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Mol Cell Biol. Dec 2012; 32(24): 4933–4945.
PMCID: PMC3510530
Functional Analysis of the NHR2 Domain Indicates that Oligomerization of Neuralized Regulates Ubiquitination and Endocytosis of Delta during Notch Signaling
Sili Liu,ab Julia Maeve Bonner,ab Soline Chanet,cd Cosimo Commisso,ab* Lara C. Skwarek,ab* François Schweisguth,cd and Gabrielle L. Bouliannecorresponding authorab
aThe Hospital for Sick Children, Program in Developmental and Stem Cell Biology, Toronto, Ontario, Canada
bDepartment of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada
cInstitut Pasteur, Developmental Biology Department, Paris, France
dCNRS, URA2578, Paris, France
corresponding authorCorresponding author.
Address correspondence to Gabrielle L. Boulianne, gboul/at/
*Present address: Cosimo Commisso, Department of Biochemistry, New York University School of Medicine, New York, New York, USA; Lara C. Skwarek, Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California, USA.
Received May 31, 2012; Revisions requested June 20, 2012; Accepted September 28, 2012.
The Notch pathway plays an integral role in development by regulating cell fate in a wide variety of multicellular organisms. A critical step in the activation of Notch signaling is the endocytosis of the Notch ligands Delta and Serrate. Ligand endocytosis is regulated by one of two E3 ubiquitin ligases, Neuralized (Neur) or Mind bomb. Neur is comprised of a C-terminal RING domain, which is required for Delta ubiquitination, and two Neur homology repeat (NHR) domains. We have previously shown that the NHR1 domain is required for Delta trafficking. Here we show that the NHR1 domain also affects the binding and internalization of Serrate. Furthermore, we show that the NHR2 domain is required for Neur function and that a point mutation in the NHR2 domain (Gly430) abolishes Neur ubiquitination activity and affects ligand internalization. Finally, we provide evidence that Neur can form oligomers in both cultured cells and fly tissues, which regulate Neur activity and, by extension, ligand internalization.
The Notch pathway is highly conserved across invertebrates and vertebrates and plays multiple and essential roles in many developmental processes, such as inhibiting differentiation by lateral signaling and regulating cell fate through inductive interactions (7, 23). Notch signaling is induced through direct cell-cell interactions between membrane-bound Notch ligands, Delta, Serrate, and Lag-2 (DSL), and the Notch receptor on adjacent cells. The activation of the Notch receptor results in the cleavage of Notch by the γ-secretase complex, leading to the translocation of the Notch intracellular domain into the nucleus, where it can activate downstream target genes (7). Recessive loss-of-function mutations in key components of the Notch pathway in Drosophila melanogaster are embryonic lethal and result in neurogenic phenotypes consisting of an overgrowth of the nervous system at the expense of the epidermis (36).
The ubiquitination and endocytosis of receptors and ligands have been shown to potentiate Notch signaling (32, 33, 54). Currently, there are two models hypothesizing how ligand endocytosis facilitates Notch activation in the signal-receiving cell. The mechanical force or pulling model suggests that Delta endocytosis exerts a force on the Delta-Notch complex that alters the conformation and promotes the cleavage of the Notch extracellular domain (NECD), which is a critical step in Notch activation (17, 41, 42, 51). The recycling model suggests that the modification of an inactive form of Delta in an endosomal compartment makes Delta a more effective ligand, which will be re-presented to the cell surface (perhaps at a microdomain of the plasma membrane) to activate Notch (2, 4, 14, 20, 47, 52). In the signal-sending cell, Neuralized (Neur) (12, 27, 43, 56) and Mindbomb1 (3, 22, 25, 28, 34, 44, 53) are two E3 ubiquitin ligases that regulate the endocytosis of the Notch ligands Delta and Serrate by ubiquitination.
Neur was one of the first five Notch pathway members identified (36). Previous analyses revealed that Neur plays an important role in all three germ layers during embryonic development (10, 18, 46). In addition, Neur is also required for the development of the adult central and peripheral nervous system, including bristle sense organ patterning and photoreceptor specification (29, 30, 57). Consistent with its role in embryogenesis and adult neurogenesis, Neur is expressed in embryonic neural tissue and in the region of larval imaginal discs that will give rise to adult sensory organs (5). Of note, Neuralized is not required for all Notch signaling events, and evidence suggests that Mind bomb, its functional homologue, performs the same role in different cellular and developmental contexts (28, 34). The presence of either Neur or Mind bomb in the signal-sending cell appears to be required for ligand endocytosis (34). In addition to its role in Notch signaling, Neur was also recently shown to regulate epithelial cell polarity in the embryo (8).
The neur locus produces two major transcripts, neur-RA and neur-RC, which give rise to two proteins, NeurPA and NeurPC, which differ only at their N termini (9). Specifically, NeurPA, which is the predominant isoform during development (5), contains a phosphoinositide (PIP)-binding motif at the N terminus, which is required for Delta endocytosis downstream of Delta ubiquitination by Neur (49). In addition, both isoforms contain three highly conserved domains, including a carboxyl-terminal RING domain and two Neuralized homology repeat (NHR) domains (NHR1 and NHR2). The RING domain is both necessary and sufficient for Neur E3 ubiquitin ligase activity and is required for the endocytosis of the Notch ligand Delta (27, 43, 56). The NHR1 domain is a protein-protein interaction module that is required for Neur to bind Delta: a point mutation in a highly conserved glycine residue at position 167 disrupts Delta binding (9). Whether the NHR1 domain also mediates the interaction between Neur and Serrate (44) is unknown.
Although several aspects of the role of Neur in Notch signaling have been characterized, the full spectrum of Neur function and regulation still remains to be elucidated, including the function of the NHR2 domain. Previous in vitro studies done with mammalian cell cultures demonstrated that the NHR2 domain of Neurl1, a mouse homologue of Drosophila Neur, mediates the interaction between Neurl1 and Jagged1 (26), a mouse homologue of Drosophila Serrate. Whether the NHR2 domain is required for Neur function in vivo and whether this function depends solely on its interaction with Serrate remain to be determined.
