Amyloid Precursor Protein and Presenilin1 Interact with the Adaptor GRB2 and Modulate ERK 1,2 Signaling

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Amyloid Precursor Protein and Presenilin1 Interact with the Adaptor GRB2 and Modulate ERK 1,2 Signaling
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  AmyloidPrecursorProteinandPresenilin1InteractwiththeAdaptorGRB2andModulateERK1,2Signaling * □ S Receivedforpublication,October30,2006,andinrevisedform,February20,2007  Published,JBCPapersinPress,February21,2007,DOI10.1074/jbc.M610146200 MarioNizzari ‡1 ,ValentinaVenezia ‡1 ,EmanuelaRepetto ‡1,2 ,ValentinaCaorsi §¶ ,RaffaellaMagrassi §¶ ,MariaCristinaGagliani ¶  ** ,PiaCarlo ‡ ,TullioFlorio ‡ ,GennaroSchettini ‡ ,CarloTacchetti ¶  ** ,TommasoRusso ‡‡ ,AlbertoDiaspro §¶ ** ,andClaudioRusso ‡§§3 Fromthe ‡ DipartimentodiOncologia,BiologiaeGenetica,Universita` diGenova,VialeBenedettoXV,2,16132Genova, § LAMBS,DipartimentodiFisica,Universita` diGenova,Genova, ¶ MicroscoBioResearchCenter,Universita` diGenova,ViaDodecaneso33,16146Genova,  DipartimentodiMedicinaSperimentale,Universita` diGenova,ViadeToni14,16132Genova, ** FIRCInstituteof MolecularOncology,ViadeToni14,16132Genova, ‡‡ CEINGEBiotecnologieAvanzate,DipartimentodiBiochimicaeBiotecnologieMediche,Universita` diNapoliFedericoII,ViaComunaleMargherita482,80131Napoli,and  §§ DipartimentodiScienzeperlaSalute,Universita` delMolise,ViaDeSanctis,86100 Campobasso, Italy  The amyloid precursor protein (APP) and the presenilins 1and 2 are genetically linked to the development of familialAlzheimerdisease.APPisasingle-passtransmembraneproteinandprecursoroffibrillarandtoxicamyloid-  peptides,which areconsidered responsible for Alzheimer disease neurodegenera-tion. Presenilins are multipass membrane proteins, involved inthe enzymatic cleavage of APP and other signaling receptorsand transducers. The role of APP and presenilins in Alzheimerdisease development seems to be related to the formation of amyloid-   peptides; however, their physiological function,reciprocal interaction, and molecular mechanisms leading toneurodegeneration are unclear. APP and presenilins are alsoinvolvedinmultipleinteractionswithintracellularproteins,thesignificance of which is under investigation. Among the differ-ent APP-interacting proteins, we focused our interest on theGRB2 adaptor protein, which connects cell surface receptors tointracellular signaling pathways. In this study we provide evi-dence by co-immunoprecipitation experiments, confocal andelectron microscopy, and by fluorescence resonance energy transfer experiments that both APP and presenilin1 interact with GRB2 in vesicular structures at the centrosome of the cell.The final target for these interactions is ERK1,2, which is acti- vated in mitotic centrosomes in a PS1- and APP-dependentmanner. These data suggest that both APP and presenilin1 canbe part of a common signaling pathway that regulates ERK1,2and the cell cycle. Alzheimerdiseaseisaheterogeneousneurodegenerativedis-order with insidious onset and irreversible progression, genet-icallylinkedtofewmoleculesasfollows:APP 4 onchromosome21andthetwopresenilins(PSs)onchromosome14(PS1)and1(PS2),respectively(1).Themolecularmechanismscausingspo-radic and familial (FAD) forms of AD are not yet known, andthe physiological functions of APP and PSs are also unclear.APPisatype1transmembraneproteinwhoseproteolyticproc-essing generates long soluble N-terminal fragments, a family of short soluble amyloid-   peptides (A  ) (2, 3), and a secondintracellular C-terminal fragment named AICD from “APPintracellular domain,” which is likely involved in gene regula-tion(4,5).A  peptides,aswater-solubleoligomersorinsolubleaggregates,aretoxicspeciessuspectedtocauseneurodegenera-tion and synaptic loss that, together with reactive gliosis andintracellular tangles of hyperphosphorylated Tau protein, rep-resent the classical neuropathological hallmarks of the