numbregulatespost-endocytictraffickinganddegradation … · inhibitor of notch and is known to...

Numb Regulates Post-endocytic Trafficking and Degradationof Notch1*

Received for publication, March 3, 2009, and in revised form, June 16, 2009 Published, JBC Papers in Press, June 30, 2009, DOI 10.1074/jbc.M109.014845

Melanie A. McGill‡§, Sascha E. Dho§, Gerry Weinmaster¶, and C. Jane McGlade‡§1

From the ‡Department of Medical Biophysics, University of Toronto, and §The Arthur and Sonia Labatt Brain Tumour ResearchCentre, The Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada and the ¶Department of Biological Chemistry, DavidGeffen School of Medicine, UCLA, Los Angeles, California 90095-1737

Notch is a transmembrane receptor that controls cell fatedecisions during development and tissue homeostasis. Bothactivation and attenuation of the Notch signal are tightly reg-ulated by endocytosis. The adaptor protein Numb acts as aninhibitor of Notch and is known to function within the intra-cellular trafficking pathways. However, a role for Numb in reg-ulating Notch trafficking has not been defined. Here we showthat mammalian Notch1 is constitutively internalized and traf-ficked to both recycling and late endosomal compartments, andwe demonstrate that changes in Numb expression alter thedynamics of Notch1 trafficking. Overexpression of Numb pro-motes sorting of Notch1 through late endosomes for degrada-tion, whereas depletion of Numb facilitates Notch1 recycling.Numb mutants that do not interact with the ubiquitin-proteinisopeptide ligase, Itch, or that lackmotifs important for interac-tion with endocytic proteins fail to promote Notch1 degrada-tion. Our data suggest that Numb inhibits Notch1 activity byregulating post-endocytic sorting events that lead to Notch1degradation.

The Notch receptor is a heterodimeric, single pass, trans-membrane protein that is an integral component of a con-served signaling pathway required for embryonic and post-natal development. Notch signaling controls cell fatedecisions, maintenance of stem cells, cellular differentiation,proliferation, and apoptosis through direct cell to cell con-tact (1). Transmission of the Notch signal is initiatedthrough binding of the Notch receptor to transmembraneligands of the Delta/Serrate/Lag2 family expressed on neigh-boring cells (2, 3). Ligand binding induces a sequential seriesof proteolytic cleavage events leading to a final intramem-brane cleavage by the �-secretase complex that releases theactive intracellular domain of Notch (4, 5). This form ofNotch functions within the nucleus as a cotransactivatorwith the CSL (CBF1/RBPj�, Suppressor of Hairless Su(H),Lag1) family of transcription factors to modulate transcrip-tion of target genes such as Hes1 (the mammalian homo-logue of the hairy and enhancer of split gene complex inDrosophila) and Herp (Hes-related) genes (6, 7).

The coordinated processing, endocytosis, and trafficking ofthe Notch receptor and its ligand are important in controllingNotch activation. Ubiquitin-dependent internalization andrecycling of Delta/Serrate/Lag2 ligands have an established rolein the activation of Notch signaling (8–19). In contrast, muchless is known regarding the role of endocytosis and traffickingof the Notch receptor itself. Studies to date suggest that endo-cytosis of Notch is important for ligand-mediated activation ofNotch.Monoubiquitination and endocytosis of a constitutivelyactive mutant form of the Notch1 receptor were shown toenhance �-secretase cleavage and activate downstream signal-ing events in a mammalian system (20). Similarly, mutationalanalysis in Drosophila showed that entry into the early endo-some is necessary for Notch activation (21).Ubiquitination and trafficking through the endocytic path-

way also play a role in the down-regulation of Notch. A con-served di-leucine sortingmotif identified in the cytoplasmic tailof the Caenorhabditis elegans Notch receptor, LIN-12, medi-ates its constitutive internalization and degradation during vul-val development (22).WWP-1, theC. elegans orthologue of theDrosophila ubiquitin ligases Suppressor of Deltex (Su(Dx)) anddNedd4, is required for LIN-12 down-regulation (23). Further-more, inDrosophilaboth Su(Dx) anddNedd4 act to limitNotchsignaling by regulating post-endocytic sorting ofNotch (24, 25).Together these studies suggest that both entry and traffickingof Notch within the endocytic pathway are important in theregulation of its activity. However, the factors involved in sort-ing Notch into activation or degradative pathways are poorlycharacterized.Genetic evidence in Drosophila has shown that the adap-

tor protein Numb acts as an inhibitor of Notch signalingduring development of both the peripheral and central nerv-ous systems and during muscle cell differentiation (26–28).Mammalian Numb orthologues appear to function in a con-served fashion within the mammalian Notch1 pathway (26,29–31). For example, overexpression of mammalian Numbantagonizes Notch1-dependent transactivation of the Hes1promoter (32) and inhibits Notch1 activity in neurite growth(33, 34). Both Drosophila and mammalian Numb are asym-metrically localized in dividing precursor cells and are prefer-entially segregated to one of the two daughter cells upon celldivision, thus ensuring cells adopt distinct cell fates, throughthe suppression of Notch signaling (26, 28, 35–40).Numb interacts with components of the endocytic machin-

ery and is localized to plasma membrane-associated and intra-cellular vesicles (41, 42). Numb binds to the �-adaptin subunit

* This work was supported by Canadian Cancer Society Grant 016125 (toC. J. M.) and, in whole or in part, by National Institutes of Health Grant R37NS031885 from NINDS (to G. W.).

1 To whom correspondence should be addressed: The Hospital for Sick Chil-dren, 555 University Ave., Toronto, Ontario M5G 1X8, Canada. Tel.: 416-813-8657; Fax: 416-813-8456; E-mail: [email protected].

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 284, NO. 39, pp. 26427–26438, September 25, 2009© 2009 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

SEPTEMBER 25, 2009 • VOLUME 284 • NUMBER 39 JOURNAL OF BIOLOGICAL CHEMISTRY 26427

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of the AP-2 complex (clathrin adaptor protein 2) in both Dro-sophila and mammalian cells (42, 43) and is required for asym-metric localization of Drosophila �-adaptin (43). Numb alsointeracts with EH domain-containing proteins Eps15 and theEHD/RME-1 family of endocytic proteins (41, 42). In mamma-lian cells, knockdownofNumbusingRNA interference or over-expression of Numb or short fragments of Numb interfereswith transmembrane protein endocytosis and traffickingthrough an unknown mechanism (41, 42, 44, 45). It has beenproposed that Numb suppression of Notch signaling is medi-ated through AP-2 recruitment and regulation of Notch endo-cytosis (43, 46, 47), although to date there is no direct evidenceto support this model. Previously, we showed that Numb alsobinds to the Su(Dx) orthologue Itch and cooperates with Itch toenhance the ubiquitination of membrane-associated Notch1(32), suggesting that Numb might function in a post-endocyticcompartment to regulate Notch trafficking.Here we investigate the role of Numb in the ligand-indepen-

dent endocytosis and trafficking ofNotch1.We show that over-expression of Numb promotes Notch1 trafficking and degrada-tion, whereas depletion of Numb facilitates Notch1 recycling.Numbmutants defective for binding the E32 ligase, Itch, or theendocytic proteins �-adaptin, Eps15 and EHD, fail to promoteNotch1 degradation. Together our data suggest that Numbsuppresses Notch1 signaling by regulating post-endocytic sort-ing events that lead to Notch1 degradation.

