BIOLOGICAL OVERVIEW

Suppressor mutations are a handy way to find interacting proteins in a biochemical pathway. The discovery of deltex illustrates the power of this method. deltex was isolated in a genetic screen aimed at identifying suppressors of Notch mutations (Xu, 1990). If a gene shows reduced function (Notch mutation for example) the resulting deficiency can often be corrected by the application of other mutations. Mutation in the deltex locus suppresses Notch mutants.

Subsequent biochemical analysis showed that Deltex does indeed function in the Notch pathway. Deltex is an intracellular protein that binds to the cytoplasmic ankyrin repeats of Notch (Diederich, 1994).

When Notch receives a signal from its ligand, it is Deltex binding to the cytoplasmic domain of Notch that frees Suppressor of Hairless from its association with the Notch receptor. Su(H) can then migrate to the nucleus where it functions as a transcription factor for the Enhancer of split complex. In turn, E(spl) acts to suppress neural development. This cascade of activity is at the heart of the lateral inhibition function characterizing the Notch pathway.

notchoid¹ (nd¹) is a viable mutant allele of Notch that causes scalloping of the wing. In a genetic screen for modifiers of Notch activity, searching for mutations that diminish the nd¹ phenotype, mutations in a gene encoding a novel WD40-repeat protein were identified. The gene, called Notchless Nle is conserved, with homologs apparent in Xenopus, mouse and humans. The sel-10 gene of C.elegans encodes a WD40-repeat-containing protein that modifies lin-12 function (lin-12 is a Notch homolog). Although SEL-10 and Notchless both contain WD40 repeats, they are not orthologs. Notchless has nine WD40 repeats rather than the seven repeats found in SEL-10, and does not contain the F-box that characterizes SEL-10 as a CDC4-related protein. SEL-10 does not share a conserved Nle domain in the N-terminus of Notchless. A different C. elegans predicted protein appears to be the ortholog of Notchless. Sequence comparison indicates that the degree of conservation in the N-terminal domain is quite high among the different family members. In the 80 amino acid region corresponding to residues 27-106 of Notchless, sequence identity ranges from 33% (between Drosophila and Saccharomyces cerevisiae) to 61% (between Drosophila and Xenopus proteins). Particular residues are identical in all species examined, suggesting that they may be important for domain structure. It is proposed that this region be called the Nle domain (Royet, 1998).

Notchless loss-of-function mutant alleles dominantly suppress the wing notching caused by nd¹ alleles. Reducing Notchless activity increases Notch activity. Overexpression of Notchless in Xenopus or Drosophila appears to have a dominant-negative effect in that it also increases Notch activity. Deltex is thought to function as a positive regulator of Notch activity. deltex mutant flies show a phenotype resembling a reduction of Notch activity: nicking of the distal region of the wing blade and thickening of the wing veins. Removing one copy of Notchless restores the deltex mutant wing to normal. Thus the effects of reducing deltex activity can be compensated for by simultaneously reducing Notchless activity. Likewise, removing one copy of Notchless enhances the effects of overexpressing Deltex using a heat-shock deltex transgene. These results suggest that Deltex and Notchless act in opposite directions. Biochemical studies show that Notchless binds to the cytoplasmic domain of Notch, suggesting that it serves as a direct regulator of Notch signaling activity (Royet, 1998).

How might Notchless act to reduce Notch activity? Genetic interactions suggest a possible link between Notchless and deltex. deltex mutants resemble weak Notch mutants, suggesting that Deltex helps to increase Notch activity. Deltex protein binds to the CDC10/Ankyrin repeats in the ICN1 domain of Notch, but does not bind to the ICN2 domain. Experiments using the yeast two-hybrid system have shown that Notchless, expressed as an activator fusion protein, binds to the ICN2 domain of Notch, but not to ICN1. This suggests that Notchless is likely to oppose Deltex function indirectly through an opposing activity on Notch, and not by direct competition for binding. Little is known about Deltex function, except that overexpression of Deltex can liberate Su(H) to translocate to the nucleus under conditions where Su(H) is artificially retained in the cytoplasm by binding to overexpressed Notch. It is possible that the balance between Deltex and Notchless activities in some way modulates processing of Notch. The observation that increasing or decreasing Nle has a similar effect on Notch activity raises the possibility that Nle forms a complex with proteins in addition to Notch. If the function of Nle is to bring other components together in a complex and if the level of any component other than Nle is limiting, it is possible that overexpression of Nle could reduce formation of the active complex by sequestering the limiting component(s) into incomplete or inactive complexes. This is easiest to imagine in a complex with several components, but it is also possible in tetramers of two components if a 1:1 stoichiometry is important for activity. Many other explanations could be proposed to account for the dominant-negative behavior of the overexpressed protein. It is worth noting that a similar phenomenon has been reported for Notch itself. Overexpression of wild-type Notch produces a phenotype of thickened veins that resembles that of reducing Notch or Delta activity. This is thought to occur by sequestration of Delta in cells overexpressing Notch, which reduces the ability of these cells to signal productively (Royet, 1998).

A Deltex-dependent pathway repressing neural fate

The Notch receptor triggers a wide range of cell fate choices in higher organisms. In Drosophila, segregation of neural from epidermal lineages results from competition among equivalent cells. These cells express achaete/scute genes, which confer neural potential. During lateral inhibition, a single neural precursor is selected, and neighboring cells are forced to adopt an epidermal fate. Lateral inhibition relies on proteolytic cleavage of Notch induced by the ligand Delta and translocation of the Notch intracellular domain (NICD) to the nuclei of inhibited cells. The activated NICD, interacting with Suppressor of Hairless [Su(H)], stimulates genes of the E(spl) complex, which in turn repress the proneural genes achaete/scute. New alleles of Notch are described that specifically display loss of microchaetae sensory precursors. This phenotype arises from a repression of neural fate, by a Notch signaling distinct from that involved in lateral inhibition. The loss of sensory organs associated with this phenotype results from a constitutive activation of a Deltex-dependent Notch-signaling event. These novel Notch alleles encode truncated receptors lacking the carboxy terminus of the NICD, which is the binding site for the repressor Dishevelled (Dsh). Dsh is known to be involved in crosstalk between Wingless and Notch pathways. These results reveal an antineural activity of Notch distinct from lateral inhibition mediated by Su(H). This activity, mediated by Deltex (Dx), represses neural fate and is antagonized by elements of the Wingless (Wg)-signaling cascade to allow alternative cell fate choices (Raiman, 2001).

In a screen for flies associated with the loss of microchaetae, a number of mutations in Notch were isolated that result in a dominant loss of thoracic microchaetae, which are called N^Mcd, where Mcd stands for microchaetae defective. These mutations are lethal, and, for this reason, their behavior was analyzed in mosaics in which clones of mutant cells are juxtaposed with wild-type territories. In these mosaics, mutant cells are recognized by the use of both bristle and epidermal markers. All mutants behave genetically in a similar manner, the strongest alleles, N^Mcd1 and N^Mcd5 (collectively N^Mcd1/5), were chosen for further analysis. In clones for N^Mcd1 and N^Mcd5, 99% of the microchaetae are absent, whereas macrochaetae are not affected (Raiman, 2001).

