Regulation of Notch1 signaling by the APP intracellular ... · AICD downregulates the levels of...

Research Article 1831

IntroductionNotch is a highly conserved transmembrane receptor that performsa key role in the determination of cell fate, differentiation, adultcell self-renewal, cancer, neurodegenerative disease, wound healing,and inflammation (Artavanis-Tsakonas et al., 1995; Egan et al.,1998; Lai, 2004; Weinmaster, 1998). The Notch1 receptor playsthe role of a membrane-bound transcription factor. Notch1 isprocessed by furin in the endoplasmic reticular Golgi complex (S1cleavage) during transport to the cell surface, where it is expressedin heterodimeric form (Lieber et al., 2002; Pan and Rubin, 1997).Upon binding to the specific ligands, Jagged and Delta, thetransmembrane C-terminal fragment of Notch is generated viaproteolytic cleavage (S2 cleavage) (Brou et al., 2000; Mumm andKopan, 2000). Cleavage of this fragment by -secretase (S3cleavage) induces the release of the Notch intracellular domain(Notch-IC) from the membrane, and induces the nucleartranslocation of Notch-IC, thus resulting in the formation of acomplex with the CSL transcription factor family [CBF1/RBP-Jk/KBF2 in mammals, Su(H) in Drosophila and Xenopus, andLag2 in Caenorhabditis elegans] (Capell et al., 2000; De Strooperet al., 1999; Mumm and Kopan, 2000; Ray et al., 1999; Steiner etal., 1999; Weinmaster, 1997). In the absence of Notch-IC, CSLinteracts with the SKIP, SMRT, CoR and HDAC proteins, resultingin the formation of a transcriptional repressor complex (Espinosaet al., 2002; Kao et al., 1998; Zhou et al., 2000; Zhou and Hayward,2001). Notch-IC dissociates the co-repressors, and Notch-ICinteracts with co-activator complexes, including the Lag-3/mastermind, p300/CBP and P/CAF/GCN5, to form a

transcriptional active complex and activates CSL-dependenttranscription (Kurooka and Honjo, 2000; Oswald et al., 2001;Petcherski and Kimble, 2000; Schuldt and Brand, 1999; Wallberget al., 2002). The RAM domain of Notch1, which mediates theinteraction of RBP-Jk/Su(H) with the Notch1-IC, induces theactivation of target gene transcription (Tamura et al., 1995; Tani etal., 2001). In addition to the enhancer of split [E(spl)] complexgenes, and the mammalian homologues of the Hairy and E(spl)genes, Hes1, Hes5, Hes7, Hey1, Hey2 and Heyl are the downstreamtarget genes of Notch signaling (Abu-Issa and Cavicchi, 1996;Bessho et al., 2001; de Celis et al., 1996; Fischer et al., 2004;Jennings et al., 1994; Jouve et al., 2000; Leimeister et al., 2000;Ligoxygakis et al., 1998; Maier and Gessler, 2000; Ohtsuka et al.,1999).

Following the transcriptional regulation of the target genes,Notch1-IC undergoes proteasomal degradation in the nucleus viathe ubiquitin-proteasome system, including Fbw7, an E3 ligaserelevant to the ubiquitylation of Notch1-IC (Gupta-Rossi et al.,2001; Lai, 2002; Minella and Clurman, 2005; Mo et al., 2007;Oberg et al., 2001; Wu et al., 2001). Several E3 ubiquitin ligaseshave been implicated in the half-life of Notch1-IC, including Fbw7,which promotes PEST-dependent Notch1-IC degradation in thenucleus, and Itch, which regulates the PEST-independentdegradation of cytoplasmic Notch protein (Lai, 2002). Wedemonstrated previously that ILK downregulates the proteinstability of Notch1-IC via the ubiquitin-proteasome pathway bymeans of Fbw7 (Mo et al., 2007).

SummaryThe Notch1 receptor is a crucial controller of cell fate decisions, and is also a key regulator of cell growth and differentiation in avariety of contexts. In this study, we have demonstrated that the APP intracellular domain (AICD) attenuates Notch1 signaling byaccelerated degradation of the Notch1 intracellular domain (Notch1-IC) and RBP-Jk, through different degradation pathways. AICDsuppresses Notch1 transcriptional activity by the dissociation of the Notch1-IC–RBP-Jk complex after processing by -secretase.Notch1-IC is capable of forming a trimeric complex with Fbw7 and AICD, and AICD enhances the protein degradation of Notch1-ICthrough an Fbw7-dependent proteasomal pathway. AICD downregulates the levels of RBP-Jk protein through the lysosomal pathway.AICD-mediated degradation is involved in the preferential degradation of non-phosphorylated RBP-Jk. Collectively, our resultsdemonstrate that AICD functions as a negative regulator in Notch1 signaling through the promotion of Notch1-IC and RBP-Jk proteindegradation.

Key words: APP, Notch1, Protein degradation

Accepted 24 January 2011Journal of Cell Science 124, 1831-1843 © 2011. Published by The Company of Biologists Ltddoi:10.1242/jcs.076117

Regulation of Notch1 signaling by the APPintracellular domain facilitates degradation of theNotch1 intracellular domain and RBP-JkMi-Yeon Kim1,*, Jung-Soon Mo1,*, Eun-Jung Ann1,*, Ji-Hye Yoon1, Jane Jung1, Yun-Hee Choi1, Su-Man Kim1,Hwa-Young Kim1, Ji-Seon Ahn1, Hangun Kim2, Kwonseop Kim2, Hyang-Sook Hoe3 and Hee-Sae Park1,‡

1Hormone Research Center, School of Biological Sciences and Technology, Chonnam National University, Gwangju 500-757, Republic of Korea2The College of Pharmacy and Research Institute for Drug Development, Chonnam National University, Gwangju 500-757, Republic of Korea3Department of Neuroscience and Neurology, Department of Neurology, Georgetown University Medical Center, Washington, DC 20007, USA*These authors equally contributed to this work‡Author for correspondence ([email protected])

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The amyloidprecursor protein (APP) is a type 1 integraltransmembrane protein composed of a large extracellular sequence,a single transmembrane region and a short intracellular fragment,which is a cytotoxic 39–43 residue peptide that performs a crucialfunction in the pathogenesis of Alzheimer’s disease (Beyreutherand Masters, 1991; Tang, 2005; Younkin, 1991). Underphysiological conditions, APP is cleaved proteolytically by secretaseactivity. APP is cleaved in a fashion similar to that of Notch, whichundergoes regulated intramembranous proteolysis induced by -secretase to release the APP intracellular domain (AICD), whichmodulates transcription (Tomita et al., 1998; Zhang et al., 2000).Indeed, AICD is capable of inducing transcriptional activation byinteracting with the adaptor protein Fe65 and the acetyltransferaseTip60 (Cao and Sudhof, 2001). AICD was initially identified in thebrains of patients with AD and was demonstrated to either sensitizeor induce cells to undergo apoptosis. We demonstrated previouslythat Notch1-IC downregulates the AICD transcriptional activitythrough physical binding with AICD, Fe65 and Tip60 (Kim et al.,2007b). We have also demonstrated that Notch1-IC is a novelsubstrate for Tip60 and acetylation is one of the key factors in theregulation of the Notch1 signaling pathway (Kim et al., 2007a).D’Adamio’s group has demonstrated that AICD binds to thecytosolic Notch inhibitors Numb and Numb-like, both of whichcan repress Notch activity (Roncarati et al., 2002). Despite the factthat AICD regulates Notch1 signaling, the precise mechanismunderlying this control remains to be clarified.

