
(see yellow-colored circles in Fig. 1B), suggesting a nuclear determinedasmajorTrkA-dependenttargetsinfluencedby
traffickingofTrkAbyanunknownmechanism. GW441756.
3.2 TrkA-mediatedcellularprocessesare
suppressedbyTrkAinhibitorGW441756
WehavepreviouslyshownthatTrkAoverexpressioninduced
various cellular processes such as ERK and JNK phospho-
rylation, PARP cleavage, and (cid:3)H2AX production in U2OS
osteosarcoma cells [28,29]. In this report, we show that
TrkAoverexpressioncausestyrosinephosphorylationofcel-
lularproteinsincludingERKandJNKinSK-N-MCneurob-
lastoma cells, and these cellular processes are blocked by
TrkA inhibitor GW441756 (Fig. 2A). It has been reported
that NGF-induced neurite outgrowth was significantly sup-
pressed by the inhibition of ERK and JNK activation in
PC12cells[30].TheseresultssuggestthattheactivatedERK
and JNK could be involved in the morphological change
of SK-N-MC neuroblastoma cells by TrkA overexpression
(Fig.1).Notably,TrkAexpressionhadnosignificantchange
by GW441756 at 12 and 36 h after tetracycline treatment
(Fig.2AandB).
3.3 2DEandimageanalysisofGW441756effectson
TrkA-dependenttargets
TofindnoveltargetsassociatedwithTrkA-mediatedtyrosine
phosphorylationsignalingpathways,weinvestigatedtheef-
Figure 3. Representative 2DE images of GW441756 effects on
fects of GW441756 at 36 h after tetracycline treatment in
TrkA-dependenttargets.SK-N-MC-TrkAcells(#9)werecultured
SK-N-MC-TrkA cells by 2DE/Commassie Blue stain analy-
intheabsenceorpresenceoftetracyclinefor12handthenun-
sis.Thisstudywascarriedoutfivetimesinanindependent treatedortreatedwith2(cid:5)MofGW441756for24h.Thecellswere
experiment, and representative 2DE images have shown in analyzedby2DEandCommassieBluestain.
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360 E.J.Jungetal. Proteomics2013,13,355–367
Table1. ProteinidentificationofGW441756-controlledTrkA-dependenttargetsbyMALDI-TOFMSanalysis
Spotno. Proteinidentification Trypticfragment MASCOTprobability UniProtKB/Swiss Protein pI
byMALDI-TOFMS coverage/matches score/expect(p) -Protaccession mass(Da)
1122 Pleckstrinhomology 15%/7fragment 78/0.00034 Q8TD55 53317 5.34
domain-containing
familyOmember2
2424 Tubulinbetachain 19%/7fragment 81/0.00015 P07437 49639 4.78
((cid:4)-tubulin)
3209 Heterogeneousnuclear 19%/7fragment 70/0.002 P07910 33650 4.95
ribonucleoproteins
C1/C2(hnRNPC1/C2)
4506 Tubulinalpha-1Cchain 25%/8fragment 97/4.2e-06 Q9BQE3 49863 4.96
((cid:2)-tubulin)
4506 Tubulinalpha-1Bchain 25%/8fragment 97/4.4e-06 P68363 50120 4.94
((cid:2)-tubulin)
4532 Tubulinalpha-1Bchain 29%/9fragment 111/1.6e-07 P68363 50120 4.94
((cid:2)-tubulin)
5416 Tubulinalpha-1Bchain 23%/8fragment 97/3.9e-06 P68363 50120 4.94
((cid:2)-tubulin)
7420 Alpha-enolase 19%/7fragment 78/0.00034 P06733 47139 7.01
((cid:2)-enolase)
8101 Peroxiredoxin-6 24%/7fragment 90/2.3e-05 P30041 25019 6.00
8105 Uncharacterizedprotein 13%/8fragment 70/0.0019 Q8IXR9 71288 9.26
C12orf56
8106 Proteasomesubunit 35%/8fragment 108/3.2e-07 P60900 27382 6.34
alphatype-6
PeptideprofilesofthetenproteinspotswereanalyzedbyMALDI-TOFMS.ThesearchprogramMASCOT(http://www.matrixscience.com)
wasusedforidentificationusingpeptidemassfingerprinting.
3.4 IdentificationofGW441756-controlled the identification results of SSP 1122 and 8105 by MALD-
TrkA-dependentproteinspots TOF/TOFMS/MSanalysiswerenotstatisticallysignificant
(datanotshown).