Here we show that Neur binds both Notch ligands via the NHR1 domain. We also show that the NHR2 domain is required for Neur-mediated Dl endocytosis and Notch signaling in vivo. Specifically, we show that the deletion of the NHR2 domain and a point mutation in a highly conserved glycine residue within the NHR2 domain reduce Dl internalization. Finally, we demonstrate that Neur can form oligomers, which regulate its ubiquitination activity and, as a result, ligand internalization.
Plasmid construction and mutagenesis.
pAC-NeurWT was previously described (49). By using pAC-NeurWT as a template, NeurΔNHR1, NeurΔNHR2, and NeurNHR2 were amplified by PCR, cloned into the pENTR vector (Life Technologies, Grand Island, NY), and subsequently flipped into the pAW vector (Murphy Laboratory Drosophila Gateway Collection via the Drosophila Genome Resource Center [DGRC], Bloomington, IN) or the pMGF vector (gift from A. McQuibban, Toronto, Ontario, Canada) by Gateway cloning (Life Technologies, Grand Island, NY). The NeurG430E and NeurΔNHR1&2 mutants were generated by using a QuikChange Lightning site-directed mutagenesis kit (Agilent Technologies, Santa Clara, CA). pMT-DlWT-Ndemyc (24) was obtained from the DGRC (Bloomington, IN). By using pUAST-Ser-myc (gift from C. Delidakis, Heraklion, Crete, Greece) as a template, cDNA encoding Ser was amplified by PCR, cloned into the pENTR vector (Life Technologies, Grand Island, NY), and subsequently flipped into the pMGF vector (gift from A. McQuibban, Toronto, Ontario, Canada) by Gateway cloning (Life Technologies, Grand Island, NY). pRM-HA-Ubiquitin was a gift from P. Rørth (48). Oligonucleotide sequences of PCR primers are available upon request.
Rabbit anti-V5 antibody (Genscript, Piscataway, NJ) was used to detect V5-tagged Neur proteins at a 1:3,000 dilution for Western blots and at 1:500 to 1:1,000 dilutions for immunostaining. Mouse anti-FLAG antibody (Sigma-Aldrich, St. Louis, MO) was used to detect FLAG-tagged Neur and Ser proteins by Western blotting (1:3,000 dilution) and immunostaining (1:1,000 dilution). Rabbit anti-myc antibody (Genscript, Piscataway, NJ) was used to detect the myc-tagged Dl protein by Western blotting (1:3,000 dilution). Mouse anti-Dl antibody (C594-9B; DSHB, Iowa City, IA) was used to detect endogenous Dl by immunostaining (1:5,000 dilution). Mouse antitubulin and mouse antiactin antibodies (E7 and JLA20, respectively; DSHB, Iowa City, IA) were used as loading controls on Western blots (1:1,000 dilution). Guinea pig anti-Sens antibody was used to mark sensory organ precursor cells (SOPs) (1:300 dilution; H. Bellen, Houston, TX). Rabbit anti-Neur antibody was used to detect endogenous Neur (1:1,000 dilution; C. Delidakis, Heraklion, Crete, Greece).
Yeast two-hybrid analysis.
A yeast two-hybrid (Y2H) analysis was performed by using the Clontech Laboratories (Mountain View, CA) pACT2 GAL4 activating domain (AD) and pGBKT7 GAL4 DNA-binding domain (DB), containing either the NHR1 domain, the NHR2 domain, or Neur Start-NHR2. The positive-control vectors used were the known interactors Jun and cFOS as well as full-length GAL4 (original constructs used for cloning were kindly provided by Charles Boone, Toronto, Ontario, Canada). Negative controls were empty vectors.
Cell culture.
Drosophila Kc167 (Kc) cells were cultured at 23°C in Schneider's medium (catalog number S9895; Sigma-Aldrich, St. Louis, MO) supplemented with 10% fetal bovine serum (PAA, Pasching, Austria). Transfections were carried out by using CellfectinII, according to the manufacturer's specifications (Life Technologies, Grand Island, NY). For pMT-DlWT-NdeMYC, pMTF-Ser, and pRM-HA-Ubiquitin, 500 μM CuSO4 was used to induce protein expression for 24 h prior to the assay.
Drosophila stocks and transgenics.
Wild-type controls were w1118 flies. Other stocks used in this study include the following: neurA101GAL4 (catalog number 6393) and ombGAL4 (catalog number 3605), obtained through the Bloomington Stock Center; neurIF65; neurP72Gal4; hs-flp, tub-Gal4, UAS-GFP; FRT82B tub-Gal80/TM6b (Flybase); UAS-neurPA-V5 (abbreviated UAS-NeurWT); UAS-neurPA5Q-V5 (49); and UAS-neurPC-V5. neurAA8-N67, obtained by the excision of the Neur-RC-specific P element from neurGE29607 (GenExel, Inc., Kaist, Daejeon, Republic of Korea), with a deletion of 1,600 bp that removes most of the first exon encoding NeurPC. Breakpoints were determined by PCR and DNA sequencing (ACTG; SickKids). UAS-NeurPAΔNHR2-V5 (abbreviated UAS-NeurΔNHR2) and UAS-NeurPAG430E-V5 (abbreviated UAS-NeurG430E) were injected into w1118 embryos, and transgenic lines were generated (BestGene, Chino Hills, CA).
