Amyloid Precursor Protein and Presenilin1 Interact with theAdaptor GRB2 and Modulate ERK1,2 Signaling*□S

Received for publication, October 30, 2006, and in revised form, February 20, 2007 Published, JBC Papers in Press, February 21, 2007, DOI 10.1074/jbc.M610146200

Mario Nizzari‡1, Valentina Venezia‡1, Emanuela Repetto‡1,2, Valentina Caorsi§¶, Raffaella Magrassi§¶,Maria Cristina Gagliani¶�**, Pia Carlo‡, Tullio Florio‡, Gennaro Schettini‡, Carlo Tacchetti¶�**, Tommaso Russo‡‡,Alberto Diaspro§¶**, and Claudio Russo‡§§3

From the ‡Dipartimento di Oncologia, Biologia e Genetica, Universita di Genova, Viale Benedetto XV, 2, 16132 Genova, §LAMBS,Dipartimento di Fisica, Universita di Genova, Genova, ¶MicroscoBio Research Center, Universita di Genova, Via Dodecaneso 33,16146 Genova, �Dipartimento di Medicina Sperimentale, Universita di Genova, Via de Toni 14, 16132 Genova, **FIRC Institute ofMolecular Oncology, Via de Toni 14, 16132 Genova, ‡‡CEINGE Biotecnologie Avanzate, Dipartimento di Biochimica e BiotecnologieMediche, Universita di Napoli Federico II, Via Comunale Margherita 482, 80131 Napoli, and §§Dipartimento di Scienze per la Salute,Universita del Molise, Via De Sanctis, 86100 Campobasso, Italy

The amyloid precursor protein (APP) and the presenilins 1and 2 are genetically linked to the development of familialAlzheimer disease. APP is a single-pass transmembrane proteinand precursor of fibrillar and toxic amyloid-� 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 ofamyloid-� peptides; however, their physiological function,reciprocal interaction, and molecular mechanisms leading toneurodegeneration are unclear. APP and presenilins are alsoinvolved inmultiple interactionswith intracellular proteins, 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 energytransfer experiments that both APP and presenilin1 interactwith 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.

Alzheimer disease is a heterogeneous neurodegenerative dis-order with insidious onset and irreversible progression, genet-ically linked to fewmolecules as follows: APP4 on chromosome21 and the two presenilins (PSs) on chromosome 14 (PS1) and 1(PS2), respectively (1). Themolecularmechanisms causing spo-radic and familial (FAD) forms of AD are not yet known, andthe physiological functions of APP and PSs are also unclear.APP is a type 1 transmembrane proteinwhose proteolytic proc-essing generates long soluble N-terminal fragments, a family ofshort 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, as water-soluble oligomers or insolubleaggregates, are toxic species suspected to cause neurodegenera-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). PSs aremultitransmembrane proteins, whichmay accumu-late as endoproteolyzed heterodimers of N- and C-terminalfragments associated with other membrane proteins (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 isapparently linked to the enhanced APP cleavage and formationof 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, The Enrico and Roberta Tadiotto Fund (to C. R.), the San PaoloFoundation (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 The on-line version of this article (available at http://www.jbc.org) containsa supplemental figure.

1 These authors contributed equally to this work.2 Present address: Dept. of Neurosciences, University of California, San Diego,

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, glutathioneS-transferase; siRNA, short interfering RNA; PS, presenilin; FAD, familial AD;SH, Src homology; RT, reverse transcriptase; IP, immunoprecipitation; CTF,C-terminal fragment; EGFP, enhanced green fluorescent protein; A�, amy-loid-� peptides; MEK, MAPK/ERK kinase; ER, endoplasmic reticulum; 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.

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are in the center of a network of protein-protein interactionswhose significance for the regulation of A� formation and gen-erally forADdevelopment is under extensive investigation (13).In addition to their proteolytic roles culminating in A� produc-tion, PSs have been shown to interact with various proteinsinvolved in the regulation of �-secretase activity, cell survival,development, and signaling. In particular, PSs are implicated inNotch 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 focused

our interest on the GRB2 (growth factor receptor-bound pro-tein 2) adaptor protein, which usually provides a critical linkbetween cell surface growth factor receptors, Ras signaling, 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), or to proteins regulating cytoskeletal dynamics, cellcycle, and metastatic proliferation (21, 25–27). GRB2 is com-posed of a central SH2domain flanked by two SH3domains andinteracts through its SH2 domain with the C-terminal682YENPTY687motif of APP upon the specific phosphorylationof Tyr-682 (numbering on APP695 isoform) (28, 29). At pres-ent, it is unclearwhetherAPPmay have a role in someof the cellactivities in which GRB2 participates or whether GRB2 modu-lates or affects APP function or even amyloid formation.Here we provide evidence that GRB2 interacts with APP and

PS1 in vesicular structures dispersed in the cytosol and mainlyin 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 centrosomaltranslocation or phosphorylation of ERK1,2 duringmitosis andthat the activation of ERK1,2 depends on phosphorylation oftyrosine 682 of APP and onAPP cleavage. These findings there-fore raise the possibility that perturbations inMEK/ERK signal-ingmay constitute a commonmetabolic pathwaymodulated byboth gene products that cause familial AD.

