Mammalian Numb Proteins Promote Notch1 Receptor Ubiquitination and Degradation of the Notch1 Intracellular Domain*

Melanie A. McGill {ddagger} and C. Jane McGlade §

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.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The cell fate determinant Numb influences developmental decisions by antagonizing the Notch signaling pathway. However, the underlying molecular mechanism of this inhibition is poorly understood. Here we report that the mammalian Numb protein promotes the ubiquitination of membrane-bound Notch1 receptor. Furthermore, Numb expression resulted in the degradation of the Notch intracellular domain following activation, which correlated with a loss of Notch-dependent transcriptional activation of the Hes1 promoter as measured by a Hes1 luciferase reporter assay. The phosphotyrosine-binding (PTB) domain of Numb was required for both Notch1 ubiquitination and down-regulation of Notch1 nuclear activity. Numb-mediated ubiquitination of Notch1 was not dependent on the PEST region, which was previously shown to mediate Sel10-dependent ubiquitination of Notch in the nucleus, suggesting a distinct E3 ubiquitin ligase is involved. In agreement we demonstrate that Numb interacts with the cytosolic HECT domain-containing E3 ligase Itch and that Numb and Itch act cooperatively to promote ubiquitination of membrane-tethered Notch1. These results suggest that Numb recruits components of the ubiquitination machinery to the Notch receptor thereby facilitating Notch1 ubiquitination at the membrane, which in turn promotes degradation of the intracellular domain circumventing its nuclear translocation and downstream activation of Notch1 target genes.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In Drosophila, the phosphotyrosine-binding (PTB)1 domain-containing protein Numb is an intrinsic regulator of binary cell fate decisions during peripheral and central nervous system development as well as muscle cell differentiation (14). Numb is asymmetrically localized in dividing progenitor cells of these lineages, segregating preferentially to one of the two daughter cells (1, 47). A cell acquiring the Numb protein adopts a fate different from its sister cell. Genetic evidence in Drosophila indicates that Numb may influence cell fate by negatively regulating the Notch signaling pathway (3, 7, 8). Vertebrate homologues of Drosophila Numb (dNumb) have been identified in mouse, rat, chicken, and human, and studies suggest an evolutionarily conserved role for the Numb protein (7, 913). Similar to Drosophila Numb, mammalian Numb is asymmetrically localized in ventricular neural progenitor cells during mouse cortical neurogenesis (7), in dividing rat retinal neuroepithelial cells (14), and in mitotic neuroepithelial cells during avian neurogenesis (15).

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-{alpha}-converting enzyme, also know as ADAM 17) releasing the Notch extracellular domain (25). A third cleavage event by a {gamma}-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{kappa}, 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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
cDNA Constructs and Mutagenesis—All Notch1 mutant constructs were cloned into the pcDNA3.1(+) vector and were constructed from the full-length Notch1 cDNA (FL-Notch). {Delta}EC-Notch was constructed by ligating amino acids 1–21 of Notch1 obtained by polymerase chain reaction (PCR) amplification to a fragment of FL-Notch containing nucleotides from the SspI restriction enzyme site to the stop codon. The NICD mutant was amplified by PCR and encodes amino acids Val1744 to the stop codon. {Delta}EC{Delta}RV was generated by digestion of {Delta}EC-Notch construct with EcoRV, and in-frame ligation to a C-terminal Myc tag. The Numbp66 cDNA was cloned into pEF. Numb{Delta}PTBC was constructed using PCR-amplified fragments of amino acids Met1 to Lys85, and Lys174 to the stop codon, and fused in-frame to the pEF parental vector. Numb{Delta}C deletes the last forty-one amino acids. All GST-Itch fusion constructs were generated by PCR amplification of Itch cDNA template and ligated into the pGEX-4T1 vector (Amersham Biosciences). The Itch GST fusions used were GST-WW1/2 (amino acids Pro276–Glu357), WW3 (amino acids Glu392–Leu43), WW4 (amino acids Gly432–Asp476), and WWHECT (amino acids Pro276–Glu862). All cDNAs generated by PCR were verified by sequencing.

