From the Department of Medical Biophysics, University of Toronto and The Arthur and Sonia Labatt Brain Tumour Research Centre, The Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada
Received for publication, March 19, 2003 , and in revised form, April 3, 2003.
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ABSTRACT |
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INTRODUCTION |
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Notch signaling plays an important role in cellular differentiation, proliferation, and apoptotic events at all stages of development, functioning as an essential communication mechanism to direct cell fate selection of neighboring cells (Refs. 16 and 17 and references therein). Genetic abnormalities in components of the Notch signaling pathway have been implicated in a number of disease states including leukemia and neurogenerative disorders (1820).
The Notch1 receptor, the most extensively studied of the four mammalian Notch receptors, is a 300 kDa type I integral membrane protein that undergoes at least three critical proteolytic steps required for its maturation and signal transmission (21, 22). Notch is first processed in the trans-Golgi network by a furin-like convertase into two distinct fragments that interact to form a functional heterodimeric receptor on the cell surface (23, 24). In this heterodimeric form, Notch is able to bind transmembrane ligands of the DSL (Delta/Serrate/Lag2) family presented on neighboring cells. Upon ligand binding, a second cleavage event occurs by the metalloproteinase TACE (TNF--converting enzyme, also know as ADAM 17) releasing the Notch extracellular domain (25). A third cleavage event by a
-secretase whose activity is strongly dependent on the presenilins, releases the active intracellular fragment of Notch (22, 2629). The precise cellular compartment in which this activation cleavage occurs is still unclear. The released intracellular domain of Notch (NotchIC) then translocates to the nucleus where it acts as a cotransactivator with transcription factors of the CSL (CBF1/RBPj
, Suppressor of Hairless Su(H), Lag-1) family to modulate transcription of downstream target genes such as Hes1, the mammalian homologue of the Hairy and Enhancer of Split gene complex in Drosophila (16).
Several lines of evidence suggest a role for the ubiquitin/proteasome degradation pathway in controlling Notch signaling (30). The ubiquitination pathway is essential for cellular processes such as cell cycle progression, cellular differentiation, protein transport, DNA repair, and quality control in the endoplasmic reticulum (ER). Protein ubiquitination is a multistep process in which free ubiquitin is first attached to an ubiquitinactivating enzyme (E1) and subsequently transferred to an ubiquitin-conjugating enzyme (E2), which in partner with an ubiquitin ligase (E3), transfers ubiquitin to the specific protein substrate (31). The E3 ligase provides specificity by functioning as an adaptor to selectively bind substrates (32, 33). While ubiquitin often targets proteins for degradation by the proteasome, it can also serve as a signal for receptor internalization and trafficking to multivesicular bodies and the lysosome (31, 34, 35).
Several distinct classes of E3 ubiquitin ligases appear to directly regulate the Notch receptor. In Drosophila, genetic evidence suggests the HECT (Homologous to E6-AP C Terminus) domain containing E3 ubiquitin ligase Suppressor of Deltex negatively regulates Notch receptor signaling (36). The related mammalian E3 ubiquitin ligase Itch has been shown to ubiquitinate membrane-tethered Notch1 in vivo and in vitro (37), although the consequence of this ubiquitination event is unknown. Furthermore Sel10, an F-box-containing protein that interacts with a SCF ubiquitin ligase complex, also functions as a negative regulator of the Notch signaling pathway (38) by specifically targeting the active nuclear form of Notch for ubiquitination (3941).
Whereas the importance of Notch and Numb function during development is recognized, there remains a limited understanding of the molecular and biochemical mechanisms through which Numb exerts its regulatory effect on Notch signaling. In this report, we demonstrate that mammalian Numb expression promotes the ubiquitination of membranetethered Notch1 and the degradation of the intracellular domain following receptor activation. Furthermore Numb interacts with the E3 ubiquitin ligase Itch to cooperatively enhance Notch1 ubiquitination and down-regulate Notch1-dependent signal transduction.