MATERIALS AND METHODS

Antibodies and Constructs—Full-length mouse Notch1cDNA (FL-Notch1), containing a single internal Myc epitopetag cloned into the HindIII site between amino acid residuesAla2292 and Ser2293, was subcloned into pcDNA1(�) vector. AllNumb mutant constructs were generated from Numbp66cDNA and cloned into a pEF vector. Numb�PTBC was gener-ated using PCR-amplified fragments representing amino acidsMet1 to Lys85 and Lys174 to the stop codon and fused in-frameto the pEF parental vector. Numb�C has a deletion of thesequence coding for the 41 amino acids at the carboxyl termi-nus. All cDNAs generated by PCR were verified by sequencingin both directions. Itch cDNAs were the kind gift from G. Gishand T. Pawson. HA-tagged Notch1 cDNA (pBOS HA-N1) hasbeen described previously (15). Rabbit anti-NotchIC (06-808,Upstate Biotechnology Inc., Lake Placid, NY) and rabbit anti-NotchEC (06-809, Upstate Biotechnology Inc.) antibodies wereused for immunoblots. Rabbit anti-intracellular domain ofNotch (2421, Cellular Signaling), goat anti-NotchC20 (sc-6015,Santa Cruz Biotechnology, Santa Cruz, CA), anti-Myc (9E10ascites), and rabbit anti-HRS antibodies (kind gift of S. Urbe)(48) were used for immunostaining. Anti-Numb was generatedin rabbits with a synthetic peptide to the carboxyl terminus asdescribed previously (30).

Cell Culture and Transfections—HEK293T cells were grownin Dulbecco’s modified Eagle’s medium (DMEM;Wisent) sup-plemented with 10% fetal bovine serum (FBS) and transfectedwith Lipofectamine reagent (Invitrogen) in Opti-MEM(Invitrogen) according to the manufacturer’s instructions.Pooled transfected cells were replated in individual 6-cmdishesfor time course experiments. C2C12 cells were grown inDMEM supplemented with 10% FBS and 5% calf serum. C2C12cells that stably express full-length, HA-tagged Notch1 weregrown in 10% FBS and 5% calf serum supplemented with 2�g/ml puromycin. C2C12 cells were transfected with Lipo-fectamine 2000 reagent (Invitrogen) using 3 �g of cDNA per6-well dish and 12 �l of Lipofectamine 2000. Equal numbers ofpooled transfected cells were replated in individual 6-cmdishes. Cells were used 18 to 24 h post-transfection.For siRNA silencing experiments, two 21-nucleotide siRNA

oligomers (Dharmacon Research) were designed to regionscorresponding to the Numb coding sequence (GCACCUGC-CCAGUGGAUCC) and 3�-untranslated region sequence(GUAGCACAUUGCAACAACA). The Scramble II duplexfromDharmacon (D-001205-20) was used as a negative controlfor siRNA activity. Only C2C12 cells were used for siRNAexperiments because significant knockdown of endogenousNumb could not be achieved in HEK293T cells. Overexpres-sion or depletion of Numb was confirmed by immunoblottingcell lysates with anti-Numb.Surface Biotinylation and Trafficking Assays—Transfected

cells grown to 70% confluency were cooled on ice for 30 min,washed with cold PBS, and then labeled with EZ-LinkNHS-SS-biotin (Pierce) in biotinylation buffer (154 mM NaCl, 10 mM

HEPES, 3 mM KCl, 1 mMMgCl2, 0.1 mM CaCl2, 10 mM glucose,pH 7.6) for 1 h at 4 °C. After two washes with cold PBS, cellswere incubated with DMEM containing 10% FBS and 100 mM

glycine for 5 min on ice to quench unconjugated biotin andwashed several times with cold PBS. One sample plate wasremoved and lysed in PLC lysis buffer (50 mM HEPES, pH 7.5,150 mMNaCl, 10% glycerol, 1.5 mMMgCl2, 1% Triton X-100, 1mM EGTA, 10mM sodium pyrophosphate, 100mM sodium flu-oride containingCOMPLETEprotease inhibitor tablets (RocheApplied Science)) to measure total surface biotinylation. A sec-ond sample plate was also removed to determine the efficiencyof removal of biotinyl groups on the cell surface by three 20-mintreatments with 50 mM 2-mercaptoethanesulfonic acid(MeSNa) in pre-chilled stripping buffer (100 mM NaCl, 50 mM

Tris-HCl, 1 mM MgCl2, 0.1 mM CaCl2, pH 8.6) at 4 °C. Theremaining sampleswere then incubated at 37 °C in pre-warmedDMEM containing 10% FBS over 30 min. At indicated timepoints, cells were washed in cold PBS and stripped of surfacebiotin in MeSNa stripping buffer (as described above). Cellswere then lysed and protein lysates quantitated. Equal amountsof protein lysate were mixed with immobilized streptavidin-Sepharose beads (Pierce) overnight at 4 °C to isolate biotiny-lated proteins and then washed three times in Nonidet P-40wash buffer (50mMHEPES, pH 7.5, 150mMNaCl, 2mMEGTA,10% glycerol, 1.5mMCaCl2, 1%Nonidet P-40). The bed volumewas removed with a 30-gauge needle before resuspending inSDS-Laemmli sample buffer. Recovered biotinylated proteinswere analyzed by SDS-PAGE andWestern blot using indicated

2 The abbreviations used are: E3, ubiquitin-protein isopeptide ligase; HA,hemagglutinin; FBS, fetal bovine serum; DMEM, Dulbecco’s modifiedEagle’s medium; siRNA, small interfering RNA; PBS, phosphate-bufferedsaline; FITC, fluorescein isothiocyanate; EGFR, epidermal growth factorreceptor; PTB, phosphotyrosine binding; MeSNa, 2-mercaptoethanesulfo-nic acid.

Numb Regulates Trafficking of Notch1

26428 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 284 • NUMBER 39 • SEPTEMBER 25, 2009


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antibodies. In all experiments, whole cell lysates were analyzedby SDS-PAGE and Western blot to examine FL-Notch andNumb expression.For internalization experiments, cells were labeled with bio-

tin as described above and incubated at 18 °C in DMEM con-taining 10% FBS and 25 mM HEPES, pH 7.6. Shifting cells to18 °C results in an accumulation of endocytosed proteins inearly or sorting endosomes inmany cell types (49–52). At indi-cated time points cells were MeSNA-stripped, lysed in PLClysis buffer, and intracellular biotinylated Notch examinedusing streptavidin-Sepharose beads as described above.For recycling assays, biotinylated cells were incubated at

18 °C for 2 h in DMEM containing 25 mM HEPES, pH 7.6, and10% FBS to accumulate internalized biotinylated surface pro-teins, and then MeSNa-stripped as described above. Afterwashing with cold PBS, cells were shifted to 37 °C to resumetrafficking. At the indicated time points, one set of cells wasstripped of surface biotin and lysed, and a second set was incu-bated in stripping buffer withoutMeSNA and then lysed. Intra-cellular pools of biotinylated protein were examined asdescribed above.For protein quantification, protein bands were visualized by

enhanced chemiluminescence (Pierce) and signals quantifiedby densitometric scanning using an Alpha Innotech Fluoro-chem8000 imaging system.Datawere acquired using theAlphaInnotech Fluorochem 8000 software and exported to Excel(Microsoft) for analysis. All statistical analysis comparisonswere done with Student’s t test in Excel.ImmunofluorescentMicroscopy—For subcellular localization

studies, HEK293T and C2C12 cells were plated onto poly-L-ornithine-coated glass coverslips 18 h before use. Cells werefixed in 2% paraformaldehyde in PBS containing Ca2� and 30mM sucrose for 20 min at room temperature, washed with 100mMglycine in PBS/Ca2� for 10min, and then permeabilized for10min with either 0.2% Triton X-100 or 0.05% saponin in PBS/Ca2�. After several washes in PBS, cells were blocked with 3%normal donkey serum in PBS/Ca2� for 30min at room temper-ature and then incubated with primary antibodies diluted inblocking serum for 30 min at 37 °C. Cells were washed fivetimes for 5 min in PBS/Ca2� and primary antibodies indirectlylabeled for 30 min at 37 °C as follows: Cy5-conjugated donkeyanti-rabbit IgG (1:500) andCy3-conjugated donkey anti-mouseIgG (1:500) from Jackson ImmunoResearch Laboratories, andAlexa488-conjugated donkey anti-mouse (1:500; MolecularProbes, Eugene,OR). Cells werewashedwith PBS andmountedin DAKOmountingmedium. Cells were examined with a ZeissAxiovert 200 microscope equipped with a Hamamatsu OrcaAG CCD camera and spinning disk confocal scan head. Voloc-ity software was used to acquire and analyze images. Figureswere made using Adobe Photoshop digital image software (SanJose, CA). To label the late endosomal/lysosomal cell compart-ment, cells were incubated overnight in normal growth mediacontaining 10�g/ml FITC-conjugated 70-kDa dextran (Molec-ular Probes) followed by a 2-h chase period in dextran-freemedia containing anti-HA.Antibody Uptake and Recycling Assay—Cells expressing