Cell Culture and Transfections—HEK293T 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 24–48 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 Analysis—Transfected 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 (06–808) 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 Notch1—To 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 Assays—NIH 3T3 cells seeded on 6-well plates were cotransfected with {Delta}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)-{beta}-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). {beta}-galactosidase activity was quantitated using the chemiluminescent substrate Galacto-PLUS according to the manufacturer's instructions (Tropix). Luciferase activity was normalized for {beta}-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 Analysis—Transfected 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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Ubiquitinated Notch1 Protein Accumulates in the Presence of Numb—Ubiquitination of the Notch receptor and components of the Notch signaling pathway is thought to play a key regulatory role in Notch signal transmission. Given that Numb has recently been shown to interact with several E3 ubiquitin ligases and is implicated as a regulator of receptor endocytosis, we examined whether Numb might affect Notch1 receptor down-regulation by ubiquitination. The effect of Numb expression on the ubiquitination of a constitutively active membrane-tethered Notch1 mutant, {Delta}EC-Notch consisting of the transmembrane domain and the entire intracellular domain of Notch1, but lacking most of the extracellular (EC) domain was examined (Fig. 1A). HEK293T cells were cotransfected with HA-epitope tagged ubiquitin and {Delta}EC-Notch in the presence or absence of exogenous Numb. HA-reactive proteins were detected upon coexpression of {Delta}EC-Notch and HA-tagged ubiquitin suggesting that {Delta}EC-Notch is ubiquitinated by an endogenous E3 ubiquitin ligase (Fig. 1B). Coexpression of Numb resulted in an increase in the level of ubiquitinated proteins detected in {Delta}EC-Notch immunoprecipitations. To determine if these proteins represented Notch itself, ubiquitinated proteins were immunoprecipitated with anti-HA antibody and anti-Notch immunoblots were used to specifically detect ubiquitinated Notch1. Only a very low level of ubiquitinated Notch immunoprecipitated with anti-HA in the absence of Numb, however there was a dramatic increase upon Numb overexpression (Fig. 1C).



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FIG. 1.
Ubiquitinated Notch1 accumulates in the presence of Numb in vivo. A, schematic representation of Notch expression constructs. Constitutively active Notch mutants were constructed by deleting most of the extracellular domain. {Delta}EC-Notch is membrane-tethered and is continuously cleaved to release the intracellular domain of Notch1. In addition a mutant that deletes the C-terminal 483 amino acids including the PEST region was constructed. EGF-like, epidermal growth factor-like repeats; LNR, LIN12/Notch repeat region; TM, transmembrane domain; ANK, ankyrin repeat; PEST, proline-, glutamate-, serine-, threonine-rich sequence. B, HEK293T cells were transiently cotransfected with {Delta}EC-Notch, HA-tagged ubiquitin, and either empty vector or Numb. Equivalent amounts of cell lysates were immunoprecipitated (IP) with anti-Notch and ubiquitinated protein detected by anti-HA immunoblot (IB) (upper panel). Blots were stripped and reprobed with anti-Notch to monitor Notch expression (lower panel). C, the same lysates were immunoprecipitated with anti-HA and ubiquitinated Notch detected by anti-Notch immunoblot. D, immunoprecipitates of Notch from lysates boiled in 1% SDS lysis buffer were analyzed with anti-HA (upper panel) or anti-Notch (lower panel) antibodies. E, Numb has no effect on the ubiquitination of the EGFR receptor. Lysates from cells transiently transfected with EGFR, HA-Ub, and Numb were immunoprecipitated with anti-EGFR, and immunoblotted with anti-HA (upper panel) and reprobed with anti-EGFR (lower panel).

 

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 Ubiquitination—Previous 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. Numb{Delta}PTBC 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{Delta}C lacks the C-terminal forty-one amino acids including the EH domain binding site. HEK293T cells were cotransfected with {Delta}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 {Delta}EC-Notch species were observed (Fig. 2B, lanes 1–3). Coexpression of Numb{Delta}C enhanced Notch1 ubiquitination as efficiently as wild-type Numb (Fig. 2B, lanes 4 and 5); however the effect of Numb{Delta}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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FIG. 2.
The PTB domain of Numb is required for function. A, schematic representation of Numb mutants. Numb{Delta}PTBC lacks eighty-eight amino acids in the C terminus of the PTB domain. Numb{Delta}C deletes the extreme C-terminal forty-one amino acids. B, Numb{Delta}PTBC does not promote Notch1 ubiquitination. HEK293T cells were cotransfected with {Delta}EC-Notch, HA-Ub, and increasing amounts of NumbWT or Numb mutants. Equivalent amounts of protein lysates were immunoprecipitated with anti-Notch, and immunoblotted with anti-HA (upper panel) to detect ubiquitinated protein, and with anti-Notch (lower panel) to monitor Notch expression.