Genetic analysis indicates that the dominant effects of the N^Mcd alleles are due to antagonism of the wild-type function of Notch. The mutant phenotype of N^Mcd is enhanced when N⁺ is lowered and is partially suppressed when N⁺ is increased. Thus, these gain-of-function alleles of Notch do not induce an aberrant function of the receptor (neomorphism), but rather produce receptors that are more active on the normal function of Notch. N^Ax alleles exhibit a similar genetic behavior and a similar phenotype to the N^Mcd alleles. However, several differences distinguish N^Ax from N^Mcd. The N^Ax mutant exhibits a variable loss of both thoracic microchaetae and macrochaetae, leading to irregular patterns. In contrast, N^Mcd affects only microchaetae. Furthermore, the remaining microchaetae of the N^Mcd/+ flies are arranged in fewer rows, which are organized in a regular pattern. Finally, N^Ax/+ flies exhibit broader wings with shortened veins. In contrast, the wings of the N^Mcd/+ flies appear as those of wild-type flies. In this study of the N^Mcd alleles, focus was placed on the bristle pattern (Raiman, 2001).

A further demonstration of the specificity of the N^Mcd mutations for microchaetae is seen by analysis of N^Mcd1/5clones with impaired function of either hairy or extramacrochaetae (emc), two negative regulators of ac/sc. Flies lacking hairy or its cofactor groucho (gro) exhibit ectopic microchaetae in the scutellum region of the thorax. In clones mutant for N^Mcd1/5 and lacking gro (N^Mcd1/5 gro^-cells), ectopic microchaetae are absent. In contrast, the N^Ax mutants again behave differently, since, in Ax^59b gro^- cells, ectopic microchaetae form. The ectopic macrochaetae, which develop in emc¹clones, also arise in N^Mcd1/5 emc¹clones, even when their precursors differentiate simultaneously to those of the microchaetae (Raiman, 2001).

In the absence of any component of lateral inhibition, an excess of neural precursors occurs at the expense of epidermis. In Notch^-, Su(H)^-, and Dl^-clones (mosaic animals), the neurogenic phenotype is extreme; all mutant cells adopt the neural fate, and no cells are left to form epidermis. The lack of epidermal mutant cells leads to a wound partially skinned up by wild-type epidermal-surrounding cells. In gro^- and E(spl)^-, as well as in the hypomorphic Dl clones, the neurogenic phenotype is less severe, and such clones can differentiate tufts of densely packed sensory bristles accompanied by few epidermal cells. Furthermore, mutant cells for loss-of-function alleles of Notch have an enhanced capacity to produce an inhibitory signal that forces neighboring wild-type cells to adopt the epidermal fate. This signal is mediated by Delta. Thus, along the borders of N mutant clones, no bristles are formed by wild-type cells (Raiman, 2001).

Alleles of Notch encoding constitutively activated receptors show the opposite phenotype, with wild-type bristles forming at the border of mutant territories that adopt epidermal fate. The phenotype of the N^Mcd mutants resembles that of classic gain-of-function alleles of Notch (among which are the N^Ax alleles) and therefore might result in an activation of the lateral inhibition function. If this were the case, removal of the function of some or all of the mediators of lateral inhibition will abolish the effects of the N^Mcdalleles. To test this, double-mutant clones were made using the loss-of-function mutations Dl^RevF10, Dl^9P39, Df(3R)E(spl)b32.2, gro^E48, and Su(H)^IB115. In this case, double-mutant clones for N^Mcd1,5 and components that mediate lateral inhibition [Delta; E(spl)-C; gro; Su(H)] would be predicted to inactivate lateral signaling; they would be predicted to display the neurogenic phenotypes characterized by the lack of mutant epidermal cells. Surprisingly, in all cases, the double-mutant clones display the N^Mcd1/5 phenotype with mutant epidermis and no microchaetae differentiated. Therefore, N^Mcdcells do not require Dl, Su(H), gro, or the E(spl)-C in order to adopt the epidermal fate. In contrast, neurogenic double-mutant clones are observed using Ax^59bor Ax^SX1and at least with Dl, gro, and E(spl)-C. The N^Mcd Ser and N^Mcd Dl Ser clones display the N^Mcdphenotype, suggesting that the N^Mcdphenotype does not require Serrate, the other ligand of Notch (Raiman, 2001).

The macrochaetae can differentiate normally in clones mutant for N^Mcd. In the absence of lateral signaling (double-mutant clones for N^Mcd1,5 and one of the components of lateral inhibition [Dl; E(spl)-C; gro; Su(H)]), mutant clones would be predicted to display tufts of macrochaetae (the neurogenic phenotype). Macrochaetae differentiating as single bristles are observed rather than as a neurogenic tuft. These results confirm that the N^Mcdmutants affect a function of Notch distinct from lateral inhibition (Raiman, 2001).

Since clones of N^Mcd cells lack microchaetae, the development of their precursors was examined during pupal stages by means of neural-specific markers. The loss of microchaetae observed in N^Mcd1/5 is due to the loss of neural cells, as visualized by stainings using the neural-specific antibody 22C10, and to the loss of their precursors, as detected with the reporter neu^A101. Since the proneural Ac activity is known to promote the development of the microchaetae precursors, Ac expression was examined in the N^Mcd mutants. The loss of microchaetae precursors is associated with a severe decrease in Ac expression (Raiman, 2001).

The N^Mcd phenotype is unlikely to be due to a lack of differentiation of the outer elements of the sensory organs, since 'escaped' microchaetae have a normal morphology. Thus, these results indicate that the N^Mcdmutations disrupt the early establishment of neural precursors rather than the late lineage that permits the differentiation of the sensory bristle (Raiman, 2001).

Different lines of work have suggested that the existence of Notch-signaling events are independent of the mechanism of lateral inhibition. Some of these experiments suggest that the adaptor protein Deltex (Dx) might be involved in some of these events (Raiman, 2001).

Dx is a cytoplasmic protein that regulates Notch through binding to the ankyrin repeats. Loss-of-function alleles of dx display an excess of microchaetae, whereas overexpression of Dx inhibits neurogenesis. It has been suggested that Dx is involved in a signal transduction event downstream of Notch. Loss-of-function dx alleles behave as dominant suppressors of all the N^Mcd alleles , and N^Mcd1/5 dx^-clones display a fairly normal microchaetae pattern. The Dx effector, therefore, might represent an essential regulator of the antineural activity revealed by the N^Mcd receptors (Raiman, 2001).

In contrast, Shaggy, the Drosophila glycogen synthase kinase 3 (GSK3) is a central element in Wingless signal transduction and behaves genetically as a downstream element of the Notch pathway. Mutations in Sgg suppress the effects of N^Mcd mutants, like mutations in Dx. Altogether, these results indicate that both Dx and Sgg might be involved in the Notch-signaling event that is distinct from lateral inhibition (Raiman, 2001).

