Both inhibition and activation of Notch signaling rely on a conserved Neuralized-binding motif in Bearded proteins and the Notch ligand Delta

Lateral inhibition is one of the key functions of Notch signaling during animal development. In the proneural clusters that give rise to Drosophila mechanosensory bristles, Delta (Dl) ligand in the sensory organ precursor (SOP) cell is targeted for ubiquitination by the E3 ligase Neuralized (Neur), resulting in activation of Dl's capacity to signal to the Notch receptor on neighboring cells. The cells that receive this signal activate a genetic program that suppresses their SOP fate potential, insuring that only a single SOP develops within each cluster. Using multiple lines of investigation, we provide evidence that members of the Bearded family of proteins (BFMs) inhibit Dl activation in non-SOP cells by binding to Neur and preventing it from interacting with Dl. We show that this activity of BFMs is dependent on the conserved NXXN motif, and report the unexpected finding that several BFMs include multiple functional copies of this motif. We find that a conserved NXXN motif in the intracellular domain of Dl is responsible for its interaction with Neur, indicating direct competition between Dl and BFMs for binding to Neur, and we show that Neur-dependent endocytosis of Dl requires the integrity of its NXXN motif. Our results illuminate the mechanism of an important regulatory event in Notch signaling that appears to be conserved between insects and crustaceans.     

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.     

neuralized Encodes a peripheral membrane protein involved in delta signaling and endocytosis.

Activation of the Notch (N) receptor involves an intracellular proteolytic step triggered by shedding of the extracellular N domain (N-EC) upon ligand interaction. The ligand Dl has been proposed to effect this N-EC shedding by transendocytosing the latter into the signal-emitting cell. We find that Dl endocytosis and N signaling are greatly stimulated by expression of neuralized (neur). neur inactivation suppresses Dl endocytosis, while its overexpression enhances Dl endocytosis and Notch-dependent signaling. We show that neur encodes an intracellular peripheral membrane protein. Its C-terminal RING domain is necessary for Dl accumulation in endosomes, but may be dispensable for Dl signaling. The potent modulatory effect of Neur on Dl activity makes Neur a candidate for establishing signaling asymmetries within cellular equivalence groups.     

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.     

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.     

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.     

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.     

Fat facets and Liquid facets promote Delta endocytosis and Delta signaling in the signaling cells

Endocytosis modulates the Notch signaling pathway in both the signaling and receiving cells. One recent hypothesis is that endocytosis of the ligand Delta by the signaling cells is essential for Notch activation in the receiving cells. Here, we present evidence in strong support of this model. We show that in the developing Drosophila eye Fat facets (Faf), a deubiquitinating enzyme, and its substrate Liquid facets (Lqf), an endocytic epsin, promote Delta internalization and Delta signaling in the signaling cells. We demonstrate that while Lqf is necessary for three different Notch/Delta signaling events at the morphogenetic furrow, Faf is essential only for one: Delta signaling by photoreceptor precluster cells, which prevents recruitment of ectopic neurons. In addition, we show that the ubiquitin-ligase Neuralized (Neur), which ubiquitinates Delta, functions in the signaling cells with Faf and Lqf. The results presented bolster one model for Neur function in which Neur enhances Delta signaling by stimulating Delta internalization in the signaling cells. We propose that Faf plays a role similar to that of Neur in the Delta signaling cells. By deubiquitinating Lqf, which enhances the efficiency of Delta internalization, Faf stimulates Delta signaling.     

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.     

The neuralized homology repeat 1 domain of Drosophila neuralized mediates nuclear envelope association and delta-dependent inhibition of nuclear import

Signaling by the Notch (N) pathway is critical for many developmental processes and requires complex trafficking of both the N receptor and its transmembrane ligands, Delta (Dl) and Serrate. neuralized encodes an E3 ubiquitin ligase required for N ligand internalization. Neuralized (Neur) is conserved from worms to humans and comprises two Neur homology repeat (NHR) domains, NHR1 and NHR2, and a carboxyl-terminal RING domain. We have previously shown that the RING domain is required for ubiquitin ligase activity and that NHR1 mediates binding to Dl, a ubiquitination target. In Drosophila, Neur associates with the plasma membrane and hepatocyte responsive serum phosphoprotein-positive endosomes. Here we demonstrate that Neur also exhibits nuclear envelope localization. We have determined that Neur subcellular localization is regulated by nuclear trafficking and that inhibition of chromosome region maintenance 1, a nuclear export receptor, interferes with Neur nuclear export, trapping Neur in the nucleus. Moreover, we demonstrate that nuclear envelope localization is mediated by the Neur NHR1 domain. Interestingly, Dl expression in Schneider cells is sufficient to inhibit Neur nuclear import and inhibition occurs in an NHR1-dependent manner, suggesting that Neur nuclear localization occurs in contexts where Dl expression is either low or absent. Consistent with this, we found that Neur exhibits nuclear trafficking and associates with the nuclear envelope in the secretory cells of the larval salivary gland and that overexpression of Dl can reduce Neur localization to the nucleus. Altogether, our data demonstrate that Neur localizes to the nuclear envelope and that this localization can be negatively regulated by Dl, suggesting a possible nuclear function for Neur in Drosophila.     