 

The PEST Region of Notch1 Is Not Required for Numb-mediated Ubiquitination—Recently, 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, {Delta}EC{Delta}RV, was constructed (see Fig. 1A). An increase in the amount of ubiquitination of {Delta}EC{Delta}RV was observed upon coexpression of {Delta}EC{Delta}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{Delta}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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FIG. 3.
The PEST region of Notch1 is not required to mediate Notch1 ubiquitination by Numb. HEK293T cells were cotransfected with Myc-tagged {Delta}EC{Delta}RV and HA-Ub in the presence or absence of Numb. Cells were lysed, immunoprecipitated with anti-Myc antibody, and analyzed by Western blot with anti-HA.

 

Numb Interacts with the E3 Ubiquitin Ligase Itch in Vitro and in Vivo—Numb 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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FIG. 4.
Interaction between Numb and the E3 ligase Itch in vitro and in vivo. A, schematic representation of full-length Itch or GST fusion constructs of the Itch WW domains and binding specificity to Numb. (++) indicates greater binding. B, Numb binds to WW1/2 of Itch. HEK293T cells were transfected with Numb, lysed in 1% Nonidet P-40 lysis buffer, and incubated with equivalent amounts of immobilized GST protein or GST fusion proteins of the individual Itch WW domains. Precipitates were analyzed by Western blot with anti-Numb antisera. Membranes were stained with Coomassie Blue to ensure equal amounts of fusion protein were used in each reaction (data not shown). C, cell lysates from C2C12 cells were incubated with GST and GST-WW domain fusion proteins as above and Western blotted with anti-Numb antisera. D, Numb coimmunoprecipitates with full-length Itch in vivo. HEK293T cells cotransfected with Numb and Myc-tagged Itch were lysed, immunoprecipitated with anti-Numb or preimmune antisera as a control, and immunoprecipitates Western blotted with anti-Myc antibody (upper panel). The blots were reprobed with anti-Numb to confirm Numb expression (lower panel). E, lysates were prepared as above, immunoprecipitated with anti-Myc and blotted with anti-Numb antisera (upper panel) or anti-AIP4/Itch (lower panel).

 

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 Notch1—To examine the biological significance of the interaction between Numb and Itch, HEK293T cells were cotransfected with {Delta}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 {Delta}EC-Notch however, coexpression of Numb and Itch together further enhanced Notch ubiquitination, suggesting that they acted cooperatively to ubiquitinate membrane-tethered {Delta}EC-Notch (Fig. 5A). In contrast, the Numb{Delta}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{Delta}PTBC no longer bound GST-Itch (Fig. 5B).



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FIG. 5.
Itch and Numb cooperatively promote the ubiquitination of Notch. A, the PTB domain of Numb is required to ubiquitinate Notch in cooperation with Itch. HEK293T cells were cotransfected with {Delta}EC-Notch and HA-Ub in the presence and absence of NumbWT or Numb{Delta}PTBC and/or ItchWT. Equivalent amounts of lysates were immunoprecipitated with anti-Notch, and ubiquitinated proteins detected with anti-HA antibody (upper panel). {Delta}EC-Notch expression was monitored by reprobing membranes with anti-Notch (lower panel). B, Numb{Delta}PTBC no longer binds to Itch in vitro. Equivalent amounts of cell lysates expressing NumbWT or Numb{Delta}PTBC were incubated with immobilized GST or GST-WW domains of Itch fusion proteins. Precipitates were immunoblotted with anti-Numb antibody (left panel). Input lysates were examined for equal expression of Numb constructs with anti-Numb antibody (right panel). C, {Delta}EC-Notch nuclear activity is down-regulated upon coexpression of NumbWT but not Numb{Delta}PTBC. Hes1 luciferase reporter assays were utilized to measure Notch nuclear activity. NIH 3T3 cells plated in 6-well dishes were transiently cotransfected with {Delta}EC-Notch, a Hes1 luciferase reporter construct, and a CMV-{beta}-galactosidase reporter construct to control for transfection efficiency. Luciferase activity was measured 24 h post-transfection and normalized for {beta}-galactosidase activity. The mean luciferase activation relative to empty vector transfected cells was calculated and presented as fold activation. Data represent the mean ± S.D. of a representative experiment of five independent experiments each performed in triplicate.