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MATERIALS AND METHODS |
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Cell Culture and TransfectionsHEK293T cells were grown in Dulbecco's modified Eagle's medium (DMEM) (Wisent) supplemented with 10% fetal bovine serum and transfected with LipofectAMINE reagent (Invitrogen) in OptiMEM (Invitrogen) according to the manufacturer's instructions. Cells were cultured at 37 °C for 2448 h before lysing. NIH 3T3 cells were grown in DMEM supplemented with 10% calf serum and transfected with LipofectAMINE PLUS reagent (Invitrogen). C2C12 cells were grown in DMEM supplemented with 10% fetal bovine serum and 5% calf serum and transfected using LipofectAMINE2000 reagent (Invitrogen). For siRNA silencing experiments, a 21-nucleotide siRNA oligomer (Dharmacon Research) was designed to a region homologous to the Numb C-terminal coding sequence. As a negative control for siRNA activity, the Scramble II duplex from Dharmacon (D-001205-20) was used.
Immunoprecipitations and Western Blot AnalysisTransfected cells were grown to confluency and lysed in PLC lysis buffer (50 mM Hepes (pH 7.5), 150 mM NaCl, 10% glycerol, 1.5 mM MgCl2, 1% Triton X-100, 1 mM EGTA, 10 mM sodium pyrophosphate, 100 mM sodium fluoride containing COMPLETE protease inhibitors tablets (Roche Applied Science)). For cells lysed in 1% SDS buffer (50 mM Hepes (pH 7.5), 150 mM NaCl, 1 mM EGTA, 1% Nonidet P-40, 1% deoxycholate, 1% SDS containing COMPLETE protease inhibitors tablets), lysates were boiled for 5 min then diluted to 0.1% SDS prior to immunoprecipitation. For immunoprecipitations, an equivalent amount of protein was incubated with anti-Notch and 20% (w/v) protein G-Sepharose bead slurry for 3 h and then washed with PLC lysis buffer. Immunoprecipitated proteins were analyzed by Western blot. Numb-specific antisera were as previously described (9). Anti-Notch (M-20) from Santa Cruz Biotechnology (sc-6015) was used for immunoprecipitations. Anti-Notch cytoplasmic domain from Upstate Biotechnology Institute (06808) and anti-ICD from Cellular Signaling (2421) were used for immunoblots. Anti-HA (clone 12CA5) (Roche Applied Science) and anti-AIP4/Itch (Santa Cruz Biotechnology), anti-EGFR (Santa Cruz Biotechnology), and anti-Myc 9E10 (Developmental Studies Hybridoma Bank, University of Iowa) were obtained from commercial sources.
For proteasome inhibition, transfected cells were incubated for 4 h prior to lysis at 37 °C with either 50 µM MG132 or Me2SO as a control in DMEM-containing serum. Cells were lysed in PLC lysis buffer containing 10 µM MG132.
For coimmunoprecipitation reactions and GST pull-down experiments, transfected HEK293T cells were lysed in 1% Nonidet P-40 lysis buffer (50 mM Hepes (pH 7.5), 150 mM NaCl, 1.5 mM MgCl2, 10% glycerol, 1% Nonidet P-40, 1 mM EGTA, 10 mM sodium pyrophosphate, 100 mM sodium fluoride) containing protease inhibitors. GST fusion proteins were prepared as described previously (42).
Activation of Notch1To activate the Notch1 receptor, transfected C2C12 cells were washed with 1x PBS, and incubated for 0 min or 15 min with prewarmed 5 mM EDTA in 1x PBS. Treated cells were then washed with PBS and chased for indicated time points in regular culture medium. Cells were lysed in PLC lysis buffer as described above.
Hes1 Luciferase Reporter AssaysNIH 3T3 cells seeded on 6-well plates were cotransfected with EC-Notch (400 ng) and Hes1 luciferase reporter construct (200 ng) in the presence or absence of pEF-Numb construct (400 ng) as described above. Included in each transfection was a cytomegalovirus (CMV)-
-galactosidase reporter (100 ng) to control for transfection efficiency. The total amount of transfected DNA was equalized with empty parental vector. 24 h post-transfection, cells were lysed in 1x Reporter Lysis Buffer (Promega). Luciferase activity was quantitated using firefly luciferin according to the manufacturer's instructions (Promega).
