An obligatory role of mind bomb-1 in notch signaling of mammalian development

Background

The Notch signaling pathway is an evolutionarily conserved intercellular signaling module essential for cell fate specification that requires endocytosis of Notch ligands. Structurally distinct E3 ubiquitin ligases, Neuralized (Neur) and Mind bomb (Mib), cooperatively regulate the endocytosis of Notch ligands in Drosophila. However, the respective roles of the mammalian E3 ubiquitin ligases, Neur1, Neur2, Mib1, and Mib2, in mammalian development are poorly understood.

Methodology/Principal Findings

Through extensive use of mammalian genetics, here we show that Neur1 and Neur2 double mutants and Mib2^−/− mice were viable and grossly normal. In contrast, conditional inactivation of Mib1 in various tissues revealed the representative Notch phenotypes: defects of arterial specification as deltalike4 mutants, abnormal cerebellum and skin development as jagged1 conditional mutants, and syndactylism as jagged2 mutants.

Conclusions/Significance

Our data provide the first evidence that Mib1 is essential for Jagged as well as Deltalike ligand-mediated Notch signaling in mammalian development, while Neur1, Neur2, and Mib2 are dispensable.     

Mind bomb-2 is an E3 ligase for Notch ligand

The zebrafish gene, mind bomb (mib), encodes a protein that positively regulates of the Delta-mediated Notch signaling. It interacts with the intracellular domain of Delta to promote its ubiquitination and endocytosis. In our search for the mouse homologue of zebrafish mind bomb, we cloned two homologues in the mouse genome: a mouse orthologue (mouse mib1) and a paralogue, named mind bomb-2 (mib2), which is evolutionarily conserved from Drosophila to human. Both Mib1 and Mib2 have an E3 ubiquitin ligase activity in their C-terminal RING domain and interact with Xenopus Delta (XD) via their N-terminal region. Mib2 is also able to ligate ubiquitin to XD and shift the membrane localization of Delta to intracellular vesicles. Importantly, Mib2 rescues both the neuronal and vascular defects in the zebrafish mib(ta52b) mutants. In contrast to the functional similarities between Mib1 and Mib2, mib2 is highly expressed in adult tissues, but almost not at all in embryos, whereas mib1 is abundantly expressed in both embryos and adult tissues. These data suggest that Mib2 has functional similarities to Mib1, but might have distinct roles in Notch signaling as an E3 ubiquitin ligase.     

Functional Analysis of the NHR2 Domain Indicates that Oligomerization of Neuralized Regulates Ubiquitination and Endocytosis of Delta during Notch Signaling

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.     

Distinct intracellular motifs of Delta mediate its ubiquitylation and activation by Mindbomb1 and Neuralized

DSL proteins are transmembrane ligands of the Notch receptor. They associate with a RING (really interesting new gene) family E3 ubiquitin ligase, either Neuralized (Neur) or Mindbomb 1 (Mib1), as a prerequisite to signaling. Although Neur and Mib1 stimulate internalization of DSL ligands, it is not known how ubiquitylation contributes to signaling. We present a molecular dissection of the intracellular domain (ICD) of Drosophila melanogaster Delta (Dl), a prototype DSL protein. Using a cell-based assay, we detected ubiquitylation of Dl by both Neur and Mib1. The two enzymes use distinct docking sites and displayed different acceptor lysine preferences on the Dl ICD. We generated Dl variants that selectively perturb its interactions with Neur or Mib1 and analyzed their signaling activity in two in vivo contexts. We found an excellent correlation between the ability to undergo ubiquitylation and signaling. Therefore, ubiquitylation of the DSL ICD seems to be a necessary step in the activation of Notch.     

Targeted disruption of Mib2 causes exencephaly with a variable penetrance

Mib1 and Mib2 ubiquitin ligases are very similar in their domain construction. They partake in the Notch signaling pathway by ubiquitinating the Notch receptors Delta and Jagged prior to endocytosis. We have created a targeted mutation of Mib2 and show that its phenotype is a variable penetrance, failure to close the cranial neural tube. The penetrance depends on the genetic background but it appears that Mib2 is not completely essential in mouse development.     