disease(6).PSsaremultitransmembraneproteins,whichmayaccumu-late as endoproteolyzed heterodimers of N- and C-terminalfragmentsassociatedwithothermembraneproteins( i.e. nicas-trin, APH-1, and PEN-2) to form high molecular weight com-plexes (7) responsible for the intramembranous    -secretasecleavage of APP, low density lipoprotein receptor-related pro-tein, E-cadherin, Notch-1, CD44, and ErbB4 (8–12). Thehypothesized role of PSs mutations in the genesis of FAD isapparentlylinkedtotheenhancedAPPcleavageandformationof A  -(  x -42) isoforms, increasing the activity of the multipro-teic   -secretase complex (6).If we consider that there is a significant phenotypical heter-ogeneity among patients, and even among familial patientsbearing the same genetic mutation, it seems likely that otherproteins modulate APP and PS functions. In fact, APP and PSs *  This work was supported by European Community Contract LSHM-CT-2003-503330/APOPIS, Alzheimer Association Grant IIRG-02-3976, Minis-tero dell’Universita` e Ricerca Grant FIRB RBNE01ARR4-08, The CARIGEFoundation,TheEnricoandRobertaTadiottoFund(toC. R.),theSanPaoloFoundation (to C. T.), and by Ministero dell’Universita` e Ricerca Grant FIRBRBNE01FEJ7-006 (to P. C.). The costs of publication of this article weredefrayed in part by the payment of page charges. This article must there-fore be hereby marked “ advertisement  ” in accordance with 18 U.S.C. Sec-tion 1734 solely to indicate this fact. □ S  Theon-lineversionofthisarticle(availableathttp://www.jbc.org)containsa supplemental figure. 1  These authors contributed equally to this work. 2 Presentaddress:Dept.ofNeurosciences,UniversityofCalifornia,SanDiego,CA 92093-0662. 3  To whom correspondence should be addressed. E-mail: Claudio.Russo@unimol.it. 4  The abbreviations used are: APP, amyloid precursor protein; AD, Alzheimerdisease; ERK, extracellular signal-regulated kinase; MEF, mouse embryofibroblast; FRET, fluorescent resonance energy transfer; Tricine,  N  -[2-hy-droxy-1,1-bis(hydroxymethyl)ethyl]glycine; PBS, phosphate-bufferedsaline; Pipes, 1,4-piperazinediethanesulfonic acid; GST, glutathione S -transferase;siRNA,shortinterferingRNA;PS,presenilin;FAD,familialAD;SH,Srchomology;RT,reversetranscriptase;IP,immunoprecipitation;CTF,C-terminalfragment;EGFP,enhancedgreenfluorescentprotein;A  ,amy-loid-  peptides;MEK,MAPK/ERKkinase;ER,endoplasmicreticulum;APLP,APP-like protein.  THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 282, NO. 18, pp. 13833–13844, May 4, 2007© 2007 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. MAY 4, 2007• VOLUME 282•NUMBER 18  JOURNAL OF BIOLOGICAL CHEMISTRY   13833   b  y  g u e s  t   , onM ar  c h 2 4  ,2  0 1  3 www. j   b  c . or  gD  ownl   o a d  e d f  r  om  http://www.jbc.org/content/suppl/2007/02/21/M610146200.DC1.html Supplemental Material can be found at:  are in the center of a network of protein-protein interactionswhosesignificancefortheregulationofA  formationandgen-erallyforADdevelopmentisunderextensiveinvestigation(13).InadditiontotheirproteolyticrolesculminatinginA  produc-tion, PSs have been shown to interact with various proteinsinvolved in the regulation of     -secretase activity, cell survival,development,andsignaling.Inparticular,PSsareimplicatedinNotch and Wnt signaling, cell-cell adhesion, vesicular trans-port, apoptosis, calcium signaling, phosphorylation and degra-dation of    -catenin, modulation of ERK1,2, and phosphatidyl-inositol 3-kinase activity (13–17). On the other side, the APPC-terminal domain is recognized by a plethora of adaptors andsignaling molecules, the role of which for AD development isstill unclear (18, 19).Among the different APP-interacting proteins, we focusedour interest on the GRB2 (growth factor receptor-bound pro-tein 2) adaptor protein, which usually provides a critical linkbetweencellsurfacegrowthfactorreceptors,Rassignaling,andcell proliferation (20, 21), and whose interaction with APP isenhanced in