In this study, we demonstrate that signal crosstalk occurs betweenAICD and Notch1 signaling after their processing by -secretase.

We have now evaluated the mechanism relevant to the AICD-mediated regulation of Notch signaling. Our data indicate thatAICD inhibits the transcriptional activity of Notch1-IC by aninduced reduction in the protein stability of Notch1-IC and RBP-Jk. Interestingly, the level of the Notch1-IC protein wasdownregulated markedly in the presence of AICD by theproteasomal degradation of Notch1-IC through Fbw7. Additionally,the level of RBP-Jk protein was downregulated markedly in thepresence of AICD by the lysosomal degradation of RBP-Jk.Collectively, our findings demonstrate that AICD functions as anegative regulator of the protein turnover of Notch1-IC and RBP-Jk.

ResultsAICD inhibits Notch1 transcriptional activityTo evaluate the possible function of AICD in Notch1 signaling, areporter assay was conducted with HEK293 cells, using luciferasereporter genes. HEK293 cells were transfected with 4�CSL-Luc,and either the active Notch1 mutant EN1 or an empty vector. Asanticipated, EN1-mediated transcription activity was found tohave increased in these samples. We determined that AICDattenuated the ability of EN1 to stimulate transcription (Fig. 1A).The basic helix-loop-helix (bHLH) proteins, Hes1 and Hes5, bothof which harbor several RBP-Jk-binding sequences on theirpromoters, were identified as essential targets of Notch in epithelialcells (Kageyama and Ohtsuka, 1999). Therefore, we confirmed theeffects of AICD on the Notch1 signaling pathway, using the Hes1reporter system. The expression of the active form of Notch1

1832 Journal of Cell Science 124 (11)

Fig. 1. AICD inhibits Notch transcriptional activity. (A,B)HEK293 cells were transfected with expression vectors for 4�CSL-Luc (A), Hes-1-Luc (B) and -galactosidase, along with EN1, as indicated. (C,D)HEK293 cells were transfected with expression vectors for 4�CSL-Luc (C), Hes-1-Luc (D) and -galactosidase, along with Notch1-IC, as indicated. (E)HEK293 cells were transfected with expression vectors for GAL4–Luc, and -galactosidase, along withAPP–GAL4, as indicated. After 42 hours of transfection, the cells were pretreated with 1M DAPT and exposed to 1–3M PMA for 6 hours, as indicated.(F)HEK293 cells were transfected with expression vectors for 4�CSL-Luc, APP and -galactosidase, along with Notch1-IC, as indicated. After 42 hours oftransfection, the cells were pretreated with 1M DAPT and then exposed to 1M PMA for 6 hours. (G)HEK293 cells were transfected for 48 hours withexpression vectors for 4�CSL-Luc, APP, siAPP and -galactosidase, along with Notch1-IC. After 42 hours of transfection, the cells were treated with 1M PMAfor 6 hours. In A–G, the cells were lysed and the luciferase activity was determined. The data were normalized to -galactosidase. These results are expressed as themean ± s.d. of three independent experiments. RLU, relative luciferase unit. The data were evaluated for significant differences by Student’s t-test; *P<0.001.

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significantly induced the activation of the Hes1 reporter system(Fig. 1B). Overexpression of AICD inhibits constitutively activeNotch1-induced natural Hes1 promoter transcriptional activity (Fig.1B). We then attempted to determine whether the overexpressionof AICD influences Notch1-IC-mediated signaling. HEK293 cellswere transfected with 4�CSL-Luc or Hes1-Luc and either theNotch1 intracellular domain (Notch1-IC) or an empty vector.Expression of Notch1-IC was found to significantly induceactivation of the 4�CSL and Hes1 reporter systems (Fig. 1C,D).Overexpression of AICD inhibited Notch1-IC-induced 4�CSL andnatural Hes1 promoter transcription activities (Fig. 1C,D).

The phorbol ester phorbol 12-myristate 13-acetate (PMA) hasbeen shown to trigger -secretase-mediated APP cleavage and weattempted to determine whether PMA could modulate APP cleavagethrough -secretase, thereby modulating Notch1 signaling(Fukushima et al., 1993; Kume et al., 2004; Suh and Checler,2002). We used APP–Gal4/VP16 fusion proteins to measure the -secretase-mediated cleavage of APP (Biederer et al., 2002; Herranzet al., 2006; May et al., 2002; Wiley et al., 2010). HEK293 cellswere transfected with APP–Gal4/VP16 and Gal4–Luc, and treatedwith either PMA or DAPT, a -secretase inhibitor. As anticipated,PMA triggered the ability of APP–Gal4/VP16 to stimulatetranscription in a dose-dependent fashion (Fig. 1E). Upon treatmentwith a -secretase inhibitor, -secretase activity was attenuatedsignificantly (Fig. 1E). In an effort to delineate the possible role ofAICD, which was generated from APP by -secretase, in theregulation of Notch1-IC signaling, we determined the effects ofPMA on Notch1-IC transcriptional activity. Whereas Notch1-IC-mediated transcriptional activity was repressed in the PMA-exposedHEK293 cells, the PMA-induced suppression of Notchtranscriptional activity was restored by treatment with DAPT orcoexpression with APP siRNA (Fig. 1F,G). According to theseresults, it was assumed that PMA can reduce Notch1-IC-mediatedtranscriptional activity by the upregulation of -secretase-dependentAPP cleavage.