EXPERIMENTAL PROCEDURES

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 ofAPP. Themutated DNAwas digested and ligated into the SacIIand AgeI site of the pEGFP-N1 vector (Clontech). Human PS1

DNA was cloned into the HindIII, XhoI site of the expressionvector pcDNA3.1-V5 (Invitrogen). The PS1 mutants H163Rand L286Vwere prepared bymutagenesis reaction (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�; L286V sense 5�-CGCTTTTTCCAGCTgTCATTTACTCC-TCAAC-3� and L286V antisense 5�-GTTGAGGAGTAAATG-AcAGCTGGAAAAAGCG-3�. BACE1 cDNA was kindlyprovided by Dr. R. Nitsch and was cloned into the pcDNA3.1vector. Human GRB2 gene was cloned into the BamHI/XbaIsite of the expression vector pcDNA3.1-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 expressionvector pGEX-4T-2 (GE Healthcare). siRNAs corresponding to�-secretase BACE1 was done as described previously (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, for theNandC terminus ofAPPwere fromZymedLaboratories Inc. andwere used at a dilution of 1:150 for immu-noprecipitation and 1:1,000 for immunodetection. The anti-body C-20 for the C terminus of APP was from Santa CruzBiotechnology (SantaCruz, CA), and the rabbit polyclonal anti-body for theN terminus ofAPPwas fromSigma andwas used inimmunofluorescence 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-cence experiments. PS1N-terminal antibody fromCalbiochemwas used at 1:200 in immunofluorescence, and the C-terminalpolyclonal antibody C-20 for PS1 (Santa Cruz Biotechnology)was used 1:100 in immunofluorescence. Antibodies for �-tubu-lin (Sigma) and pericentrin-specific antibody (Abcam Cam-

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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-FLAGM2 monoclonal antibody (Sigma) were used at1:100 in immunoprecipitation. Polyclonal antibody F25608antibody specific for the C terminus of APP and CTFs (kindlyprovided by Dr. P. Gambetti, CaseWestern Reserve University,Cleveland, OH) was used at 1:100 for immunoprecipitation.The polyclonal antibody anti-BACE1 (custommade) recogniz-ing the C-terminal region (amino acids 486–501) was used at1:1,000 for immunodetection.Anti-CD26 antibody (SantaCruzBiotechnology) was used at 1:100 for immunoprecipitation.Alexa Fluor� 568 and Alexa Fluor� 488 (Invitrogen) were used1:250 for immunofluorescence.Anti-VHis antibody conjugatedwith 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 expressing

BACE1 with a C-terminal FLAG tag were kindly provided byProf. RogerM. Nitsch (University of Zurich) andmaintained inthe presence of 500 �g/ml G418. MEF APP/APLPs null cellswere kindly provided byDr.U.Mueller, andMEF cells wild typeand 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 were

lysed in a buffer containing 1 mM sodium orthovanadate, 1�Complete� (Roche Applied Science), 0.5% Nonidet P-40, 0.5%cholic acid, 100 mM NaCl, 10 mM Tris, and 10 mM EDTA, pH7.6. After a 10-min centrifugation at 1,500 rpm, cell lysateswereeither cold methanol-precipitated, and the resulting pelletsanalyzed by Western blotting after protein counting (Bio-Radprotein assay), or immunoprecipitatedwith different antibodiesas specified under “Results.” The antigen-antibody complexeswere collected by protein G-agarose beads (GE Healthcare),which were then electrophoresed by Tris-Tricine SDS-PAGE,and electroblotted onpolyvinylidene difluoridemembrane, 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 centrosomes

from cells was done as described previously (34). Cells (1–3 �107) were treated with 5 �g/ml cytochalasin B and 10 �g/mlnocodazole for 2 h at 37 °C and then harvested, centrifuged, andwashed with a buffer containing 10 mM Tris, pH 7.4, 150 mMNaCl, and 8% (w/v) sucrose. Cells were then lysed in 10 ml oflysis buffer containing 1mMHepes, pH 7.2, 0.5%Nonidet P-40,0.5 mM MgCl2, 0.1% 2-mercaptoethanol, and protease inhibi-

tors (Complete� from Roche Applied Science) and centrifugedat 2,500� g for 10min. The supernatant was adjusted to 10mMHepes, digested with 2 units/ml DNase I for 30min on ice, thenunderlaid with 60% sucrose solution (in 10 mM 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 discontinuousgradient consisting of 70% sucrose at the bottom followed by 50and 40% layers and centrifuged at 120,000� g for 1 h. Fractionsof 600 �l were collected and diluted in 1 ml of 10 mM Pipesbuffer, pH 7.2. Centrosomes were obtained by centrifugation at15,000 rpm for 10 min (35).Immunoelectron Microscopy—H4 cells were embedded in

12% 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) and 10nmproteinA-gold-conjugated.Allsamples are examined on a Philips CM10 or Fei Tecnai 12G2electron microscope.Immunocytochemistry, FRET Studies—Wild type H4 cells

and 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-cific secondary antibodies (Alexa 488- or 568-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 measuredusing the acceptor photobleaching method. FRET experimentshave been performed on a Leica TCS SP2, AOBS spectral sys-tem operating with a 20-milliwatt argon laser (for 488 nm laserline) and a 1.2-milliwattHe-Ne laser (for 543 nm laser line). TheFRET couples used were Alexa 488 as donor (�ex499 nm,�em520 nm) and Alexa 568 as acceptor (�ex579 nm, �em603nm), the Forster radius being 62 Å, or EGFP as donor (�ex499nm, �em520 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-orescence spectrum from500 to 700 nmwith a bandwidth of 10nm to check the presence of acceptor fluorescence because ofenergy 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 changeof the donor peak depending on the energy transfer. To controlthe 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