We performed MALDI-TOF MS to identify the ten TrkA-
dependentproteinspotsregulatedbyGW441756.Thepeptide
massspectratreatedwithtrypsinwereclearlyacquiredforthe 3.5 QuantitativeinhibitioneffectsofGW441756
massrangeof800–3500Da(datanotshown)andanalyzed onTrkA-dependenttargets
by peptide mass fingerprinting using the search program
MASCOT (http://www.matrixscience.com). As shown in Protein expressions of the identified (cid:4)-tubulin (SSP 2424),
Table1,thetenproteinspotswereidentifiedaspleckstrinho- hnRNP C1/C2 (SSP 3209), and (cid:2)-tubulin (SSP 4506, 4532,
mologydomain-containingfamilyOmember2(SSP1122), 5416)spotsweremorethantwofoldupregulatedbyTrkAwith
(cid:4)-tubulin(SSP2424),hnRNPC1/C2(SSP3209),(cid:2)-tubulin astatisticalsignificance(*p<0.05),andeachTrkA-dependent
(SSP4506,4532,5416),(cid:2)-enolase(SSP7420),peroxiredoxin- upregulation was significantly inhibited by GW441756
6(SSP8101),uncharacterizedproteinC12orf56(SSP8105), (Fig. 4A). In contrast, protein expressions of the identified
andproteosomesubunitalphatype-6(SSP8106)withasta- (cid:2)-enolase(SSP7420)andperoxiredoxin-6(SSP8101)spots
tisticalsignificance(p<0.05). weremorethantwofolddownregulatedbyTrkAwithasta-
ToconfirmtheidentificationresultsbyMALDI-TOFMS tistical significance (*p < 0.05), and it was inhibited by
analysis, the ten protein spots were reanalyzed by MALDI- GW441756(Fig.4B).2DEprofilesofthesevenGW441756-
TOF/TOFMS/MSinanindependentexperiment.Thus,the controlled TrkA-dependent protein spots have shown in
identification results of seven protein spots have shown in Fig.4C.Notably,theidentifiedthreespots(SSP4506,4532,
Table2withastatisticalsignificance(p<0.05),andtheywere 5416)weredeterminedas(cid:2)-tubulinwithadifferentmobil-
wellmatchedwiththoseanalyzedbyMALDI-TOFMS.How- ity, suggesting certain posttranslational modifications of (cid:2)-
ever, SSP 8106 was identified as glutathione S-transferase tubulinasdescribedpreviously[31,32].
byMALDI-TOF/TOFMS/MSanalysis(datanotshown)but Inaddition,weshowedherethatSSP1122,8105,and8106
proteosomesubunitalphatype-6byMALDI-TOFMSanaly- were potentially identified as pleckstrin homology domain-
sis.Judgingfromtheoreticalandexperimentalpropertiesof containing family O member 2, uncharacterized protein
molecularweightandpISSP8106seemstobe,atpresent, C12orf56andproteosomesubunitalphatype-6,respectively,
potentiallyproteosomesubunitalphatype-6.Unfortunately, by MALDI-TOF MS analysis. As shown in Fig. 5, these
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Proteomics2013,13,355–367 361
Table2. ProteinidentificationofGW441756-controlledTrkA-dependenttargetsbyMALDI-TOF/TOFMS/MSanalysis
Spotno. Proteinidentification Trypticfragment MASCOTprobability UniProtKB/ Protein pI
byMALDI-TOF/TOF coverage/matches score/expect(p) EMBLaccession mass(Da)
MS/MS
2424 Tubulinbetapolypeptide 53%/35fragment 407/3e-034 Q5JP53 48135 4.70
((cid:4)-tubulin)
3209 Heterogeneousnuclear 16%/6fragment 91/0.011 G3V576 25298 9.82
ribonucleoproteins
C1/C2(hnRNPC1/C2)
4506 cDNAFLJ32131fis 40%/24fragment 419/1.9e-035 B3KPS3 46725 4.99
((cid:2)-tubulin)
4532 cDNAFLJ32131fis 38%/20fragment 314/5.9e-025 B3KPS3 46725 4.99
((cid:2)-tubulin)
5416 cDNAFLJ32131fis 38%/20fragment 267/3e-020 B3KPS3 46725 4.99
((cid:2)-tubulin)
7420 Alpha-enolase 38%/25fragment 237/3e-017 G2HEB0 47516 6.63
((cid:2)-enolase)
8101 Peroxiredoxin-6 36%/9fragment 183/7.5e-012 Q5R7E0 25119 6.00
TheproteinspotsidentifiedbyMALDI-TOFMSwerereanalyzedbyMALDI-TOF/TOFMS/MSinanindependentexperimentasdescribedin
Section2.
proteinspotsweremorethantwofoldupregulatedordown- ing17cmIPGstrip(pH4–7)andanalyzedbyWesternblot.