AICD downregulates the level of Notch1-IC and RBP-JkproteinsWe, and others, have reported previously that AICD might interactwith Notch1-IC (Fassa et al., 2005; Fischer et al., 2005; Kim et al.,2007b; Oh et al., 2005). To delineate more precisely the manner inwhich AICD prevents Notch1-IC and RBP-Jk-mediatedtranscription, we conducted a series of in vitro binding andcoimmunoprecipitation experiments. Lysates from cells expressingMyc–Notch1-IC and FLAG–RBP-Jk were immunoprecipitated andsubsequently incubated with either GST or with GST–AICD. Theformation of the Notch1-IC and RBP-Jk complex was suppressedsubstantially by AICD in vitro (Fig. 2A). Notch1-IC harbors aCDC domain that incorporates a RAM domain, seven ankyrinrepeats (ANK), an OPA domain, and a PEST domain within itsstructure. Therefore, we attempted to determine which, if any, ofthese domains are involved in the interaction between Notch1-ICand AICD. Our results showed that AICD bound to the RAM–ANK domain of Notch1, but not to the OPA and PEST domains(Fig. 2B). We also attempted to confirm that the physical associationof endogenous AICD with endogenous Notch1-IC or RBP-Jk inintact cells. Coimmunoprecipitation assays with endogenousNotch1-IC and RBP-Jk revealed binding with endogenous AICDin intact cells (Fig. 2C,D). To evaluate the effects of AICD on themolecular interactions between Notch1-IC and RBP-Jk in intactcells, coimmunoprecipitation was conducted in HEK293 cells by

cotransfection of Myc–Notch1-IC, FLAG–RBP-Jk and GFP–AICD. Notch1-IC and RBP-Jk were coimmunoprecipitated, butwhen they were cotransfected with AICD, the band of Notch1-ICthat interacted with RBP-Jk disappeared (Fig. 2E). Surprisingly,the protein levels of both Notch1-IC and RBP-Jk were significantlydownregulated upon cotransfection with AICD as determined byimmunoblotting (Fig. 2E, lanes 4 and 6), which demonstrates thatAICD might regulate the steady state levels of the Notch1-IC andRBP-Jk proteins. Because the AICD protein is known to accumulateupon treatment with PMA, we attempted to determine whetherPMA could modulate endogenous protein levels of AICD, Notch1-IC or RBP-Jk (Fukushima et al., 1993; Kume et al., 2004; Suh andChecler, 2002). We observed that AICD protein accumulated in adose-dependent manner upon PMA treatment. However, the steadystate levels of Notch1-IC and RBP-Jk proteins were graduallydecreased by PMA treatment (Fig. 2F). To delineate the possiblerole for AICD in the regulation of Notch1-IC protein stability, weassessed the effects of APP knockdown on Notch1-IC proteinstability. We determined that APP siRNA was able to block thesuppressive effects of PMA on downregulation of Notch1-ICprotein level (Fig. 2G). Furthermore, as demonstrated in Fig. 2B,the PEST domain was just detectable when coexpressed withAICD, probably as a result of the rapid turnover of the protein.Otherwise, AICD modulated neither the Tip60 nor Fe65 proteinlevels (data not shown). Therefore, we could assume thatattenuation of Notch1 transcription activity by AICD results fromthe downregulation of steady state Notch1-IC and RBP-Jk proteinlevels.

Notch1-IC is downregulated by AICD in a proteasome-dependent mannerWe attempted to determine whether Notch1-IC could be subjectedto proteasome-mediated proteolysis, as previously reported (Gupta-Rossi et al., 2001; Lai, 2002; Minella and Clurman, 2005; Mo etal., 2007; Oberg et al., 2001; Wu et al., 2001). We transfected theHEK293 cells with Myc–Notch1-IC and GFP–AICD, and thequantity of remaining Notch1-IC was evaluated after variousperiods of cycloheximide treatment. We determined the proteinstability of Notch1-IC in HEK293 cells by cycloheximide treatmentwith or without AICD. Cycloheximide interacts with the translocaseenzyme and blocks protein synthesis in eukaryotic cells. Aftercycloheximide treatment, the level of Notch1-IC protein graduallydropped, with approximately half of the protein degraded after 3hours without AICD (Fig. 3A). Upon cycloheximide treatment, thereduced level of Notch1-IC protein dropped rapidly, withapproximately half of the protein being degraded after 1.5 hours inthe presence of AICD (Fig. 3A). No reduction was noted in thelevel of actin used as a control (Fig. 3A). This result demonstratesthat Notch1-IC is rapidly turned over in the presence of AICD.

To determine whether degradation of Notch1-IC proteins ismediated by the proteasome pathway, the proteasome inhibitorMG132 was used to treat Notch1-IC-expressing and AICD-expressing cells. MG132 can reversibly block all activities of the26S proteasome (Rock et al., 1994). ALLN inhibits neutral cysteineproteases and the proteasome (Drexler, 1997). The cells weretreated with proteasome inhibitors for 6 hours and Notch1-ICprotein was detected via an immunoblot assay. The results of ourstudies demonstrated that the proteasome inhibitor significantlyincreased the level of Notch1-IC (Fig. 3B), which was reduced inthe presence of AICD, but was restored by treatment with MG132or ALLN (Fig. 3B,C).

1833Negative regulation of Notch1 signaling by APP

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We then attempted to determine whether lysosomal inhibitorsexerted any detectable effect on the degradation of Notch1-IC.NH4Cl is a very effective inhibitor of lysosomal function andinhibits the function of lysosomal proteases by an induced increasein the intralysosomal pH (Dean et al., 1984; Ohkuma and Poole,1978). Owing to its relatively low cytotoxicity, NH4C1 wasprimarily used in our study (Dean et al., 1984). We transfectedHEK293 cells with Myc–Notch1-IC and GFP–AICD, and thequantity of remaining Notch1-IC was analyzed after various periods

of NH4Cl treatment. Our studies demonstrated that lysosomalinhibitor could not regulate the steady state level of Notch1-ICproteins (Fig. 3D). Moreover, compared with the proteasomeinhibitor, the lysosomal inhibitor exerted no detectable effect onthe AICD-induced downregulation of Notch1-IC (Fig. 3D). Toconfirm the role of MG132, NH4Cl and chloroquine in theregulation of known target protein, we introduced c-Myc forproteasomal degradation and Notch3-IC for lysosomal degradation(Bahram et al., 2000; Gregory and Hann, 2000; Jia et al., 2009).