AD brain (22). Besides its involvement in signaltransduction pathways mediated by tyrosine kinase receptors,GRB2 may also anchor to a number of proteins involved in cellsignaling and vesicular trafficking, such as dynamin and synap-sin(23,24),ortoproteinsregulatingcytoskeletaldynamics,cellcycle, and metastatic proliferation (21, 25–27). GRB2 is com-posedofacentralSH2domainflankedbytwoSH3domainsandinteracts through its SH2 domain with the C-terminal 682 YENPTY 687 motifofAPPuponthespecificphosphorylationof Tyr-682 (numbering on APP695 isoform) (28, 29). At pres-ent,itisunclearwhetherAPPmayhavearoleinsomeofthecellactivities in which GRB2 participates or whether GRB2 modu-lates or affects APP function or even amyloid formation.HereweprovideevidencethatGRB2interactswithAPPandPS1 in vesicular structures dispersed in the cytosol and mainly in the pericentriolar material at the centrosome, a crucialregion for microtubule nucleation, cell cycle progression,migration, cytokinesis, and cellularization (30). We also pro- vide evidence that APP and PS1 modulates the centrosomaltranslocationorphosphorylationofERK1,2duringmitosisandthat the activation of ERK1,2 depends on phosphorylation of tyrosine682ofAPPandonAPPcleavage.Thesefindingsthere-foreraisethepossibilitythatperturbationsinMEK/ERKsignal-ingmayconstituteacommonmetabolicpathwaymodulatedby both gene products that cause familial AD. EXPERIMENTALPROCEDURES  Recombinant Constructs —APP695 wild type, APPY653F,APPY682A, Y687A, and Y682A/Y687A mutants have beendescribed previously (31). The APP-EGFP construct was madeas follows. The human APP695 cloned into the pRc/CMV vec-tor (Invitrogen), as described previously, was the template formutagenesis reaction (QuikChange   mutagenesis kit, Strat-agene, La Jolla, CA). A set of primers, 5  -GAGCAGATGCAA-CCGGTCCCCCGCCACAGC-3   sense and 5  -GCTCAAGA-ACTTGGCGGTTGGATTTTCG-3  antisense, were designedto add the AgeI site and remove the stop codon to the 3  end of APP.ThemutatedDNAwasdigestedandligatedintotheSacIIand AgeI site of the pEGFP-N1 vector (Clontech). Human PS1DNA was cloned into the HindIII, XhoI site of the expression vector pcDNA3.1-V5 (Invitrogen). The PS1 mutants H163RandL286Vwerepreparedbymutagenesisreaction(Stratagene)using pcDNA3.1-PS1-V5 (Invitrogen) as template and the fol-lowing specific primers: H163R sense 5  -GGTGCTATAAG-GTCATCgtTGCCTGGCTTATTA-3  and H163R antisense5  -TAATAAGCCAGGCAacGATGACCTTATAGCACC-3  ;L286Vsense5  -CGCTTTTTCCAGCTgTCATTTACTCC-TCAAC-3  andL286Vantisense5  -GTTGAGGAGTAAATG-AcAGCTGGAAAAAGCG-3  . BACE1 cDNA was kindly provided by Dr. R. Nitsch and was cloned into the pcDNA3.1 vector. Human  GRB2  gene was cloned into the BamHI/XbaIsiteoftheexpressionvectorpcDNA3.1-V5/Hist(Invitrogen)toobtain the histidine tag. To get a fusion protein between GRB2and glutathione  S  -transferase (GRB2-GST), the human GRB2cDNA was cloned into the EcoRI/AgeI site of the expression vector pGEX-4T-2 (GE Healthcare). siRNAs corresponding to  -secretaseBACE1wasdoneasdescribedpreviously(32),withminor modifications, using Silencer siRNA construction kit(Ambion Inc. Austin, TX). The following set of oligonucleo-tides was designed: siRNA1 sense 5  -CATCCTGGTGGATA-CAGGC-3  and antisense 5  -GCCTGTATCCACCAGGATG-3  ; siRNA2 sense 5  -TGGACTGCAAGGAGTACAA-3   andantisense 5  -TTGTACTCCTTGCAGTCCA-3  ; siRNA3 sense5  -TTGGCTTTGCTGTCAGCGC-3   and antisense 5  -GCG-CTGACAGCAAAGCCA-3  . Results obtained from siRNA2are shown. RT-PCR was done in H4 wild type cells andH4-BACE1 transfected cells grown to confluence, and totalRNA was isolated using the RNeasy mini kit from Qiagen(Valencia, CA). Two micrograms of total RNA were subjectedto reverse transcriptase (RT) first strand synthesis using theSuperscript kit (Invitrogen) according to the manufacturer’sinstructions. Equal amounts of the RT product were then usedfor PCR of BACE1 or BACE2 using specific primers.  