Investigations at the molecular level show that all N^Mcd alleles, except N^Mcd5, encode receptors with C-terminal truncations. N^Mcd5 is associated with a single C739Y change that disrupts the median disulphide bridge of the 18^th EGF repeat of the extracellular domain. The 114 amino acid common region deleted in all the truncated receptors contains a PEST sequence, which is conserved in the Notch family and is involved in protein degradation. The loss of microchaetae is accentuated with the decreasing length of the NICD. In addition to the PEST sequence, the NICD includes additional elements such as the CcN domain. Deletion of different combinations of these elements might therefore explain differences in the severity of the phenotypes observed (Raiman, 2001).

Since Achaete/Scute expression is required for the establishment of the neural fate, the novel Notch pathway revealed by the N^Mcd mutants must be repressed during wild-type neural development. One candidate to exert this repression is Dishevelled (Dsh), a component of the Wingless-signaling cascade, which has been shown to bind Notch and block some of its activities. Using a yeast two-hybrid assay, it has been found that Dsh does bind to the C-terminal 114 amino acids of the NICD that are absent in the truncated receptors. Therefore, the Dx-dependent repressive effect of the N^Mcd receptors appears as the consequence of the loss of the Dsh binding site (Raiman, 2001).

Therefore, Notch associates in vitro with Dsh through its C-terminal 114 amino acids. In order to test the functional significance of this C-terminal domain of Notch in vivo, the effect of overexpressed Dsh on the development of microchaetae was examined either in wild-type or in N^Mcd8 flies lacking the Dsh binding site. Flies carrying four copies of a hsp70-Dsh transgene were analyzed. One 15-min heat pulse (37°C) at the onset of pupariation leads to an increase of 5.8% of the number of microchaetae in a wild-type background. In contrast, the pulse has no effect on N^Mcd8 flies. These experiments suggest that Dsh binds the 114 amino acid C terminus of Notch in vivo to antagonize the Dx-dependent signaling of the receptor. The effects of overexpressed Dsh were examined in Notch mutant-carrying lesions in the extracellular EGF repeats (nd³; spl;Ax^9B2; Ax^E2). In each case, an increase in the number of microchaetae was observed after heat treatment (Raiman, 2001).

Dsh and Dx display antagonistic activities. Overexpressed Dx inhibits neurogenesis, whereas overexpressed Dsh increases the number of microchaetae in wild-type flies. Furthermore, this latter excess of microchaetae is accentuated when the dosage of Dx is lowered (Raiman, 2001).

N^Mcd2, N^Mcd3, N^Mcd7, and N^Mcd8 characteristically produce hemizygous escapers showing a strong reduction in the number of microchaetae. However, lateral inhibition is not abolished in these mutants, since the remaining microchaetae are evenly spaced. Consistent with this, Western blot analysis of protein extracts prepared from mutant animals reveals that all N^Mcd proteins are processed. The resulting NICDs carry intact ankyrin repeats, known to bind Su(H), and therefore could mediate lateral inhibition (Raiman, 2001).

Loss-of-Su(H) alleles behave as dominant enhancers of the N^Mcd alleles. Dx is a cytoplasmic protein whose activity also relies on binding to the ankyrin repeats. The antagonism between Dx and Su(H) could be explained by a binding competition for the ankyrin repeats of the NICD. Thus, when Su(H) concentration is reduced, Dx signaling is increased and the N^Mcd phenotype is accentuated. This observation suggests that activity of the Notch receptor depends on the balance between Dx and Su(H) (Raiman, 2001).

Although Deltex has been interpreted as being involved in lateral inhibition, the results of this study make it more likely that it is associated with an alternative signaling event. Dx is a ubiquitous cytoplasmic protein that regulates Notch through binding to the NICD. During lateral inhibition, upon activation by the ligand Dl, the NICD is translocated to the nucleus where it interacts with Su(H) to regulate target genes. However, Su(H) is also present in the cytoplasm, where it displays antagonism with Dx, reflecting a competition to associate to the ankyrin repeats of Notch. Consistently, it has been suggested that Dx may maintain an activated state of Notch indirectly by interfering with the retention of Su(H) in the cytoplasm by virtue of its interaction with the ankyrin repeats of Notch. Moreover, loss-of-functions alleles of Su(H) and loss-of-functions alleles of dx behave, respectively, as dominant enhancers and dominant suppressors of the phenotype of N^Mcd/+ heterozygous flies. This observation demonstrates that Su(H) and Dx display antagonist activities during N signaling (Raiman, 2001).

The loss of microchaetae in N^Mcd mutants is accentuated when the number of putative functional domains removed in the NICD is increased. The truncated receptors lack a 114 amino acid fragment required for Dsh to bind to the NICD. This fragment also contains a PEST sequence, which is conserved in the Notch family and which is likely to be involved in protein degradation. Furthermore, a CcN motif is located between the ankyrin repeats and the PEST sequence in the different Notch receptors. It has been shown that the activity of the morphogen Dorsal is negatively regulated by heterodimerization of Dorsal with the ankyrin repeats of the Cactus inhibitor. The proteolysis of Cactus controlled by a PEST domain associated with a CKII site is an essential step for the nuclear translocation of Dorsal and the patterning of the Drosophila embryo. Interestingly, N^Mcd1 displays the most severe phenotype correlated with the deletion of the CcN motif. The CcN motif contains nuclear-targeting information, and its deletion may explain a reduction of the nuclear import of the NICD, leading to the reinforced cytoplasmic activity of Dx (Raiman, 2001).

When Su(H) concentration is reduced, the cytoplasmic activity of Dx is increased and the N^Mcd phenotype is accentuated. This observation suggests that the activity of the Notch receptor depends on the balance between Dx and Su(H), and, consequently, any factor that modifies the activity of either pathway would affect bristle pattern. One can speculate that Dsh might play such a role and regulate this balance. Furthermore, the cytoplasmic activity of Su(H) has been reported to stabilize the NICD associated with the membrane, possibly by preventing both Notch ubiquitinylation and the entry of the NICD into the nucleus. Dsh may modulate the phosphorylation status of the NICD, which may favor the binding of Su(H) to the ankyrin repeats and consequently repress the Dx activity (Raiman, 2001).

Potentially, Dsh could exert its repressing effect by modulating the proteasome-dependent proteolysis of Notch or the phosphorylation state versus cytoplasmic/nuclear distribution of the NICD. Interestingly, Dsh contains two proline-rich sequences, PPLP and PPXY, putative binding sites for Su(dx), a cytoplasmic ubiquitin ligase involved in ubiquitinylation/turnover of proteins. When binding to Notch, Dsh could serve as a docking protein for Su(Dx) and could regulate the activity of Dx in targeting the proteasome activity to the C terminus of Notch (Raiman, 2001).