Notch ligand activity is modulated by glycosphingolipid membrane composition in Drosophila melanogaster

Endocytosis of the transmembrane ligands Delta (Dl) and Serrate (Ser) is required for the proper activation of Notch receptors. The E3 ubiquitin ligases Mindbomb1 (Mib1) and Neuralized (Neur) regulate the ubiquitination of Dl and Ser and thereby promote both ligand endocytosis and Notch receptor activation. In this study, we identify the α1,4-N-acetylgalactosaminyltransferase-1 (α4GT1) gene as a gain of function suppressor of Mib1 inhibition. Expression of α4GT1 suppressed the signaling and endocytosis defects of Dl and Ser resulting from the inhibition of mib1 and/or neur activity. Genetic and biochemical evidence indicate that α4GT1 plays a regulatory but nonessential function in Notch signaling via the synthesis of a specific glycosphingolipid (GSL), N5, produced by α4GT1. Furthermore, we show that the extracellular domain of Ser interacts with GSLs in vitro via a conserved GSL-binding motif, raising the possibility that direct GSL–protein interactions modulate the endocytosis of Notch ligands. Together, our data indicate that specific GSLs modulate the signaling activity of Notch ligands.     

Neuralized-like 1 (Neurl1) targeted to the plasma membrane by N-myristoylation regulates the Notch ligand Jagged1

Notch signaling constitutes an evolutionarily conserved mechanism that mediates cell-cell interactions in various developmental processes. Numerous regulatory proteins interact with the Notch receptor and its ligands and control signaling at multiple levels. Ubiquitination and endocytosis followed by endosomal sorting of both the receptor and its ligands is essential for Notch-mediated signaling. The E3 ubiquitin ligases, Neuralized (Neur) and Mind Bomb (Mib1), are crucial for regulating the activity and stability of Notch ligands in Drosophila; however, biochemical evidence that the Notch ligands are directly targeted for ubiquitination by Neur and/or Mib1 has been lacking. In this report, we explore the function of Neurl1, a mouse ortholog of Drosophila Neur. We show that Neurl1 can function as an E3 ubiquitin ligase to activate monoubiquitination in vitro of Jagged1, but not other mammalian Notch ligands. Neurl1 expression decreases Jagged1 levels in cells and blocks signaling from Jagged1-expressing cells to neighboring Notch-expressing cells. We demonstrate that Neurl1 is myristoylated at its N terminus, and that myristoylation of Neurl1 targets it to the plasma membrane. Point mutations abolishing either Neurl1 myristoylation and plasma membrane localization or Neurl1 ubiquitin ligase activity impair its ability to down-regulate Jagged1 expression and to block signaling. Taken together, our results argue that Neurl1 at the plasma membrane can affect the signaling activity of Jagged1 by directly enhancing its ubiquitination and subsequent turnover.     

Zebrafish Mib and Mib2 are mutual E3 ubiquitin ligases with common and specific delta substrates

It was already known that both mind bomb (mib) and mind bomb-2 (mib2) encode E3 ubiquitin ligases that target Delta in Notch activation. Here we further demonstrated that zebrafish Mib and Mib2, similar to their mouse orthologs, have a C-terminal-most RING finger-dependent E3 ubiquitin ligase activity. Mib and Mib2 are reciprocal E3 ubiquitin ligases and substrates. They function similarly in Notch signaling by using DeltaC as a common substrate. However, Mib2 behaves differently from Mib in DeltaD internalization. In addition, Mib and Mib2 bind differently to extracellular and intracellular parts of DeltaA and DeltaC. Finally, mutant Mibs, Mib(ta52b) with a missense mutation in the C-terminal-most RING finger (M1013R) and Mib(m132) with a premature stop codon that leads to a deletion of three RING fingers (C785stop), act dominant-negatively and compete with Mib2 in DeltaC ubiquitylation and internalization, suggesting a molecular basis for the antimorphic phenotypes (stronger than the null phenotypes) observed in zebrafish mib(ta52b) and mib(m132) alleles.     