Fig. 2. AICD downregulates the level ofNotch1-IC and RBP-Jk proteins. (A)HEK293cells were transfected for 48 hours withexpression vectors for Myc–Notch1-IC andFLAG–RBP-Jk. The cell lysates were thensubjected to immunoprecipitation with anti-FLAG antibody, and the resultant precipitateswere then incubated with either GST or GST-AICD for 1 hour on ice. The immunopelletswere additionally subjected to immunoblottinganalysis with anti-Myc antibody. GST or GST–AICD proteins were visualized by staining withCoomassie Brilliant Blue (CBB). (B)HEK293cells were transfected for 48 hours withexpression vectors encoding FLAG–Notch1-IC,FLAG–RAM-ANK, FLAG–OPA, FLAG–PEST and GFP–AICD. After transfection, thecell lysates were subjected toimmunoprecipitation with anti-GFP antibody.The immunoprecipitates were thenimmunoblotted with anti-FLAG antibody. Thecell lysates were also immunoblotted with anti-FLAG and anti-GFP antibodies. (C,D)HEK293cell lysates were then subjected toimmunoprecipitation with anti-AICD or IgGantibody, and the resulting precipitates weresubjected to immunoblotting analysis with anti-Notch1-IC (C) or anti-RBP-Jk (D) antibody.The cell lysates were also subjected toimmunoblotting analysis with the indicatedantibodies. (E)HEK293 cells were transfectedfor 48 hours with the indicated combinations ofexpression vectors for Myc–Notch1-IC, FLAG–RBP-Jk and GFP–AICD. The cell lysates werethen immunoprecipitated with anti-FLAGantibody, and the resultant precipitates weresubjected to immunoblotting analysis with anti-Myc antibody. The cell lysates were alsosubjected to immunoblotting analysis with theindicated antibodies. (F)HEK293 cells weretreated with the indicated amounts of PMA for 6hours. The cell lysates were also subjected toimmunoblotting analysis with anti-AICD, anti-Notch1-IC and anti-RBP-Jk antibodies.(G)HEK293 cells were transfected for 42 hourswith the indicated combinations of expressionvectors encoding for Myc–Notch1-IC, APP andsiAPP. The cells were then treated with 1MPMA, as indicated. The cell lysates were alsosubjected to immunoblotting analysis with anti-Myc and anti-AICD antibodies. Equal amountsof protein from each sample are immunoblottedwith anti--actin antibody as a loading control.Molecular size markers in kDa are indicated onthe left of all blots.

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Coincident with previous reports, we found the accumulation ofthose proteins in a dose-dependent manner (Fig. 3E–G). Theseresults reveal that the stability of the Notch1-IC protein isdownregulated by AICD through the proteasome-dependentpathway.

RBP-Jk is downregulated by AICD in a lysosome-dependent mannerNevertheless, despite the many studies of RBP-Jk conducted thusfar, the mechanism underlying degradation of the protein remainsincompletely understood. In an effort to evaluate the possible roleof AICD in the regulation of RBP-Jk protein stability, we transfectedHEK293 cells with FLAG–RBP-Jk and GFP–AICD, and thequantity of remaining RBP-Jk was analyzed after various periodsof cycloheximide treatment. We determined the protein stability ofRBP-Jk in HEK293 cells by cycloheximide treatment with orwithout AICD. After cycloheximide treatment, the level of RBP-Jk protein declined gradually with approximately half of the proteinbeing degraded after 5 hours without AICD (Fig. 4A). The level ofthe RBP-Jk protein was reduced significantly withoutcycloheximide treatment; after treatment, the reduced level ofRBP-Jk protein also declined rapidly, with approximately half of

the protein being degraded after 2 hours in the presence of AICD(Fig. 4A). No reduction was detected in the level of actin used asa control (Fig. 4A). This result demonstrates that RBP-Jk is rapidlyturned over in the presence of AICD.

In an effort to determine whether the degradation of RBP-Jkproteins is mediated by the proteasome pathway, the proteasomeinhibitor MG132 was administered to cells expressing RBP-Jk andAICD. The cells were then subjected to 6 hours of treatment withproteasome inhibitor, and the RBP-Jk protein was detected usingan immunoblot assay. Our studies demonstrated that the proteasomeinhibitor induced a significant increase in the RBP-Jk protein level(Fig. 3B), which was reduced in the presence of AICD, but wasnot significantly restored by treatment with MG132 (Fig. 4B).However, we found a band shift of RBP-Jk: the upper band clearlyremained in the presence of AICD (Fig. 4B). Severalphosphorylation events might be required to induce a band shift toa new level. Thus, we attempted to determine whether the upperband was a phosphorylated form of RBP-Jk using the generalphosphatase, CIP. In the same samples as shown in Fig. 4B, wedetermined that the upper band was downshifted in the presenceof CIP, thereby suggesting that the two bands appear to correspondto the phosphorylation of RBP-Jk by an unknown kinase (Fig. 4C).


Fig. 3. Notch1-IC is downregulated by AICD in aproteasome-dependent manner. (A)HEK293cells were transfected for 48 hours with theindicated combinations of expression vectorsencoding Myc–Notch1-IC and GFP–AICD. Thecells were then treated with 100M cycloheximide(CHX), as indicated. (B,C)HEK293 cells weretransfected for 42 hours with the indicatedcombinations of expression vectors encoding Myc–Notch1-IC and GFP–AICD. The cells were thentreated with the indicated amount of MG132 orALLN for 6 hours. (D)HEK293 cells weretransfected for 42 hours with the indicatedcombinations of expression vectors encoding Myc–Notch1-IC and GFP–AICD. The cells were thentreated with the indicated amounts of NH4Cl for 6hours. (E)HEK293 cells were transfected for 42hours with expression vector encoding GFP–Myc.The cells were then treated with 1, 5 or 10M ofMG132 for 6 hours. (F)HEK293 cells weretransfected for 42 hours with expression vectorencoding FLAG–Notch3-IC. The cells were thentreated with 10, 20, or 50 mM NH4Cl for 6 hours.(G)HEK293 cells were transfected for 42 hourswith the indicated combinations of expressionvectors encoding for FLAG–Notch3-IC. The cellswere then treated with 50, 100 or 200Mchloroquine for 6 hours. (A–G) The cell lysateswere also subjected to immunoblotting analysiswith the indicated antibodies. Equal amounts ofprotein from each sample are immunoblotted withanti--actin antibody as a loading control.

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We then attempted to determine whether the lysosomal inhibitorexerted any effect on RBP-Jk degradation. Chloroquine is anotherlysosomotrophic agent, and is commonly used to inhibit lysosomefunction (Welman and Peters, 1977). Chloroquine is a weak base,which can disrupt lysosomal function by the blockade of acidification.We transfected HEK293 cells with FLAG–RBP-Jk and GFP–AICD,and the quantity of remaining RBP-Jk was analyzed after various

periods of chloroquine treatment. The results of our studies revealedthat chloroquine also induced an increase in RBP-Jk protein levels(Fig. 4D). The RBP-Jk protein levels were reduced in the presenceof AICD, but were significantly restored after treatment withchloroquine (Fig. 4D) and NH4Cl (Fig. 4E).