How the Dx-dependent transduction is achieved in the cells is poorly understood. One could speculate that the repressing activity of Dsh may also rely on a direct effect on the Dx-dependent signaling. Thus, Dsh and Dx antagonistically regulate a common target, JNK (JUN N-terminal kinase), and Sgg antagonizes JNK-dependent activation of the JUN transcription factor. dJUN might therefore represent an element mediating the antineural activity of Dx (Raiman, 2001).

The Dx-dependent antineural activity of Notch is regulated by elements of the Wingless-signaling cascade, e.g., the cytoplasmic protein Dsh or the kinase Sgg. Overexpression of Dsh generates extrasensory organs in wild-type flies and fails to elicite ectopic bristles in the N^Mcdmutants lacking the Dsh binding site. The kinase Sgg is negatively regulated by Dsh in the Wingless-signaling cascade. Dsh and Sgg have opposite effects on the Dx-dependent Notch pathway. Loss-of-function alleles of sgg lead to a constitutive derepression of Wingless signaling and elicit the same number of ectopic bristles in wild-type and N^Mcd mutant flies (Raiman, 2001).

This analysis of the N^Mcd mutants supports the idea that Dsh, an effector of the Wingless pathway, directly interacts with Notch in wild-type flies in order to maintain the neural potential. Dsh antagonizes the cytoplasmic activity of Dx and then represses the antineural Dx-dependent function of Notch. In wild-type flies, crosstalks between Wingless and Notch allow stimulation of the ac/sc expression in the equivalent cells of the proneural clusters until a given threshold. It has been reported that Su(H) functions as the core of a molecular switch, acting as a repressor of Notch target genes in the absence of nuclear NICD. Thus, prior to the onset of lateral signaling, the repressive activity of Su(H) is compatible with the activation of ac/sc by the Wingless-dependent pathway. When a given level is reached, ac/sc can activate the Dl gene, and cells can compete with each other for the choice of the neural precursor via lateral signaling. At this stage, the Wg and the Su(H)-dependent Notch signalings have opposite effects on the expression of ac/sc. ac/sc is repressed in the inhibited cells, suggesting that the Su(H)-dependent Notch signaling overrides the Wingless pathway (Raiman, 2001).

Though the N^Mcd5 allele shares the same loss-of-microchaetae phenotype as other N^Mcd and affects the same developmental pathway, the N^Mcd5 mutant receptor carries a single point mutation, leading to the C739Y substitution that disrupts the 18^th EGF repeat of the extracellular domain, whereas the other N^Mcdalleles encode truncated receptors lacking the C terminus of the intracellular domain. Experiments with N^Mcd5 suggest that the region of the 18^th EGF is instrumental for the regulation of alternative Notch signaling. The extracellular EGF domain is known to physically bind Wingless. Further experiments are necessary to determine whether the N^Mcd5 lesion in the 18^th EGF repeat specifically alters the binding of Wingless, Fringe, or other unknown effector(s) (Raiman, 2001).

The present study of N^Mcd alleles demonstrates that a Deltex-mediated function of Notch represses the proneural activity during establishment of the neural precursors of the thoracic microchaetae. This repressive activity precedes and is distinct from that which mediates lateral inhibition and is constitutively active in N^Mcd mutants. The N^Mcd alleles encode truncated receptors that lack the binding domain of the repressor Dishevelled, which is involved in functional interactions between Notch and Wingless signalings. The results suggest a model in which Dishevelled is used to alleviate this initial repressive function of Notch in wild-type development, thereby permitting lateral inhibition to generate the regularly spaced sensory microchaetae. In the absence of ligands or effectors, the repressive function of the Dx-dependent activity of Notch could therefore maintain the cells in an uncommited state. In the presence of effectors like Dsh (Wingless signaling) that repress this antineural activity, cells become competent for further choice between two alternative fates (lateral inhibition). It is proposed that Notch acts during development either as a repressor preventing cell differentiation or as a receptor involved in the choice of cell fate during lateral signaling. This dual function is likely to be regulated in a ligand-dependent manner by crosstalk between the Notch and Wingless pathways. It will be important to find out the different components of this new Dx-dependent repressive cascade of Notch (Raiman, 2001).

GENE STRUCTURE

Bases in 5' UTR - 353

Exons - three

Bases in 3' UTR - 1198

PROTEIN STRUCTURE

Amino Acids - 737

Structural Domains and Evolutionary homologs

Deltex is a novel cytoplasmic protein (Busseau, 1994), with no known homologs. The protein is rich in glutamine, histidine and serine. The Deltex protein contains three domains separated by stretches of glutamine-rich sequence. The N-terminal domain is responsible for binding to the intracellular domain of Notch. The middle section contains a proline-rich sequence that has been proposed to be an SH3 domain-binding site, and the C terminus contains a ring zinc-finger motif.

E47 (Drosophila homolog: Daughterless) is a widely expressed transcription factor that activates B-cell-specific immunoglobulin gene transcription and is required for early B-cell development. In an effort to identify processes that regulate E47, and potentially B-cell development, it was found that activated Notch1 and Notch2 effectively inhibit E47 activity. Only the intact E47 protein is inhibited by Notch. Fusion proteins containing isolated DNA binding and activation domains are unaffected. Although overexpression of the coactivator p300 partially reverses E47 inhibition, results of several assays indicate that p300/CBP is not a general target of Notch. Notch inhibition of E47 does not correlate with its ability to activate CBF1/RBP-Jkappa, the mammalian homolog of Suppressor of Hairless, a protein that associates physically with Notch and defines the only known Notch signaling pathway in Drosophila (Ordentlich, 1998).

E47 is inhibited by mammalian Deltex, a second Notch-interacting protein. Evidence is provided that Notch and Deltex may act on E47 by inhibiting signaling through Ras. The EGR-1 promoter (see Huckebein) is known to be stimulated by Ras through the action of mitogen-activated protein kinases (MAPKs) on a ternary complex involving ETS proteins (e.g., ELK1) and Serum response factor. The activity of a CAT reporter under the control of the EGR-1 promoter is inhibited by Deltex, both in the presence and in the absence of Ras stimulation by platelet-derived growth factor. To reduce the complexity of the effects, a series of GAL4 promoter fusions were used and their abilities to activate a minimal promoter containing GAL4 binding sites was assessed. GAL4-Jun includes a portion of the c-Jun protein whose activity is dependent on signaling from Ras to SAPK/JNK. A promoter fragment lacking the CBF1 interaction domain inhibits GAL4-Jun activity but has no effect on GAL4-CREB. Similarly, Deltex inhibits GAL4-Jun activity and has no effect on GAL4-CREB. Although it is likely that N2-IC and Deltex have somewhat different effects on cells, these results clearly show that both Notch and Deltex inhibit signaling by Ras, as measured by the ability to stimulate SAPK/JNK activity. It is proposed that this is the mechanism by which Notch and Deltex inhibit E47 (Ordentlich, 1998).