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collecting donor and acceptor emission intensities measured indifferent cells. To acquire donor fluorescence we opened thedonor channel from 500 to 540 nm (D�em520 nm), and for theacceptor channel we collected the fluorescence from 590 to 630nm (A�em603 nm). By analyzing donor fluorescence intensitybefore (IDA,pre) and after the complete bleaching (IDA,post) of theacceptormolecules, it is possible to evaluate the efficiency (E) asshown in Equation 1,

E � 1 �IDA,pre

IDA,post� IDA,post � IDA,pre (Eq. 1)

No FRET was observed if the primary or secondary antibodywas 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 ofAPP (51-2700) and, after Tris-Tricine SDS-PAGE, were immu-noblotted with the same antibody.Theseexperiments showed thatAPP695 is fullyprocessed inde-

pendently on the tyrosinemutation tested, as demonstratedby thepresence ofmature and immature APP isoforms (Fig. 1A). On theother hand, the co-IP of APP with an anti-GRB2 antibody wasdependent on Tyr-682 but not on Tyr-687 (Fig. 1A). Accordingly,the mutation Y653F does not hamper the possibility to interactwith GRB2, whereas the double mutant Y682A/Y687A abolishedthe interaction (Fig.1A).Conversely, IPwithantibodies specific forthe C terminus of APP (51-2700 and F25608) co-immunoprecipi-tated GRB2 (Fig. 1B, top panel). As expected, this interactionappears to be dependent on Tyr-682 and not on Tyr-687 (Fig. 1B,bottom panel), thus confirming the specific interaction of APP-GRB2and the importanceof theTyr-682phospho-site.To furtherconfirm the occurrence of APP-GRB2 direct interaction, we per-formed pulldown experiments using a Sepharose-conjugatedGRB2-GST purified fusion protein. As shown in Fig. 1C, GRB2-GST fusion protein pulled down APP695 only in cell lysates fromAPP695-transfected MEFapp cells. To test whether the APP-GRB2 interaction also occurs between endogenous proteins, weperformed IP experiments in H4 human cells. In this cell line, theAPP 51-2700 antibody co-precipitated all isoforms of APP andGRB2. Conversely, the antibody to GRB2 co-precipitated bothAPP and GRB2 as well (Fig. 1D).Localization of APP-GRB2 Interaction into the Cell—In order

to 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. 2A).GRB2 is present at the centrosome, in the cytosol, and also atthe plasmamembrane in any cell typewe have so far tested (H4,

SH-SY5Y,HEK293, C6, rat cortical astrocytes and neurons, 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. 2A).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. 2A). 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). Immunostainingwith an antibody specific for thehistidine tag Alexa 568-conjugated showed that a fraction ofboth transfected proteins co-localized in the perinuclear regionand at the centrosome (Fig. 2A). 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 originand composition are unclear. Previous reports have identifiedsignaling proteins and membrane proteins in this region (for areview see Ref. 30). To better define the localization of GRB2 atthe 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. 2B, top panels). Double immunogold label-ing with antibodies to �-tubulin and GRB2 on cross-sections ofcentrioles in the centrosome area confirmed these results (Fig.2B, 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 nullcells upon transfection. APP is co-IP by GRB2 antibody, except in cells express-ing the Y682A mutant. Y653F mutant, vector (vec), mock (mo), and doublemutant (dm)-transfected cells are shown as controls. B, antibodies for GRB2,ShcA (both used as positive controls), and for APP (51-2700 and F25608), co-IPGRB2 protein except in cells expressing the APP Y682A mutant. C, pulldownexperiment with a GRB2-GST fusion protein that binds to a subset of APP695only in APP695-expressing cells. The immunoprecipitation with the antibody51-2700 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.

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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, and antibodies for GRB2 (either C23 or E1) coupled to aspecific secondary antibody Alexa 568 conjugated as acceptormolecule.Spectral analysis of immunolabeled centrosomes showed

that 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-Alexa568 upon energy transfer from the donormolecules, suggestingthe occurrence of FRET (Fig. 3A, 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 donorpeak above 500 nm (Fig. 3A, red line). These results implied thatAPP-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 differentcells showed that the APP-GRB2 interaction at the centrosome

has an efficiency of 25 � 4% (n � 20 cells on at least threedifferent experiments).Controls for FRET experiments included the following: (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. 3B); (c) we checkedthe fluorescence intensity inside the bleached region but out-side the centrosome (Fig. 3C); (d) wemeasured the fluorescenceintensity in a region outside the bleaching regionwithin the cell(Fig. 3D); (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 ofH4 cells.Fig. 4A shows an example of FRET reflecting the interaction

between APP and GRB2 in the centrosome. H4 cells wereimmunolabeled as described above and analyzed before andafter photobleaching by confocal analysis. Upon photobleach-ing, a significant enhancement of donor (APP-Alexa 488) emis-sion intensity, with a parallel decrease of acceptor (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 experiments

using �-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,

FIGURE 2. APP-GRB2 co-localize in the centrosome. A, immunofluorescence microscopy on H4 cells. Labeling for GRB2 and APP (top) co-localizes in a discreteperinuclear 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 theassociation to the centriole (asterisks) and the pericentriolar centrosome area. Bar: top, from left to right 349, 214, and 251 nm, respectively; bottom, from left toright 368, 342, and 342 nm, respectively.