-galactosidase activity was quantitated using the chemiluminescent substrate Galacto-PLUS according to the manufacturer's instructions (Tropix). Luciferase activity was normalized for
-galactosidase activity and represented as fold activation. Protein expression was monitored by Western blot. Each condition was performed in triplicate and experiments repeated at least three times.
Pulse-Chase AnalysisTransfected C2C12 cells were grown to confluency, washed with PBS, and incubated at 37 °C for 1 h in serum-free DMEM without methionine and cysteine (depletion medium). Cells were pulsed for 1.5 h in depletion medium containing 200 µCi ProMix L-[35S]methionine and cysteine (Amersham Biosciences), washed extensively with unlabeled DMEM, and chased at 37 °C for up to 6 h in DMEM containing serum and supplemented with unlabeled methionine and cysteine (Sigma). At indicated time points cells were washed with PBS and lysed in PLC lysis buffer. Protein lysates were quantified, and equal amounts of lysates were immunoprecipitated with anti-Notch. Immunoprecipitates were washed with PLC lysis buffer, separated by SDS-PAGE, and autoradiographed to visualize labeled protein. For pulse-chase analysis of the NICD mutant, HEK293T cells were pulsed for 30 min and chased over a period of 3 h.
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RESULTS |
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As Notch1 has been shown to interact with a number of intracellular proteins including Numb that are themselves substrates for ubiquitination, cells were lysed in 1% SDS lysis buffer and boiled to disrupt protein complexes. Ubiquitinated Notch was detected in immunoprecipitates from boiled lysates indicating the HA reactive proteins observed was Notch1 and not associated ubiquitinated proteins (Fig. 1D). Numb expression resulted in an increase in the levels of Notch1 ubiquitination under these conditions, indicating that Numb enhanced ubiquitination of Notch1 itself.
Finally, we examined the effect of Numb on the ubiquitination of another cell surface receptor, the epidermal growth factor receptor (EGFR) to assess the specificity of Numb with respect to Notch1 ubiquitination. Coexpression of Numb with EGFR had no effect on its ubiquitination in transiently transfected HEK293T cells (Fig. 1E) indicating Numb has a specific effect on the Notch1 receptor, rather than affecting membrane proteins in general.
A Functional PTB Domain of Numb Is Required to Promote Notch1 UbiquitinationPrevious studies have demonstrated that the Numb PTB domain and sequence motifs near the C terminus are important for Numb function by mediating its interaction with other proteins (8, 13, 4249).2 To determine the region of Numb required to promote Notch1 ubiquitination, two Numb mutants were generated. NumbPTBC lacks eighty-eight amino acids at the C-terminal end of the PTB domain and is similar to a deletion that abolishes dNumb function in Drosophila (Fig. 2A and Ref. 46). Numb
C lacks the C-terminal forty-one amino acids including the EH domain binding site. HEK293T cells were cotransfected with
EC-Notch and HA-Ub in the absence or presence of increasing amounts of wild-type or mutant forms of Numb. In the presence of increasing amounts of NumbWT, increasing amounts of ubiquitinated
EC-Notch species were observed (Fig. 2B, lanes 13). Coexpression of Numb
C enhanced Notch1 ubiquitination as efficiently as wild-type Numb (Fig. 2B, lanes 4 and 5); however the effect of Numb
PTBC on Notch1 ubiquitination was dramatically attenuated (Fig. 2B, lanes 6 and 7), indicating that an intact PTB domain is required to mediate Notch1 ubiquitination.
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The PEST Region of Notch1 Is Not Required for Numb-mediated UbiquitinationRecently, the E3 ubiquitin ligase, Sel10 has been shown to associate with and ubiquitinate the NotchIC domain within the nucleus and target it for proteasomal degradation (3941). The C terminus of Notch contains a PEST sequence that is required to mediate Sel10 binding and ubiquitination of nuclear Notch. To determine if the PEST region of Notch1 is required for Numb to promote ubiquitination, a C-terminal deletion mutant that removes the last 483 amino acids of Notch1, EC
RV, was constructed (see Fig. 1A). An increase in the amount of ubiquitination of
EC
RV was observed upon coexpression of
EC
RV and Numb in HEK293T cells as compared with coexpression with empty vector (Fig. 3). In addition, deletion of the same region in a non-membrane-tethered Notch1 mutant, NICD
RV had no effect on Numb-mediated ubiquitination (data not shown). Therefore the C terminus of Notch1 is dispensable for Numb-mediated ubiquitination and suggests that an E3 ubiquitin ligase other than Sel10 is required for Numb-mediated ubiquitination of Notch1.