Two of the positive regulators of the Notch pathway of Drosophila are encoded by the Suppressor of hairless ([Su(H)]) and deltex (dx) genes. Drosophila dx encodes a ubiquitous, novel cytoplasmic protein of unknown biochemical function. A human deltex homolog has been cloned and characterized in parallel with its Drosophila counterpart, in biochemical assays to assess Deltex function. Both human and Drosophila Deltex bind to Notch across species and carry putative SH3-binding domains. Using the yeast interaction trap system, it has been found that Drosophila and human Deltex bind to the human SH3-domain containing protein Grb2. Results from two different reporter assays demonstrate the association of Deltex with Notch-dependent transcriptional events. Evidence is presented linking Deltex to the modulation of basic helix-loop-helix (bHLH) transcription factor activity (Matsuno, 1998).

A partial cDNA has been isolated that encodes a novel chicken homolog of human Deltex (DTX1), a member of the Notch signaling pathway. The cDtx2 sequence shows higher homology to KIAA0937 protein (92% identical) than to DTX1 (68% identical). cDtx2 is expressed widely in the epiblast at stage 4. Later in development it is expressed in many neural and sensory structures, such as neural tube, migrating neural crest cells, epidermal placodes, cranial ganglia, and the optic and otic vesicles. Expression of cDtx2 is uniformly distributed in the prospective spinal cord at earlier stages from stages 8 to 15. At later stages (22-25) expression of cDtx2 becomes restricted to the ventricular zone, which contains proliferating precursor cells. At all stages there is minimal expression in the floor plate. At stages 9, 10 and 12 cDtx2 expression is seen in the neural and surface ectoderm. As development proceeds, cDtx2 expression becomes stronger in the closed neural tube and weaker in the ectoderm. At stages 9 and 10 cDtx2 is expressed throughout the brain with higher expression in the forebrain and midbrain. From stages 12 to 15 cDtx2 mRNA levels increase in the optic vesicle and hindbrain. From stages 15 to 24, signal in the diencephalon and telencephalon decreases. At the same time, signal in the mesencephalon, myelencephalon and metencephalon increases and becomes restricted to the ventricular zone as in spinal cord. Staining for cDtx2 is not detected in the floor plate and roof plate. In the spinal cord and brain the area of the expression of cDtx2 is broader in the dorsal part and narrowed in more ventral regions. At stage 12 cDtx2 expression is seen in the outer half of the optic vesicle and the thickened lens placode. As the optic cup forms at stage 15 cDtx2 expression increases and is present in the prospective neural retina, invaginating lens placode, and optic stalk. At this stage signal is not detected in the prospective retinal pigmented epithelium. At stages 22 and 25 cDtx2 expression disappears from the lens but continues to be expressed in the neural retina. cDtx2 is detected in migrating cranial and trunk neural crest cells. Stripes of cDtx2-positive cells, apparently migrating toward the branchial arches and cranial ganglia, are seen at all stages examined. cDtx2 is also expressed in the epibranchial placodes, the placodal epithelial cells that contribute to the cranial ganglia, and in migrating presumptive neuroblasts derived from those placodes. At later stages cDt2x transcripts are detected in the cranial ganglia and cranial nerves. Migrating trunk neural crest and dorsal root ganglion cells also express cDtx2 mRNA. Strong expression of cDtx2 is seen in the otic placode and the developing vesicle. cDtx2 is detected in the nasal placodes as they begin to invaginate. Transient expression of cDtx2 is also seen in the somites soon after they form. Expression of cDtx2 is observed in developing kidney at stages 22 and 25, in the liver and around the dorsal aorta (Frolova, 2000).

Genetic studies have identified human Itch, which is homologous to the E6-associated protein carboxyl terminus (Hect) domain-containing E3 ubiquitin-protein ligase that is disrupted in non-agouti lethal mice or Itchy mice. Itch is a homolog of Drosophila Suppressor of Deltex. Itch-deficiency results in abnormal immune responses and constant itching in the skin. Itch associates with Notch, a protein involved in cell fate decision in many mammalian cell types, including cells in the immune system. Itch binds to the N-terminal portion of the Notch intracellular domain via its WW domains and promotes ubiquitination of Notch through its Hect ubiquitin ligase domain. Thus, Itch may participate in the regulation of immune responses by modifying Notch-mediated signaling (Qiu, 2000).

Intercellular signaling through the cell-surface receptor Notch plays important roles in a variety of developmental processes as well as in pathogenesis of some human cancers and genetic disorders. However, the mechanisms by which Notch signals are transduced into cells still remain elusive. The signaling mechanisms for Notch in the cell fate control of neural progenitor cells has been investigated. Deltex-1 (DTX1), a mammalian homolog of Drosophila Deltex, is shown to mediate a Notch signal to block differentiation of neural progenitor cells. A significant fraction of DTX1 proteins are localized in the nucleus and physically interact with the transcriptional coactivator p300. Through its binding to p300, DTX1 inhibits transcriptional activation by the neural-specific helix-loop-helix-type transcription factor MASH1, and this mechanism is likely responsible for the differentiation inhibition of neural progenitor cells. These results further suggest that DTX1 regulates transcription independently of the previously characterized Notch signaling pathway involving RBP-J and HES1/HES5. Thus, DTX1 serves as an important signaling component downstream of Notch that regulates transcription in the nucleus (Yamamoto, 2001).

Axon-derived molecules are temporally and spatially required as positive or negative signals to coordinate oligodendrocyte differentiation. Increasing evidence suggests that, in addition to the inhibitory Jagged1/Notch1 signaling cascade, other pathways act via Notch to mediate oligodendrocyte differentiation. The GPI-linked neural cell recognition molecule F3/contactin is clustered during development at the paranodal region, a vital site for axoglial interaction. F3/contactin acts as a functional ligand of Notch. This trans-extracellular interaction triggers gamma-secretase-dependent nuclear translocation of the Notch intracellular domain. F3/Notch signaling promotes oligodendrocyte precursor cell differentiation and upregulates the myelin-related protein MAG in OLN-93 cells. This can be blocked by dominant negative Notch1, Notch2, and two Deltex1 mutants lacking the RING-H2 finger motif, but not by dominant-negative RBP-J or Hes1 antisense oligonucleotides. Expression of constitutively active Notch1 or Notch2 does not upregulate MAG. Thus, F3/contactin specifically initiates a Notch/Deltex1 signaling pathway that promotes oligodendrocyte maturation and myelination (Hu, 2003).

Transcriptional Regulation

Suppressor of Hairless exhibits allele-specific interactions with deltex, indicating that this Notch pathway may regulate deltex transcripition by means of Su(H) (Fortini, 1994).

Post-transcriptional Regulation

The 5' non-coding region of deltex has an unusual arrangement of five ATG codons that are part of a short open reading frame of 14 codons. This organization resembles that of genes known to be under transcriptional control. Removal of these ATG codons increases the level of Deltex protein accumulation (Busseau, 1994).

Protein Interactions

There is a direct interaction between Notch ankyrin repeats and Deltex (Diederich, 1994).