Antibodies —Polyclonal antibodies 13-0200 and 51-2700,respectively,fortheNandCterminusofAPPwerefromZymedLaboratoriesInc.andwereusedatadilutionof1:150forimmu-noprecipitation and 1:1,000 for immunodetection. The anti-body C-20 for the C terminus of APP was from Santa CruzBiotechnology(SantaCruz,CA),andtherabbitpolyclonalanti-bodyfortheNterminusofAPPwasfromSigmaandwasusedinimmunofluorescence at a 1:100 dilution. The antibody R3659specific to the N terminus of A  and C99 was a gift from Dr. P.Gambetti (Case Western Reserve University, Cleveland, OH)was used 1:100 in immunoprecipitation. Antibodies for GRB2used in Western blotting (1:5000) or in immunofluorescence(1:100) were from BD Transduction Laboratories and SantaCruz Biotechnology (C-23 and E-1), respectively. Anti-ShcAantibody (Upstate Biotechnology, Inc., Lake Placid, NY) wasused at 1:200 for immunoprecipitation. The phospho-p44/42ERK and unphosphorylated p44/42 ERK antibodies from CellSignaling Technology (Danvers, MA) were used at 1:1,000 and1:100, respectively, for immunoblotting and immunofluores-cenceexperiments.PS1N-terminalantibodyfromCalbiochemwas used at 1:200 in immunofluorescence, and the C-terminalpolyclonal antibody C-20 for PS1 (Santa Cruz Biotechnology)wasused1:100inimmunofluorescence.Antibodiesfor   -tubu-lin (Sigma) and pericentrin-specific antibody (Abcam Cam-  APPandPS1ModulateERK1,2 13834  JOURNAL OF BIOLOGICAL CHEMISTRY   VOLUME 282•NUMBER 18• MAY 4, 2007   b  y  g u e s  t   , onM ar  c h 2 4  ,2  0 1  3 www. j   b  c . or  gD  ownl   o a d  e d f  r  om   bridge, UK) were used at 1:200 and 1:500, respectively, inimmunofluorescence experiments. Antibody anti-Golgin97specific for the Golgi compartment was used at 1:2,000 inimmunoblotting (Invitrogen). Anti-V5 antibody (Invitrogen)and anti-FLAG M2 monoclonal antibody (Sigma) were used at1:100 in immunoprecipitation. Polyclonal antibody F25608antibody specific for the C terminus of APP and CTFs (kindly providedbyDr.P.Gambetti,CaseWesternReserveUniversity,Cleveland, OH) was used at 1:100 for immunoprecipitation.The polyclonal antibody anti-BACE1 (custom made) recogniz-ing the C-terminal region (amino acids 486–501) was used at1:1,000forimmunodetection.Anti-CD26antibody(SantaCruzBiotechnology) was used at 1:100 for immunoprecipitation.Alexa Fluor   568 and Alexa Fluor   488 (Invitrogen) were used1:250forimmunofluorescence.Anti-VHisantibodyconjugatedwith Alexa 568 (Invitrogen) was used at 1:100 for immunoflu-orescence. Substances and specific drugs used in this paperwere from Sigma. Cell Cultures —H4 cells and H4 cells stably expressingBACE1 with a C-terminal FLAG tag were kindly provided by Prof.RogerM.Nitsch(UniversityofZurich)andmaintainedinthe presence of 500   g/ml G418. MEF APP/APLPs null cellswerekindlyprovidedbyDr.U.Mueller,andMEFcellswildtypeand  PS   /  (genetically deficient in  PS1  and  PS2 ) have beendescribed previously (33) and were provided by Dr. B. DeStrooper. All cell lines were grown in Dulbecco’s modifiedEagle’s medium (EuroClone, Paignton-Devon, UK) supple-mented with 10% fetal bovine serum (Invitrogen),  L -glutamine(EuroClone, Paignton-Devon, UK), and antibiotics (penicillinand streptomycin) (EuroClone, Paignton-Devon, UK), unlessexplicitly stated otherwise. Transient transfections were car-ried out using FuGENE 6 reagent (Roche Diagnostics) and JetPei transfection reagent (PolyPlus transfection France) andthen incubated for 48 h.  