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Numb Interacts with the E3 Ubiquitin Ligase Itch in Vitro and in VivoNumb itself does not possess intrinsic E3 ubiquitin ligase activity (data not shown), and therefore likely mediates Notch ubiquitination through recruitment of components of the ubiquitination machinery to the Notch1 receptor. To date, three E3 ubiquitin ligases Sel10, Itch, and Cbl, target different forms of Notch1 for ubiquitination.
Similar to Numb-mediated ubiquitination of Notch, the HECT domain containing E3 ubiquitin ligase Itch was shown to ubiquitinate membrane-tethered Notch1 independent of the C-terminal PEST sequence (37). Given the similarity between the action of Numb and Itch we investigated whether Numb and Itch proteins interact as part of a functional complex. HEK293T cells were transfected with Numb, and lysates were incubated with glutathione-Sepharose beads bound with GST alone or GST fusions of Itch WW domains and HECT domain (Fig. 4A). Transfected Numb interacted with both GST-WW1/2 and GST-WWHECT (Fig. 4B), but not GST-WW3, GST-WW4, or GST alone. Similarly endogenous Numb from C2C12 cells (Fig. 4C) and HEK293T cells (data not shown) bound preferentially to GST-WW1/2.
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To further confirm the interaction between Numb and Itch, HEK293T cells were cotransfected with Numb and Myc-tagged Itch, and prepared lysates were immunoprecipitated with either anti-Numb or anti-Myc antibody. Itch was detected in anti-Numb immunoprecipitations, but not in the preimmune control immunoprecipitates (Fig. 4D). In the reciprocal experiment, Numb co-immunoprecipitated with Itch (Fig. 4E), suggesting that Numb can interact with the WW domains of the E3 ligase Itch both in vivo and in vitro.
Numb and the E3 Ligase Itch Act Cooperatively to Enhance the Ubiquitination of Notch1To examine the biological significance of the interaction between Numb and Itch, HEK293T cells were cotransfected with EC-Notch and HA-Ub in the presence or absence of Numb and/or Itch. Expression of either Numb or Itch alone enhanced the ubiquitination of
EC-Notch however, coexpression of Numb and Itch together further enhanced Notch ubiquitination, suggesting that they acted cooperatively to ubiquitinate membrane-tethered
EC-Notch (Fig. 5A). In contrast, the Numb
PTBC mutant was unable to cooperate with Itch in promoting Notch1 ubiquitination (Fig. 5A). GST pull-down experiments showed that in addition to impairing the effect of Numb on Notch1 ubiquitination, Numb
PTBC no longer bound GST-Itch (Fig. 5B).
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In mammalian cells, signaling by activated Notch1 induces expression of target genes including HES1 through an association of the Notch intracellular domain with CSL family of transcription factors (16). A luciferase reporter assay in which the production of luciferase is under control of the Hes1 promoter was utilized to examine the downstream effect of mammalian Numb on Notch1 nuclear activity. NIH 3T3 cells were cotransfected with the Hes1 luciferase reporter and constitutively active EC-Notch expression construct in the presence or absence of either wild type Numb or Numb
PTBC. A
-galactosidase reporter was included in each transfection to control for transfection efficiency. Transfection with
EC-Notch alone resulted in a 5-fold increase in Hes1 promoter activation as compared with transfections with empty vector (Fig. 5C). Coexpression with NumbWT resulted in a 60% decrease in the level of Notch1-induced Hes1 transcriptional activation. The Nb
PTBC mutant, which is impaired in its ability to enhance Notch1 ubiquitination, was unable to antagonize Notch nuclear activity as efficiently as NumbWT. The ability of Numb to ubiquitinate Notch1 therefore correlates with its functional inhibition of Notch1 signaling.