RBP-Jk is localized mainly in the nucleus, and the intracellulardistribution of LAMP2 is in the lysosome. To determine the effect


Fig. 4. RBP-Jk is downregulated byAICD in a lysosome-dependentmanner. (A)HEK293 cells weretransfected for 48 hours with theindicated combinations of expressionvectors encoding for FLAG–RBP-Jkand GFP–AICD. The cells were thentreated with 100M cycloheximide(CHX) for the indicated times.(B)HEK293 cells were transfectedfor 42 hours with the indicatedcombinations of expression vectorsencoding for FLAG–RBP-Jk andGFP–AICD. The cells were thentreated with the indicated amounts ofMG132 for 6 hours. (C)HEK293cells were transfected for 42 hourswith the indicated combinations ofexpression vectors encoding forFLAG–RBP-Jk and GFP–AICD. Thecells were then treated with theindicated amounts of MG132 for 6hours. The cell lysates were treatedwith 10 U CIP for 1 hour.(D)HEK293 cells were transfectedfor 42 hours with the indicatedcombinations of expression vectorsencoding for FLAG–RBP-Jk andGFP–AICD. The cells were thentreated with the indicated amounts ofchloroquine for 6 hours. (E)HEK293cells were transfected for 42 hourswith the indicated combinations ofexpression vectors encoding FLAG–RBP-Jk and GFP–AICD. The cellswere then treated with the indicatedamounts of NH4Cl for 6 hours (A–E).The cell lysates were also subjectedto immunoblotting analysis using theindicated antibodies. (F)HEK293cells were transfected for 48 hourswith expression vectors encodingAICD or siAPP. Endogenous RBP-Jkand LAMP2 were double-stainedwith Alexa Fluor 488 (green) andAlexa Fluor 546 (red) and examinedby confocal microscopy. The DNAdye ToPro3 was used to visualizenuclei. For each experiment, at least300 cells were examined, and theimages shown here represent thetypical staining pattern for a majorityof cells and quantify the foldenrichment at the indicated region(white bar).

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of siRNA encoding AICD or APP on RBP-Jk localization, cellswere transfected with the respective vectors. The expression ofAICD significantly facilitated accumulation of RBP-Jk in theLAMP2-positive lysosome. However, APP siRNA did not affectthe cellular localization of RBP-Jk; however, the intensity of RBP-Jk fluorescence was moderately increased by transfection withAPP siRNA compared with the control (Fig. 4F). These resultsdemonstrated that the stability of the RBP-Jk protein isdownregulated by AICD through the lysosomal-dependent pathway.

GSK-3 does not affect AICD-mediated suppression ofNotch1 signalingOur cumulative observations revealed that AICD activates GSK-3 in vitro (Ghosal et al., 2009) and in vivo (Ryan and Pimplikar,2005; von Rotz et al., 2004) and that GSK-3 modulates Notch1signaling and stability (Espinosa et al., 2003; Lai, 2002; Lee et al.,2008). Therefore, we attempted, using GSK-3 inhibitors andGSK-3-knockout mouse embryonic fibroblast cells, to determinewhether AICD downregulates Notch1 transcription activity andstability via GSK-3. To confirm the role of lithium chloride in theregulation of GSK-3, we introduced -catenin, a specific targetof GSK-3. We found that -catenin was accumulated in a dose-dependent manner upon treatment with lithium chloride (Fig. 5A).To evaluate any involvement of GSK-3 in the downregulation ofthe Notch1-IC protein by AICD, HEK293 cells were transfectedwith Myc–Notch1-IC and GFP–AICD with the GSK-3 inhibitorslithium chloride and SB216763. The results demonstrate that thedownregulated level of Notch1-IC protein was not restored to asufficient degree by the inhibition of GSK-3 (Fig. 5B,C).Additionally, the transcriptional activation of the Notch1-IC targetgene was suppressed by cotransfection with AICD in GSK-3

wild-type mouse embryonic fibroblast cells and to a similar extentin GSK-3-knockout cells, thus demonstrating the GSK-3-independent negative regulation of Notch1 by AICD (Fig. 5D).These results show that the negative regulation of the transcriptionalactivity and protein stability of Notch1-IC by AICD occurs via aGSK-3-independent pathway.

Phosphorylation of AICD by JNK3 is required for thesuppression of Notch1 signalingThe phosphorylation of AICD at T668 by JNK3 regulates APPsignaling by accumulation in the cytoplasm, amyloidogenicprocessing, and the destabilization of AICD (Chang et al., 2006;Colombo et al., 2009; Lee et al., 2003; Santos et al., 2010; Shin etal., 2007; Sodhi et al., 2008). We subsequently attempted tocharacterize the involvement of JNK3 in the regulation of Notch1signaling by using a reporter assay. HEK293 cells were transfectedwith 4�CSL-Luc and either the Notch1 intracellular domain(Notch1-IC) or an empty vector. The expression of Notch1-IC wasshown to induce significant activation of the 4�CSL reportersystems (Fig. 6A). Whereas Notch1-IC-mediated transcriptionalactivity was repressed in the AICD-expressing HEK293 cells, theAICD-induced suppression of Notch1 transcriptional activity wasrestored by the coexpression of JNK3, but not JNK1 (Fig. 6A).The Notch1-IC and RBP-Jk protein levels were reduced in thepresence of AICD, but were restored to a moderate degree bycoexpression with JNK3, but not with JNK1 (Fig. 6B).

We then evaluated the involvement of APP phosphorylation atT668 in the regulation of Notch1-IC transcriptional activity andprotein stability using the T668A mutant. Whereas AICD inhibitedNotch1-IC induced transcriptional activity and was restored byJNK3, AICD (T668A) did not prevent Notch1-IC-induced


Fig. 5. GSK-3 does not affect AICD-mediatedsuppression of Notch1 signaling. HEK293 cellswere transfected for 42 hours with the indicatedcombinations of expression vectors encoding (B,C)Myc–Notch1-IC and GFP–AICD or (A) GFP–-catenin. The cells were then treated with (A,B) 10mM LiCl or (C) 10M SB216763 for 6 hours. Thecell lysates were also subjected to immunoblottinganalysis with the indicated antibodies. (D)WTGSK-3and GSK-3-knockout MEF cells weretransfected for 48 hours with expression vectors for4�CSL-Luc, AICD and -galactosidase, alongwith Notch1-IC, as indicated. The cells were lysed,and the luciferase activity was determined. Thedata were normalized with -galactosidase. Theseresults are expressed as the mean ± s.d. of threeindependent experiments. RLU, relative luciferaseunit. The data were evaluated for significantdifference using the Student’s t-test; *P<0.001.

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transcriptional activity (Fig. 6C). The Notch1-IC and RBP-Jkprotein levels were reduced in the presence of AICD, but were notinfluenced by coexpression with AICD (T668A) (Fig. 6D). Theseresults reveal that the JNK3-mediated phosphorylation of AICD iscritically relevant to its ability to regulate Notch1 signaling.