The rescue studies were performed as double-blind experiments. Embryos from w1118 UAS-Neurtransgene/+ neurA101GAL4/TM3Ser self-crosses were collected. Numbers of wild-type (WT) and neurogenic embryos were scored, and the neurogenic percentage was calculated. Student's t test was performed for statistical comparisons. We disregarded those embryos that carried two copies of the balancer chromosome, which are easily identifiable because they are highly disorganized and positive for green fluorescent protein (GFP). Given this protocol, Mendelian inheritance predicts that one-third of embryos will be homozygous for neurA101GAL4 and therefore neurogenic in the absence of any transgenes.
neur mutant clones overexpressing versions of V5-tagged Neur were generated by using hs-flp, tub-Gal4, UAS-GFP; UAS-NeurV5/+; FRT82B neurIF65/FRT82B tub-Gal80 flies. Mitotic clones were induced by a 45-min heat shock at 36.5°C during the first-instar larval stage. All crosses were performed at 25°C.
For immunostaining, Kc cells were grown on 25-mm circular coverslips. Cells were fixed at 48 h after transfection with fresh 4% paraformaldehyde. After fixation, cells were permeabilized, incubated with primary and secondary antibodies, and then mounted in Dako mounting medium (Dako, Burlington, Ontario, Canada). Images were acquired on a Quorum Spinning Disk confocal microscope (Quorum, Guelph, Ontario, Canada). For the quantification of localization, cells were scored for cytoplasmic or membrane localization; a total of ~100 cells were examined per genotype from 3 individual transfections. To examine localization in embryos, fly crosses were set up in cages on grape plates, and 0- to 18-h embryo collections were obtained, followed by standard methanol fixation. Immunostaining protocols were the same as those described above for cell culture. For imaginal disc staining, wandering third-instar larvae were dissected in cold phosphate-buffered saline (PBS). Wing imaginal discs were obtained and treated as described above for cell culture staining.
Endocytosis assay.
The endocytosis protocol was modified from a method described previously by Le Borgne et al. (35). Wing discs were dissected in Schneider's Drosophila medium (catalog number S9895; Sigma-Aldrich, St. Louis, MO) containing 1% fetal bovine serum (PAA, Pasching, Austria). After dissection, the medium was replaced and supplemented with 1 μg/ml 20-hydroxyecdysone (Sigma-Aldrich, St. Louis, MO). Wing discs were cultured for 10 min in the presence of the mouse monoclonal anti-Dl antibody (1:100 dilution) (C594-9B; DSHB, Iowa City, IA). Following washes, wing discs were fixed and subjected to secondary antibody treatment.
Western blot analysis and immunoprecipitation.
Embryo lysates were prepared from 0- to 18-h collections. Embryos were dechorionated and then lysed in 2× SDS loading buffer. For immunoprecipitation (IP) experiments, transfected cells (~1 × 107 cells) were lysed in 300 μl radioimmunoprecipitation assay (RIPA) buffer with protease inhibitors (Roche, Basel, Switzerland). The input was precleared with 25 μl 50% RIPA buffer plus 50% protein G-Sepharose (Sigma-Aldrich, St. Louis, MO) for 1 h at 4°C and incubated with 30 μl 50% RIPA buffer plus 50% protein G and 3 μl anti-V5 antibody (Life Technologies, Grand Island, NY) overnight. Protein G beads were washed and resuspended in 2× SDS loading buffer. For the ubiquitination IP, cells were treated with 50 μM calpain inhibitor IV (MG-132; American Peptide Company, Sunnyvale, CA) for 3 h prior to lysis.
The NHR2 domain is required for Neur function in vivo.
Neur is comprised of three functional domains: a C-terminal RING finger that is required for E3-ligase activity and two highly conserved Neur homology repeats, NHR1 and NHR2 (Fig. 1A). We have previously shown that the NHR1 domain mediates Neur binding to its ubiquitination target Dl (9). Specifically, we showed that the deletion of the entire NHR1 domain or the mutation of a highly conserved glycine residue (Gly167) within the NHR1 domain disrupts binding to Dl (9). To date, however, the function of the NHR2 domain remains unknown. To determine whether the NHR2 domain is required for Neur function, we generated transgenic flies carrying a deletion of the entire NHR2 domain (UAS-NeurΔNHR2) or a mutation at Gly430 (UAS-NeurG430E), which corresponds to the Gly167 residue found in the NHR1 domain (9), under the transcriptional control of the GAL4 upstream activation sequence (UAS) system (Fig. 1A) (6). To ensure that all of our transgenes were expressed at comparable levels, we performed Western blots with embryonic extracts derived from transgenic flies expressing NeurWT (49), NeurΔNHR2, or NeurG430E using neurA101GAL4. We found that all three transgenes were expressed at comparable levels, with slightly higher levels of expression observed for the NeurG430E line (Fig. 1B).
Fig 1
Fig 1
The NHR2 domain and the conserved Gly430 residue are required for Neuralized function in vivo. (A) Various V5-tagged Neur constructs used in this study. The NHR1 domain is shown in dark blue, the NHR2 domain is shown in purple, and the RING domain is (more ...)