Numb Promotes the Ubiquitination of Notch1 Prior to ActivationTo further elucidate the functional relevance of Numb-mediated ubiquitination, we analyzed the fate of ubiquitinated EC-Notch. HEK293T cells coexpressing
EC-Notch and empty vector or Numb were treated with the proteasome inhibitor MG132 for 4 h prior to lysis. In cells treated with MG132 an increase in higher molecular weight HA reactive Notch1 species was observed as compared with untreated cells suggesting a subpopulation of ubiquitinated
EC-Notch is targeted for degradation by the proteasome pathway (Fig. 6A).
EC-Notch is constitutively activated and spontaneously undergoes cleavage at the S3 site releasing the active intracellular domain. Therefore the ubiquitinated species stabilized by MG132 treatment could represent the 120-kDa
EC-Notch protein or the 110-kDa cleaved intracellular domain.
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To investigate whether Numb effects the ubiquitination of membrane bound Notch1 prior to activation we examined the effect of Numb on full-length Notch1. Upon coexpression of FL-Notch and Numb there was an increase in the ubiquitination of both the unprocessed 300 and 120 kDa heterodimeric form of full-length Notch1 protein as compared with coexpression of FL-Notch and empty vector (Fig. 6B) suggesting that Numb acts on Notch receptors prior to ligand activation. Furthermore, the ubiquitination of endogenous Notch1 was enhanced by overexpression of Numb but not GFP in C2C12 cells (Fig. 6C).
To examine whether Numb and Itch together promote the ubiquitination of FL-Notch prior to activation, HEK293T cells were cotransfected with FL-Notch, HA-Ub, and Numb and/or Itch. Numb and Itch cooperatively promoted the ubiquitination of FL-Notch, and this effect was lost when a form of Itch containing a mutation of the active site cysteine in the HECT domain, ItchC830A was transfected (Fig. 6D).
To directly examine whether Numb-mediated ubiquitination of full-length Notch1 promoted its degradation we performed pulse-chase analysis. C2C12 cells expressing either GFP or Numb proteins were pulse-labeled with 35S-labeled methionine and cysteine and chased with normal culture medium for 6 h. Endogenous Notch1 was immunoprecipitated and analyzed by SDS-PAGE. Overexpression of Numb did not significantly alter the steady-state levels of the 300 kDa unprocessed full-length nor the 120 kDa heterodimeric form of Notch1 protein (Fig. 6E) suggesting Numb does not promote the degradation of membrane-bound Notch1.
Numb Promotes Ubiquitination and Proteasome-dependent Degradation of the Notch1 Intracellular DomainFurther investigation of the stability of EC-Notch suggested that similar to FL-Notch, Numb does not effect the stability of the 120-kDa form of
EC-Notch (data not shown). However these experiments did reveal that Numb expression resulted in the reduction in the level of the 110-kDa intracellular cleavage product of
EC-Notch suggesting a specific effect on the degradation of the Notch1 intracellular domain.
To examine whether Numb promotes the ubiquitination of the intracellular domain of Notch1, a constitutively active Notch1 mutant encoding only the intracellular domain of Notch1, NICD was constructed. HEK293T cells transfected with NICD in the presence and absence of Numb protein and HA-Ub were treated with 50 µM MG132 for 4 h prior to lysis. Ubiquitinated NICD was detected only in cells treated with the proteasome inhibitor MG132 (Fig. 7A, lanes 58) suggesting it is rapidly degraded by the proteasome. Coexpression of Numb increased the level of ubiquitinated NICD detected in the MG132 treated cells as compared with coexpression with empty vector (Fig. 7A, compare lanes 7 and 8). Therefore overexpression of Numb promotes the ubiquitination of the isolated intracellular domain of Notch in addition to membrane-bound Notch1. We also examined whether Itch was able to ubiquitinate the NICD mutant. Ubiquitination assays demonstrate that Itch also targets NICD for ubiquitination and that coexpression of Numb and Itch further enhances this ubiquitination (Fig. 7B).
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The effect of Numb on NICD stability was examined using pulse-chase analysis. HEK293T cells transfected with NICD and empty vector or Numb were pulsed with 35S-labeled methionine and cysteine and chased for 3 h in normal culture medium. Overexpression of Numb caused a reduction in the half-life of NICD as compared with cells transfected with empty vector (Fig. 7C).