During development, the Notch receptor regulates many cell fate decisions by a signaling pathway that has been conserved during evolution. One positive regulator of Notch is Deltex, a cytoplasmic, zinc finger domain protein, which binds to the intracellular domain of Notch. Phenotypes resulting from mutations in deltex resemble loss-of-function Notch phenotypes and are suppressed by the mutation Suppressor of deltex [Su(dx)]. Homozygous Su(dx) mutations result in wing-vein phenotypes and interact genetically with Notch pathway genes. Su(dx) has been defined genetically as a negative regulator of Notch signaling. This study presents the molecular identification of the Su(dx) gene product. Su(dx) belongs to a family of E3 ubiquitin ligase proteins containing membrane-targeting C2 domains and WW domains that mediate protein-protein interactions through recognition of proline-rich peptide sequences. A seven-codon deletion has been identified in a Su(dx) mutant allele; expression of Su(dx) cDNA rescues Su(dx) mutant phenotypes. Overexpression of Su(dx) also results in ectopic vein differentiation, wing margin loss, and wing growth phenotypes and enhances the phenotypes of loss-of-function mutations in Notch, evidence that supports the conclusion that Su(dx) has a role in the downregulation of Notch signaling (Cornell, 1999).

Cytoplasmic retention of Su(H) requires the intracellular cdc10/ankyrin repeats of Notch, which associate with Su(H) in the absence of ligand signaling. The Deltex protein regulates the subcellular localization of Suppressor of Hairless via antagonistic interactions with Notch ankyrin repeats (Fortini, 1994 and Matsuno, 1995).

The Notch (N) pathway defines an evolutionarily conserved cell signaling mechanism that governs cell fate choices through local cell interactions. The ankyrin repeat region of the Notch receptor is essential for signaling and has been implicated in the interactions between Notch and two intracellular elements of the pathway: Deltex (Dx) and Suppressor of Hairless (Su[H]). The function of the Notch cdc10/ankyrin repeats (ANK repeats) was examined by transgenic and biochemical analysis. In vivo expression of the Notch ANK repeats reveals a cell non-autonomous effect and elicits mutant phenotypes that indicate the existence of novel downstream events in Notch signaling. The intracellular domain induces five cone cells, a phenotype consistent with the idea that this truncated form of Notch inhibits the R7 precursor which acquires R7 fate; instead, it turns into an additional cone cell. The intracellular domain induces the activation of Mdelta, a bHLH member of the E(spl) complex. In contrast, an ankyrin repeat transgene suppresses ectopic Mdelta expression. It is thought that the suppression of Mdelta by ANK repeats does not reflect a dominant negative behavior associated with an overexpressin of the ANK repeats, and instead suggests that ankyrin repeats exert their action independent of Su(H). Su(H) binds to both a subtransmembrane region that excludes the ankyrin repeats; it also binds the ANK repeats themselves. However, a peptide encompassing just the ANK repeats does not bind independently to Su(H) but is capable of binding to Deltex. The ANK repeats also mediate homotypic interactions, a property that may underly the biological function of the repeats. The simplest way to explain the non-autonomous, antagonistic action of the ANK sequences is to suggest that the ANK-expressing cells down-regulate the endogenous Delta activity. These results suggest the existence of yet unidentified Notch pathway components (Matsuno, 1997).

The Drosophila deltex gene regulates Notch signaling in a positive manner, and its gene product physically interacts with the intracellular domain of Notch through its N-terminal domain. Deltex has two other domains that are presumably involved in protein-protein interactions: a proline-rich motif that binds to SH3-domains, and a RING-H2 finger motif. Using an overexpression assay, the functional involvement of these Deltex domains in Notch signaling has been analyzed. The N-terminal domain of Deltex that binds to the CDC10/Ankyrin repeats of the Notch intracellular domain is indispensable for the function of Deltex. A mutant form of Deltex that lacked the proline-rich motif behaves as a dominant-negative form. This dominant-negative Deltex inhibits Notch signaling upstream of an activated, nuclear form of Notch and downstream of full-length Notch, suggesting that the dominant-negative Deltex might prevent the activation of the Notch receptor. Deltex forms a homo-multimer, and mutations in the RING-H2 finger domain abolish this oligomerization. The same mutations in the RING-H2 finger motif of Deltex disrupts the function of Deltex in vivo. However, when the same mutant is fused to a heterologous dimerization domain (Glutathione-S-Transferase), the chimeric protein has normal Deltex activity. Therefore, oligomerization mediated by the RING-H2 finger motif is an integral step in the signaling function of Deltex (Matsuno, 2002).

A proline-rich motif in the middle region of Deltex shows homology to a consensus amino acid sequence of a binding site for SH3-domain proteins. Indeed, human Grb-2, an SH3-domain protein, binds to Deltex. Deltex lacking the proline-rich motif (Dx^DeltaPRM) behaves as a dominant-negative form. Based on these observations, it is speculated that an as-yet-unidentified SH3-domain protein interacts with the proline-rich motif of Deltex and is an integral part of Deltex activity (Matsuno, 2002).

Nonetheless, the mechanism of the dominant-negative action of this mutant Deltex remains to be elucidated. Because proline-rich motifs are also found in the human, chicken and mouse Deltex homologs, the underlying mechanisms of this dominant-negative behavior may be evolutionarily conserved. The expression of Deltex domain I fragment (amino acids 1-303), which lacks approximately two-thirds of the C-terminal region of the molecule, rescues a loss-of-function deltex phenotype and does not show dominant-negative function. Therefore, in addition to the absence of the proline-rich motif, the presence of some other part(s) of the Deltex domain II-III is required for the Dx^DeltaPRM mutant to act as a dominant-negative form of the Deltex protein (Matsuno, 2002).

While Dx^DeltaPRM behaves as a dominant-negative protein during wing margin development, overexpression of Dx^DeltaPRM under the control of a heat-shock promoter during early embryogenesis does not result in a neurogenic phenotype, which is an indication that Notch signaling was not disrupted. Therefore, the dominant-negative action of Dx^DeltaPRM may depend on the developmental context of cells, although the cellular component(s) responsible for this context-dependence remains to be identified. In this regard, it is noteworthy that none of the existing deltex alleles show the neurogenic phenotype (Matsuno, 2002).

Deltex forms homo-oligomers, and this oligomerization is integral for Deltex function. GST-mediated dimerization can substitute for the function of the Deltex RING-H2 finger motif. The activity of Dx^mRZF+GST does not seem to be neomorphic, because the loss-of-function deltex phenotype is rescued by the expression of Dx^mRZF+GST. Furthermore, the partial loss of wing veins was observed, which resembles the phenotypes of gain-of-function Notch mutants, or is also seen under the circumstances of constitutive activation of the Notch signal. However, the fusion of wild-type Deltex to GST (Dx^full+GST) does not show substantial activity in the system examined. Therefore, the GST-mediated dimerization may abolish the activity of wild-type Deltex; it is also possible that Dx^full+GST is not functional because the fusion to GST leads to some nonspecific disruption of the protein structure. Sequential oligomerization taking place in the proper order may be also important for Deltex function. However, the factor that might relieve Deltex from its inhibitory oligomeric state remains to be identified (Matsuno, 2002).