AICD facilitates a stable association between Notch1-ICand E3 ligase Fbw7 by the formation of a trimeric complexNotch1-IC is ubiquitylated by the F-box protein Fbw7, which wasinitially isolated in a genetic screening process for negativeregulators of Notch in C. elegans (Oberg et al., 2001; Wu et al.,2001). We anticipated that Fbw7 might function as a mediator forthe negative regulation of Notch1-IC by AICD. Therefore, weevaluated the involvement of Fbw7 using the F-box-deleted, andhence the dominant-negative mutant form of Fbw7 (Fbw7F). Wealso attempted to determine whether AICD could regulate the levelof Notch1-IC ubiquitylation through Fbw7. Using dominant-negative Fbw7, the immunoblot analysis of the Notch1-ICprecipitated with anti-Myc antibodies demonstrated that the levelsof polyubiquitylated Notch1-IC were increased upon coexpressionof AICD (Fig. 7A). The AICD-mediated upregulation of Notch1-

IC ubiquitylation was inhibited by coexpression with Fbw7F(Fig. 7A).

We subsequently evaluated the involvement of AICD in thephysical association between Fbw7 and Notch1-IC in acoimmunoprecipitation experiment. HEK293 cells werecotransfected with vectors encoding Myc–Notch1-IC, FLAG–Fbw7and GFP–AICD, and were then subjected to coimmunoprecipitationanalysis (Fig. 7B). Immunoblot analysis using the anti-FLAGantibody on anti-Myc immunoprecipitates from the transfectedcells showed that AICD facilitates the physical association betweenFbw7 and Notch1-IC in the cells (Fig. 7B). These resultsdemonstrate that the downregulation of the Notch1-IC protein byAICD occurs in an Fbw7-dependent pathway. At this point, weevaluated the formation of a trimeric complex between AICD andNotch1-IC or Fbw7, in an effort to define more precisely the roleof AICD in the negative regulation of Notch1 signaling. We detectedbinding between Notch1-IC and AICD, but not between AICD andFbw7, although the trimeric complex was detected in this case(Fig. 7C). Therefore, the results demonstrate that AICD interactswith Fbw7 in the presence of Notch1-IC, thereby forming a trimericcomplex.


Fig. 6. Phosphorylation of AICD by JNK3 is required for the suppression of Notch1signaling. (A)HEK293 cells were transfected for 48 hours with expression vectors for4�CSL-Luc, AICD, JNK1, JNK3 and -galactosidase, along with Notch1-IC and RBP-Jk,as indicated. (B)HEK293 cells were transfected for 48 hours with expression vectors forMyc–Notch1-IC, FLAG–RBP-Jk, GFP–AICD, HA–JNK1 and HA–JNK3, as indicated.(C)HEK293 cells were transfected for 48 hours with expression vectors for 4�CSL-Luc,AICD, AICD(T668A), JNK3 and -galactosidase, along with Notch1-IC and RBP-Jk, asindicated. (D)HEK293 cells were transfected for 48 hours with expression vectors forMyc–Notch1-IC, FLAG–RBP-Jk, GFP–AICD, GFP–AICD(T668A) and HA–JNK3, asindicated. (A,B)The cells were lysed, and the luciferase activity was determined. The datawere normalized using -galactosidase. These results are expressed as the mean ± s.d. ofthree independent experiments. RLU, relative luciferase unit. (C,D). The data wereevaluated for significant difference by Student’s t-test; *P<0.001. The cell lysates were alsosubjected to immunoblotting analysis using the indicated antibodies.

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AICD restores the suppression of muscle differentiationby Notch1MyoD is basic helix-loop-helix transcription factor that has keybut redundant roles in myogenesis (Li and Olson, 1992; Troucheet al., 1993). Notch1 signaling inhibits muscle cell differentiationthrough inhibition of MyoD expression (Anant et al., 1998; Bakerand Schubiger, 1996; Kopan et al., 1994; Shawber et al., 1996).Because AICD suppressed the Notch1 signaling, we decided toexamine the transcription activity of MyoD. As expected, Notch1-IC inhibited the expression of a MyoD promoter-luciferase reportergene (MyoD-Luc) in C2C12 cells. We also found that AICDrestored the suppression of MyoD reporter activity by Notch1-IC(Fig. 8A).

We next analyzed MyoD and Notch1-IC levels in the presenceand absence of AICD at different time points during muscledifferentiation in C2C12 cells. MyoD expression was detected inearly muscle differentiation and gradually disappeared. Comparedwith the control, AICD ectopic expression modulated the expressionpattern of MyoD proteins during muscle differentiation.Furthermore, AICD ectopic expression reduced the expressionlevels of Notch1-IC proteins during muscle differentiation (Fig.8B). These results indicate that decreased Notch1-IC protein levelscorrelate with AICD and MyoD protein levels. Therefore, it islikely that the induction of AICD is involved in the regulation ofNotch1 signaling.

DiscussionIn this study, we have demonstrated that AICD promotes thedegradation of Notch1-IC and RBP-Jk by different pathways.AICD inhibits Notch1 transcription activity by dissociating the

Notch1-IC–RBP-Jk complex. Furthermore, Notch1-IC is capableof forming a trimeric complex with Fbw7 and AICD; AICD therebyenhances the protein degradation of Notch1-IC in an Fbw7-dependent proteasomal pathway. AICD-mediated degradation isinvolved in the preferential degradation of the non-phosphorylatedRBP-Jk through the lysosomal pathway.

In our recent report, we showed that expression of Notch1-ICdownregulates transcriptional activity mediated by the AICD–Fe65–Tip60 (AFT) complex, ROS generation and cell death (Kimet al., 2007b). Our results also represent the functional crosstalkbetween Tip60 and Notch1 through acetylation (Kim et al., 2007a).Fe65 has recently been determined to be involved in the regulationof Notch1 signaling in an AICD- or Tip60-independent manner(our unpublished results). AICD harbors several internalizationand trafficking motifs and might possess transcriptional activitythat resembles the Notch1-IC of Notch1 (Baek et al., 2002; Caoand Sudhof, 2001; Gao and Pimplikar, 2001). AICD regulatesphosphoinositide-mediated calcium signaling in vitro (Leissring etal., 2002) and also induces apoptosis and cytotoxicity in neurons(Lee et al., 2000). Previous reports have suggested the possibilityof crosstalk between the Notch and APP signaling pathways, whichwould manifest as -secretase substrate competition (Berezovskaet al., 2001; Lleo et al., 2003). However, we and other groups havedemonstrated that negative crosstalk occurs between Notch andAPP signaling, in a -secretase-independent fashion (Kim et al.,2007b; Petit et al., 2002). That is, Notch1-IC-mediated geneexpression is regulated negatively by AICD, by some currentlyunknown mechanism (Roncarati et al., 2002). The functionalinvolvement of AICD in Notch1 signaling, therefore remains amatter of some controversy. Our results demonstrate that Notch1-