neurA101GAL4 is a hypomorphic allele of neur, which shows an embryonic neurogenic phenotype due to the insertion of a P element containing the transcriptional activator GAL4 within the neur promoter. To determine if the NHR2 domain was required for Neur function, we examined whether the expression of the NHR2 mutant transgenes could rescue the neurogenic phenotype of neurA101GAL4 mutant homozygous embryos (Fig. 1C), which we scored by using an anti-horseradish peroxidase (HRP) antibody (50). We observed that 32.91% of embryo progeny of neurA101GAL4 heterozygotes were neurogenic in the absence of any transgene (Fig. 1C and andG).G). In the presence of the NeurWT transgene, only 7.32% of embryos appeared neurogenic (referred to as rescue) (Fig. 1D and andG),G), as previously shown (49). In contrast, both NeurΔNHR2 and NeurG430E failed to rescue the neur mutant phenotype, resulting in significantly higher percentages of embryos appearing neurogenic: 24.46% and 45.10%, respectively (Fig. 1E to toG).G). Although the number of neurogenic embryos in the presence of NeurΔNHR2 (24.46%) was less than that observed for neurA101GAL4 alone (32.91%), it remained significantly higher than what we observed with NeurWT (7.32%). This finding suggests that this construct may retain some residual activity albeit not enough to fully rescue the neur mutant phenotype. The observation that NeurG430 resulted in a significant increase in the percentage of neurogenic embryos (Fig. 1G) suggests that this construct may have a dominant negative effect, by inactivating zygotic Neur in the heterozygous progeny. Consistent with this interpretation, the ectopic expression of NeurG430E in a heterozygous background (UAS-NeurG430E/+; neurA101GAL4/+) also resulted in a similar increase in the percentage of neurogenic embryos, while the overexpression of NeurWT or NeurΔNHR2 had no effect compared to the effect of heterozygous neurA101GAL4 by itself (Fig. 1H). Altogether, these data indicate that the deletion of the NHR2 domain significantly reduces Neur activity, and the Gly430 residue is essential for Neur function during embryonic neurogenesis. In addition, our data suggest that the deletion and point mutation in the NHR2 domain have distinct effects on Neur function.
The NHR2 domain is not required for Neur localization at the plasma membrane or its interaction with Notch ligands.
Given that the NHR2 domain is important for Neur function, we then asked whether the inability of the NHR2 mutant transgenes to rescue neur mutant embryos was due to protein mislocalization in vivo. We first analyzed the subcellular localization of the transgenic protein in embryos using neurA101GAL4 (Fig. 1D″, E″, and F″) and in wing imaginal discs using ombGAL4 (see Fig. 5J′, K′, and L′) and found no significant difference between the localizations of the mutant proteins and WT Neur. NeurWT, NeurΔNHR2, and NeurG430E seemed to be enriched at the plasma membrane.
Fig 5
Fig 5
The NHR2 domain is required for Neuralized-mediated Delta endocytosis. (A to D) Third-instar larval wing discs expressing UAS-enhanced GFP (EGFP) (A), UAS-NeurWT (B), UAS-NeurΔNHR2(C), and NeurG430E (D) in the developing wing pouch, using ombGAL4 (more ...)
In previous studies, we showed that in singly transfected S2 cells, Neur and Delta were found primarily at the plasma membrane (49). We also showed that a mutant form of Neur lacking a PIP-binding motif was localized primarily to the cytoplasm in singly transfected cells but could be recruited to the membrane in the presence of Dl (49). To determine if mutations in the NHR2 domain altered the subcellular localization of Neur or its ability to be recruited to the membrane in the presence of Delta, we transfected V5-tagged Neur and/or Dl-myc into Kc cells and examined their localization. We found that in singly transfected cells, Dl was localized to the plasma membrane, as previously described (49) (Fig. 2E and andO).O). However, in contrast to what we previously observed for S2 cells, we found that Neur proteins were predominantly cytoplasmic in the absence of Dl (Fig. 2A to toCC and andO)O) (9, 49). This discrepancy may reflect differences between Kc and S2 cell lines. Nonetheless, when cells were cotransfected with Dl and either WT or NHR2 mutant Neur constructs, Neur relocated to the plasma membrane (Fig. 2F to toHH and andO),O), demonstrating that the NHR2 domain is not required for Neur localization to the plasma membrane when Dl is present. In contrast, and consistent with our previous findings (9), a mutation of a highly conserved glycine residue in the NHR1 domain (NeurG167E) disrupts the interaction between Neur and Dl, leading to the accumulation of Neur within the cytoplasm (Fig. 2I and andO)O) and a concomitant retention of Dl at the membrane (Fig. 2I′ and andO)O) (9). The introduction of either NeurWT or NeurΔNHR2 resulted in an increase in the cytoplasmic Dl level (Fig. 2F′ and G′), although NeurΔNHR2 was not as efficient as the WT (Fig. 2O). These results are consistent with our rescue data demonstrating that while NeurΔNHR2 does not have wild-type function, it may still have residual activity. In contrast, Dl remained mostly membrane bound in the presence of NeurG430E (Fig. 2H′ and andO).O). These data suggest that the NHR2 domain is not required for Neur to bind to Delta; however, the Gly430 residue may be required for Dl internalization. To confirm that the NHR2 domain is not required for Neur to bind Dl, we cotransfected Dl and Neur into Kc cells and performed coimmunoprecipitation (co-IP) experiments. As was shown previously, NeurG167E disrupts Dl binding in this assay. We found that Dl coimmunoprecipitated with both NHR2 mutants (Fig. 3A, IP lanes 5 and 6 on the anti-myc blot), further demonstrating that mutations in NHR2 do not affect the interaction between Neur and Dl.
Fig 2
Fig 2
The NHR2 domain is not required for ligand-dependent membrane recruitment of Neur. (A to I) Single confocal slices of Kc cells expressing myc-Dl (E) or NeurWT, NeurΔNHR2, NeurG430E, or NeurG167E alone (A to D) or in combination with myc-Dl (F (more ...)
Fig 3
Fig 3
The G167E mutation in the NHR1 domain disrupts the Ser-Neur interaction. (A) NeurWT, NeurΔNHR2, and NeurG430E can coimmunoprecipitate full-length Dl from Kc cell lysates. NeurG167E failed to coimmunoprecipitate full-length Dl. (B) Ser can coimmunoprecipitate (more ...)