HA-N1, grown on polyornithine-coated coverslips (�50% con-fluent), were incubated with Alexa488-conjugated anti-HA

(1:1000) in growth media for 30 min. After cooling on ice andwashing with cold growthmedia, anti-mouse IgG-binding siteson cell surface Alexa488 anti-HA were blocked by incubationwith excess unlabeled anti-mouse (1:100; Jackson Immuno-Research) for 15 min at 4 °C. Cells were washed and then re-warmed to 37 °C in growth media between 0 and 30 min.Alexa488 anti-HA that had re-inserted into the plasma mem-brane during this incubation period was identified by incuba-tion with Cy3-labeled anti-mouse (1:500) at 4 °C for 15 minfollowedbywashing and fixation in 2%paraformaldehyde.Cellswere imaged using a 25�water objective (0.8NA) and spinningdisk confocalmicroscope as described above. The total cellular,endocytosed HA-Notch1 (Alexa488 anti-HA fluorescenceintensity) and cell surface Alexa488 anti-HA labeled HA-N1(Cy3-�mouse fluorescence intensity) were quantified using themeasurement functions of Volocity imaging software.

RESULTS

Notch1 Is Constitutively Internalized and Recycled—Toexamine constitutiveNotch1 receptor trafficking, wemeasuredthe internalization and subsequent trafficking of surface-la-beled endogenousNotch1 in C2C12 cells. Subconfluent C2C12cells were surface-labeled with biotin at 4 °C and then incu-bated at 37 °C to allow internalization and post-endocytic traf-ficking of biotinylated Notch1. At specific time points, the cellswere treated with MeSNa to remove any biotin label on pro-teins remaining at the cell surface. Intracellular biotinylatedNotch1 protein was recovered with streptavidin beads andquantified byWestern blot analysis using anti-Notch1 antibod-ies. Biotinylation of both subunits of the Notch1 heterodimerwas observed confirming that the processedNotch1 receptor ispresent at the plasma membrane (Fig. 1A, upper panels). Fol-lowing incubation at 37 °C, a steady increase in intracellularbiotinylated Notch1 was observed (Fig. 1A, lower panel) indi-cating that endogenous Notch1 is constitutively internalizedfrom the cell surface. A similar analysis of HEK293T cellsexpressing the full-length Notch1 receptor (FL-Notch1) wasalso performed. In HEK293T cells, both subunits of the Notch1heterodimer were biotinylated at the plasma membrane indi-cating that ectopically expressed Notch1 is processed and pres-ent at the cell surface (Fig. 1B, upper and lower panels, respec-tively). Similar to endogenous Notch1 in C2C12 cells,biotinylated FL-Notch1 was endocytosed and accumulatedintracellularly in 293T cells (Fig. 1B) demonstrating that bothendogenous and ectopically expressed Notch1 are constitu-tively trafficked. To examine post-endocytic trafficking ofNotch1, HEK293T cells expressing FL-Notch1 were labeledwith biotin at 4 °C and then incubated at 18 °C to allow inter-nalization of biotinylated FL-Notch1 from the plasma mem-brane under conditions that block recycling to the cell surface.Cells were then treated withMeSNa to remove biotin at the cellsurface and returned to 37 °C to resume trafficking. At the indi-cated time points, cells were either lysed to examine the totalamount of biotinylated protein or treated a second time withMeSNa before lysis to remove biotin from proteins that hadreturned to the cell surface. A steady loss in the intracellularpool of biotinylated FL-Notch1, with almost complete loss after30 min, was observed (Fig. 1C, compare lane 1 and lanes 2–4,




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lower panel) suggesting that internalized FL-Notch1 was eitherbeing returned to the cell surface or degraded. To assess FL-Notch1 degradation, the rate of change in the total amount ofbiotinylated FL-Notch1 was measured in cells shifted to 37 °Cbut not stripped of surface biotin a second time. Under theseexperimental conditions, the total amount of biotinylated FL-Notch1 remained relatively unchanged at 5 min (Fig. 1C, com-pare lane 1 and lane 2, upper panel) suggesting that the loss ofbiotinylated FL-Notch1 from the intracellular pool at 5 min isthe result of recycling to the cell surface. However, by 30 minthe total cellular, biotinylated FL-Notch1 had decreased sug-gesting that a portion of biotinylated FL-Notch1 is targeted fordegradation at later times. A similar trend was observed whenan antibody against the extracellular domain of the FL-Notchheterodimer (Fig. 1D) was used, indicating that the intactreceptor is constitutively trafficked.We used immunocytochemistry and antibody uptake exper-

iments to further investigate the constitutive internalizationand trafficking of Notch1 toward a degradative pathway orrecycling to the plasma membrane. HEK293T cells were tran-siently transfected with pBOS HA-N1, which encodes full-length Notch1 with an extracellular amino-terminal HA tag(15). Cells were surface-labeled with Alexa488-conjugatedanti-HA (Al488�HA) while at 4 °C to prevent endocytosis (Fig.2A, left panel).When the cells werewarmed to 37 °C for 60min,surface-labeled HA-Notch1 was greatly reduced with a con-comitant labeling of many intracellular vesicles (Fig. 2A, rightpanel). To characterize theHA-Notch1-containing vesicles, wepreincubated cells overnight with the fluid-phase markerFITC-dextran followed by anti-HA for 2 h at 37 °C. Under theseconditions, some surface-labeled HA-N1 colocalized withFITC-dextran positive vesicles (Fig. 2B). This suggests that con-stitutively endocytosed Notch1 is sorted to late endosomes anddegraded in a lysosomal compartment. In addition, whenHEK293T cells expressing HA-N1 were incubated withAl488�HA at 37 °C for 2 h, fixed and immunostainedwith anti-HRS, a marker for late endosomes, we observed colocalizationbetween the labeled HA-Notch1 and HRS (Fig. 2C).To examine whether Notch1 is also recycled back to the

plasma membrane, we used C2C12 cells that stably expressHA-N1 (C2C12 HA-N1 (15)). C2C12 HA-N1 cells were incu-bated with Al488�HA at 37 °C to allow uptake of labeledHA-N1 into intracellular vesicles, and any remaining extracel-lular Al488�HA bound to the cell surface was blocked to sub-sequent binding of Cy3-conjugated anti-mouse (Cy3-anti-mouse) by incubation with excess unlabeled anti-mouse IgG at4 °C. Cells were re-warmed to 37 °C to allow trafficking to con-tinue for 10, 20, or 30 min, and we detected the re-appearanceofAl488�HA-labeledHA-N1 at the plasmamembrane by incu-bating nonpermeabilized cells with Cy3 anti-mouse, which wasmeasured using spinning disk confocal microscopy (Fig. 3). Asteady increase in Cy3-anti-mouse labeling of the cell surfaceHA-N1 was observed from 0 to 30 min, although total HA-N1remained constant, indicating that constitutively internalizedantibody labeled HA-N1 is also recycled (Fig. 3).Numb Regulates Notch1 Intracellular Trafficking—The varia-

bility in HA-N1 expression levels in cell lines precluded a quanti-tative analysis of Notch antibody uptake and trafficking events in