The loss-of-function deltex phenotype can be rescued by the expression of an activated form of Notch. This observation suggests that Deltex might act upstream of the activated form of Notch. The present study shows that the dominant-negative form of Deltex acts upstream of an activated form of Notch and downstream of wild-type Notch. Although care must be taken in using a dominant-negative form of a protein to speculate about an epistatic relationship, the above two results are consistent. Therefore, it is speculated that this dominant-negative form of Deltex may inhibit the activation or maturation of the Notch receptor. For example, possible target steps include the ligand-dependent cleavage of Notch, the processing of Notch to its mature form or the ligand susceptibility of Notch. Alternatively, it is possible that the dominant-negative Deltex specifically decreases the stability of full-length Notch (Matsuno, 2002).

Overexpression of Dx^full induces an ectopic wing margin-like structure, which is similar to the consequence of the ectopic expression of N^act. However, these two proteins appear to have distinct inductive properties in the wing pouch. Dx^full induces SOPs only in the ventral compartment of the wing pouch, while N^act induces SOPs in both the dorsal and ventral compartments. Furthermore, Dx^full induces SOPs in cells other than and distant from those expressing Dx^full. From these results, it is speculated that induction of Serrate may be a part of these events. N^act has been shown to induce Serrate within the wing pouch, and Serrate effectively activates Notch only in the ventral compartments. The activation of Notch results in the Wg induction that in turn induces SOPs in the neighboring cells. Furthermore, high-level expression of Serrate autonomously inhibits the induction of the genes within the wing pouch that are dependent upon Notch signaling. Thus, the induction of Serrate would explain, at least in part, the result that Dx^full induces SOPs only in the ventral compartment, and ectopic SOPs are formed slightly removed from the cells expressing Dx^full (Matsuno, 2002).

The dominant-negative behavior of Dx^DeltaPRM suggests that putative factor(s) that interact with the proline-rich motif might be essential for Deltex function. Suppressor of deltex [Su(dx)] is a good candidate. Su(dx) genetically suppresses deltex and Notch mutant phenotypes and encodes an E3-ubiquitin ligase. Su(dx) has WW domains that bind to proline-rich motifs in general. In a mammalian system, a mammalian homolog of Su(dx), Itch, binds to the intracellular domain of Notch and ubiquitinates it. Therefore, Deltex may function to suppress Su(dx), a negative regulator of Notch signaling, through an interaction that may be mediated by the proline-rich motif of Deltex and the WW domain of Su(dx) (Matsuno, 2002).

Drosophila Deltex mediates Suppressor of Hairless-independent and late-endosomal activation of Notch signaling

Notch (N) signaling is an evolutionarily conserved mechanism that regulates many cell-fate decisions. deltex (dx) encodes an E3-ubiquitin ligase that binds to the intracellular domain of N and positively regulates N signaling. However, the precise mechanism of Dx action is unknown. Dx is required and sufficient to activate the expression of gene targets of the canonical Su(H)-dependent N signaling pathway. Although Dx requires N and a cis-acting element that overlaps with the Su(H)-binding site, Dx activates a target enhancer of N signaling, the dorsoventral compartment boundary enhancer of vestigial (vgBE), in a manner that is independent of the Delta (Dl)/Serrate (Ser) ligands or Su(H). Dx causes N to be moved from the apical cell surface into the late-endosome, where it accumulates stably and co-localizes with Dx. Consistent with this, the dx gene was required for the presence of N in the endocytic vesicles. Finally, blocking the N transportation from the plasma membrane to the late-endosome by a dominant-negative form of Rab5 inhibits the Dx-mediated activation of N signaling, suggesting that the accumulation of N in the late-endosome was required for the Dx-mediated Su(H)-independent N signaling (Hori, 2004).

Recent studies suggest that Dx might not participate in the canonical N pathway. In Drosophila, it was suggested that Dx has a Su(H)-independent function in the development of bristles on the notum and the eye. In the current study it was shown that a null mutation of Su(H) prevents N^ICD from activating vgBE, but the same mutation does not interfere with the Dx-dependent activation of the same vgBE construct. This finding indicates that the Dx-induced signaling occurs by a mechanism that is independent of Su(H), although the results do not exclude the possibility that Dx also contributes to Su(H)-dependent N signaling. In contrast, it was also found that vgBE Su(H)m, which has mutations in the Su(H)-binding site, is not activated by either N^ICD or Dx. Thus, it is speculated that Dx signaling is mediated by another factor that recognizes a DNA sequence that overlaps with the Su(H)-binding site. Investigation of another protein that binds to the DNA sequence around the Su(H)-binding site of vgBE may allow the identification of a novel effector protein involved in Dx-mediated N signaling. Based on the mutant phenotypes of dx and Su(H), the Dx-mediated Su(H)-independent pathway is probably critical only in a small subset of N functions in Drosophila, although a null mutation allele of dx has not been reported (Hori, 2004).

Overexpression of Dx depletes N from the apical cell surface and increases the number of endocytic vesicles containing N. Dx extends the half-life of N, although it is not clear whether this is due to the prolonged half-life of the vesicles or to stabilization of the N protein itself inside them. N accumulates in the late-endosomal compartment, which was identified by the Rab7-GFP marker. Several models could explain this accumulation of N. (1) Dx may promote the initiation of endocytic vesicle formation. However, this is thought unlikely, because no increase in N-containing vesicles is observed at the early stage of hs-N⁺-GV3 [a heat-shock promoter-inducible chimeric protein of N that contains GAL4-VP16 (GV) inserted after the transmembrane domain in an otherwise wild-type N protein] turnover. (1) Dx may interfere with membrane-trafficking, consequently preventing N from becoming degraded, or sustaining the half-life of vesicles containing N. There is accumulating evidence that the degradation of many transmembrane receptors, which leads to the downregulation of signaling, occurs in the lysosome. Thus, it is speculated that Dx interferes with the delivery of N to the lysosome. In dx mutant cells, a reduced number of N-containing vesicles is observed; this is consistent with the idea that in wild-type cells, Dx also prevents N from relocating to the lysosomes, where it would be degraded. In Drosophila, it is known that Scabrous and Gp150, which localize to the late-endosome, negatively regulate N signaling; however, whether there is any functional relationship between Dx and these proteins remains to be studied. In addition, Dx may play a role in receptor recycling, another process known to involve protein sorting to multivesicular bodies (MVBs), given that N at the apical plasma membrane is significantly depleted by Dx overexpression. However, the precise functions of Dx in these poorly understood processes remain to be addressed (Hori, 2004).