Fig. 7. AICD facilitate stable associationbetween Notch1-IC and E3 ligase Fbw7through the formation of a trimeric complex.(A)HEK293 cells were transfected withexpression vectors for Myc–Notch1-IC, GFP–AICD, FLAG–Fbw7, FLAG–Fbw7F, and HA–Ub, as indicated. After 42 hours of transfection,the cells were treated with 10M MG132 for 6hours and the cell lysates wereimmunoprecipitated with anti-Myc antibody andthe immunoprecipitates were immunoblotted withanti-HA antibody. (B)HEK293 cells weretransfected with expression vectors for Myc–Notch1-IC, FLAG–Fbw7 and GFP–AICD, asindicated. After 42 hours of transfection, the cellswere treated with 10M MG132 for 6 hours andthe cell lysates were immunoprecipitated withanti-Myc antibody, after which theimmunoprecipitates were immunoblotted withanti-FLAG antibody. (C)HEK293 cells weretransfected with expression vectors for Myc–Notch1-IC, FLAG–Fbw7, and GFP–AICD, asindicated. After 42 hours of transfection, the cellswere treated with 10M MG132 for 6 hours andthe cell lysates were immunoprecipitated withanti-GFP antibody and the immunoprecipitateswere immunoblotted with anti-Myc or anti-Flagantibody. The cell lysates were also subjected toimmunoblotting analysis with the indicatedantibodies.

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IC transcriptional activity is attenuated in the presence of AICD,which suggests that AICD is also involved in suppression ofNotch1-IC transcriptional activity.

Several previous reports have suggested that Notch might directlyinteract with APP in intact cells (Fassa et al., 2005; Fischer et al.,2005; Kim et al., 2007b; Oh et al., 2005). We have determined thatNotch1-IC interacts directly with AICD through the RAM–ANKdomain. Intriguingly, our results demonstrate that the inhibitorymechanism suppresses the interaction of Notch1-IC and RBP-Jk,as a result of the downregulation of Notch1-IC and RBP-Jk proteinstability. Several groups have demonstrated that Sel-10/Fbw7,through its WD40 domains, binds to phosphorylated Notch1-ICand mediates its ubiquitylation and subsequent rapid degradation(Gupta-Rossi et al., 2001; O’Neil et al., 2007; Wu et al., 2001). Inthis study, we determined that AICD stimulates the proteasomaldegradation of Notch1-IC and the lysosomal degradation of RBP-

Jk. AICD-mediated degradation is involved in the preferentialdegradation of non-phosphorylated RBP-Jk through the lysosomalpathway. Collectively, our findings demonstrate that thephosphorylation of RBP-Jk by unknown kinases regulates its half-life in either a positive or negative manner.

Thus AICD activates GSK-3 in vitro (Ghosal et al., 2009) andin vivo (Ryan and Pimplikar, 2005; von Rotz et al., 2004) andGSK-3 modulates Notch1 signaling and stability (Espinosa et al.,2003; Lai, 2002; Lee et al., 2008). Our results demonstrated thatthe AICD-mediated degradation of the Notch1-IC protein occursindependently of GSK-3. The phosphorylation of AICD at T668by JNK3 contributes to the neuronal degeneration inherent toAlzheimer’s disease (AD) by regulating its translocation into thecytoplasm, amyloidogenic processing and destabilization of AICD(Chang et al., 2006; Colombo et al., 2009; Lee et al., 2003; Santoset al., 2010; Shin et al., 2007; Sodhi et al., 2008). For that reason,it was anticipated that JNK3 might function as a possible regulatorfor Notch1 signaling by the deregulation of AICD. The Notch1-ICtranscriptional activity and protein levels were reduced in thepresence of AICD, but were restored by coexpression with JNK3,but not with JNK1. The phosphorylation-deficient mutant of AICD(T668A) was not influenced by the regulation of Notch1-ICtranscriptional activity and protein stability. Moreover, wedetermined that AICD negatively regulates the transcriptionalactivation of the Notch1-IC target genes and the stability of theNotch1-IC protein in an Fbw7- and proteasome-dependent manner.Notch1-IC, Fbw7 and AICD form a trimeric complex, andenhancement of the interaction occurring between Notch1-IC andFbw7 might be a possible mechanism underlying the AICD-mediated proteasomal degradation of Notch1-IC (Fig. 9).

In summary, our results demonstrate that AICD performs thefunction of a negative regulator in Notch1 signaling by thepromotion of Notch1-IC and RBP-Jk protein degradation.


Fig. 8. AICD restores the suppression of muscle differentiation by Notch1.(A)C2C12 cells were transfected with expression vectors for MyoD-Luc,Notch1-IC, AICD and -galactosidase, along with MyoD, as indicated. Thecells were lysed, and the luciferase activity was determined. The data wasnormalized with -galactosidase. These results are expressed as the mean ±s.d. of three independent experiments. RLU, relative luciferase unit. The datawe evaluated for significant differences by Student’s t-test; *P<0.001.(B)C2C12 cells were transiently transfected with GFP–AICD. After switchingthe culture medium with differentiation medium, differentiating C2C12 cellswere observed at 1, 2, 3, 4, 5 and 6 days of differentiation. The cell lysateswere also subjected to immunoblot analysis with the indicated antibodies.Equal amounts of protein from each sample are immunoblotted with anti--actin antibody as a loading control.

Fig. 9. Proposed model for the role of AICD in the regulation of Notch1signaling. Notch1 and APP are processed by -secretase and translocated intothe nucleus, where they function as an activator of transcription. Notch1-ICforms a complex with RBP-Jk and activates the transcription of target genes.AICD suppresses Notch1 signaling by accelerating the degradation of Notch1-IC and RBP-Jk. Notch1-IC is capable of forming a trimeric complex withFbw7 and AICD; AICD enhances the protein degradation of Notch1-IC by theFbw7-dependent proteasomal pathway. AICD downregulates the protein levelof non-phosphorylated RBP-Jk through the lysosomal pathway.

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Henceforth, the findings of this study might begin to shed somelight onto what may be a signal crosstalk mechanism of Notch1and APP, or might point to the existence of -secretase-independentcrosstalk.