FIGURE 1. Notch1 is constitutively trafficked independent of ligand. A, subcon-fluent C2C12 cells expressing endogenous Notch1 receptor were labeled with bio-tin at 4 °C. After 1 h, one plate of cells was lysed to examine total surface biotin-labeled Notch1 (Tot), and a second was treated with MeSNa (0�) to strip the biotinlabel. Cells were then incubated at 37 °C and at the indicated times stripped ofbiotin remaining at the cell surface. Intracellular pools of biotinylated Notch1were recovered from whole cell lysates using streptavidin beads and analyzed byWestern blot using antisera specific for the carboxyl terminus of Notch1, anti-NotchIC, or for the extracellular epidermal growth factor-like repeat region ofNotch1, anti-NotchEC, as indicated. IB, immunoblot. B, HEK293T cells transfectedwith FL-Notch1 were labeled with biotin, and intracellular pools of biotin-labeledFL-Notch1 were examined as described above. C and D, FL-Notch1 is recycled.HEK293T cells expressing FL-Notch1 were biotinylated, incubated at 18 °C for 2 hto internalize biotin-labeled FL-Notch1, and then treated with MeSNa to removesurface biotin. Cells were then returned to 37 °C to resume trafficking. One set ofcells was treated a second time with MeSNa followed by lysis (intracellular, lowerpanel), although the second set was lysed without MeSNa treatment (surface �IC, intracellular; upper panel). Pools of biotinylated FL-Notch1 were recoveredwith streptavidin beads and analyzed by Western blot using antisera specific foreither the intracellular (C) or extracellular domain (D) of Notch1.




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the presence of excess Numb orNumb depletion. Therefore, we useda biochemical approach to examinethe effects of Numb on constitutiveNotch1 receptor trafficking. Weoverexpressed p66 Numb in C2C12cells and measured intracellularaccumulation of surface-biotiny-lated, endogenous Notch1 over 30min (Fig. 4A). Overexpression ofNumb resulted in a reduction of theinternalized pool of biotinylatedNotch1 that accumulated after 30min (Fig. 4A) compared with con-trol cells. Similarly, whenNumbandFL-Notch1 were coexpressed inHEK293T cells, the amount of intra-cellular biotinylated FL-Notch1 pro-tein that accumulated after 30 min ofinternalization (Fig. 4B, lower panel)was decreased compared withFL-Notch1 alone (Fig. 4B, upperpanel). Under identical conditions,overexpression of Numb had noeffect on the intracellular accumula-tion of surface-biotinylated epider-mal growth factor receptor (EGFR)or transferrin receptor (Fig. 4C anddata not shown) indicating thatNumb overexpression has a specificeffect on Notch1 trafficking.To assess Notch1 receptor traf-

ficking in the absence of Numb,C2C12 cells were transfected withNumb-specific RNA duplexes orwith control scrambled RNAduplexes. In Numb siRNA trans-fected cells, accumulation of inter-nalized, biotinylated Notch1 wasmarkedly reduced compared withcells transfected with scrambledsiRNA, suggesting that depletionof Numb protein also alters thetrafficking of Notch1 (Fig. 4D).Together these data suggest thataltering the protein levels of Numbwithin the cell disrupts constitutivesteady state trafficking of Notch1.Numb Endocytic Motifs and

Interaction with Itch Are Requiredto Regulate Notch1 Trafficking—Todetermine the regions of Numbrequired for modulating Notch1trafficking, we examined the effectsof the Numb mutant (Nb�C) lack-ing the last 41 amino acids of Numb,which includes the NPF and DPFmotifs required for Numb binding

FIGURE 2. Surface-labeled Notch1 is constitutively endocytosed into vesicles that colocalize with FITC-dextran and HRS. A, C2C12 cells that stably express HA-tagged Notch1 were surface-labeled withAl488anti-HA at 4 °C (left panel), washed, and incubated at 37 °C for 60 min to allow for endocytosis (right panel).B, HEK293T cells transiently expressing HA-Notch1 were incubated for 18 h with 1 mg/ml FITC-dextran (70,000molecular weight; Molecular Probes) in normal growth media at 37 °C. The cells were washed and chased withmedia containing anti-HA for 2 h, fixed, permeabilized, and then stained with Cy3-�-mouse to identify �-HA-labeled Notch1 and with rabbit �-NbC (Cy5-�-rabbit secondary) to mark the plasma membrane. C, HEK293Tcells transiently expressing HA-Notch1 were incubated with Alexa488-�-HA (green) at 37 °C for 1 h. Followingfixation with 2% paraformaldehyde, cells were permeabilized and immunostained with rabbit �HRS (Cy5-labeled donkey �-rabbit) and guinea pig �NbC (Cy3 labeled donkey anti-guinea pig). Images were capturedusing a Zeiss Axiovert 200 microscope equipped with a Hamamatsu Orca AG CCD camera and spinning diskconfocal scan head. Images were processed using Volocity software, and figures were prepared using Photo-shop. Scale bars represent 10 �m. Images are representative of three independent experiments.




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to �-adaptin, Eps15, and EHD4 (41, 42), and the Nb�PTBCmutant, which lacks 88 amino acids within the carboxyl-termi-nal region of the PTB domain required for binding to the E3ligase, Itch (32) (Fig. 5A). FL-Notch1 was cotransfected withwild typeNumb,Nb�C, orNb�PTBC, and the accumulation of

intracellular biotinylated FL-Notch1 was examined. Althoughwild typeNumb (NbWT) decreased the amount of biotinylatedFL-Notch1 within intracellular pools compared with controlcells (Fig. 5B, top andmiddle panels), expression of Nb�C hadno effect on the amount of biotinylated FL-Notch1 at the cell

FIGURE 3. Surface-labeled Notch1 is constitutively recycled to the plasma membrane. A, C2C12 HA-N1 cells were incubated with Al488anti-HA (green) ingrowth media at 37 °C for 30 min to accumulate surface-labeled HA-Notch1 in intracellular vesicles (0 min No Block). Cy3-labeled anti-mouse-binding sites onsurface Al488anti-HA were blocked with excess unlabeled anti-mouse IgG at 4 °C (0 min). Cells were then re-warmed to 37 °C to allow for continued traffickingof internalized HA-Notch1 (10 min and 20 min). Prior to fixation, all cells were incubated with Cy3-labeled anti-mouse (red) to visualize unblocked, Al488-anti-HAtagged, and HA-Notch1 at the cell surface. Cells were imaged using a �25 water objective and spinning disk confocal microscope as described under “Materialsand Methods.” Scale bars represent 20 �m. Images are representative of three independent experiments. B, average total cellular fluorescence intensity in eachchannel was measured in 45–55 cells for each condition shown in A. The mean intensities for each condition are shown, allowing comparison of the changesin the total labeled HA-Notch1 (green bars, left) and surface-labeled HA-Notch1 (red bars, right).