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confirming the presence of these two proteins at the centro-some (Fig. 4, B and C). Spectral analysis and acceptor photo-bleaching experiments indicated that there was no FRETbetween �-tubulin and APP. In fact, also from Fig. 4B it is evi-dent that the APP emission intensity did not rise after GRB2photobleaching. Similar negative results were obtained analyz-ing the couple �-tubulin and GRB2 (Fig. 4C), confirming thespecificity of the APP-GRB2 interaction in the centrosome.PS1-GRB2 Interaction at the Centrosome—Previous reports

have described the presence of PS1 in the centrosome (40, 41).To verify whether PS1 co-localizes and interacts with GRB2 incentrosomes of H4 human cells, we performed double immu-nofluorescence studies. Antibodies directed to both the N-ter-minal (Fig. 5A) and C-terminal (Fig. 5B) regions of PS1 con-

firmed the localization of PS1 in centrosomes, as well as inGolgi-like structures, and at the cell surface. As expected, boththe polyclonal C23 and the monoclonal E1 antibodies to GRB2co-localized with the N terminus (Fig. 5A) and the C terminus(Fig. 5B) of PS1, at the centrosome. In addition, the co-localiza-tionwith �-tubulin further confirmed the PS1 expression in thecentrosome (Fig. 5C). To test whether the PS1-GRB2 co-local-ization in the centrosome was associated with a direct interac-tion between the two proteins, we transfected MEF cells PS1and PS2 null (MEFps�/�) with vectors encoding for either PS1wild type or with PS1 carrying familial mutations of Alzheimerdisease (i.e. H163R and L286V), tagged with a C-terminal V5tag. Cell lysates were analyzed by a combination of IP andimmunoblotting. These experiments showed that the antibodyfor GRB2 was able to precipitate the V5-tagged C-terminal PS1fragment migrating at 20 kDa (Fig. 5D). Similarly, the antibodyto the PS1-C-terminal V5 tag co-precipitated GRB2 proteinmigrating at 25 kDa (Fig. 5D). The expression of mutated PS1proteins carrying familial mutations did not hamper the inter-action with GRB2 (Fig. 5D). In parallel experiments we verifiedby crossed IP and immunoblotting the reciprocal interactionbetween endogenous PS1 and GRB2 in H4 cells. The IP with anantibody for PS1 co-precipitated GRB2 at 25 kDa and similarlythe IP with GRB2 co-precipitated PS1 C-terminal fragmentmigrating at 19 kDa (Fig. 5E). A parallel IP performed with anunrelated antibody (CD26) was done as a control for the spec-ificity of the interaction. The relative amount of PS1 co-precip-itated byGRB2 corresponds to the 1.24� 0.33% of the total PS1content, whereas the relative amount of GRB2 co-precipitatedby the antibody for PS1 (N terminus) is 1.8� 0.2%.However,wemust consider that under our conditions, the efficiency ofGRB2 IP is around 7.8% and for PS1 IP is 6.5%.Altogether, theseexperiments confirm the presence of PS1 in the centrosomalarea and suggest a relevant interaction with GRB2.As shown previously for APP, to prove the centrosomal

interaction PS1-GRB2, we performed FRET experiments asdescribed above (Fig. 3). H4 cells were immunolabeled with anantibody for GRB2 coupled to a secondary antibody Alexa 568conjugated as acceptor, and the two antibodies for the C termi-nus of PS1 (C20) or for PS1 N terminus both conjugated toAlexa 488 and used alternatively as donors. Spectral analysisshowed a significant increase of the donor peak after a completeacceptor photobleaching at the centrosome (Fig. 6, A and C).PS1 C-terminal GRB2 interaction displayed a FRET efficiencyof 23� 5% (n� 25 cells) at the centrosome, whereas the couplePS1 N-terminal GRB2 showed an efficiency of 18 � 6 (n � 13cells). The same experiments were done also with the couplesPS1 C-terminal Alexa 488 or PS1 N-terminal Alexa 488 asdonors, and �-tubulin-Alexa 568 as acceptor. Besides their co-localization, after photobleaching of �-tubulin, the intensity ofthe signal from PS1C or N-terminal labeling did not change(Fig. 6, B and D), confirming the specificity of the interactionPS1-GRB2. C-terminal PS1 and �-tubulin (Fig. 6B) showed anonsignificant increase of the donor peak (E � 4 � 4% n � 18cells). Therefore, PS1-GRB2 directly and specifically interactsin the centrosome.Toobtain direct biochemical proof of the presence ofAPP, PS1,

and GRB2 in the centrosome, we performed Western blotting

FIGURE 3. Emission spectra of immunolabeled centrosomes. f, emissionbefore acceptor photobleaching; F, after emission acceptor photobleaching.Background spectra obtained before (Œ) and after (�) acceptor photobleach-ing, respectively. Left column, donor excitation wavelength (488 nm). Rightcolumn, acceptor excitation wavelength (543 nm). A, intensity measured in aregion of interest centered on a centrosome where we performed acceptorphotobleaching experiment. After acceptor photobleaching (GRB2-Alexa568), the second peak is extinguished, whereas the donor peak (APP-Alexa488) increases demonstrates the occurrence of FRET. B, intensity measured ina region of interest centered on a centrosome nearby where we did not per-form acceptor photobleaching. C, intensity measured inside the bleachingregion but outside the centrosome. There is no increase in the donor peak. D,intensity measured outside the bleaching region elsewhere in the cell to con-trol for any possible change in fluorescence because of phenomena otherthan FRET. Error bars are part of each experimental point.