regulatedbyTrkAwithastatisticalsignificance(*p<0.05), AsshowninFig.9,certainmodificationsof(cid:2)-tubulin(SSP
and it was significantly suppressed by GW441756 (Fig. 5A 4532,4506,5416)andhnRNPC1/C2(SSP3209)byTrkAwas
andB). significantly downregulated by GW441756. Taken together,
ourresultssuggestthattheseTrkA-dependentnoveltargets
couldplayanimportantroleinTrkA-mediatedtyrosinephos-
3.6 CharacterizationofnovelTrkA-dependent phorylation signaling pathways via regulation of their post-
targetsassociatedwithtyrosine translationalmodifications.
phosphorylationsignalingpathways
4 Discussion
To probe the identification results, each protein spot and a
pieceof(cid:4)-actinspotasanegativecontrolwerepickedfrom Paradoxically, TrkA plays an essential role in either cell
CommassieBluestainedgelsandanalyzedbyWesternblot. survival or death depending on a cellular circumstance
AsshowninFig.6,fiveproteinspotsweredemonstratedas in an NGF-dependent or NGF-independent mechanism.
(cid:2)-tubulin(6A,SSP4532,and4506),(cid:4)-tubulin(6B,SSP2424), Moreover, activation of TrkA via Y-490 phosphorylation is
hnRNPC1/C2(6C,SSP3209),and(cid:2)-enolase(6D,SSP7420). required for both TrkA-induced cell survival [33,34] and
In Fig. 4A and B, we showed that these protein spots were death[7,11,27].Untilnow,itisstillunclearaboutthemech-
either upregulated or downregulated by TrkA, and it was anisms why TrkA induces cell survival or death via Y-490
suppressed by GW441756. To probe this, the protein sam- phosphorylationinanNGF-dependentorNGF-independent
pleswereperformedby10%SDS-PAGEfollowedbyWestern mechanism. To solve this puzzle, it seems to be very im-
blotanalysis.Asexpected,TrkAphosphorylatonattyrosine- portanttorevealthedifferencesofsignaltransductionpath-
490 was significantly downregulated by GW441756 (Fig. 7, waysbetweenTrkA-inducedcellsurvivalanddeath.Wehave
first panel). However, the identified major TrkA-dependent previously showed that TrkA overexpression induced apop-
targetssuchas(cid:2)-tubulin,(cid:4)-tubulin,hnRNPC1/C2,and(cid:2)- toticcellularprocessesincludingPARPandBaxcleavagesin
enolasewerenotaffectedbyTrkAandGW441756,andacety- bothneuronalSK-N-MCcellsandnonneuronalU2OScells,
lationof(cid:2)-tubulinappearedtohavenosignificanteffectby leading to the decrease of colony formation by cancer cells
TrkA (Fig. 7). Thus, the protein samples were performed and induction of a significant cell death [23,28]. Interest-
by 2DE using 7 cm IPG strip (pH 4–7) and analyzed by ingly,TrkA-inducedcelldeathwassuppressedbytheexpres-
Westernblot.Theresultsshowedthatcertainmodifications sionofcaveolin-1viadownregulationofY-490phosphoryla-
of(cid:2)-tubulinandhnRNPC1/C2wereupregulatedbyTrkA, tion[35],whereasitwasenhancedbytheexpressionofH2AX
whereas(cid:2)-enolasemodificationwasdownregulatedbyTrkA viaupregulationofY-490phosphorylation[36],suggestinga
(Fig.8,comparearrowareasbetween–Tetand+Tetpanel). critical role of Y-490 phosphorylation in TrkA-induced cell
Finally,toprovethatthesemodifiedproteinspotsareassoci- death.TobetterunderstandthemechanismsofTrkA-induced
atedwithTrkA-mediatedtyrosinephosphorylationsignaling celldeath,weidentifiednoveltargetsassociatedwithTrkA-
pathways, the protein samples were performed by 2DE us- mediatedtyrosinephosphorylationsignalingpathwaysusing
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362 E.J.Jungetal. Proteomics2013,13,355–367
Figure4. QuantitativeanalysisofGW441756inhibitoryeffectsonTrkA-dependentproteinspots.PDQuestimageanalysisofGW441756
effects on TrkA-dependent protein spots was performed with two sets of Commassie Blue stained 2DE images. Seven protein spots,
whichhadsameidentificationresultsinMALDI-TOFMSandMALDI-TOF/TOFMS/MSanalysis,weremorethantwofoldupregulated(A)
ordownregulated(B)byTrkAoverexpression,anditwassuppressedbyGW441756(compareproteinspotsindicatedbyarrowsinleft
fourpanels).RelativeintensitiesoftheproteinspotsdeterminedbyPDQuestsoftwarehaveshowninrightpanels.Eachbarrepresents
themean±SDfortwoindependentexperiments,anddatasignificancewasevaluatedwithaStudent’st-test,*p<0.05.(C)2DEprofileof
GW441756-controlledTrkA-dependentproteinspotsthathadmatchedidentificationresultsinMALDI-TOFMSandMALDI-TOF/TOFMS/MS
analysis.