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surface or accumulating within intracellular pools (Fig. 5B, bot-tom panel). This suggests that interaction with endocytic pro-teins �-adaptin, Eps15, and/or EHD/Rme1 is required forNumb to regulate Notch1 trafficking. Overexpression of

Nb�PTBC also failed to cause a reduction in intracellular poolsof Notch1 and, in contrast to wild type Numb, resulted inan increased accumulation of intracellular biotinylated FL-Notch1 compared with control cells (Fig. 5C). These observa-tions indicate a distinct role for the Numb PTB domain in reg-ulating intracellular FL-Notch1 trafficking. Previously we haveshown that Nb�PTBC is defective for binding to Itch, and oth-ers have demonstrated a role for Itch in Notch1 trafficking tothe lysosome for degradation (32, 53, 54). Together these datasuggest that Numb-mediated sorting of Notch1 also requiresNotch1 ubiquitylation. In support of this, we observed thatoverexpression of wild type Itch promoted depletion of intra-cellular pools of biotinylated FL-Notch1 (Fig. 5D) similar tothat observed with Numb overexpression. Expression of aligase-dead mutant form of Itch, ItchC830A, had a reducedeffect on FL-Notch1 trafficking but did cause a reduction inintracellular pools at 30 min.Numb Promotes Post-endocytic Trafficking and Degradation

of Notch1—The reduction in the pool of internalized Notch1observed could reflect changes in either the rate of internaliza-tion, degradation, or recycling. To determine the step in theendocytic pathway influenced by Numb expression levels, wefirst examined Notch1 internalization. HEK293T cells express-ing FL-Notch1were biotinylated and then incubated at 18 °C toallow for internalization but not the subsequent intracellulartrafficking of biotinylated FL-Notch1.A steady accumulation ofintracellular biotinylated Notch1 was observed over 4 h (Fig.6A, upper panel). Overexpression of Numb had no effect onFL-Notch1 internalization compared with control cells (Fig.6A, lower panel). Similarly, when Numb protein levels weredepleted in C2C12 cells usingNumb-specific RNA interferenceduplexes, no change in Notch1 internalization was observedcompared with control (Fig. 6B).We next examined the effect of Numb on post-endocytic

trafficking. Using a procedure similar to that described in Fig.1C, biotinylated FL-Notch1 was allowed to accumulate inHEK293T cells, with or without overexpressed Numb. Follow-ing the removal of surface biotin with MeSNa, we measuredchanges in the total pool of biotinylated Notch1 (intracellularplus surface Notch1), and we compared this to changes in theintracellular pool of biotinylated FL-Notch1 remaining over 30min. In cells expressing FL-Notch1 alone, a steady decrease inthe amount of intracellular biotinylated FL-Notch1 wasobserved in theMeSNa-treated cells over 30min (Fig. 7A, com-pare lane 1 with lanes 2–4, lower panel), whereas the totalamount of biotinylated FL-Notch1 remained relatively stable at5 and 15 min and then decreased after 30 min (Fig. 7A, lane 1with lanes 2–4, upper panel). Because the total pool of biotin-ylated FL-Notch1 remained constant at 5 and 15 min, indicat-ing that minimal degradation had occurred, the decrease in theintracellular biotinylated FL-Notch1 (in the MeSNa treatedsamples) likely represents loss because of receptor recyclingback to the cell surface. Coexpression of FL-Notch1 and Numbresulted in an accelerated loss of both the intracellular pool ofbiotinylated FL-Notch1 (Fig. 7B, compare lane 1 with lanes2–4, lower panels) and the total biotinylated FL-Notch1 (Fig.7B, compare lane 1 with lanes 2–4, upper panels) comparedwith cells expressing FL-Notch1 alone, indicating that the loss

FIGURE 4. Numb regulates the dynamics of Notch1 receptor trafficking.A, C2C12 cells expressing endogenous Notch1 receptor transfected withempty vector or Numb were labeled with biotin at 4 °C for 1 h, allowed totraffic at 37 °C, and the intracellular pools of biotin-labeled Notch1 (biotin-N1)examined using streptavidin beads and Western blot analysis as describedpreviously. IB, immunoblot; Tfxn, transfection. B, HEK293T cells were trans-fected with FL-Notch1 alone or FL-Notch1 and Numb and the trafficking ofthe FL-Notch1 receptor examined. C, Numb does not alter trafficking of EGFR.HEK293T were cotransfected with EGFR alone or with Numb, and accumula-tion of intracellular biotin-labeled EGFR examined as described above. D, lossof Numb protein disrupts intracellular trafficking of Notch1. C2C12 cells weretransfected with Numb-specific RNA duplexes or control scrambled RNAduplexes, and the trafficking of endogenous Notch1 receptor was examined48 h post-transfection as described above. Whole cell lysates were examinedfor Numb protein expression using antisera specific for Numb. Scram, Scram-bled siRNA. Representative Western blots are shown. Films from three inde-pendent experiments were scanned and quantitated using an Alpha Inno-tech Fluorochem 8000 imaging system (see “Material and Methods”), andresults are shown in graphs (right). Data are expressed relative to total (Tot)surface-biotinylated Notch1 (mean � S.D.; n � 3). Significance was deter-mined by Student’s t test; **, p � 0.01; *, p � 0.05 compared with control.




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FIGURE 5. PTB domain and tripeptide endocytic motifs of Numb are required to regulate Notch1 trafficking. A, schematic representation of Numb andNumb mutants. Nb�PTBC is missing 88 amino acids in the carboxyl-terminal half of the PTB domain. Nb�C lacks the carboxyl-terminal 41 amino acids,including NPF and DPF motifs. Tfxn, transfection; IB, immunoblot. B, Nb�C does not affect Notch1 receptor trafficking. HEK293T cells were cotransfected withFL-Notch1 and wild type Numb or Nb�C and labeled with biotin at 4 °C. The total amount of biotin-labeled FL-Notch1 (biotin-N1) at the cell surface (Tot) andthe efficiency of MeSNa treatment (0�) were monitored as described previously. Cells were incubated at 37 °C, and surface biotin stripped at indicated timepoints. Equivalent amounts of protein lysates were incubated with streptavidin beads and the intracellular pool of biotin-labeled FL-Notch1 analyzed byWestern blot. Whole cell lysates were examined for equal expression of NbWT and Nb�C. Tfxn, transfection. C, accumulation of intracellular FL-Notch1 uponoverexpression of Nb�PTBC. HEK293T cells were cotransfected with FL-Notch1 and NbWT or Nb�PTBC, and the intracellular pools of biotin-labeled FL-Notch1were examined as described above. Whole cell lysates were examined for equal expression of NbWT and Nb�PTBC. D, Itch disrupts trafficking of FL-Notch1.HEK293T cells expressing FL-Notch1 and wild type Itch or a ligase-dead mutant were biotinylated, and trafficking of FL-Notch1 was examined. Whole celllysates were examined for equal expression of ItchWT and ItchC830A. Representative Western blots are shown. Films from three independent experimentswere scanned and quantitated using an Alpha Innotech Fluorochem 8000 imaging system (see “Materials and Methods”) and results are shown in the graphs(right). Data are expressed relative to total (Tot) surface-biotinylated Notch1 (mean � S.D.; n � 3). Significance was determined by Student’s t test; **, p � 0.01;*, p � 0.05 compared with control.




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of intracellular biotinylated FL-Notch1 under these conditionsis primarily because of degradation of FL-Notch1. These resultsimply that overexpression of Numb reduces intracellular poolsof biotinylated Notch1 by promoting Notch1 degradation.Next we examined the effect of Numb depletion on post-

endocytic trafficking of endogenous Notch1. In C2C12 cellsexpressing a nonspecific scrambled RNA duplex, the totalamount of biotinylated Notch1 remained steady after 5 and 15min (Fig. 7C, compare lane 1 with lanes 2–4, upper panel),whereas the intracellular pool of biotinylated endogenousNotch1 protein was steadily lost over 30 min (Fig. 7C, comparelane 1 with lanes 2–4, lower panel), similar to the trendobserved in Fig. 7A. Numb depletion by siRNA resulted in anaccelerated loss of the accumulated biotinylated Notch1 atearly time points (Fig. 7D, compare the lower panels of Fig. 7, Cand D). However, the total amount of biotinylated Notch1remained steady after 5 and 15 min (Fig. 7, C and D, comparelane 1 with lane 2–4, upper panels) suggesting that the accel-erated loss of intracellular biotinylated Notch1 in Numbdepleted cells is a consequence of Notch1 recycling rather thandegradation. Together these results support a role for Numbin promoting the routing of Notch1 from a constitutive recy-cling pathway into an endocytic compartment that leads todegradation.