 

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 {Delta}EC-Notch expression construct in the presence or absence of either wild type Numb or Numb{Delta}PTBC. A {beta}-galactosidase reporter was included in each transfection to control for transfection efficiency. Transfection with {Delta}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{Delta}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 Activation—To further elucidate the functional relevance of Numb-mediated ubiquitination, we analyzed the fate of ubiquitinated {Delta}EC-Notch. HEK293T cells coexpressing {Delta}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 {Delta}EC-Notch is targeted for degradation by the proteasome pathway (Fig. 6A). {Delta}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 {Delta}EC-Notch protein or the 110-kDa cleaved intracellular domain.



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FIG. 6.
Numb promotes the ubiquitination but not the degradation of membrane-bound Notch1. A, proteasome inhibition has no effect on membrane-bound Notch1 ubiquitination. HEK293T cells overexpressing {Delta}EC-Notch, HA-Ub, and empty vector or Numb were treated with 50 µM MG132 for 4 h prior to lysis. Lysates were immunoprecipitated with anti-Notch and analyzed by Western blot. Ubiquitinated Notch1 was visualized by anti-HA immunoblots (upper panel), and Notch1 expression with anti-Notch immunoblots (lower panel). B, Numb mediates the ubiquitination of full-length Notch1. Cells expressing FL-Notch alone or coexpressing Numb were immunoprecipitated with anti-Notch and immunoblotted with anti-HA (upper panel) or reprobed with anti-Notch (lower panel). C, Numb promotes the ubiquitination of endogenous Notch1 from C2C12 cells. C2C12 cells were cotransfected with HA-Ub and GFP or cotransfected with Numb. Lysates were immunoprecipitated with anti-Notch and immunoblotted with anti-HA. D, Itch and Numb can target FL-Notch1 for ubiquitination. HEK293T cells cotransfected with FL-Notch, HA-Ub, and/or Numb, and either ItchWT or Itch C830A were lysed, immunoprecipitated with anti-Notch, and immunoblotted for ubiquitinated Notch using anti-HA antibody (upper panel) and reprobed with anti-Notch (lower panel) to monitor protein expression. E, C2C12 cells expressing GFP or Numb were pulse-labeled for 1.5 h with [35S]methionine and cysteine then chased for up 6 h in culture medium supplemented with non-labeled methionine and cysteine. Cells were harvested at specific time points and immunoprecipitated with anti-Notch antibody. Immunoprecipitates were separated by SDS-PAGE and autoradiographed to visualize labeled Notch1. Arrowheads indicate the unprocessed 300-kDa full-length Notch1 receptor and 120-kDa fragment of the Notch1 surface heterodimer.

 

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 Domain—Further investigation of the stability of {Delta}EC-Notch suggested that similar to FL-Notch, Numb does not effect the stability of the 120-kDa form of {Delta}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 {Delta}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 5–8) 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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FIG. 7.
Numb promotes the ubiquitination and degradation of the intracellular domain of Notch1. A, HEK293T cells overexpressing NICD, HA-Ub and empty vector or Numb were treated with Me2SO or 50 µM MG132 for 4 h and then lysed in PLC lysis buffer containing MG132. Lysates were then immunoprecipitated with anti-Notch antibody and analyzed by Western blot. B, Itch ubiquitinates NICD. HEK293T cells overexpressing NICD, HA-Ub in the presence or absence of Numb and/or Itch were treated with MG132 4 h prior to lysis, immunoprecipitated with anti-Notch, and immunoblotted for ubiquitinated Notch using anti-HA antibody (upper panel) and reprobed with anti-Notch (lower panel) to monitor protein expression. C, overexpression of Numb targets NICD for degradation. HEK293T cells transfected with NICD and empty vector or Numb were pulse-labeled for 30 min with [35S]methionine and cysteine and then chased for up3hin culture medium supplemented with non-labeled methionine and cysteine. At 1-h intervals cells were lysed and immunoprecipitated with anti-Notch antibody. Immunoprecipitates were separated by SDS-PAGE and autoradiographed to visualize labeled NICD protein.