To investigate whether Numb-mediated ubiquitination affected the stability of the endogenous Notch intracellular domain, NotchIC, following receptor activation, C2C12 cells were transfected with GFP or Numb. Twenty-four hours post-transfection, cells were treated with 5 mM EDTA to induce activation and cleavage of endogenous Notch1. EDTA treatment activates the Notch1 receptor by forcing the dissociation of the heterodimeric surface receptor thereby relieving the inhibition associated with the extracellular domain (50). After 15 min of EDTA treatment, cells were washed with PBS and incubated in normal culture medium for up to 2 h. Cells were lysed at the time points indicated and analyzed by Western blot. The activation cleavage event can be monitored by the appearance of a 110-kDa band representing the released intracellular domain of Notch1. In GFP-transfected cells the NotchIC domain appeared after 15 min of EDTA treatment and was greatly reduced after 2 h (Fig. 8A). The identity of this band was confirmed using an antibody specific for the active intracellular domain of Notch1 starting at the S3 cleavage site Val1744 (Fig. 8A, lower panel). In cells overexpressing Numb, the amount of the NotchIC domain observed after 15 min of activation was reduced compared with GFP-transfected cells and was undetectable after 1 h (Fig. 8A). A similar effect on the stability of activated NotchIC domain was also observed in HEK293T cells transfected with FL-Notch (data not shown).
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To further examine the effect of Numb on the stability of NotchIC domain following activation we used siRNA interference to knockdown levels of endogenous Numb in C2C12 cells. Cells were transfected with RNA duplexes targeted for Numb or with a control scrambled RNA duplex. Forty-eight hours after transfection endogenous Notch1 was activated and the appearance of the NotchIC domain was examined by immunoprecipitation (Fig. 8B, upper panel) and whole cell lysates (Fig. 8B, lower panels). Upon transfection with Numb RNA duplexes but not scrambled RNA duplexes we observed a greater than 60% percent reduction in the levels of Numb protein (Fig. 8B, bottom panel). In Numb siRNA-transfected cells the amount of the Notch intracellular domain detected was increased compared with cells transfected with scrambled siRNA (Fig. 8B). These results further confirm a role for Numb in promoting the degradation of the Notch intracellular domain following activation.
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DISCUSSION |
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In order to study the biochemical properties of the Numb/Notch1 interaction we have used both transfected cell lines and cell lines expressing the endogenous Notch1 receptor, and therefore our proposed model may include certain caveats. For example, Itch likely represents only one of several E3 ligases that may work in concert with Numb within distinct cellular and developmental contexts. In addition, the study of exogenously expressed proteins and epitope-tagged ubiquitin molecules may mask our ability to distinguish qualitatively different forms of ubiquitin modification on Notch1. However, despite these limitations our findings provide for the first time, insight into the molecular mechanisms of Numb-mediated Notch down-regulation and provide a framework for future investigations.
Notch signaling mediates a broad spectrum of cell fates and developmental decisions in both vertebrate and invertebrate systems. Increasing evidence highlights the importance of ubiquitin-mediated protein degradation in controlling Notch signaling. In Drosophila, temperature-sensitive mutations of the proteasome enhanced Notch activity and stabilized an ectopically expressed nuclear form of Notch (52). Recently the F-box containing protein Sel10 was shown to mediate the ubiquitination of Notch in the nucleus, and target it for proteasome-dependent degradation (3941). Sel10 antagonizes Notch nuclear activity, and a mutant Sel10 defective in its E3 ligase activity failed to inhibit Notch signaling supporting a functional role for ubiquitin-dependent proteasomal degradation in regulation of the Notch pathway. In contrast to Sel10, which acts in the nucleus to terminate signaling by activated Notch1, we found that Numb promotes the ubiquitination of membrane-tethered Notch1 and the degradation of the cleavage product following receptor activation. Rapid degradation of the activated intracellular domain of Notch1 would prevent it from entering the nucleus and activating downstream targets. In the presence of Numb therefore, cell surface Notch1 is modified such that it is primed for destruction following activation.