Neur binds to both Notch ligands Dl and Ser (16, 49). We have previously shown that the NHR1 domain is both necessary and sufficient to bind to Dl in Drosophila (9). Others have shown that the NHR2 domain in mouse Neurl1 mediates the interaction with Jagged1, the mouse orthologue of Ser (26). To determine whether the NHR2 domain is also required for Neur to interact with Ser in Drosophila, we first transfected tagged Neur and/or Ser into Kc cells and examined its subcellular localization. Similarly to Dl, Ser was localized to the plasma membrane in singly transfected cells (Fig. 2J and andO).O). When Ser was cotransfected with either WT Neur or the NHR2 mutant, Neur was also concentrated at the plasma membrane (Fig. 3K to toMM and andO).O). Similarly to what we observed with Delta, the expression of NeurWT and NeurΔNHR2 resulted in a dramatic increase in the cytoplasmic Ser level (Fig. 2K′, L′, and andO),O), while NeurG430E did not (Fig. 2M′ and andO).O). Interestingly, NeurG167E remained cytoplasmic in the presence of Ser (Fig. 2N and andO),O), while Ser was predominantly retained at the plasma membrane (Fig. 2N′ and andO),O), suggesting that the G167E mutant is not recruited to the plasma membrane, where it is required to induce the internalization of Ser. Furthermore, in coimmunoprecipitation experiments with Neur and Ser, we found that both NHR2 mutants of Neur could be coimmunoprecipitated with Ser (Fig. 3B, IP lanes 9 and 10 on the anti-V5 blot) but not with NeurG167E (Fig. 3B, IP lane 8 on the anti-V5 blot), suggesting that the G167E mutation in the NHR1 domain disrupts Neur-Ser binding. Of note, the interaction between WT Neur and Ser in our co-IP experiment appeared to be very weak; however, given that the expression of NeurWT caused an increase in the cytoplasmic Ser level and other researchers previously reported an interaction between NeurWT and Ser using co-IP experiments (16), we speculated that the weak binding in our experiments might reflect technical differences in co-IP protocols. Taken together, our data show that the NHR2 domain is not required for the plasma membrane localization of Neur in the presence of either Dl or Ser, nor is it required to bind to either ligand. In contrast, the conserved Gly167 residue in the NHR1 domain is required for Neur to bind to both Dl and Ser in Drosophila.
The G430E mutation in the NHR2 domain disrupts Neur E3 ubiquitination activity.
Given that the NHR2 domain is not required for Neur to interact with its targets Dl and Ser, we asked whether the NHR2 domain or the Gly430 residue is required for Neur E3-ubiquitin ligase activity. To this end, we performed immunoprecipitation experiments with cells cotransfected with Neur-V5, Dl-myc, and hemagglutinin (HA)-ubiquitin. High levels of HA-tagged ubiquitin were detected in the anti-V5 immunoprecipitate in the presence of NeurWT (Fig. 4A, IP lane 5 on the anti-HA blot). In contrast, in the presence of NeurC701S, a Neur mutant that has no E3 ligase activity (56), the levels of ubiquitinated proteins were significantly reduced (Fig. 4A, compare IP lane 6 to lane 5 on the anti-HA blot). Both the NeurG167E and NeurΔNHR2 mutants appeared to be as efficient at ubiquitination as NeurWT (Fig. 4A, compare IP lanes 7 and 8 to lane 5 on the anti-HA blot). Of note, this assay does not distinguish whether the ubiquitinated proteins correspond to Delta, Neur, or other unidentified partners. Given that NeurG167E does not bind to Dl but we still observed high levels of ubiquitin in the IP, it is possible that Neur is autoubiquitinated or has other targets besides Dl. Consistent with this idea, in a similar co-IP experiment, where we expressed Neur and HA-ubiquitin on their own, we observed high levels of HA-tagged ubiquitin coimmunoprecipitating with Neur (Fig. 4B, lanes 3 and 9 on the anti-HA blot), further suggesting that Dl may not be the only target of Neur. Nonetheless, the ubiquitination activity was completely abolished in NeurG430E (Fig. 4A, compare IP lane 9 to lane 5 on the anti-HA blot, and B, compare IP lane 10 to lane 9 on the anti-HA blot), which still binds to Dl (Fig. 4A, IP lane 9 on the anti-myc blot), while the deletion of either the NHR1 or the NHR2 domain had no effect (Fig. 4A, lane 8 on the anti-HA blot, and B, lanes 4 and 6 on the anti-HA blot), demonstrating that the two mutations in NHR2 have distinct effects on Neur function. Interestingly, when the NHR1 domain was removed from NeurG430E, ubiquitination activity was restored (Fig. 4B, lane 5).
Fig 4
Fig 4
The G430 residue in the NHR2 domain is required for Neuralized ubiquitination activity. Neur variants and HA-ubiquitin were cotransfected into Kc cells either with Dl (A) or without Dl (B), followed by anti-V5 immunoprecipitation. The immunoprecipitation (more ...)
The NHR2 domain is required for Neur-mediated Dl endocytosis in vivo.
We have shown that the deletion of the NHR2 domain does not affect the ability of Neur to bind to Notch ligands or its ubiquitination activity. Furthermore, in Kc cells, NeurΔNHR2 was still able to induce Dl and Ser internalization albeit with reduced activity. Switching from cultured cells, we wanted to assess the role of the NHR2 domain in Dl endocytosis in flies, a more physiologically relevant context. To address this, we first assessed the effect of the expression of Neur transgenes on Dl localization throughout the presumptive wing pouch using ombGAL4. Of note, Neur is not endogenously expressed in these cells at this stage of development. In WT larvae, Dl is expressed at elevated levels in the wing vein primordia (Fig. 5E, arrow). The ectopic expression of NeurWT resulted in an increase in the amount of internalized Dl, as observed in z sections of wing discs (compare Fig. 5F′ to E′). In contrast, we observed relatively normal levels of internalized Dl in the presence of either NeurΔNHR2 or NeurG430E (compare Fig. 5G and andHH to toE),E), suggesting that NHR2 mutant proteins failed to facilitate Dl endocytosis.