Immunoprecipitation Experiments —Cultured cells werelysed in a buffer containing 1 m M  sodium orthovanadate, 1  Complete   (Roche Applied Science), 0.5% Nonidet P-40, 0.5%cholic acid, 100 m M  NaCl, 10 m M  Tris, and 10 m M  EDTA, pH7.6.Aftera10-mincentrifugationat1,500rpm,celllysateswereeither cold methanol-precipitated, and the resulting pelletsanalyzed by Western blotting after protein counting (Bio-Radproteinassay),orimmunoprecipitatedwithdifferentantibodiesas specified under “Results.” The antigen-antibody complexeswere collected by protein G-agarose beads (GE Healthcare),which were then electrophoresed by Tris-Tricine SDS-PAGE,andelectroblottedonpolyvinylidenedifluoridemembrane,andproteins were probed with specific antibodies diluted in PBSplus 1% normal goat serum, hybridized with a horseradish per-oxidase-conjugated secondary antibody, and detected by ECL(GE Healthcare). Centrosome Purification —The isolation of centrosomesfrom cells was done as described previously (34). Cells (1–3  10 7 ) were treated with 5   g/ml cytochalasin B and 10   g/mlnocodazolefor2hat37 °Candthenharvested,centrifuged,andwashed with a buffer containing 10 m M  Tris, pH 7.4, 150 m M NaCl, and 8% (w/v) sucrose. Cells were then lysed in 10 ml of lysisbuffercontaining1m M Hepes,pH7.2,0.5%NonidetP-40,0.5 m M  MgCl 2 , 0.1% 2-mercaptoethanol, and protease inhibi-tors (Complete  from Roche Applied Science) and centrifugedat2,500   g  for10min.Thesupernatantwasadjustedto10m M Hepes,digestedwith2units/mlDNaseIfor30minonice,thenunderlaid with 60% sucrose solution (in 10 m M  Pipes, pH 7.2,0.1% Triton X-100, 0.1% 2-mercaptoethanol), and centrifugedat 10,000    g   for 30 min. Centrosomes sedimented onto thecushion were resuspended and loaded onto a discontinuousgradientconsistingof70%sucroseatthebottomfollowedby50and40%layersandcentrifugedat120,000   g  for1h.Fractionsof 600   l were collected and diluted in 1 ml of 10 m M  Pipesbuffer,pH7.2.Centrosomeswereobtainedbycentrifugationat15,000 rpm for 10 min (35).  Immunoelectron Microscopy —H4 cells were embedded in12% gelatin, 2.3  M  sucrose and frozen in liquid nitrogen. Ultra-thin cryosections are obtained by a Reichert-Jung Ultracut Ewith FC4E cryoattachment and collected on copper-Formvarcarbon-coated grids. Immunogold localization was performedusing the antibody for GRB2 C23 from Santa Cruz Biotechnol-ogy(SantaCruz,CA)and10nmproteinA-gold-conjugated.Allsamples are examined on a Philips CM10 or Fei Tecnai 12G2electron microscope.  Immunocytochemistry, FRET Studies —Wild type H4 cellsand transfected H4 cells grown to 80% confluence on slideswere fixed with 4% paraformaldehyde for 15 min, blocked with0.1  M  glycine, and treated with 0.1% Triton X-100 in PBS for 3min. After removal of the detergent, cells were then incubatedwith the different primary antibodies in PBS plus 3% bovineserum albumin for 1 h and finally incubated with antigen-spe-cificsecondaryantibodies(Alexa488-or568-conjugated,fromInvitrogen). Cells were then mounted by Mowiol and analyzedon MRC 1024 ES confocal microscope (Bio-Rad), equippedwith a Nikon Eclipse TE 300 inverted microscope with a  60objective lens. Fluorescent resonance energy transfer (FRET)was investigated through spectral analysis and then measuredusingtheacceptorphotobleachingmethod.FRETexperimentshave been performed on a Leica TCS SP2, AOBS spectral sys-tem operating with a 20-milliwatt argon laser (for 488 nm laserline)anda1.2-milliwattHe-Nelaser(for543nmlaserline).TheFRET couples used were Alexa 488 as donor (  ex 499 nm,  em 520 nm) and Alexa 568 as acceptor (  ex 579 nm,   em 603nm), the Forster radius being 62 Å, or EGFP as donor (  ex 499nm,  em 520 nm) and Alexa 568 as acceptor (Forster radius  60 Å). Emission spectra were collected by exciting the donormolecules at 488 nm (laser power, 10%) and acquiring the flu-orescencespectrumfrom500to700nmwithabandwidthof10nm to check the presence of acceptor fluorescence because of energy transfer. To verify the occurrence of FRET, we per-formed acceptor photobleaching using 543 nm laser line (laserpower, 100%) for 5–10 min, the variability depending from cellto cell. Comparing the donor emission spectrum before andafter the complete acceptor photobleaching (acquired in thesame conditions), it is possible to observe a significant changeofthedonorpeakdependingontheenergytransfer.Tocontrolthe bleaching, we also collected acceptor emission spectraexciting at 543 nm (laser power, 15%) and collecting the fluo-rescence from 550 