TrkA inhibitor GW441756 by proteomic analysis (Tables 1 identified [38,39], and each isotype can undergo various
and2). posttranslational modifications such as acetylation, tyrosi-
Microtubules, formed by the repeated connection of (cid:2)- nation, detyrosination, (cid:7)2 modification, polyglutamylation,
and (cid:4)-tubulin heterodimer, are a major component of the polyglycylation,palmitoylation,andphosphorylation[31,32].
cytoskeletonandplayanimportantroleinthemaintenance These modifications of tubulin affect microtubule function
of cell shape and integrity, mitosis, and intracellular trans- andorganization[40].Moreover,ithasbeenreportedthat(cid:2)-
port [37]. Isotypes of human (cid:2)- and (cid:4)-tubulin have been tubuliniscolocalizedwithphosphorylatedTrkA,suggestinga
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Proteomics2013,13,355–367 363
Figure 5. Quantitative analysis of
GW441756 inhibitory effects on a poten-
tial TrkA-dependent protein spots. Three
proteinspotsofSSP1122,8105,and8106
were more than twofold upregulated (A)
ordownregulated(B)byTrkA,anditwas
significantlysuppressedbyGW441756.
Figure7. InvestigationofTrkAandGW441756effectsontheiden-
tifiedproteinsby1DE/Westernblotanalysis.Proteinsamples(5
(cid:5)g each) prepared for 2DE were separated by 10% SDS-PAGE
andanalyzedbyWesternblotusingantibodiesagainstphospho-
TrkA,(cid:2)-tubulin,acetylated(cid:2)-tubulin,(cid:4)-tubulin,hnRNPC1/C2,(cid:2)-
enolase,and(cid:4)-actin.
Figure6. VerificationofproteinspotsidentifiedbyMALDI-TOF
MSandMALDI-TOF/TOFMS/MSanalysis.Eachproteinspotanda
pieceof(cid:4)-actinspotwereexcisedfromCommassieBluestained possibility of (cid:2)-tubulin phosphorylation by TrkA [41]. We
gels and performed by 10% SDS-PAGE. The proteins were an- showedherethatcertainmodificationsof(cid:2)-tubulinweresig-
alyzed by Western blot using antibodies against (cid:2)-tubulin (A), nificantlyupregulatedbyTrkA(Fig.8,comparepanelsaand
(cid:4)-tubulin(B),hnRNPC1/C2(C),and(cid:2)-enolase(D).TheNCmem- b),anditwassignificantlysuppressedbyGW441756(Fig.9,
braneswerereprobedby(cid:4)-actin.
compare panels a and b), indicating that TrkA enzyme
(cid:2)C 2013WILEY-VCHVerlagGmbH&Co.KGaA,Weinheim www.proteomics-journal.com
364 E.J.Jungetal. Proteomics2013,13,355–367
Figure8. CharacterizationofTrkA
effects on the identified proteins
by2DE/Westernblotanalysis.Pro-
tein samples (10 (cid:5)g each) pre-
paredfor2DEwereperformedby
1Disoelectricfocusingusing7cm
IPG strip (pH 4–7), separated by
10% SDS-PAGE and transferred
to NC membrane. Panels a and
b were detected by Western blot
analysisusing(cid:2)-tubulinantibody,
andtheblotswerereprobedwith
(cid:2)-tubulin and (cid:4)-actin antibodies
(panelsa`andb`).Panelscandd
weredetectedwithhnRNPC1/C2
antibody,andpanelseandfwere
detectedwith(cid:2)-enolaseantibody.
Each arrow indicates major area
modifiedbyTrkA.
Figure 9. Confirmation of
GW441756 effects on the
identified protein spots by
2DE/Western blot analysis.
Protein samples (50 (cid:5)g each)
were performed by 1D isoelec-
tric focusing using 17 cm IPG
strip (pH 4–7) and separated
on 8.5–14% sucrose gradient
polyacrylamidegels.Theareas
of (cid:2)-tubulin and hnRNP C1/C2
proteinspotswereexcisedand
transferred to NC membrane.
Panelsaandbwereperformed
by Western blot analysis using
(cid:2)-tubulin antibody, and the
blots were reprobed with
(cid:2)-tubulinand(cid:4)-actinantibodies
(panelsa`andb`).Panelscand
d were detected with hnRNP
C1/C2 antibody, and the blots
were reprobed with hnRNP
C1/C2 and (cid:4)-actin antibodies
(panelsc`andd`).
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