Frizzled/PCP-dependent asymmetric neuralized expression determines R3/R4 fates in the Drosophila eye

Planar cell polarity (PCP) is a common feature in many epithelia, reflected in cellular organization within the plane of an epithelium. In the Drosophila eye, Frizzled (Fz)/PCP signaling induces cell-fate specification of the R3/R4 photoreceptors through regulation of Notch activation in R4. Except for Dl upregulation in R3, the mechanism of how Fz/PCP signaling regulates Notch in this context is not understood. We demonstrate that the E3-ubiquitin ligase Neuralized (Neur), required for Dl-N signaling, is asymmetrically expressed within the R3/R4 pair. It is required in R3, where it is also upregulated in a Fz/PCP-dependent manner. As is the case for Dl, N activity in R4 further represses neur expression, thus, reinforcing the asymmetry. We demonstrate that Neur asymmetry is instructive in correct R3/R4 specification. Our data indicate that Fz/PCP-dependent Neur expression in R3 ensures the proper directionality of Dl-N signaling during R3/R4 specification.     

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.     

Participation of Tom1L1 in EGF‐stimulated endocytosis of EGF receptor

Although many proteins have been shown to participate in ligand‐stimulated endocytosis of EGF receptor (EGFR), the adaptor protein responsible for interaction of activated EGFR with endocytic machinery remains elusive. We show here that EGF stimulates transient tyrosine phosphorylation of Tom1L1 by the Src family kinases, resulting in transient interaction of Tom1L1 with the activated EGFR bridged by Grb2 and Shc. Cytosolic Tom1L1 is recruited onto the plasma membrane and subsequently redistributes into the early endosome. Mutant forms of Tom1L1 defective in Tyr‐phosphorylation or interaction with Grb2 are incapable of interaction with EGFR. These mutants behave as dominant‐negative mutants to inhibit endocytosis of EGFR. RNAi‐mediated knockdown of Tom1L1 inhibits endocytosis of EGFR. The C‐terminal tail of Tom1L1 contains a novel clathrin‐interacting motif responsible for interaction with the C‐terminal region of clathrin heavy chain, which is important for exogenous Tom1L1 to rescue endocytosis of EGFR in Tom1L1 knocked‐down cells. These results suggest that EGF triggers a transient Grb2/Shc‐mediated association of EGFR with Tyr‐phosphorylated Tom1L1 to engage the endocytic machinery for endocytosis of the ligand–receptor complex.     

Neuralized-2 regulates a Notch ligand in cooperation with Mind bomb-1

Mutations in Drosophila neuralized (Dneur) result in a variety of developmental defects that closely resemble those of Notch mutants and other Notch pathway mutants. However, mice with disrupted neur1 do not show any aberrant cell fate specifications in neurogenesis and somitogenesis. Thus, we speculated that other vertebrate neur homolog(s) might compensate for loss of the neur gene. Here, we report the paralog of mouse Neur1, named Neuralized-2 (Neur2), which is a ubiquitin-protein isopeptide ligase (E3) that interacts with and ubiquitinates Delta. Both murine Neur1 and Neur2 have similar degrees of homology to DNeur, and neur2 is expressed in patterns similar to those of neur1 in embryos, suggesting potential functional redundancy. Interestingly, two distinct classes of E3 ligases, Mind bomb-1 (Mib1) and Neur2, have cooperative but distinct roles in Delta endocytosis to Hrs-positive vesicles, i.e. Mib1 functions in the initial step of Delta endocytosis, and Neur2 is required for targeting endocytosed Delta to Hrs-positive vesicles. Thus, our study provides a new insight into how distinct E3 ligases work together in the endocytic pathways for Notch signaling.     

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.