Distinct roles for Mind bomb, Neuralized and Epsin in mediating DSL endocytosis and signaling in Drosophila

Ligands of the Delta/Serrate/Lag2 (DSL) family must normally be endocytosed in signal-sending cells to activate Notch in signal-receiving cells. DSL internalization and signaling are promoted in zebrafish and Drosophila, respectively, by the ubiquitin ligases Mind bomb (Mib) and Neuralized (Neur). DSL signaling activity also depends on Epsin, a conserved endocytic adaptor thought to target mono-ubiquitinated membrane proteins for internalization. Here, we present evidence that the Drosophila ortholog of Mib (Dmib) is required for ubiquitination and signaling activity of DSL ligands in cells that normally do not express Neur, and can be functionally replaced by ectopically expressed Neur. Furthermore, we show that both Dmib and Epsin are required in these cells for some of the endocytic events that internalize DSL ligands, and that the two Drosophila DSL ligands Delta and Serrate differ in their utilization of these Dmib- and Epsin-dependent pathways: most Serrate is endocytosed via the actions of Dmib and Epsin, whereas most Delta enters by other pathways. Nevertheless, only those Serrate and Delta proteins that are internalized via the action of Dmib and Epsin can signal. These results support and extend our previous proposal that mono-ubiquitination of DSL ligands allows them to gain access to a select, Epsin-dependent, endocytic pathway that they must normally enter to activate Notch.     

Two distinct E3 ubiquitin ligases have complementary functions in the regulation of delta and serrate signaling in Drosophila

Signaling by the Notch ligands Delta (Dl) and Serrate (Ser) regulates a wide variety of essential cell-fate decisions during animal development. Two distinct E3 ubiquitin ligases, Neuralized (Neur) and Mind bomb (Mib), have been shown to regulate Dl signaling in Drosophila melanogaster and Danio rerio, respectively. While the neur and mib genes are evolutionarily conserved, their respective roles in the context of a single organism have not yet been examined. We show here that the Drosophila mind bomb (D-mib) gene regulates a subset of Notch signaling events, including wing margin specification, leg segmentation, and vein determination, that are distinct from those events requiring neur activity. D-mib also modulates lateral inhibition, a neur- and Dl-dependent signaling event, suggesting that D-mib regulates Dl signaling. During wing development, expression of D-mib in dorsal cells appears to be necessary and sufficient for wing margin specification, indicating that D-mib also regulates Ser signaling. Moreover, the activity of the D-mib gene is required for the endocytosis of Ser in wing imaginal disc cells. Finally, ectopic expression of neur in D-mib mutant larvae rescues the wing D-mib phenotype, indicating that Neur can compensate for the lack of D-mib activity. We conclude that D-mib and Neur are two structurally distinct proteins that have similar molecular activities but distinct developmental functions in Drosophila.     

Two Distinct E3 Ubiquitin Ligases Have Complementary Functions in the Regulation of Delta and Serrate Signaling in Drosophila

Signaling by the Notch ligands Delta (Dl) and Serrate (Ser) regulates a wide variety of essential cell-fate decisions during animal development. Two distinct E3 ubiquitin ligases, Neuralized (Neur) and Mind bomb (Mib), have been shown to regulate Dl signaling in Drosophila melanogaster and Danio rerio, respectively. While the neur and mib genes are evolutionarily conserved, their respective roles in the context of a single organism have not yet been examined. We show here that the Drosophila mind bomb (D-mib) gene regulates a subset of Notch signaling events, including wing margin specification, leg segmentation, and vein determination, that are distinct from those events requiring neur activity. D-mib also modulates lateral inhibition, a neur- and Dl-dependent signaling event, suggesting that D-mib regulates Dl signaling. During wing development, expression of D-mib in dorsal cells appears to be necessary and sufficient for wing margin specification, indicating that D-mib also regulates Ser signaling. Moreover, the activity of the D-mib gene is required for the endocytosis of Ser in wing imaginal disc cells. Finally, ectopic expression of neur in D-mib mutant larvae rescues the wing D-mib phenotype, indicating that Neur can compensate for the lack of D-mib activity. We conclude that D-mib and Neur are two structurally distinct proteins that have similar molecular activities but distinct developmental functions in Drosophila.     