E3 ubiquitin ligases that target membrane associated Notch1 for ubiquitination have recently been described. The RING finger containing E3 ligase Cbl regulates the ubiquitination of membrane-bound Notch1 and has been reported to result in lysosomal degradation of Notch1 (18). We did not observe an interaction between Cbl and Numb in coimmunoprecipitation experiments and therefore Cbl is unlikely to mediate Numb's effect on Notch1 (data not shown). The HECT-type E3 ligase Itch also targets a transmembrane form of Notch1 for ubiquitination (37). We have shown that Numb interacts with WW domains of Itch, and together Numb and Itch cooperate to increase the ubiquitination of Notch1. A Numb mutant, NumbPTBC that does not enhance Notch1 ubiquitination also does not interact with Itch. Our data suggest Numb may function as an adaptor for the recruitment of the E3 ligase Itch and components of the ubiquitination machinery to the Notch1 receptor thereby promoting Notch1 ubiquitination. Previous studies have shown that the Numb PTB domain mediates the interaction with the intracellular domain of Notch in vitro; however, we do not observe Numb coimmunoprecipitation with Notch1 in vivo. This may be a reflection of the affinity with which the Numb PTB domain binds to an unidentified site within the intracellular domain of Notch1 or of the transient nature of the interaction.
In our experiments, Numb promoted Notch1 ubiquitination in the absence of transfected Itch suggesting that Numb interacts with other E3 ligases present in HEK293T and C2C12 cells. Other members of the Nedd4/Rsp5 family of E3 ubiquitin ligases including Nedd4, which is highly expressed in HEK293T cells, may fulfill this role. Numb also interacts with a number of other RING-type E3 ligases including LNX, Siah1 and Mdm2 that appear to regulate levels of Numb itself rather than promoting Notch1 ubiquitination ((42, 44, 45), and data not shown). By inducing Numb degradation, these E3 ligases are predicted to potentiate Notch1 signaling. Indeed overexpression of LNX potentiates Notch1-dependent activation of a Hes1 luciferase reporter (42).
Itch belongs to the Nedd4/Rsp5p family of E3 ubiquitin ligases that have been reported to control the down-regulation of both transmembrane proteins through mono-ubiquitination, and cytoplasmic proteins by poly-ubiquitination and proteasome degradation. Mono-ubiquitination of cell surface receptors triggers receptor internalization and subsequent trafficking to the lysosome for degradation (35). While in this study we have examined the effect of Numb on Notch1 ubiquitination and degradation, accumulating evidence indicates that Numb also plays a role in subcellular trafficking events. The C terminus of Numb interacts with proteins involved in endocytic trafficking, including Eps15, the EHD/Rme-1 family of proteins, and the clathrin adaptor protein, AP2 (47, 48, 53)2 and localizes to cytoplasmic vesicles (53). Therefore, in addition to facilitating Notch1 ubiquitination, Numb may also function to assemble components of the endocytic machinery that together modulate Notch1 receptor trafficking. In agreement with this model, it has been recently proposed that Drosophila Numb serves as an adaptor to link the AP2 complex to the intracellular domain of Notch (47). Whether Numb-dependent ubiquitination of Notch1 influences trafficking of the Notch1 receptor is currently under investigation.
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FOOTNOTES |
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Supported by a studentship from the National Science and Engineering Research Council.
A research scientist of the National Cancer Institute of Canada supported by the Canadian Cancer Society. To whom correspondence should be addressed: The Hospital for Sick Children, 555 University Ave., Toronto, ON M5G 1X8, Canada. Tel.: 416-813-8657; Fax: 416-813-8456; E-mail: jmcglade{at}sickkids.on.ca.
1 The abbreviations used are: PTB, phosphotyrosine-binding; DMEM, Dulbecco's modified Eagle's medium; HEK, human embryonic kidney; PBS, phosphate-buffered saline; HA, hemagglutinin; GST, glutathione S-transferase; GFP, green fluorescent protein; EC, extracellular; EGFR, epidermal growth factor receptor; NICD, mutant coding the Notch intracellular domain.
2 C. A. Smith, S. E. Dho, and C. J. McGlade, in preparation.
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ACKNOWLEDGMENTS |
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REFERENCES |
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