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analysis on purified centrosomes. Centrosome-enriched fractionswere prepared fromH4 cells after treatment with nocodazole andcytochalasin B to depolymerize the microtubule-microfilamentnetwork. Cells were lysed, and the post-nuclear supernatant wassubjected to sucrose density gradient centrifugation procedure asdescribed previously (34) withminormodifications. The fractionswere collected and analyzed by immunoblotting with differentantibodies. In this protocol, the centrosomeswere enriched in the50–70%sucrose regionof thediscontinuousgradient correspond-ing to fractions7–9.AsshowninFig.7, the�-tubulinwasprimarilyrecovered in these fractions, although some �-tubulin also sedi-mented in the lower sucrose fractions corresponding to the cyto-plasmic pool of the protein (34). APP, PS1, and GRB2 also co-sedimented with the centrosome fractions 7–9, whereasGolgin-97 immunoreactivity was detected only in whole cell

lysates (Fig. 7,� lane) andwas absentin centrosomal fractions, indicatingthe absence ofGolgi vesicles fromoursamples, which represent the mostcommon form of contamination inthese preparations.APP Regulates Phopsho-ERK1,2

Levels—Because GRB2 is mainlyinvolved in the regulation of growthfactor signaling targeted to ERK1,2activation, we investigated whetherthe centrosomal interactions withAPP and/or PS1 would influenceERK1,2 phosphorylation. We ini-tially verified whether changes inthe levels of APP in vitro wouldaffect the phosphorylation ofERK1,2.We increased APP levels byeither transfecting H4 cells withvectors encoding for APP695 or bysilencing the mRNA of the �-secre-tase BACE1. On the contrary, wedecreased APP content overex-

pressing BACE1 to increase APP cleavage. RT-PCR revealedthat bothH4 andH4-BACE expressed BACE1mRNA (Fig. 8A).In particular, the expression of the engineered BACE1 proteinwas verified by direct immunostaining with an anti-FLAG anti-body and by an anti-BACE1 C-terminal antibody upon West-ern blotting (Fig. 8A). These data show that in H4 cells express-ing BACE1 (H4-BACE), there is effectively a higher amount ofBACE1 protein as expected.To provide further evidence of the enhanced BACE1 expres-

sion and activity on APP processing, wemonitored the amountof CTFs upon BACE1 overexpression as described previously(42). Cleavage of APP by BACE1 generates C99 and C89,whereas cleavage by -secretase results in C83, all of which canbe resolved by Tris-Tricine SDS-PAGE, along with their phos-phorylated isoforms (43) (Fig. 8B).

FIGURE 4. Example of FRET acceptor photobleaching, reflecting the interaction between APP and GRB2 in the centrosome. A, after photobleaching ofthe acceptor GRB2-Alexa 568 (GRB2post), the intensity of the donor APP-Alexa 488 (APPpost) is significantly enhanced. The signals of APP-Alexa 488 andGRB2-Alexa 568 before bleaching (APPpre and GRB2pre) are shown as reference. Purple circles and brown squares represent bleached noncentrosomal areas andbackground areas, respectively. B, similar experiments on control cells immunolabeled for �-tubulin and APP; C, �-tubulin and GRB2 show absence of FRETupon photobleaching.

FIGURE 5. PS1-GRB2 co-localization. A and B, immunofluorescence microscopy on H4 cells labeled withantibodies for GRB2 (polyclonal C23 and monoclonal E1) and PS1 (N-terminal and C-terminal C20), whichco-localize in punctate cytosolic vesicles and at the centrosome. C, immunolabeling with antibodies for the Cterminus of PS1 and �-tubulin is shown as control for the centrosomal localization of both proteins. D, crossedimmunoblotting identifies PS1 C-terminal fragments detected by anti-V5 staining upon GRB2 immunoprecipi-tation, and similarly GRB2 is co-precipitated by the antibody for the V5 tag expressed on the PS1 C terminus.The co-IP occurs in cells transfected with PS1 wild type (wt) or with the familial mutations H163R and L286V.Vector (vec) and mock (mo) transfected cells are shown as controls. E, parallel experiments on endogenousproteins on H4 cells show that GRB2 migrating at 25 kDa is co-IP by anti-PS1 antibody (left), and that the PS1C-terminal fragment migrating at 19 kDa is instead co-precipitated by the antibody for GRB2 (right). Immuno-blotting for each precipitating antibody is shown as control. An unrelated antibody (CD26) is used as furthercontrol for the IP. IgG are shown as loading control for each IP.

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In our conditions, the analysis with an antibody specific forthe very N terminus of C99 (44, 45) confirms that the first twobands represent C99 and its phosphorylated isoform (43). Theincreased expression of BACE1 resulted in a significantlyenhanced CTFs formation, with cleavages at position 1 and 11predominating (Fig. 8B), as demonstrated by the increasedamount of phospho-C99, C99, and C89 fragments in H4-BACEcells, compared with H4 wild type cells.We then determined the basal level of expression of phos-

pho-ERK1,2 (pERK1,2) upon overexpression of APP and/orBACE1. H4 cells transfected with vectors encoding forAPP695 showed a synchronous up-regulation of both APPand pERK1,2 after 16 h of starvation in medium withoutserum (Fig. 8C). The co-transfection with BACE1 and itsoverexpression decreased significantly both APP andpERK1,2 levels (Fig. 8C). The densitometric quantification indifferent experiments showed a significant enhancement of