DISCUSSION

Accumulating evidence suggests that entry of Notch1 intothe endosomal pathway and subsequent trafficking is inti-mately linked to downstream signaling outcomes (reviewed inRefs. 55–57). In Drosophila, disruption of the endocytic path-way can alter Notch localization and activity. In addition, pro-teolysis of the ligand-activated receptor by �-secretase requires

FIGURE 6. Numb regulates intracellular sorting of Notch1. A, overexpres-sion of Numb does not affect internalization of Notch1 from the cell surface.HEK293T cells expressing FL-Notch1 with or without overexpressed Numbwere biotinylated at 4 °C and then incubated at 18 °C. Total biotin-labeledFL-Notch1 (biotin-N1) at the cell surface (Tot) and the efficiency of MeSNatreatment (0�) was monitored as described previously. Biotin remaining at thecell surface was stripped at the indicated time points, and intracellular poolsof biotin-labeled FL-Notch1 were recovered using streptavidin beads andanalyzed by Western blot. Tfxn, transfection; IB, immunoblot. B, loss of Numbprotein using Numb-specific siRNA had no effect on Notch1 internalizationcompared with C2C12 cells expressing scrambled siRNA duplexes. Repre-sentative Western blots are shown. Numb overexpression or depletion wasconfirmed by immunoblotting of whole cell lysates with anti-Numb. Filmsfrom three independent experiments were scanned and quantitated usingan Alpha Innotech Fluorochem 8000 imaging system (see “Materials andMethods”) and results are shown in graphs (right). Data are expressed relativeto total (Tot) surface-biotinylated Notch1 (mean � S.D.; n � 3). Significancewas determined by Student’s t test.

FIGURE 7. Numb promotes Notch1 degradation. A and B, overexpression ofNumb promotes Notch1 degradation not recycling. HEK293T cells expressingFL-Notch1 alone (A) or FL-Notch1 and Numb (B) were biotinylated, incubatedat 18 °C for 2 h to accumulate biotin-labeled FL-Notch1 intracellularly, andthen stripped of remaining surface biotin with MeSNa. Cells were thenreturned to 37 °C to resume trafficking. One set of cells was treated a secondtime with MeSNa followed by lysis to examine intracellular pools of biotin-ylated FL-Notch1 (IC, lower panels), whereas the second set was lysed withoutMeSNa treatment to examine total pools of biotinylated FL-Notch1 remain-ing (Surf � IC, upper panels). Pools of biotinylated FL-Notch1 were recoveredwith streptavidin beads and analyzed by Western blot using antisera specificfor Notch1. Tfxn, transfection; IB, immunoblot. C and D, knockdown of Numbprotein using Numb-specific RNA interference increases FL-Notch1 recyclingback to the plasma membrane. C2C12 cells expressing a scrambled controlsiRNA duplex (C) or Numb-specific siRNA duplex (D) were analyzed asdescribed above for intracellular and total pools of biotinylated FL-Notch1.Whole cell lysates were quantified and analyzed for Numb expression. Scram,scrambled siRNA; IC, intracellular; Surf � IC, surface and intracellular. Repre-sentative Western blots are shown. Films from three independent experi-ments were scanned and quantitated using an Alpha Innotech Fluorochem8000 imaging system (see “Materials and Methods”), and results are shown ingraphs (right). Data are expressed relative to total (Tot) surface-biotinylatedNotch1 (mean � S.D.; n � 3). Significance was determined by Student’s t test;**, p � 0.01;*, p � 0.05 compared with respective controls shown in A and C.




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both mono-ubiquitination and clathrin-mediated endocytosis(20, 58). Notch signaling is also affected by the availability of thereceptor at the plasmamembrane to interact with ligand, whichcan be influenced by both its rate of removal from the mem-brane and its insertion. In this study, we investigated the role ofNumb in the constitutive endocytosis and intracellular traffick-ing of mammalian Notch1. Our data demonstrate that,althoughNumb has no effect on the constitutive endocytosis ofNotch1 from the plasmamembrane, Numb positively regulateslate sorting events in the endocytic pathway such thatNotch1 istrafficked to the late endosome/lysosomal pathway. Our resultsare consistent with a model in which Numb promotes the re-routing of Notch1 from a constitutive recycling pathway into alate endosomal compartment leading to degradation.We and others have shown that Notch1 is continuously

internalized from the plasmamembrane into endocytic sortingcompartments where it is recycled back to the cell surface orsorted to the late endosome and degraded (53). Similar obser-vations of constitutive internalization of Notch have beendescribed in Drosophila (24, 59). Internalization of Notch intosorting compartments functions as an important decision pointfor regulation of downstream signaling events (20, 23–25). Ourresults strongly support a role for Numb as a mediator of thesetrafficking decisions. In the presence of Numb, Notch may bediverted from returning to the plasma membrane where it caninteract with ligand, and instead is routed through the lateendosome to the lysosome where it is degraded. Numb there-fore could regulate the pool of activation-competent Notch.Although this model implies that Numb regulates the steadystate trafficking of Notch, upstream of activation events, wecannot exclude the possibility that Numb might also influencethe trafficking of ligand-bound Notch.InDrosophila, Numb is required for asymmetric localization

of the �-adaptin subunit of AP-2 in dividing sensory organ pre-cursor cells (43), and both vertebrate and Drosophila Numbbind �-adaptin. In mammalian cells �-adaptin is required forNumb localization in cortical membrane patches (60). Giventhese interactions with the AP-2 complex, it has been proposedthat Numb regulates cell fate decisions through asymmetricsegregation of the endocytic machinery, which in turn facili-tates Notch receptor internalization (47). Our data suggest thatNumb directs the intracellular routing of the Notch1 receptorthrough endocytic sorting compartments and late endosomes,and this function is dependent on the presence of both the�-adaptin and EH domain binding sites. This suggests that theNumb interaction with AP-2 is important for the post-endo-cytic trafficking of Notch1. In keeping with this notion, a rolefor AP-2 in the post-endocytic trafficking of cargo through aclathrin-independent pathway has recently been described(61).Trafficking of receptors for lysosomal degradation is a well

characterized mode of receptor down-regulation (reviewed inRefs. 62, 63). Alternatively, membrane proteins can be routedthrough an endocytic sorting compartment where theyundergo complex internal cycling between the recycling endo-somes and late endosomes before being returned to the cellsurface or shuttled into a degradative pathway. The EHdomain-containing protein, EHD/RME-1, functions to posi-

tively regulate recycling and promotes the exit of cargo fromendocytic recycling compartment (64–70). Previously, wereported a conserved interaction betweenNumbandEHD fam-ily members and demonstrated that Numb knockdown dis-rupted the post-endocytic trafficking of the interleukin-2 �receptor (Tac) (41). A recent study in C. elegans examined thefunction of RME-1 and the Numb orthologue, NUM-1A, andprovided evidence that NUM-1A inhibits endosomal recyclingby negatively regulating RME-1 (71). This suggests that theenhanced Notch1 recycling that we observed in Numb-de-pleted cells may reflect the loss of EHD inhibition. Furtherexamination of the Numb-EHD interaction and how this mayaffect Notch1 recycling will be important in understanding themechanism by which Numb influences endocytic sorting.Our observations are also congruent with the previously