To further confirm that the differences that we saw in the localization of Dl were due to a defect in endocytosis, we used an antibody uptake assay (35) to monitor the endocytosis of Dl in the presence of exogenous Neur. Specifically, we used ombGAL4 to drive the expression of Neur transgenes and examined Dl endocytosis using an anti-Dl antibody generated against the extracellular domain of Dl (DlECD). Consistent with our previously reported observations (49), we found that the ectopic expression of NeurWT resulted in an increase in Dl-antibody internalization (Fig. 5J). NeurΔNHR2 showed significantly decreased levels of Dl-antibody internalization (Fig. 5K), indicating that the deletion of the NHR2 domain does affect Dl endocytosis in vivo. As expected, minimal levels of internalized Dl were observed for the NeurG430E mutant (Fig. 5L), since the G430E mutant has no ubiquitination activity.
Neur forms oligomers.
RING domain E3 ligases often function as oligomers (13). Evidence for the oligomerization of Neur in vivo was obtained by examining the localization of the two isoforms of Neur (NeurPA and NeurPC) during the asymmetric division of sensory organ precursor cells (SOPs). Neur is localized asymmetrically at the anterior cortex when SOPs divide along the anterior-posterior axis to produce a posterior pIIa cell and an anterior pIIb cell (Fig. 6A) (15, 35). We found that the anterior cortical localization of NeurPA in dividing SOPs is not affected in a NeurPC-specific mutant, neurAA8-N67, that deletes the first exon of NeurPC specifically (Fig. 6B), suggesting that the asymmetrical localization of NeurPA is independent of NeurPC. Of note, NeurPC, the minor isoform, is essentially an N-terminal truncation of NeurPA. Importantly, NeurPC lacks a PIP-binding motif that was previously shown to be required for the plasma membrane association and function of NeurPA (49) and is cytoplasmic in vivo (data not shown).
Fig 6
Fig 6
Asymmetric localization of Neur lacking a PIP-binding motif depends on endogenous Neur. (A) Neur (red) localizes asymmetrically in an anterior crescent in wild-type dividing SOPs (Senseless [Sens], a marker for SOPs, is shown in green). (B) Neur-PA localizes (more ...)
We then examined the distributions of V5-tagged NeurPA and NeurPC expressed in SOPs using neurA101GAL4 and found that both NeurPA and NeurPC localized asymmetrically (Fig. 6C and andD).D). However, in the absence of endogenous Neur, i.e., in clones of neurIF65 mutant cells, we observed that NeurPA but not NeurPC localized asymmetrically (Fig. 6F and andG).G). We therefore conclude that the localization of NeurPC depends on the presence of NeurPA. This observation suggested that NeurPA recruits NeurPC at the anterior cortex.
NeurPA differs from NeurPC by the presence of a PIP-binding motif. We therefore tested the role of this motif in the asymmetric localization of NeurPA. To do so, we examined the distribution of NeurPA5Q, a NeurPA version with a mutated PIP-binding motif (49). In the presence of endogenous Neur, NeurPA5Q localized to an anterior crescent in the metaphase and segregated into the anterior cell (Fig. 6E). However, similarly to NeurPC, this asymmetric distribution of NeurPA5Q was found to require endogenous Neur (Fig. 6H). In contrast, the asymmetric localization of Numb was Neur independent (35). Thus, our analysis of both NeurPC and NeurPA5Q revealed that the asymmetric localization of Neur proteins lacking the N-terminal PIP-binding motif required endogenous Neur. These results suggest that NeurPA can oligomerize in vivo with NeurPC and NeurPA5Q to promote their asymmetric localization.
We next verified the ability of Neur to form oligomers in vitro. We generated V5- and FLAG-tagged NeurWT constructs (Fig. 7A), transfected these constructs into Kc cells, and performed anti-V5 immunoprecipitations. We found that FLAG-tagged Neur could be immunoprecipitated with NeurWT-V5 (Fig. 7B, IP lane 3 on the anti-FLAG blot), further demonstrating that Neur can form homo-oligomers.
Fig 7
Fig 7
Neuralized forms oligomers in Kc cells. (A) Schematic of tagged wild-type and deletion mutants of Neur for coimmunoprecipitation. Kc cells were cotransfected with differently tagged Neur protein variants, followed by an anti-V5 immunoprecipitation. (B (more ...)
To further define the role of the NHR domains in oligomerization, we performed immunoprecipitation experiments with lysates obtained from Kc cells cotransfected with various wild-type and truncated Neur constructs (Fig. 7A). We observed a strong interaction between NeurΔNHR1-V5 and NeurΔNHR1-FLAG (Fig. 7B, IP lane 7 on the anti-FLAG blot), suggesting that the NHR1 domain cannot be the sole mediator of oligomerization. Similarly, we found that the deletion of the NHR2 domain alone does not affect Neur oligomerization (Fig. 7B, IP lane 9 on the anti-FLAG blot). We also found that NeurΔNHR1-FLAG and NeurΔNHR2-V5 could be coimmunoprecipitated from Kc cells (Fig. 7B, IP lane 5 on the anti-FLAG blot). Moreover, when both NHR1 and NHR2 were deleted, the remaining RING domain and the interstitial region could interact with WT Neur (Fig. 7C, IP lanes 5 and 6 on the anti-FLAG blot) and self-associate as well (Fig. 7C, IP lane 7 on the anti-FLAG blot). To determine whether the NHR domains alone are capable of mediating Neur dimerization, we also performed a direct yeast two-hybrid analysis (Fig. 7D). Results from two independent trials indicated that Neur can dimerize via the NHR1 domain (Fig. 7E). Moreover, a construct containing both the NHR1 and NHR2 domains is capable of binding to the NHR2 domain alone (Fig. 7E). Although we did not observe an interaction between the NHR1 and NHR2 domains in Y2H experiments, it is possible that factors required for the NHR1-NHR2 interaction were missing in the in vitro system. When we performed immunoprecipitation experiments with lysates obtained from Kc cells cotransfected with NHR1-V5 (9) and NHR2-FLAG, we found that NHR1 can interact with NHR2 (Fig. 7F). These data suggest that the NHR domains are sufficient but not necessary for oligomerization, and there may be multiple ways for oligomers to be assembled and maintained.