pERK1,2 levels upon overexpression of APP695. Wheninstead BACE1 was overexpressed along with APP, we meas-ured a significant reduction of pERK1,2 activation (Fig. 8D).The overall effect resulted in a significant up-regulation ofcell growth, measured by [3H]thymidine incorporation, incells overexpressing APP695, which is negatively modulatedby BACE1 (Fig. 8E). To verify the specificity of this signaling,we measured the levels of pERK1,2 in cells transfected withthe APP mutant Y682A, which is processed and cleaved toform CTFs as the wild type and the Y687A mutant (Fig. 9A),but does not interact with GRB2 (Fig. 1, A and B). In theseexperiments we observed that the expression of the APP695wild type or the Y687A mutant enhanced pERK1,2, whereasthe tyrosine mutation at residues 682 significantly decreasedthe phosphorylation of ERK1,2 (Fig. 9B). These experimentsruled out also the possibility that the positive effect onpERK1,2 exerted by APP695 would be generated followingan enhanced formation of soluble APP N-terminal frag-ments, by a sort of paracrine effect. In fact, the APP mutantY682A, which is normally processed and cleaved as the wildtype isoform, does not activate ERK1,2, thus suggesting thatthe mechanism, rather than a proliferative effect of solubleAPP, is linked to the C-terminal region of APP and it isGRB2-related. We then verified the effect of the overexpres-sion of BACE1 compared with its silencing by RNA interfer-ence. The overexpression of BACE1 diminished pERK1,2,and its silencing with a specific siRNA (32) significantly up-regulated ERK1,2 phosphorylation (Fig. 9C), suggesting adirect correlation between APP amount, BACE1 activity,and activation of pERK1,2. The treatment with PD98059, aspecific inhibitor of MEK kinase, was used as control for thephosphorylation of ERK1,2 (Fig. 9C). Altogether these datasuggest that APP, through its interaction with GRB2,enhances ERK1,2 phosphorylation in a manner that in partdepends on its cleavage by BACE1.

FIGURE 6. Emission spectra of immunolabeled centrosomes. f, emissionbefore acceptor photobleaching; F, emission after acceptor photobleaching.Background spectra obtained before (Œ and after (�) acceptor photobleach-ing, respectively. Left column, donor excitation wavelength (488 nm); rightcolumn, acceptor excitation wavelength (543 nm). Intensity is measured in aregion of interest centered on a centrosome where we performed acceptorphotobleaching experiments for the couples. A, PS1C-terminal/GRB2; B,PS1C-terminal/�-tubulin; C, PS1N-terminal/GRB2; and D, PS1N-terminal/�-tu-bulin. Evidence of FRET is statistically significant only for the couples PS1C-terminal/GRB2 and PS1N-terminal/GRB2. (Error bars are part of each experi-mental point.)

FIGURE 7. H4 cell lysates were fractionated by sucrose density gradient.Fractions 1–10 are the 40 –70% sucrose fractions. Equal amount of each frac-tion was subjected to immunoblot analysis. Fractions 7–9 corresponding tothe 50 –70% interface sucrose densities are enriched in centrosome proteins.APP, PS1, and GRB2 co-sediments with fractions 7–9. A whole cell lysate wasloaded as a positive (�) control for each antibody. The Golgin-97 marker forGolgi was detected in whole extract only, and �-tubulin is highly enriched infractions 7–9. Similar results were obtained from six independentexperiments.

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Presence and Localization of Phospho-ERK1,2 at the Centro-some in Mitotic Cells in a PS1- and APP-dependent Manner—Our data suggest that a target for the signaling of APP andPS1 is the kinase ERK1,2, which is known to be down-regu-

lated by PSs loss (16, 17), and isup-regulated by APP overexpres-sion and down-regulated byBACE1 overexpression (thispaper). We therefore analyzed thestatus of ERK1,2 at the centro-some, where the phosphorylatedform of ERK1,2 is detected duringmitosis (46), hypothesizing thatboth APP and PS1, which interactwith GRB2 at the centrosome,would influence ERK1,2 activationat this site. By analyzing theimmunofluorescence in MEFAPP/APLPs null or PSs null cellswith antibodies specific forpERK1,2 and �-tubulin, weobserved that the phosphorylatedisoforms of ERK1,2 were detecta-ble at the centrosome in mitoticcells and co-localized with �-tubu-lin (Fig. 10). The amount of cen-trosomal pERK1,2 was very low inAPP-null cells and in PSs-nullcells, in comparison with APP wildtype- or PS1 wild type-expressingcells, thus suggesting a direct cor-relation between the presence ofAPP or PS1 and the amount ofpERK1,2 at the centrosome. It isworth noting that in APP-null andPSs-null cells, the occurrence ofextranumerary centrosomes oraberrant mitotic fuses is very com-mon and is significantly enhancedin comparison with wild type cells.

DISCUSSION

Our data show that APP and PS1 modulate the centrosomalmitotic activity of ERK1,2, through their interaction withGRB2. Although APP and PS1 are pivotal proteins in AD gen-eration, at present it is unclear whether this signaling has arelevance for AD, or if it is rather involved in the normal cellcycle regulation.To date the most relevant role for APP or PSs on AD develop-

ment is linked to the “amyloid hypothesis,” which considers theformation of A� peptides the central event of the neurodegenera-tion (2,6). In thisview,PSsareconsideredessentiallyas�-secretaseregulatorymolecules, APP the substrate of the �-secretase action,and corollary or secondary events are Tau-linked pathology andgliosis. The theory foresees that familial mutations in both pro-teins enhance and accelerate A� formation, triggering the patho-genic cascade that leads to neurodegeneration (47). A second the-ory, complementary to the amyloidhypothesis, proposes thatAPPand PSs may modulate a yet unclear cell signal, the disruption ofwhich may induce cell cycle abnormalities, neuronal death, even-tually A� formation, and finally dementia (14). In support of thistheory are the following: (a) cell cycle defects represent a major