 

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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FIG. 8.
Numb targets the intracellular domain of Notch1 for degradation following activation. A, lower levels of activated Notch1 are observed upon overexpression of Numb. C2C12 cells transfected with GFP or Numb were activated by treatment with 5 mM EDTA and chased in culture medium for up to 2 h. Cells were lysed at indicated time points, immunoprecipitated with anti-Notch antibody, and analyzed by Western blot with anti-Notch to visualize total Notch1 protein (upper panel) and with anti-ICD to visualize activated Notch1 (lower panel). The cleaved Notch1 intracellular domain (ICD) is labeled with an arrowhead. B, an increase in the amount of activated Notch1 is observed when levels of endogenous Numb are knocked down. C2C12 cells were transfected with an RNA duplex specific for Numb or with a scrambled RNA duplex. Forty-eight hours post-transfection, cells were treated with 5 mM EDTA then lysed, and immunoprecipitated with anti-Notch. Immunoprecipitation reactions were separated and immunoblotted with anti-Notch (top panel). Cell lysates were analyzed with anti-Notch (second panel) and anti-ICD (third panel) to monitor Notch1 expression and activation and anti-Numb immunoblots to monitor Numb protein levels (bottom panel).

 

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.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Studies in both Drosophila and vertebrate systems strongly suggest Numb functions to antagonize Notch receptor signaling, although the biochemical mechanism of inhibition is unknown (3, 4, 8, 46). Studies examining Drosophila Numb function in S2 cells suggest that Numb prevents nuclear translocation of the Notch cotransactivator Su(H), and thereby down-regulates the Notch signal (46). Consistent with our data, an intact PTB domain of Numb was required for Numb function in these assays. Similarly overexpression of avian Numb results in a loss of nuclear staining of a constitutively active avian Notch mutant (15, 51). These studies suggested that Numb interaction with the intracellular domain of Notch prevents its nuclear localization and transmission of the Notch signal. In the present study we provide a molecular explanation for these observations, and suggest that by promoting the ubiquitination and rapid degradation of the NotchIC domain, Numb prevents the translocation of activated Notch to the nucleus.

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, Numb{Delta}PTBC 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.


    FOOTNOTES
 
* This work was supported by the National Cancer Institute of Canada with funds from the Canadian Cancer Society. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} Supported by a studentship from the National Science and Engineering Research Council. Back

§ 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. Back

2 C. A. Smith, S. E. Dho, and C. J. McGlade, in preparation. Back


    ACKNOWLEDGMENTS
 
We thank J. Nye for the Notch1 cDNA, R. Kageyama for the Hes1 luciferase reporter construct, D. Bohmann for the HA-ubiquitin expression plasmid, G. Gish and T. Pawson for the Itch cDNA, and A. Guha for the EGFR expression construct. Also we wish to thank members of the McGlade laboratory for helpful discussions and comments on the manuscript.