Mind bomb 1 is essential for generating functional Notch ligands to activate Notch

The Delta-Notch signaling pathway is an evolutionarily conserved intercellular signaling mechanism essential for cell fate specification. Mind bomb 1 (Mib1) has been identified as a ubiquitin ligase that promotes the endocytosis of Delta. We now report that mice lacking Mib1 die prior to embryonic day 11.5, with pan-Notch defects in somitogenesis, neurogenesis, vasculogenesis and cardiogenesis. The Mib1-/- embryos exhibit reduced expression of Notch target genes Hes5, Hey1, Hey2 and Heyl, with the loss of N1icd generation. Interestingly, in the Mib1-/- mutants, Dll1 accumulated in the plasma membrane, while it was localized in the cytoplasm near the nucleus in the wild types, indicating that Mib1 is essential for the endocytosis of Notch ligand. In accordance with the pan-Notch defects in Mib1-/- embryos, Mib1 interacts with and regulates all of the Notch ligands, jagged 1 and jagged 2, as well as Dll1, Dll3 and Dll4. Our results show that Mib1 is an essential regulator, but not a potentiator, for generating functional Notch ligands to activate Notch signaling.     

The characterization of zebrafish antimorphic mib alleles reveals that Mib and Mind bomb-2 (Mib2) function redundantly

Both mind bomb (mib) and mind bomb-2 (mib2) encode RING E3 ubiquitin ligases that promote Delta ubiquitylation and endocytosis in Notch activation. Detailed morphological and molecular examinations revealed that zebrafish mib(ta52b) (missense mutation in the C-terminal RING Finger (RF), M1013R) and mib(m132) (nonsense mutation resulting in a truncated protein that loses all three RFs, C785stop) are strong and weak antimorphic alleles, respectively, compared to the null allele, mib(tfi91) (nonsense mutation resulting in a truncated protein of only 60 amino acids, Y60stop). Zebrafish mib2 ortholog was identified in this study. Zebrafish Mib and Mib2 are colocalized in transfected cells and function redundantly in regulating Notch signaling in embryos. Mib(ta52b) and Mib(m132) have a dosage-dependent dominant-negative effect, at least, on Mib2, which is a molecular basis for the antimorphic phenotypes. It was also shown that Notch signaling negatively regulates mib expression in a Su(H)-dependent manner, forming a negative feedback loop in modulating Notch activation.     

Structural and Functional Characterization of the NHR1 Domain of the Drosophila Neuralized E3 Ligase in the Notch Signaling Pathway

The Notch signaling pathway is critical for many developmental processes and requires complex trafficking of both Notch receptor and its ligands, Delta and Serrate. In Drosophila melanogaster, the endocytosis of Delta in the signal-sending cell is essential for Notch receptor activation. The Neuralized protein from D. melanogaster (Neur) is a ubiquitin E3 ligase, which binds to Delta through its first neuralized homology repeat 1 (NHR1) domain and mediates the ubiquitination of Delta for endocytosis. Tom, a Bearded protein family member, inhibits the Neur-mediated endocytosis through interactions with the NHR1 domain. We have identified the domain boundaries of the novel NHR1 domain, using a screening system based on our cell-free protein synthesis method, and demonstrated that the identified Neur NHR1 domain had binding activity to the 20-residue peptide corresponding to motif 2 of Tom by isothermal titration calorimetry experiments. We also determined the solution structure of the Neur NHR1 domain by heteronuclear NMR methods, using a ¹⁵N/¹³C-labeled sample. The Neur NHR1 domain adopts a characteristic β-sandwich fold, consisting of a concave five-stranded antiparallel β-sheet and a convex seven-stranded antiparallel β-sheet. The long loop (L6) between the β6 and β7 strands covers the hydrophobic patch on the concave β-sheet surface, and the Neur NHR1 domain forms a compact globular fold. Intriguingly, in spite of the slight, but distinct, differences in the topology of the secondary structure elements, the structure of the Neur NHR1 domain is quite similar to those of the B30.2/SPRY domains, which are known to mediate specific protein-protein interactions. Further NMR titration experiments of the Neur NHR1 domain with the 20-residue Tom peptide revealed that the resonances originating from the bottom area of the β-sandwich (the L3, L5, and L11 loops, as well as the tip of the L6 loop) were affected. In addition, a structural comparison of the Neur NHR1 domain with the first NHR domain of the human KIAA1787 protein, which is from another NHR subfamily and does not bind to the 20-residue Tom peptide, suggested the critical amino acid residues for the interactions between the Neur NHR1 domain and the Tom peptide. The present structural study will shed light on the role of the Neur NHR1 domain in the Notch signaling pathway.     