to 750 nm, bandwidth of 10 nm, before andafter acceptor photobleaching. To determine FRET efficiency,we performed acceptor photobleaching experiments simply   APPandPS1ModulateERK1,2 MAY 4, 2007• VOLUME 282•NUMBER 18  JOURNAL OF BIOLOGICAL CHEMISTRY   13835   b  y  g u e s  t   , onM ar  c h 2 4  ,2  0 1  3 www. j   b  c . or  gD  ownl   o a d  e d f  r  om   collectingdonorandacceptoremissionintensitiesmeasuredindifferent cells. To acquire donor fluorescence we opened thedonor channel from 500 to 540 nm (  D  em 520 nm), and for theacceptorchannelwecollectedthefluorescencefrom590to630nm (  A  em 603 nm). By analyzing donor fluorescence intensity before(I  DA ,pre )andafterthecompletebleaching(I  DA ,post )oftheacceptormolecules,itispossibletoevaluatetheefficiency(  E  )asshown in Equation 1, E   1  I DA ,pre I DA ,post  I DA ,post  I DA ,pre  (Eq. 1) No FRET was observed if the primary or secondary antibody was omitted. Further controls are described under “Results.” RESULTS  APP-GRB2 Interaction in Cultured Cells —To characterizethe interaction occurring between APP and GRB2, we usedmouse embryonal fibroblasts not expressing endogenous APPor APP-like proteins (MEFapp). As a first approach we trans-fected MEFapp cells with human APP695, or its Y682A andY687A tyrosine mutants, which may modulate the interactionwith GRB2 (29), and we performed a series of immunoprecipi-tations (IP) and co-IP experiments. Cell lysates were immuno-precipitated with an antibody specific for the C terminus of APP(51-2700)and,afterTris-TricineSDS-PAGE,wereimmu-noblotted with the same antibody.TheseexperimentsshowedthatAPP695isfullyprocessedinde-pendentlyonthetyrosinemutationtested,asdemonstratedbythepresenceofmatureandimmatureAPPisoforms(Fig.1  A ).Ontheother hand, the co-IP of APP with an anti-GRB2 antibody wasdependentonTyr-682butnotonTyr-687(Fig.1  A ).Accordingly,the mutation Y653F does not hamper the possibility to interactwith GRB2, whereas the double mutant Y682A/Y687A abolishedtheinteraction(Fig.1  A ).Conversely,IPwithantibodiesspecificfortheCterminusofAPP(51-2700andF25608)co-immunoprecipi-tated GRB2 (Fig. 1  B ,  top panel  ). As expected, this interactionappearstobedependentonTyr-682andnotonTyr-687(Fig.1  B , bottom panel  ), thus confirming the specific interaction of APP-GRB2andtheimportanceoftheTyr-682phospho-site.Tofurtherconfirm the occurrence of APP-GRB2 direct interaction, we per-formed pulldown experiments using a Sepharose-conjugatedGRB2-GST purified fusion protein. As shown in Fig. 1 C  , GRB2-GSTfusionproteinpulleddownAPP695onlyincelllysatesfromAPP695-transfected MEFapp cells. To test whether the APP-GRB2 interaction also occurs between endogenous proteins, weperformedIPexperimentsinH4humancells.Inthiscellline,theAPP 51-2700 antibody co-precipitated all isoforms of APP andGRB2. Conversely, the antibody to GRB2 co-precipitated bothAPPandGRB2aswell(Fig.1  D ).  LocalizationofAPP-GRB2InteractionintotheCell  —Inorderto identify the intracellular site of APP-GRB2 interaction, weperformed immunofluorescence experiments and confocalmicroscopy analysis (36) in human H4 cells. The two antigensco-localized in a discrete, intensely fluorescent spot in thenuclear area reminiscent of the centrosomal region (Fig. 2  A ).GRB2 is present at the centrosome, in the cytosol, and also attheplasmamembraneinanycelltypewehavesofartested(H4,SH-SY5Y,HEK293,C6,ratcorticalastrocytesandneurons,andmouse and human fibroblasts), although with heterogeneousdistribution among each different cell type. APP, detected by aspecific antibody directed to the N-terminal region, was alsopresent at the centrosome, in structures resembling the ER, inthe perinuclear region, and at the cell surface (Fig. 2  A ).Although the peak of co-localization is at the centrosome, weobserved a co-localization also in the punctate cytosolic stain-ing, in the perinuclear region, and also partially at the plasmamembrane. The proof that GRB2 localized in the centrosomearea stemmed from a double immunofluorescence experimentusing antibodies for    -tubulin, a typical centrosomal marker(37).    -Tubulin co-localized with GRB2 in a large dot next tothe nuclear region (Fig. 2  A ). Similar data were obtained usingpericentrin as a centrosomal marker. To further prove thatGRB2 and APP co-localized in the centrosome area, we trans-fected H4 cells with vectors encoding for a histidine-taggedGRB2 construct (GRB2-his) and an EGFP-tagged APP695(APP-EGFP).Immunostainingwithanantibodyspecificforthehistidine tag Alexa 568-conjugated showed that a fraction of bothtransfectedproteinsco-localizedintheperinuclearregionand at the centrosome (Fig. 2  A ). These experiments indicatethat in human H4 cells APP and GRB2 co-localize in scatteredcytosolic structures and mainly at the centrosome.The centrosome is a very peculiar region of the cell, charac-terized by a pair of centrioles surrounded by a cloud of amor-phous material ( i.e.  the pericentriolar material) whose srcinand composition are unclear. Previous reports have identifiedsignaling proteins and membrane proteins in this region (for areviewseeRef.30).TobetterdefinethelocalizationofGRB2atthe centrosome, we carried out immunogold labeling and elec-tron microscopy studies using a specific antibody for GRB2(C23). These experiments showed that GRB2 antibody labeledthe outer periphery of centrioles, the pericentriolar electron-dense material, membrane-bound vesicles, and tubulo-vesicu-lar structures (Fig. 2  B ,  top panels ). Double immunogold label-ingwithantibodiesto   -tubulinandGRB2oncross-sectionsof centrioles in the centrosome area confirmed these results (Fig.2  B ,  bottom panels ). FIGURE 1.  APP-GRB2 co-IP.  A,  APP695 wild type ( wt  ) and tyrosine mutantsY682A and Y687A are expressed and fully processed in MEF APP/APLPs nullcellsupontransfection.APPisco-IPbyGRB2antibody,exceptincellsexpress-ing the Y682A mutant. Y653F mutant, vector ( vec  ), mock ( mo ), and doublemutant ( dm )-transfected cells are shown as controls.  B,  antibodies for GRB2,ShcA(bothusedaspositivecontrols),andforAPP(51-2700andF25608),co-IPGRB2 protein except in cells expressing the APP Y682A mutant.  C,  pulldownexperimentwithaGRB2-GSTfusionproteinthatbindstoasubsetofAPP695onlyinAPP695-expressingcells.Theimmunoprecipitationwiththeantibody51-2700 to APP is shown as positive control.  D,  crossed IP in H4 cells withantibodies for APP (51-2700) and GRB2, which co-precipitate endogenousGRB2 and APP, respectively.  APPandPS1ModulateERK1,2 13836  JOURNAL OF BIOLOGICAL CHEMISTRY   VOLUME 282•NUMBER 18• MAY 4, 2007   b  y  g u e s  t   , onM ar  c h 2 4  ,2  0 1  3 www. j   b  c . or  gD  ownl   o a d  e d f  r  om   Characterization by Fluorescence Resonance Energy Transfer (FRET) of APP-GRB2 Interaction at the Centrosome —The co-localization studies suggested that the GRB2-APP interactionmay occur in the centrosome area. We sought for more directevidence of this site-specific interaction by the way of FRETexperiments combining spectral analysis with acceptor photo-bleaching (38). FRET allows measuring the vicinity betweentwo molecules within 10 nm, because of the radiationlessenergy transfer between the donor and the acceptor. We usedantibodies for APP (N terminal (Sigma), C20, and 13-0200),coupled to a secondary antibody conjugated to Alexa 488 asdonor,andantibodiesforGRB2(eitherC23orE1)coupledtoaspecific secondary antibody Alexa 568 conjugated as acceptormolecule.Spectral analysis of immunolabeled centrosomes showedthat excitation of the donor (APP) at 488 nm allowed theappearance of a first emission peak above 500 nm ( i.e.  the nor-mal emission peak for Alexa 488), followed by a second peakabove 600 nm that represents the emission from GRB2-Alexa568uponenergytransferfromthedonormolecules,suggestingthe occurrence of FRET (Fig. 3  A ,  black line ). To ascertain theincidence and the amount of FRET, we