described function of two Itch-related E3 ligases, dNedd4 andSuppressor of Deltex, Su(Dx), that regulate endosomal sortingof Notch in the absence of ligand activation (24, 25). Overex-pression of these E3 ligases did not affect Notch internalizationbut did promote the sorting of Notch into vesicular compart-ments positive forHrs andRab7. Similarly, theC. elegans ortho-logue,WWP1, is important for degradation but not internaliza-tion of LIN12/Notch (23). More recently, the mammalianorthologue of Su(Dx), AIP4/Itch, was also shown to be involvedin controlling post-endocytic degradation of constitutivelyinternalized Notch1 via a process that requires ubiquitination(53). These studies suggest that ubiquitination of Notch isrequired for a post-endocytic sorting event leading to Notchdegradation. Consistent with these observations, we found thata form of Numb that does not interact with Itch causes accu-mulation rather than degradation of internalized Notch1. Thissuggests that without the Numb-Itch interaction, Notch1 isrouted into an endocytic sorting compartment but then fails totransit to the late endosome andmultivesicular body, a step thatis thought to require receptor ubiquitination (24, 25). A recentstudy demonstrated the important role of ubiquitination at thislate endocytic step in preventing ligand-independent activationof Notch (72).This study provides evidence that Numb may function as an

antagonist of endocytic recycling in mammalian cells, and oth-ers have demonstrated such a role inC. elegans. However, a rolefor Numb in regulating post-endocytic trafficking in Drosoph-ila has not yet been demonstrated. Genetic interaction betweenthe Drosophila EHD orthologue Past1 and the Notch pathwayhas recently been reported, andwe previously showed thatDro-sophilaNumb interacts with Past1 (41, 73). These observationssuggest that Drosophila Numb is likely to have a similar post-endocytic function. In Drosophila, Numb binds to and regu-lates the subcellular distribution of another transmembraneprotein, Sanpodo, that functions at the cell surface as an activa-tor of Notch-dependent cell fate selection (74, 75). Therefore,Drosophila Numb could influence Notch activity indirectlythrough regulation of Sanpodo trafficking.The role ofNumb in post-endocytic trafficking likely extends

to transmembrane cargo proteins in addition to Notch1. Arecent study examining the intracellular trafficking and proc-essing of the amyloid precursor protein, a molecule that under-goes similar proteolytic processing to Notch1, showed that




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overexpression of Numb influenced amyloid precursor proteinendosomal degradation (76). Interestingly, this effect wasdependent on the presence of the alternatively spliced insertwithin the PTB domain of Numb (the same isoform of Numbused in this study). PC12 cells, which overexpressed forms ofNumb having the PTB insert, showed decreased intracellularaccumulation of amyloid precursor protein. This effect wasabrogated by treatment with inhibitors of the lysosomal, degra-dative pathway but unaffected by inhibition of the proteosomalpathway for degradation.Numb has also been shown to influence endocytosis of other

transmembrane proteins, including �1-integrin, and the neu-ronal cell adhesion molecule L1 (44, 45). These studies demon-strated effects of Numb on the intracellular accumulation ofinternalizedmolecules; however, post-endocytic events such asrecycling and degradation were not assessed. Based on ourobservationswe suggest thatNumbmay also influence the con-stitutive trafficking of �1-integrins and L1 toward recycling ordegradation. Further studies are required to identify additionalNumb-specific cargo proteins and determine the mechanismby which Numb regulates their post-endocytic trafficking.

Acknowledgments—We thank J. Nye for the Notch1 cDNA; T. Pawsonfor the Itch cDNA; S. Urbe for the HRS antibody; Mike Woodside andPaul Paroutis of the SickKids Imaging Facility for help with confocalMicroscopy, and members of the McGlade lab for helpful discussionsand comments on the manuscript.

REFERENCES1. Artavanis-Tsakonas, S., Rand, M. D., and Lake, R. J. (1999) Science 284,

770–7762. Schweisguth, F. (2004) Curr. Biol. 14, R129–R1383. Lai, E. C. (2004) Development 131, 965–9734. Kidd, S., Lieber, T., and Young, M. W. (1998) Genes Dev. 12, 3728–37405. Schroeter, E. H., Kisslinger, J. A., and Kopan, R. (1998) Nature 393,

382–3866. Iso, T., Kedes, L., and Hamamori, Y. (2003) J. Cell. Physiol. 194, 237–2557. Davis, R. L., and Turner, D. L. (2001) Oncogene 20, 8342–83578. Seugnet, L., Simpson, P., and Haenlin, M. (1997) Dev. Biol. 192, 585–5989. Klueg, K. M., and Muskavitch, M. A. (1999) J. Cell Sci. 112, 3289–329710. Parks, A. L., Klueg, K. M., Stout, J. R., and Muskavitch, M. A. (2000)

Development 127, 1373–138511. Wang, W., and Struhl, G. (2004) Development 131, 5367–538012. Overstreet, E., Fitch, E., and Fischer, J. A. (2004) Development 131,

5355–536613. Pavlopoulos, E., Pitsouli, C., Klueg, K. M., Muskavitch, M. A., Moschonas,

N. K., and Delidakis, C. (2001) Dev. Cell 1, 807–81614. Lai, E. C., Deblandre, G. A., Kintner, C., and Rubin, G. M. (2001)Dev. Cell

1, 783–79415. Nichols, J. T., Miyamoto, A., Olsen, S. L., D’Souza, B., Yao, C., and Wein-

master, G. (2007) J. Cell Biol. 176, 445–45816. Itoh, M., Kim, C. H., Palardy, G., Oda, T., Jiang, Y. J., Maust, D., Yeo, S. Y.,

Lorick, K., Wright, G. J., Ariza-McNaughton, L., Weissman, A. M., Lewis,J., Chandrasekharappa, S. C., and Chitnis, A. B. (2003) Dev. Cell 4, 67–82

17. Emery, G., Hutterer, A., Berdnik, D., Mayer, B., Wirtz-Peitz, F., Gaitan,M. G., and Knoblich, J. A. (2005) Cell 122, 763–773

18. Commisso, C., and Boulianne, G. L. (2007)Mol. Biol. Cell 18, 1–1319. Yeh, E., Dermer, M., Commisso, C., Zhou, L., McGlade, C. J., and Bou-

lianne, G. L. (2001) Curr. Biol. 11, 1675–167920. Gupta-Rossi, N., Six, E., LeBail, O., Logeat, F., Chastagner, P., Olry, A.,

Israel, A., and Brou, C. (2004) J. Cell Biol. 166, 73–8321. Vaccari, T., Lu, H., Kanwar, R., Fortini, M. E., and Bilder, D. (2008) J. Cell

Biol. 180, 755–76222. Shaye, D. D., and Greenwald, I. (2002) Nature 420, 686–69023. Shaye, D. D., and Greenwald, I. (2005) Development 132, 5081–509224. Wilkin, M. B., Carbery, A. M., Fostier, M., Aslam, H., Mazaleyrat, S. L.,

Higgs, J., Myat, A., Evans, D. A., Cornell, M., and Baron, M. (2004) Curr.Biol. 14, 2237–2244

25. Sakata, T., Sakaguchi, H., Tsuda, L., Higashitani, A., Aigaki, T., Matsuno,K., and Hayashi, S. (2004) Curr. Biol. 14, 2228–2236