As shown previously, the Gly430 residue in the NHR2 domain is essential for Neur ubiquitination activity. To test whether Gly430 plays a role in protein oligomerization, we cotransfected NeurG430E with NeurWT-FLAG into Kc cells, followed by anti-V5 immunoprecipitation. We found that the G430E mutant could still form oligomers with WT Neur but was less efficient than the WT control (Fig. 7G, compare IP lanes 3 and 4 on the anti-FLAG blot). Of note, the difference in binding is unlikely to be due to differences in protein expression levels or immunoprecipitation efficiencies, since we observed comparable levels of wild-type and mutant proteins in both the input (Fig. 7G, input lanes 3 and 4) and immunoprecipitated baits (Fig. 7G, IP lanes 3 and 4 on the anti-V5 blot). Furthermore, when we repeated the co-IP experiments with Kc cells cotransfected with NeurG430E-V5 and NeurG430E-FLAG followed by anti-V5 immunoprecipitation, we found that the oligomerization of NeurG430E was compromised (Fig. 7G, lane 9).
We also examined whether the presence of the G430E mutation in the NHR1 deletion protein (NeurΔNHR1G430E-FLAG) affected the binding to Neur lacking the NHR2 domain (NeurΔNHR2-V5). As before, we observed similar levels of input (Fig. 7B, input lanes 5 and 11) and immunoprecipitated V5-tagged baits (Fig. 7B, IP lanes 5 and 11), but the level of coimmunoprecipitated FLAG-tagged NeurΔNHR1G430E was increased compared to that of NeurΔNHR1 (Fig. 7B, compare IP lane 11 to lane 5 on the anti-FLAG blot), suggesting that the G430E mutation allows NeurΔNHR1G430E to bind more efficiently with NeurΔNHR2 than with NeurΔNHR1. Taken together, our data suggest that Neur forms oligomers via the two NHR domains; however, without the two NHR domains, the remaining RING domain with the interstitial regions could still direct Neur oligomerization. Nevertheless, the Gly430 residue in NHR2 plays an important role in this oligomerization process.
Neur is an E3-ubiquitin ligase that plays an essential role in Notch signaling by regulating the endocytosis of Notch ligands. It contains NHR domains, which are rare and conserved between vertebrates and invertebrates but not present in viruses, bacteria, fungi, or plants. In the Drosophila proteome, besides Neuralized, there are two other NHR-containing proteins, CG3894 and Bluestreak. In mammals, proteins containing NHR domains (also known as NEUZ) include the β-catenin regulator OzzE3 (40) and lung-inducible Neuralized-related C3HC4 RING protein (LINCR) (21). Recent studies reported that the human homologue of Bluestreak serves to localize to the centrosome (1, 37). Although the general role of the NHR domains is unclear, these domains tend to cluster, and most proteins contain two to six NHR domains. The significance of having more than one NHR domain in one protein is to yet be determined.
Here we have investigated the role of the highly conserved NHR domains in Neur function. We show that the NHR1 domain alone mediates the interaction between Neur and both Delta and Serrate. We also show that the NHR2 domain is required for Neur function, and while it is not required for the interaction with Notch ligands, it is involved in Dl internalization, a critical step in Notch activation. Moreover, the NHR domains play a role in Neur oligomerization, which in turn could contribute to Neur ubiquitination activity and ligand endocytosis.
The NHR1 domain mediates the interaction between Neur and the Notch ligands Delta and Serrate.
We have previously shown that the NHR1 domain of Neur is both necessary and sufficient for the interaction with the Notch ligand Dl (9). Specifically, we found that a point mutation in a highly conserved glycine residue within the NHR1 domain (G167E) abolishes the ability of Neur to bind Delta. Whether the NHR1 domain was also required to bind to Serrate, however, was unknown. In fact, in vitro studies in vertebrates suggested that the NHR2 domain in mouse Neuralized-like 1 (Neurl1) was sufficient to bind to Jagged1, the mouse orthologue of Serrate (26). In contrast, we find that the NHR2 domain is not required for Neur to bind Serrate in Drosophila and that the interaction is mediated entirely by the NHR1 domain. Other studies reported previously that the motifs on Dl and Ser that mediate the interaction with Neur are conserved (11, 16). In comparisons of protein sequences, Jagged1 and Serrate share 40.7% similarity overall, while the overall similarity between Jagged1 and Dl is 33.8% (39). The NHR1 and NHR2 domains from Drosophila Neuralized have the same degree of amino acid similarity with the mouse Neurl1 NHR2 domain (33%). Since there is no clear correlation between protein sequence similarity and the ability of either the NHR1 or the NHR2 domain to interact with Notch ligands, whether NHR1 or NHR2 is important for interacting with ligands is likely to be species dependent. We found that the Neur-Ser interaction is abrogated by the G167E mutation in the NHR1 domain. Given that the NeurG167E mutant still retains ubiquitination activity, it is unlikely to affect overall protein folding. A previously reported structural analysis of the Drosophila NHR1 domain suggested that Gly167 resides in a hydrophobic core and that the Gly167 mutation presumably destabilizes the surrounding microenvironment (19). Therefore, the Gly167 mutation may result in spatial changes in the neighboring residues of the core, thus abolishing binding to ligands.
The G430E mutation reveals a distinct role for the NHR2 domain in the regulation of Neur activity and Delta trafficking.
Our data demonstrate that the NHR2 domain is required for Neur function in vivo. NeurG430E fails to rescue neur mutant embryos, while NeurΔNHR2 has some residual activity, which suggests that they affect different aspects of Neur function. The expression of NeurG430 in a heterozygous background, which does not have a neurogenic phenotype on its own, resulted in a significant increase in the percentage of neurogenic embryos, suggesting that NeurG430E has a negative effect on Neur function. In contrast, NeurΔNHR2 overexpression did not have any effect on heterozygous embryos, suggesting that it behaves as a loss-of-function allele.