then performed accep-tor photobleaching experiments in the same conditions (39).After photobleaching of the acceptor (GRB2-Alexa 568), and asecond scan at 488 nm, we observed an increase of the donorpeakabove500nm(Fig.3  A , redline ).TheseresultsimpliedthatAPP-Alexa 488 molecules can no longer transfer their photonsto the photobleached acceptor, suggesting that the two mole-cules were in close proximity at the centrosome. Statisticalanalysis and measurement of FRET by steady state acceptorphotobleaching experiments in centrosomes from differentcellsshowedthattheAPP-GRB2interactionatthecentrosomehas an efficiency of 25    4% ( n    20 cells on at least threedifferent experiments).ControlsforFRETexperimentsincludedthefollowing:( a )to verify the complete acceptor photobleaching, we collected theemission spectra directly exciting the acceptor molecules (543nm) before and after the bleaching (Fig. 3,  right panels ); ( b ) toexclude phenomena other than FRET, we collected the inten-sity in a region centered in another centrosome in closeproximity (either in the same cell or in a second cell nearby),where we did not bleach the acceptor (Fig. 3  B ); ( c ) we checkedthe fluorescence intensity inside the bleached region but out-sidethecentrosome(Fig.3 C  );( d  )wemeasuredthefluorescenceintensityinaregionoutsidethebleachingregionwithinthecell(Fig. 3  D ); ( e ) we controlled background regions (background isreported for each graph). FRET was observed in none of theseconditions. Altogether these data indicate that APP and GRB2co-localize and interact with each other in the centrosome of H4 cells.Fig. 4  A  shows an example of FRET reflecting the interactionbetween APP and GRB2 in the centrosome. H4 cells wereimmunolabeled as described above and analyzed before andafter photobleaching by confocal analysis. Upon photobleach-ing,asignificantenhancementofdonor(APP-Alexa488)emis-sionintensity,withaparalleldecreaseofacceptor(GRB2-Alexa568) emission intensity, indicated resonance and interactionbetween APP and GRB2 in the centrosome. Analogous experi-ments using the FRET couple APP-EGFP and GRB2-his conju-gated to Alexa 568 gave similar results (data not shown).Under the same conditions, we performed also experimentsusing    -tubulin-Alexa 488 as donors and APP-Alexa 568 orGRB2-Alexa 568 as acceptors. We observed that the centroso-mal marker    -tubulin co-localized with both APP and GRB2, FIGURE2. APP-GRB2co-localizeinthecentrosome.  A ,immunofluorescencemicroscopyonH4cells.LabelingforGRB2andAPP( top )co-localizesinadiscreteperinuclear area ( arrows ) and in punctate cytosolic vesicles in the perinuclear region and close to the membrane ( arrowheads ). Labeling for GRB2 and for thecentrosome marker   -tubulin ( middle ) shows the co-localization of both proteins in a discrete perinuclear area identified as a centrosome ( arrows ).  Bottom, APP-EGFP- and GRB2-His-transfected cells are labeled with an antibody to the His tag. Transfected APP and GRB2 co-localize in a discrete perinuclear area( arrows ) and close to the membrane ( arrowheads ).  B , immunogold labeling on ultrathin cryo-sections of H4 cells.  Top,  labeling for GRB2 (15 nm gold) onlongitudinal sections of centrioles and centrosome area. Gold is associated with the periphery of the centriole ( asterisks ), vesicles, and tubulo-vesicularstructures surrounding the centrioles.  Bottom,  double labeling for GRB2 (15 nm gold) and    -tubulin (10 nm gold). Cross-sections of centrioles confirm theassociationtothecentriole( asterisks )andthepericentriolarcentrosomearea. Bar:top ,from lefttoright  349,214,and251nm,respectively; bottom ,from lefttoright   368, 342, and 342 nm, respectively.  APPandPS1ModulateERK1,2 MAY 4, 2007• VOLUME 282•NUMBER 18  JOURNAL OF BIOLOGICAL CHEMISTRY   13837   b  y  g u e s  t   , onM ar  c h 2 4  ,2  0 1  3 www. j   b  c . or  gD  ownl   o a d  e d f  r  om 
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