Mind bomb1 is a ubiquitin ligase essential for mouse embryonic development and Notch signaling

The Notch-Delta signaling pathway controls many conserved cell determination events. While the Notch end is fairly well characterized, the Delta end remains poorly understood. Mind bomb1 (MIB1) is one of two E3 ligases known to ubiquitinate Delta. We report here that a targeted mutation of Mib1 in mice results in embryonic lethality by E10.5. Mutants exhibit multiple defects due to their inability to modulate Notch signaling. As histopathology revealed a strong neurogenic phenotype, this study concentrates on characterizing the Mib1 mutant by analyzing Notch pathway components in embryonic neuroepithelium prior to developmental arrest. Premature neurons were observed to undergo apoptosis soon after differentiation. Aberrant neurogenesis is a direct consequence of lowered Hes1 and Hes5 expression resulting from the inability to generate Notch1 intracellular domain (NICD1). We conclude that MIB1 activity is required for S3 cleavage of the Notch1 receptor. These results have direct implications for manipulating the differentiation of neuronal stem cells and provide a putative target for the modulation of specific tumors.     

Bearded family members inhibit Neuralized-mediated endocytosis and signaling activity of Delta in Drosophila

Endocytosis of Notch receptor ligands in signaling cells is essential for Notch receptor activation. In Drosophila, the E3 ubiquitin ligase Neuralized (Neur) promotes the endocytosis and signaling activity of the ligand Delta (Dl). In this study, we identify proteins of the Bearded (Brd) family as interactors of Neur. We show that Tom, a prototypic Brd family member, inhibits Neur-dependent Notch signaling. Overexpression of Tom inhibits the endocytosis of Dl and interferes with the interaction of Dl with Neur. Deletion of the Brd gene complex results in ectopic endocytosis of Dl in dorsal cells of stage 5 embryos. This defect in Dl trafficking is associated with ectopic expression of the single-minded gene, a direct Notch target gene that specifies the mesectoderm. We propose that inhibition of Neur by Brd proteins is important for precise spatial regulation of Dl signaling.     

Drosophila neuralized is a ubiquitin ligase that promotes the internalization and degradation of delta.

The Drosophila gene neuralized (neur) has long been recognized to be essential for the proper execution of a wide variety of processes mediated by the Notch (N) pathway, but its role in the pathway has been elusive. In this report, we present genetic and biochemical evidence that Neur is a RING-type, E3 ubiquitin ligase. Next, we show that neur is required for proper internalization of Dl in the developing eye. Finally, we demonstrate that ectopic Neur targets Dl for internalization and degradation in a RING finger-dependent manner, and that the two exist in a physical complex. Collectively, our data indicate that Neur is a ubiquitin ligase that positively regulates the N pathway by promoting the endocytosis and degradation of Dl.     

The ubiquitin ligase Drosophila Mind bomb promotes Notch signaling by regulating the localization and activity of Serrate and Delta

The receptor Notch and its ligands of the Delta/Serrate/LAG2 (DSL) family are the central components in the Notch pathway, a fundamental cell signaling system that regulates pattern formation during animal development. Delta is directly ubiquitinated by Drosophila and Xenopus Neuralized, and by zebrafish Mind bomb, two unrelated RING-type E3 ubiquitin ligases with common abilities to promote Delta endocytosis and signaling activity. Although orthologs of both Neuralized and Mind bomb are found in most metazoan organisms, their relative contributions to Notch signaling in any single organism have not yet been assessed. We show here that a Drosophila ortholog of Mind bomb (D-mib) is a positive component of Notch signaling that is required for multiple Neuralized-independent, Notch-dependent developmental processes. Furthermore, we show that D-mib associates physically and functionally with both Serrate and Delta. We find that D-mib uses its ubiquitin ligase activity to promote DSL ligand activity, an activity that is correlated with its ability to induce the endocytosis and degradation of both Delta and Serrate (see also Le Borgne et al., 2005). We further demonstrate that D-mib can functionally replace Neuralized in multiple cell fate decisions that absolutely require endogenous Neuralized, a testament to the highly similar activities of these two unrelated ubiquitin ligases in regulating Notch signaling. We conclude that ubiquitination of Delta and Serrate by Neuralized and D-mib is an obligate feature of DSL ligand activation throughout Drosophila development.