FIGURE 8. APP modulates ERK1,2 activation. A, H4 cells were engineered to overexpress BACE1 as shown byRT-PCR for BACE1 and BACE2, by Western blotting for BACE1, and for its C-terminal FLAG; B, analysis of CTFs bydirect Western blotting after being IP with the antibody 51-2700 for the C terminus of APP or with the antibodyR3659 for the N terminus of C99. Cells in which BACE1 is overexpressed either transiently (tr) or in a stable (st)manner show an increment of phospho-C99 (phC99), C99, and C89, in comparison with the wild type (wt) cells.C83 levels are unaffected. C, upon overexpression of APP695, both APP and pERK1,2 levels are up-regulated,whereas are both down-regulated when a BACE1-expressing vector is co-transfected. D, almost 2-fold incre-ment in phospho-ERK1,2 levels is measured by densitometric analysis in six different experiments uponAPP695 transfection. This increment is instead significantly down-regulated if BACE1 is co-transfected.E, [3H]thymidine incorporation assay shows that DNA synthesis is significantly enhanced in H4 cells uponAPP695 overexpression, although it is reduced if BACE1 levels are simultaneously enhanced.

FIGURE 9. ERK1,2 up-regulation depends on APP. A, mutants Y682A andY687A of APP695 are processed and cleaved as the wild type (wt) isoformupon transfection in APP/APLPs null MEF cells. B, expression of APP695 wildtype (wt) or Y687A mutant results in a parallel up-regulation of phospho-ERK1,2. The expression of the Y682A mutant instead results in a significantlyreduced activation of pERK1,2. C, phosphorylation of ERK1,2 is enhanced bythe overexpression of APP695 and by the RNA silencing of the constitutiveBACE1 and is down-regulated by the overexpression of BACE1 and by thetreatment with the MEK kinase inhibitor PD98059 (PD). ERK1,2 and tubulin areshown as loading controls, whereas the transfection with an unrelated vector(vec) is shown as control for the specificity.

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neuropathological feature in AD and in transgenic animalmodelsof AD (48–51); (b) APP and PSs have been associated with chro-mosome missegregation, trisomy 21 mosaicism (41, 52); and (c)bothproteins interactwith a complexproteinnetworkwith a clearrelevance for signal transduction mechanisms (13, 19, 53, 54). Inthis context, the C-terminal domain of APP has a central rolebecause of the presence of the 682YENPTY687 motif that repre-sents the docking site formultiple interacting proteins involved incell signaling (22, 29, 31, 55–64). Among different APP-interact-ing proteins, only ShcAandGRB2act through a specific phospho-rylation step at Tyr-682 (29), the intimate regulation of which isstill under investigation. GRB2 and ShcA, which usually connectgrowth factor receptors to mitogen-activated protein kinase(MAPK) signaling, may act independently from each other, andAPP has the capability to recruit GRB2 directly or indirectlythrough ShcA (21, 29, 65).Our data show that APP regulates pERK1,2 levels, its phos-

phorylation/translocation to the centrosome, and the cell pro-liferation rate. We demonstrated that the proteolysis of APP

regulates ERK1,2 activation as well, because BACE1 overex-pression enhances APP cleavage and CTFs formation andreduces ERK1,2 phosphorylation in H4 cells. Noteworthy,APP-GRB2 interaction is present in proliferating cells, and it isabolished in apoptotic conditions, where the interaction C99-GRB2 instead prevails (65). Considering that both complexesmight influence pERK1,2 activity, it would be interesting toobserve if selective �-secretase inhibitors may modulatepERK1,2 activity in apoptotic models. Altogether these datasuggest a dual and opposite pERK1,2 function as observed pre-viously in other systems, where enhanced pERK1,2 levels caneither enhance proliferation or cause growth arrest modulatingdifferent cyclin kinases (66). As experienced in other tyrosinekinase receptor-dependent signaling, a selective phosphoryla-tion at Tyr-682 of APP regulates both the interaction withGRB2 and ERK1,2 phosphorylation, suggesting a typical recep-tor/transducer-like activity for APP aimed at the regulation ofERK1,2 through its interaction with GRB2.In this scenario, the signaling activity of APP is complicated

by the fact that PS1 as well interacts with GRB2 and modulatesERK1,2 signaling as shown here and in previous works (16, 17).Thus, the hypothesis is that both APP and PS1 participate onthe same signaling pathway through GRB2, which become thefirst identified signaling protein that interacts with both geneproducts responsible for the generation of familial AD.Although the presence of PSs in the centrosome has beenreported previously (40, 41), the simultaneous presence of PS1,APP, and GRB2, andmost importantly their reciprocal interac-tion, represents a new finding thatmight opennewperspectivesfor the investigation of PSs and APP function, as well as for theunderstanding of GRB2 role in cell signaling and cell cycle reg-ulation. In fact, the centrosome is a crucial region for microtu-bule nucleation, cell cycle progression, migration, cytokinesis,and cellularization (30).The centrosome is a central component of the cell cycle

machinery of eukaryotic cells, and centrosome amplificationsor perturbations of centrosomal proteins are linked to cell cyclemisregulation and cancer (67, 68). The microtubule cytoskele-ton provides a highly dynamic intracellular framework uponwhich trafficking of vesicles, individual proteins, and macro-molecular complexes can occur. Estimates suggest that the cen-trosome includes hundreds of proteins involved in cell cycleregulation, protein degradation, transporters, kinases andphosphatases, microtubule-associated proteins, signaling pro-teins, membrane receptor signaling and scaffold proteins thatserve as docking sites for a growing number of regulatory activ-ities (30). Our immuno-EM study (Fig. 2B) shows the presenceof 50–70 nm vesicles in the pericentriolar material, whoseorigin is still unclear, although the presence of late endo-some recycling vesicles, Rab11, and caveolin-positive vesi-cles has been described previously in the pericentriolar com-partment (69, 70). These vesicles may be carriers of proteinsand receptors stemming from the cell surface or from othercell compartments.The centrosomes are also an important location for proteo-

lytic degradation of proteins known as “aggresomes,” which areproteinaceous inclusions formed at the centrosome in responseto proteolytic stress (68, 71). At present our observations