    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Rhyu, M. S., Jan, L. Y., and Jan, Y. N. (1994) Cell 76, 477–491[Medline] [Order article via Infotrieve]
  2. Ruiz, G. M., and Bate, M. (1997) Development 124, 4857–4866[Abstract/Free Full Text]
  3. Spana, E. P., and Doe, C. Q. (1996) Neuron 17, 21–26[Medline] [Order article via Infotrieve]
  4. Uemura, T., Shepherd, S., Ackerman, L., Jan, L. Y., and Jan, Y. N. (1989) Cell 58, 349–360[Medline] [Order article via Infotrieve]
  5. Knoblich, J. A., Jan, L. Y., and Jan, Y. N. (1995) Nature 377, 624–627[CrossRef][Medline] [Order article via Infotrieve]
  6. Spana, E. P., Kopczynski, C., Goodman, C. S., and Doe, C. Q. (1995) Development 121, 3489–3494[Abstract/Free Full Text]
  7. Zhong, W., Feder, J. N., Jiang, M. M., Jan, L. Y., and Jan, Y. N. (1996) Neuron 17, 43–53[Medline] [Order article via Infotrieve]
  8. Guo, M., Jan, L. Y., and Jan, Y. N. (1996) Neuron 17, 27–41[Medline] [Order article via Infotrieve]
  9. Verdi, J. M., Schmandt, R., Bashirullah, A., Jacob, S., Salvino, R., Craig, C. G., Program, A. E., Lipshitz, H. D., and McGlade, C. J. (1996) Curr. Biol. 6, 1134–1145[Medline] [Order article via Infotrieve]
  10. Zhong, W., Jiang, M. M., Schonemann, M. D., Meneses, J. J., Pedersen, R. A., Jan, L. Y., and Jan, Y. N. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 6844–6849[Abstract/Free Full Text]
  11. Zilian, O., Saner, C., Hagedorn, L., Lee, H. Y., Sauberli, E., Suter, U., Sommer, L., and Aguet, M. (2001) Curr. Biol. 11, 494–501[CrossRef][Medline] [Order article via Infotrieve]
  12. Dho, S. E., French, M. B., Woods, S. A., and McGlade, C. J. (1999) J. Biol. Chem. 274, 33097–33104[Abstract/Free Full Text]
  13. Zhong, W. M., Jiang, M. M., Weinmaster, G., Jan, L. Y., and Jan, Y. N. (1997) Development 124, 1887–1897[Abstract/Free Full Text]
  14. Cayouette, M., Whitmore, A. V., Jeffery, G., and Raff, M. (2001) J. Neurosci. 21, 5643–5651[Abstract/Free Full Text]
  15. Wakamatsu, Y., Maynard, T. M., Jones, S. U., and Weston, J. A. (1999) Neuron 23, 71–81[CrossRef][Medline] [Order article via Infotrieve]
  16. Artavanis-Tsakonas, S., Rand, M. D., and Lake, R. J. (1999) Science 284, 770–776[Abstract/Free Full Text]
  17. Miele, L., and Osborne, B. (1999) J. Cell Phys. 181, 393–409[CrossRef][Medline] [Order article via Infotrieve]
  18. Dievart, A., Beaulieu, N., and Jolicooeur, P. (1999) Oncogene 18, 5973–5981[CrossRef][Medline] [Order article via Infotrieve]
  19. Ellisen, L. W., Bird, J., West, D. C., Soreng, A. L., Reynolds, T. C., Smith, S. D., and Sklar, J. (1991) Cell 66, 649–661[Medline] [Order article via Infotrieve]
  20. Joutel, A., and Tournier-Lasserve, F. (1998) Sem. Cell Dev. Biol. 9, 619–625[CrossRef][Medline] [Order article via Infotrieve]
  21. Kopan R., and Turner, D. L. (1996) Curr. Opin. Neurobiol. 6, 594–601[CrossRef][Medline] [Order article via Infotrieve]
  22. Schroeter, E. H., Kisslinger, J. A., and Kopan, R. (1998) Nature 393, 382–386[CrossRef][Medline] [Order article via Infotrieve]
  23. Blaumueller, C. M., Qi, H., Zagouras, P., and Artivanis-Tsakonas, S. (1997) Cell 90, 281–291[Medline] [Order article via Infotrieve]
  24. Logeat, F., Bessia, C., Brou, C., LeBail, O., Jarriault, S., Seidaah, N. G., and Israel, A. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 8108–8112[Abstract/Free Full Text]
  25. Brou, C., Logeat, F., Gupta, N., Bessia, C., LeBail, O., Doedens, J. R., Cumano, A., Roux, P., Black, R. A., and Israel, A. (2000) Mol. Cell 5, 207–216[Medline] [Order article via Infotrieve]
  26. Berezovska, O., Jack, C., McLean, P., Aster, J. C., Hicks, C., Xia, W., Wolfe, M. S., Kimberly, W. T., Wienmaster, G., Selkoe, D. J., and Hyman, B. T. (2000) J. Neurochem. 75, 583–593[CrossRef][Medline] [Order article via Infotrieve]