Despite the fact that the two NHR2 mutant proteins NeurG430E and NeurΔNHR2 behave differently, they both localize to the plasma membrane in the presence of Delta both in vitro and in vivo, and they are both capable of binding to the Notch ligands Delta and Serrate. However, both mutant proteins affect the extent of Dl internalization to various degrees. NeurG430E exhibits severely compromised ubiquitination activity and is no longer capable of inducing Delta internalization. NeurΔNHR2, on the other hand, retains ubiquitination activity but is much less efficient than WT Neur at directing Dl internalization in vivo or in Kc cells. The precise mechanism by which NeurΔNHR2 affects Delta endocytosis is unclear.
One possibility is that the NHR2 domain is required for Neur oligomerization. We have shown that Neur forms NHR domain-mediated oligomers by coimmunoprecipitation experiments. Therefore, the deletion of the NHR2 domain (NeurΔNHR2) may simply reduce the oligomerization potential of Neur, leading to a decrease in ligand endocytosis. In contrast, the point mutation (NeurG430) might disrupt the overall structure of the NHR2 domain, preventing oligomerization and resulting in a protein that has no ubiquitination activity and therefore can no longer internalize ligands. However, this model cannot fully explain our data, which show that ΔNHR2 does not prevent Neur oligomerization and that Neur oligomerization still occurs in the absence of any NHR domains, suggesting that while the NHR domains may play a role in oligomerization, they are not necessary for this process. Our data also show that although the G430E mutant loses ubiquitination activity, the double mutant containing the NHR1 deletion and the G430E mutation retains ubiquitination activity, which argues that G430E does not affect the overall folding of the NHR2 domain.
Another possibility is that the NHR1 and NHR2 domains initially form an intramolecular structure that is inactive and must be resolved for ubiquitination to promote ligand internalization (Fig. 8B). The G430E mutation may lock Neur into an intramolecular conformation through an NHR1-NHR2 interaction, such that this inactive form can still bind to Dl and Ser but cannot form oligomers and has no ubiquitination activity as a consequence of dysfunctional oligomerization (Fig. 8C). This model, in contrast to the former one, is supported by our data, which demonstrate that the G430E mutant no longer forms oligomers and has no ubiquitination activity. Furthermore, when the NHR1 domain was removed from the G430E mutant (NeurΔNHR1G430E), the ubiquitination activity was restored, suggesting that the intramolecular loop can no longer form, whereas intermolecular interactions between NHR1 and NHR2 domains can occur. It may also explain the negative effect of NeurG430E on NeurWT: although NeurG430E has a reduced ability to bind WT Neur, it is still be able to sequester some portion of NeurWT into a nonfunctional intermolecular oligomer that can no longer ubiquitinate targets. In contrast, the deletion of the NHR2 domain would prevent intramolecular interactions but would be expected to have a reduced ability to form productive oligomers, leading to a defect in ligand internalization. Whether the NHR2 domain has additional roles in recruiting a protein(s) (marked as “X?” in Fig. 8) that promotes Notch ligand ubiquitination and endocytosis remains to be determined.
Fig 8
Fig 8
Possible mechanisms for Neur oligomerization. (A) Schematic of wild-type Neur. (B) NeurWT activity is inactivated by the intramolecular interactions between NHR1 and NHR2 in a looped-back conformation. The resolution of the intramolecular interactions (more ...)
Like Neuralized, other RING domain E3 ligases often function as oligomers, and they multimerize in different ways: some form heterodimers, such as Mdm2-MdmX (38), and some can form homo-oligomers, such as TRAF (55). The functional significance of RING E3 oligomerization is poorly defined. One previously proposed model is that the oligomerization of E3 ligases may functionally resemble the dimerization of receptor tyrosine kinases in such a way that autoubiquitination yields a mark that serves as a platform to assemble a signaling complex (31). Consistent with this idea, we did see ubiquitination in anti-Neur IPs when Dl was not present, consistent with the idea that Neur may be autoubiquitinated (12). Whether this autoubiquitination can initiate a cascade of further downstream ubiquitination events remains to be determined. It is possible that oligomerization is mediated via autoubiquitination and the interaction of ubiquitinated Neur with itself through a ubiquitin-binding motif (UIM). If so, then the NHR1-NHR2 interaction could also function to keep Neur in an inactive state by occluding the putative UIM. Such a model of Ubi-UIM complex formation was previously proposed for other endocytic proteins (45).
In summary, we have shown that NHR domains are protein-protein interaction modules that are required for many aspects of Neur function. The NHR1 domain mediates the interaction between Neur and its targets Dl and Ser. Both NHR domains appear to regulate Neur activity by affecting its ability to form oligomers and/or interact with proteins required for the endocytosis of Notch ligands. Interestingly, NHR domains have been identified in several other proteins that are conserved between flies and humans. Whether Neur can form heterodimers with these other NHR-containing proteins and whether these heterodimers play a role in Notch signaling and other developmental processes remain to be determined.
We thank S. Egan and F. Sicheri as well as members of the Boulianne laboratory for helpful discussions and critical comments on the manuscript. We are particularly grateful to C. Delidakis, H. Bellen, P. Rørth, the DSHB, and the Bloomington Stock Center for reagents.
S.L. was the recipient of a University of Toronto open scholarship and is supported through a studentship, in part, by the Research Training Competition (Restracomp) Hospital for Sick Children Foundation Student Scholarship Program. J.M.B. is the recipient of a CIHR graduate scholarship. G.L.B. holds a tier 1 Canada research chair in molecular and developmental neurobiology. This work was funded by a grant from the Natural Sciences and Engineering Council of Canada to G.L.B.
Published ahead of print 8 October 2012
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