Materials and MethodsCell culture and transfectionHEK 293 and GSK-3 wild-type and GSK-3-knockout mouse embryonic fibroblastscells and mouse skeletal muscle C2C12 myoblast cells were cultured in Dulbecco’smodified Eagle’s medium (DMEM-Gibco) containing 10% fetal bovine serum and1% penicillin-streptomycin, in a humidified incubator with an atmosphere containing5% CO2. The cultured cells were transiently transfected using calcium phosphate forHEK293, GSK-3 wild type and GSK-3-knockout mouse embryonic fibroblastcells. Cells were grown to ~80% confluence and transfected with the plasmids (Chenand Okayama, 1987; Mo et al., 2007). C2C12 cells were grown to 80–90%confluence, and induced for differentiation by switching from growth medium todifferentiation medium with DMEM containing 2% horse serum. Differentiationmedium was replenished every day. The cultured cells were transiently transfectedusing the calcium phosphate method or Lipofectamine-plus reagent. For plasmidDNA transfection, the cells were grown to ~80% confluence and transfected withthe plasmids (Chen and Okayama, 1987; Mo et al., 2007).

Luciferase reporter assayHEK293 cells were co-transfected with 4�CSL-Luc (a repeat section of the RBP-Jk target sequence, CGTGGGAA, with the luciferase gene) and -galactosidasecoupled with the indicated vector constructs. PMA was added 2 hours before theaddition of DAPT, which was present for the final 6 hours. After 48 hours oftransfection, the cells were lysed in chemiluminescent lysis buffer (18.3% of 1 MK2HPO4, 1.7% of 1 M KH2PO4, 1 mM phenylmethylsulfonyl fluoride and 1 mMdithiothreitol), and were analyzed using a Luminometer (Berthold). The luciferasereporter activity in each sample was normalized in relation to the -galactosidaseactivity in the same lysate (Kim et al., 2007a).

In vitro binding assayThe recombinant GST–AICD protein was expressed in Escherichia coli BL21 strain,using the pGEX system as indicated (Kim et al., 2007b). The GST fusion proteinwas then purified using glutathione–agarose beads (Sigma), in accordance with themanufacturer’s instructions. An equal quantity of GST or GST–AICD fusion proteinwas incubated with lysates of HEK293 cells, which were transfected withcombinations of expression vectors at 4°C on a rotator. The supernatants weresubjected to immunoprecipitation with anti-FLAG antibody. After incubation for 1hour, Protein-A–agarose was added, and the samples were incubated for 3 hours at4°C on the rotator. After incubation, the beads were washed three times in ice-coldphosphate-buffered saline and boiled with 20 l Laemmli sample buffer. Theprecipitates were then resolved by SDS-PAGE, and the immunoprecipitates weredetected by immunoblotting with specific antibodies.

Immunoblot analysisAfter 48 hours of transfection, the cultured cells were harvested and lysed in RIPAbuffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodiumdeoxycholate, 0.1% SDS, 1 mM PMSF, 1 mM DTT, and 2 g/ml each of leupeptinand aprotinin) for 30 minutes at 4°C. The cell lysates were then subjected to 20minutes of centrifugation at 12,000 g at 4°C. The resultant soluble fraction wasboiled in Laemmli buffer and subjected to SDS-PAGE. After gel electrophoresis, theseparated proteins were transferred by electroblotting onto polyvinylidene difluoride(PVDF) membranes (Millipore). The membranes were then blocked with phosphate-buffered saline solution (pH 7.4) containing 0.1% Tween-20 and 5% non-fat milk.The blotted proteins were subsequently probed with anti-Myc antibody (9E10), anti-HA (12CA5) antibody, or anti-FLAG M2 antibody (Sigma), followed by incubationwith anti-mouse horseradish-peroxidase-conjugated secondary antibodies(Amersham). The blots were then developed using enhanced chemiluminescence(ECL).

Co-immunoprecipitation48 hours after transfection, the cells were lysed for 10 minutes in 1 ml of RIPA lysisbuffer at room temperature. After 20 minutes of centrifugation at 12,000 g, thesupernatants were subjected to immunoprecipitation with specific antibodies. Afterovernight incubation, Protein-A–agarose was added, and the samples were incubatedfor 3 hours at 4°C on the rotator. The beads were subsequently washed three timesin ice-cold PBS, and any proteins that remained bound to the beads were eluted byboiling in 5� protein sample buffer. The samples were separated by SDS-PAGE,and visualized by immunoblotting.

Protein accumulation assayCells were treated with the proteasomal inhibitor MG-132 (Sigma), ALLN(Calbiochem) or the lysosomal inhibitors NH4Cl (Sigma) and chloroquine (Sigma),or the translational inhibitor cycloheximide (Sigma). MG-132 was used at 0, 5 and

10 M for 6 hours for the dosage assay of the proteasomal inhibitors. NH4Cl wasused at 0, 10, 20, and 50 mM for 6 hours for the dosage assay of lysosomalinhibitors. Chloroquine was used at 0, 50 and 200 M for 6 hours for the dosageassay of lysosomal inhibitors. Cycloheximide was used at 1 mM for 0, 1, 2, 4 and 6hours for the time-course assay of the translational inhibitors. Protein levels wereanalyzed by immunoblotting.

Protein degradation assayHalf-life experiments using the cycloheximide-mediated inhibition of proteinsynthesis were conducted as previously described (Mo et al., 2007). Proteasomeinhibitors were added 1 hour before cycloheximide treatment, and the cell lysateswere subjected to SDS-PAGE and immunoblotting with the respective antibodies.

Immunofluorescence stainingAssays were conducted as previously described with HEK293 cells plated at 1�105

cells per well onto coverslips (Fisher). The cultured cells were fixed with 4%paraformaldehyde in phosphate-buffered saline (PBS), and then permeabilized with0.1% Triton X-100 in PBS. Cells were blocked in 1% BSA in PBS. anti-LAMP2antibody (Santa Cruz) and anti-RBP-Jk antibody (Santa Cruz) were used as theprimary antibodies at a dilution of 1:100, washed three times in PBS. Mousesecondary antibodies conjugated to Alexa Fluor 488 or rabbit secondary antibodiesconjugated to Alexa Fluor 546 (Invitrogen, 1:100) were added and the DNA dyeToPro3 was used for nuclear localization. The stained cells were evaluated forlocalization using confocal microscopy (LeicaTCS SPE). Each image is a single zsection at the same cellular level. The final images were obtained and analyzed usingconfocal microscopy with LAS AF software (Leica).

We would like to thank Raphael Kopan (Washington UniversityMedical School) for the Notch-related constructs, Thomas Südhof(University of Texas Southwestern) for providing us with the APP-related constructs, Yoo-Hun Suh (Seoul National University) for theAICD and AICD (T668A) constructs, Qubai Hu (WashingtonUniversity) for the AICD construct and Bruce E. Clurman (FredHutchinson Cancer Research Center) for the Fbw7 construct. GSK-3-knockout cells were kindly provided by James R. Woodgett (OntarioCancer Institute). This study was supported by a grant from the KoreaHealthcare Technology R&D Project, Ministry of Health & Welfare,Republic of Korea (A080441).

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