  27. Kidd, S., Lieber, T., and Young, M. W. (1998) Genes Dev. 12, 3728–3740[Abstract/Free Full Text]
  28. Mumm, J. S., Schroeter, E. H., Saxena, M. T., Griesemer, A., Tian, X., Pan, D. J., Ray, W. J., and Kopan, R. (2000) Mol. Cell 5, 197–206[Medline] [Order article via Infotrieve]
  29. Struhl, G., and Adachi, A. (1998) Cell 93, 649–660[Medline] [Order article via Infotrieve]
  30. Lai, E. C. (2002) Curr. Biol. 12, R74–78[CrossRef][Medline] [Order article via Infotrieve]
  31. Hershko, A., and Ciechanover, A. (1998) Annu. Rev. Biochem. 67, 425–479[CrossRef][Medline] [Order article via Infotrieve]
  32. Joazeiro, C. A., and Weissman, A. M. (2000) Cell 102, 549–552[Medline] [Order article via Infotrieve]
  33. Tyers, M., and Willems, A. R. (1999) Science 284, 601, 603–604[Free Full Text]
  34. Hicke, L. (1999) Trends Cell Biol. 9, 107–112[CrossRef][Medline] [Order article via Infotrieve]
  35. Rotin, D., Staub, O., and Haguenauer-Tsapis, R. (2000) J. Membr. Biol. 176, 1–17[CrossRef][Medline] [Order article via Infotrieve]
  36. Cornell, M., Evans, D. A., Mann, R., Fostier, M., Flasza, M., Monthatong, M., Artavanis-Tsakonas, S., and Baron, M. (1999) Genetics 152, 567–576[Abstract/Free Full Text]
  37. 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, 35734–35737[Abstract/Free Full Text]
  38. Hubbard, E. J., Wu, G., Kitajewski, J., and Greenwald, I. (1997) Genes Dev. 11, 3182–3193[Abstract/Free Full Text]
  39. Gupta-Rossi, N., Le Bail, O., Gonen, H., Brou, C., Logeat, F., Six, E., Ciechanover, A., and Israel, A. (2001) J. Biol. Chem. 276, 34371–34378[Abstract/Free Full Text]
  40. Oberg, C., Li, J., Pauley, A., Wolf, E., Gurney, M., and Lendahl, U. (2001) J. Biol. Chem. 276, 35847–35853[Abstract/Free Full Text]
  41. Wu, G., Lyapina, S., Das, I., Li, J., Gurney, M., Pauley, A., Chui, I., Deshaies, R. J., and Kitajewski, J. (2001) Mol. Cell. Biol. 21, 7403–7415[Abstract/Free Full Text]
  42. Nie, J., McGill, M. A., Dermer, M., Dho, S. E., Wolting, C. D., and McGlade, C. J. (2002) EMBO J. 21, 93–102[Abstract/Free Full Text]
  43. Dho, S. E., Jacob, S., Wolting, C. D., French, M. B., Rohrschneider, L. R., and McGlade, C. J. (1998) J. Biol. Chem. 273, 9179–9187[Abstract/Free Full Text]
  44. Juven-Gershon, T., Shifman, O., Unger, T., Elkeles, A., Haupt, Y., and Oren, M. (1998) Mol. Cell. Biol. 18, 3974–3982[Abstract/Free Full Text]
  45. Susini, L., Passer, B. J., Amzallag-Elbaz, N., Juven-Gershon, T., Prieur, S., Privat, N., Tuynder, M., Gendron, M. C., Israel, A., Amson, R., Oren, M., and Telerman, A. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 15067–15072[Abstract/Free Full Text]
  46. Frise, E., Knoblich, J. A., Younger-Shepherd, S., Jan, L. Y., and Jan, Y. N. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 11925–11932[Abstract/Free Full Text]
  47. Berdnik, D., Torok, T., Gonzalez-Gaitan, M., and Knoblich, J. (2002) Dev. Cell 3, 221[Medline] [Order article via Infotrieve]
  48. Salcini, A. E., Confalonieri, S., Doria, M., Santolini, E., Tassi, E., Minenkova, O., Cesareni, G., Pelicci, P. G., and Difiore, P. P. (1997) Genes Dev. 11, 2239–2249[Abstract/Free Full Text]
  49. Santolini, E., Salcini, A. E., Kay, B. K., Yamabhai, M., and Di Fiore, P. P. (1999) Exp. Cell Res. 253, 186–209[CrossRef][Medline] [Order article via Infotrieve]
  50. Rand, M. D., Grimm, L. M., Artavanis-Tsakonas, S., Patriub, V., Blacklow, S. C., Sklar, J., and Aster, J. C. (2000) Mol. Cell. Biol. 20, 1825–1835[Abstract/Free Full Text]
  51. Sestan, N., Artavanis-Tsakonas, S., and Rakic, P. (1999) Science 286, 741–746[Abstract/Free Full Text]
  52. Schweisguth, F. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 11382–11386[Abstract/Free Full Text]
  53. Santolini, E., Puri, C., Salcini, A. E., Gagliani, M. C., Pelicci, P. G., Tacchetti, C., and Di Fiore, P. P. (2000) J. Cell Biol. 151, 1345–1352[Abstract/Free Full Text]