FIGURE 10. Localization of pERK1,2 at the centrosome in mitotic cells.Mitotic wild type (APPwt) and APP/APLPs null MEF cells (APP KO) were ana-lyzed by immunofluorescence with antibodies for �-tubulin and pERK1,2. Thecentrosomal localization of pERK1,2 is patent in wild type cells and is reducedin the absence of APP. Similar experiments in mitotic MEF cells PS1 and PS2-null (PSdKO), PS1-null (PS1KO), or PS wild type (PSwt) show an analogousresult with centrosomal pERK1,2 levels reduced in the absence of both PSs orof PS1, in comparison with wild type cells.

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exclude that the complex between PS1, APP, and GRB2 repre-sents a degradative pathway localized at the centrosome. Ourevidence shows that APP, PS1, and GRB2 are not ubiquitinatedand are detectable in the centrosome at different stages of themitotic cycle in cells that do not overexpress proteins, inde-pendently on treatment with proteasome inhibitors and in amanner that instead depends onmicrotubule stability (data notshown).Because many regulatory molecules are found at centro-

somes, it has been proposed that centrosomes serve as signalingmachines that modulate different cell functions (30). In partic-ular, considering that GRB2, APP, PS1, and pERK1,2 are alldetectable in mitotic centrosomes, it is conceivable that thesestructures might anchor signal transduction pathways, inte-grate signals, and facilitate the conversion of these signals intocellular functions. Our data suggest that PSs and APPmay takepart in these signaling complexes, whose function is related tothe activation of ERK1,2 and to modulate the cell cycle.In summary this study represents an initial step toward the

comprehension of a novel signaling mechanism, in which piv-otal proteins for AD genesis are involved. It is tempting to spec-ulate that ERK1,2 activation, mediated by both APP and PS1,may be relevant for Tau phosphorylation and neurofibrillarytangles formation, considering that centrosomes are essentialfor microtubule stability and assembly. Nonetheless, thehypothesis that pERK1,2 is involved in the activation of a par-allel proapoptotic pathway, possibly through an enhanced A�formation, must be considered as well. Further analyses areneeded to identify the effect of APP or PSs FADmutants on thecentrosomal phenotype, to verify whether PS1 and APP com-pete each other for the GRB2-linked signal, and to understandthe main consequences of their centrosomal signaling activityin the human brain. Among the different centrosomal func-tions, it will be of importance to also verify the effect on glialproliferation and cell cycle regulation in postmitotic neuronswhere aberrant cell cycle re-entry may occur (14). It alsoremains to be elucidated whether the centrosomal pool of APPand PS1, together with GRB2, is involved in intracellular amy-loid-� formation as well, and whether other components of the�-secretase complex are implicated in the interaction, becausein our conditions APP and PS1 are in close proximity also at thecentrosome.However, wemust bear inmind that differentAPPor PS1 interactors are able to modulate the formation of A�(72–75), and therefore that parallel signals, often connectedeach other, may modulate this event that is difficult to studywhen we look at each single interactor. The formation of amy-loid-� peptides has been previously described in different com-partments, from early endosomes toGolgi, ER, and also close tothe cell surface, and only recently a close interaction APP-PS1was described and characterized essentially near the cell mem-brane rather than in the Golgi-ER (76). Nonetheless, to ourknowledge the centrosomal area was never analyzed as a possi-ble site of interaction for these proteins or for the formation ofintracellular amyloid.The co-localization of APP-GRB2 and PS1-GRB2 is distrib-

uted in punctate and vesicular structures scattered in differentportions of the cell, and mainly centered at the centrosome.Therefore, the interactions described abovemay also take place

in other cell compartments, where APP and PS1 may also co-localize and interact. This hypothesis is also suggested by thesignificant amount of proteins engaged in co-IP experiments. Adeeper comprehension of these aspects of APP and PS1 life isnecessary and would open new possibilities for a therapeuticintervention for AD. In this respect it is of particular impor-tance that the Tyr-682 phosphorylation site regulates GRB2interaction and ERK1,2 activation. The investigation on kinasesand phosphatases that activate this site would represent a puta-tive spot of intervention to facilitate or block the underlyingsignal.The last aspect, which is unrelated to AD development,

regards the central role of GRB2 in cancer. The etiology andprogression of a variety of humanmalignancies are linked to thederegulation of receptor tyrosine kinases that may recruiteither ShcA or GRB2 adaptor proteins to activate their signal-ing. In this study we show that GRB2, besides its well knowncytosolic localization, is also recruited at the centrosome of thecell. Considering the pivotal role of GRB2 in oncogenic prolif-eration and metastasis, and considering the complexity of thecross-talk between receptors in tumorigenesis, we cannotexclude that APP and PSs might influence GRB2 activity orlocalization andmodulate themitogenic ormetastatic potentialof tumor cells, especially in view of the potential involvement ofPS1 in oncogenic pathways, which is suggested by the fact thatreceptors and transducers cleaved by �-secretase are involvedin oncogenic signaling such as Notch (77, 78), ErbB-4 (10, 79),Wnt (12, 80, 81), and CD44 (9).

Acknowledgments—MEF APP/APLP null cells were a gift from Prof.U.Mueller, andMEF PS1/PS2 null and PS1 null cells were a gift fromProf. Bart De Strooper.

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