26. Zhong, W., Feder, J. N., Jiang, M. M., Jan, L. Y., and Jan, Y. N. (1996)Neuron 17, 43–53

27. Guo, M., Jan, L. Y., and Jan, Y. N. (1996) Neuron 17, 27–4128. Spana, E. P., and Doe, C. Q. (1996) Neuron 17, 21–2629. Verdi, J. M., Schmandt, R., Bashirullah, A., Jacob, S., Salvino, R., Craig,

C. G., Program, A. E., Lipshitz, H. D., andMcGlade, C. J. (1996)Curr. Biol.6, 1134–1145

30. Dho, S. E., French, M. B., Woods, S. A., and McGlade, C. J. (1999) J. Biol.Chem. 274, 33097–33104

31. Zhong, W., Jiang, M. M., Weinmaster, G., Jan, L. Y., and Jan, Y. N. (1997)Development 124, 1887–1897

32. McGill, M. A., andMcGlade, C. J. (2003) J. Biol. Chem. 278, 23196–2320333. Sestan, N., Artavanis-Tsakonas, S., and Rakic, P. (1999) Science 286,

741–74634. Berezovska, O., McLean, P., Knowles, R., Frosh, M., Lu, F. M., Lux, S. E.,

and Hyman, B. T. (1999) Neuroscience 93, 433–43935. Rhyu, M. S., Jan, L. Y., and Jan, Y. N. (1994) Cell 76, 477–49136. Spana, E. P., Kopczynski, C., Goodman, C. S., and Doe, C. Q. (1995) De-

velopment 121, 3489–349437. Ruiz Gomez, M., and Bate, M. (1997) Development 124, 4857–486638. Uemura, T., Shepherd, S., Ackerman, L., Jan, L. Y., and Jan, Y. N. (1989)

Cell 58, 349–36039. Cayouette,M.,Whitmore, A.V., Jeffery,G., andRaff,M. (2001) J. Neurosci.

21, 5643–565140. Wakamatsu, Y., Maynard, T. M., Jones, S. U., and Weston, J. A. (1999)

Neuron 23, 71–8141. Smith, C. A., Dho, S. E., Donaldson, J., Tepass, U., and McGlade, C. J.

(2004)Mol. Biol. Cell 15, 3698–370842. Santolini, E., Puri, C., Salcini, A. E., Gagliani, M. C., Pelicci, P. G., Tac-

chetti, C., and Di Fiore, P. P. (2000) J. Cell Biol. 151, 1345–135243. Berdnik, D., Torok, T., Gonzalez-Gaitan, M., and Knoblich, J. A. (2002)

Dev. Cell 3, 221–23144. Nishimura, T., Fukata, Y., Kato, K., Yamaguchi, T., Matsuura, Y., Kamigu-

chi, H., and Kaibuchi, K. (2003) Nat. Cell Biol. 5, 819–82645. Nishimura, T., and Kaibuchi, K. (2007) Dev. Cell 13, 15–2846. Le Borgne, R., Bardin, A., and Schweisguth, F. (2005) Development 132,

1751–176247. Shen, Q., and Temple, S. (2002) Sci. STKE 2002, PE5248. Urbe, S., Sachse, M., Row, P. E., Preisinger, C., Barr, F. A., Strous, G.,

Klumperman, J., and Clague, M. J. (2003) J. Cell Sci. 116, 4169–417949. Sorkin, A., Krolenko, S., Kudrjavtceva, N., Lazebnik, J., Teslenko, L.,

Soderquist, A. M., and Nikolsky, N. (1991) J. Cell Biol. 112, 55–6350. Czekay, R. P., Orlando, R. A., Woodward, L., Lundstrom, M., and Far-

quhar, M. G. (1997)Mol. Biol. Cell 8, 517–53251. Le, T. L., Yap, A. S., and Stow, J. L. (1999) J. Cell Biol. 146, 219–23252. Le, T. L., Joseph, S. R., Yap, A. S., and Stow, J. L. (2002) Am. J. Physiol. Cell

Physiol. 283, C489–C49953. Chastagner, P., Israel, A., and Brou, C. (2008) PLoS ONE 3, e273554. Qiu, L., Joazeiro, C., Fang, N., Wang, H. Y., Elly, C., Altman, Y., Fang, D.,

Hunter, T., and Liu, Y. C. (2000) J. Biol. Chem. 275, 35734–3573755. Wilkin, M. B., and Baron, M. (2005)Mol. Membr. Biol. 22, 279–28956. Le Borgne, R. (2006) Curr. Opin. Cell Biol. 18, 213–22257. Bray, S. J. (2006) Nat. Rev. Mol. Cell Biol. 7, 678–68958. Vaccari, T., and Bilder, D. (2005) Dev. Cell 9, 687–69859. Jaekel, R., and Klein, T. (2006) Dev. Cell 11, 655–66960. Dho, S. E., Trejo, J., Siderovski, D. P., and McGlade, C. J. (2006)Mol. Biol.

Cell 17, 4142–415561. Lau, A. W., and Chou, M. M. (2008) J. Cell Sci. 121, 4008–401762. Di Fiore, P. P., and De Camilli, P. (2001) Cell 106, 1–463. Katzmann, D. J., Odorizzi, G., and Emr, S. D. (2002) Nat. Rev. Mol. Cell




http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

Biol. 3, 893–90564. Caplan, S., Naslavsky, N., Hartnell, L. M., Lodge, R., Polishchuk, R. S.,

Donaldson, J. G., and Bonifacino, J. S. (2002) EMBO J. 21, 2557–256765. Grant, B. D., and Caplan, S. (2008) Traffic 9, 2043–205266. Naslavsky, N., Rahajeng, J., Sharma, M., Jovic, M., and Caplan, S. (2006)

Mol. Biol. Cell 17, 163–17767. Lin, S. X., Grant, B., Hirsh, D., and Maxfield, F. R. (2001)Nat. Cell Biol. 3,

567–57268. Park, S. Y., Ha, B. G., Choi, G. H., Ryu, J., Kim, B., Jung, C. Y., and Lee, W.

(2004) Biochemistry 43, 7552–756269. Rapaport, D., Auerbach,W., Naslavsky, N., Pasmanik-Chor,M., Galperin,

E., Fein, A., Caplan, S., Joyner, A. L., and Horowitz, M. (2006) Traffic 7,52–60

70. Picciano, J. A., Ameen, N., Grant, B. D., and Bradbury, N. A. (2003) Am. J.Physiol. Cell Physiol. 285, C1009–C1018

71. Nilsson, L., Conradt, B., Ruaud, A. F., Chen, C. C., Hatzold, J., Bessereau,J. L., Grant, B. D., and Tuck, S. (2008) Genetics 179, 375–387

72. Wilkin,M., Tongngok, P., Gensch, N., Clemence, S., Motoki, M., Yamada,K., Hori, K., Taniguchi-Kanai,M., Franklin, E.,Matsuno, K., and Baron,M.(2008) Dev. Cell 15, 762–772

73. Olswang-Kutz, Y., Gertel, Y., Benjamin, S., Sela, O., Pekar, O., Arama, E.,Steller, H., Horowitz, M., and Segal, D. (2009) J. Cell Sci. 122, 471–480

74. O’Connor-Giles, K. M., and Skeath, J. B. (2003) Dev. Cell 5, 231–24375. Hutterer, A., and Knoblich, J. A. (2005) EMBO Rep. 6, 836–84276. Kyriazis, G. A., Wei, Z., Vandermey, M., Jo, D. G., Xin, O., Mattson, M. P.,

and Chan, S. L. (2008) J. Biol. Chem. 283, 25492–25502




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Melanie A. McGill, Sascha E. Dho, Gerry Weinmaster and C. Jane McGladeNumb Regulates Post-endocytic Trafficking and Degradation of Notch1

doi: 10.1074/jbc.M109.014845 originally published online June 30, 20092009, 284:26427-26438.J. Biol. Chem.

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