It is known that receptor-mediated signaling can be upregulated by the inhibition of receptor degradation by preventing its endosome-to-lysosome delivery. Although Dx overexpression results in the accumulation of N in the late-endosome, the results suggest that this triggers a signaling event that is distinct from canonical N signaling, rather than merely upregulating signaling by increasing the availability of N. Indeed, the consequence of overexpressing full-length N is very different from that of overexpressing Dx. In this respect, it is notable that two contradictory views have been reported regarding the intracellular compartments where Presenilin cleaves N in mammalian cells, although this issue has not been addressed in Drosophila. One view is that the cleavage of N occurs at the plasma membrane, while another is that Presenilin has a low optimal pH, raising the possibility that it is active in the acidic endocytic compartments, such as late-endosomes. This discrepancy can be resolved by a hypothesis that two distinct N signaling pathways are executed in different membrane-bound compartments. Namely, the Su(H)-dependent canonical pathway and the Dx-mediated signaling pathway occur at the plasma membrane and the late-endosome, respectively. However, the biochemical mechanism of N activation in the late-endosomal compartment is virtually unknown. It was also found that the ectopic activation of N signaling associated with Dx overexpression does not depend on the Dl/Ser ligands. However, it has recently been reported that F3/Contactin, a novel ligand for mammalian N, specifically activates Dx-mediated N signaling (Hu, 2003). Therefore, Drosophila Dx may need an F3/Contactin ortholog to activate vgBE. It is possible that the Su(H)-dependent and -independent N pathways are selectively activated by specific sets of N ligands, such as Dl/Ser and F3/Contactin (Hori, 2004).

In Drosophila, the dx wing-margin phenotype is completely suppressed by mutations of Suppressor of deltex [Su(dx)], which encodes a HECT domain E3 ubiquitin ligase, and this product binds to the intracellular domain of N. Indeed, itch, a mouse homolog of Su(dx), binds to the intracellular domain of mouse notch-1 through its WW domains and promotes the ubiquitination of N. Recently, it was shown that the mono-ubiquitination of transmembrane proteins facilitates their incorporation into endocytic vesicles and lysosomal delivery. Given that Dx is also an E3 ubiquitin ligase and affects membrane trafficking, a balance between Dx and Su(dx) activity may be important for controlling the rate of lysosomal delivery. Studies in progress should increase the understanding of the trafficking of N protein, which is probably a pivotal element in both the positive and negative regulation of N signaling (Hori, 2004).

Structure and Notch receptor binding of the tandem WWE domain of Deltex

Deltex is a cytosolic effector of Notch signaling thought to bind through its N-terminal domain to the Notch receptor. The structure of the Drosophila Deltex N-terminal domain contains two tandem WWE sequence repeats. The WWE repeats, which adopt a novel fold, are related by an approximate two-fold axis of rotation. Although the WWE repeats are structurally distinct, they interact extensively and form a deep cleft at their junction that appears well suited for ligand binding. The two repeats are thermodynamically coupled; this coupling is mediated in part by a conserved segment that is immediately C-terminal to the second WWE domain. Although the Deltex WWE tandem is monomeric in solution, it forms a heterodimer with the ankyrin domain of the Notch receptor. These results provide structural and functional insight into how Deltex modulates Notch signaling, and how WWE modules recognize targets for ubiquitination (Zweifel, 2005).

Surface features provide some clues as to how domain 1 of Deltex recognizes targets such as the Notch ankyrin domain. Deltex domain 1 has large patches of positive charge on its surface, reflecting the high pI of this protein, whereas the Notch ankyrin domain has substantial negative charge on its surface, reflecting its low pI. Thus, the heterodimeric Notch-Deltex complex may be stabilized electrostatically. Enhancement of binding by charge-charge interaction is supported by observation that the Kd decreases by a factor of 5 when the sodium chloride concentration is decreased from 300 to 200 mM. Enhancement of binding affinity may also be provided by the proposed Deltex oligomerization mediated by domain, which may allow domain 1 to form a polyvalent complex with membrane-bound Notch receptors (Zweifel, 2005).

Another surface feature of domain 1 of Deltex suggestive of binding is a large cleft formed between the two WWE modules. The floor of this cleft is made primarily from beta strands 1 and 2 from the first module and from the short alpha helix of the second module. The sides of the cleft are made from one end of the long alpha helix of the first module, the loop connecting beta strands 3 and 4 in the first WWE module, and the loop connecting beta strands 1 and 2 in the second WWE module. The walls of the cleft are composed of polar and charged residues, whereas the floor of the cleft is composed largely of nonpolar residues. Many of the residues lining this cleft are conserved, either among all WWE sequences or among Deltex-specific WWE sequences. One of the Deltex-specific Trp-Glu-Arg motifs of the second WWE module is contiguous with one end of this cleft, also suggesting a role in molecular recognition. This cleft appears to be well suited for binding to an extended polypeptide segment. Although the first ankyrin repeat of the Notch receptor, which is disordered in the crystal structure and contributes little to the structural stability of the Notch ankyrin domain, would fit into this cleft, velocity AUC demonstrates that a Notch ankyrin construct lacking the first repeat retains at least some ability to bind to domain 1 of Deltex. Thus, if the first repeat is involved in binding to this cleft, it is not the sole determinant of binding. Ankyrin repeats 2-7 of the Notch receptor form a rigid domain that, although extended, is not narrow enough to fit into the Deltex cleft described above. Given the large number of irregular surface features on Deltex, including clusters of conserved and basic residues, it seems likely that the Notch-Deltex domain 1 interaction may be mediated by contacts outside this large cleft, and that the cleft may be involved in forming higher order complexes with other components of the Notch pathway (Zweifel, 2005).

Drosophila HOPS and AP-3 complex genes are required for a Deltex-regulated activation of Notch in the endosomal trafficking pathway

DSL ligands promote proteolysis of the Notch receptor, to release active Notch intracellular domain (N^ICD). Conversely, the E3 ubiquitin ligase Deltex can activate ligand-independent Notch proteolysis and signaling. This study shows that Deltex effects require endocytic trafficking by HOPS (homotypic fusion and vacuole protein sorting consisting of VPS41, VPS33A, VPS18) and AP-3 (δ, μ3A, β3A) complexes. The data suggest that Deltex shunts Notch into an endocytic pathway with two possible endpoints. If Notch transits into the lysosome lumen, it is degraded. However, if HOPS and AP-3 deliver Notch to the limiting membrane of the lysosome, degradation of the Notch extracellular domain allows subsequent Presenilin-mediated release of N^ICD. This model accounts for positive and negative regulatory effects of Deltex in vivo. Indeed, HOPS/AP-3 contributions to Notch signaling were uncover during Drosophila midline formation and neurogenesis. Ways are discussed in which these endocytic pathways may modulate ligand-dependent and -independent events, as a mechanism that can potentiate Notch signaling or dampen noise in the signaling network (Wilkins, 2008).

A model of Notch activation involving ligand-directed regulated intramembrane cleavage (RIP) at the cell surface has recently been complicated by several reports showing that Notch endocytosis can precede signal activation. Dx is an E3 ubiquitin ligase, which is required for the full activity of Notch in a subset of developmental contexts including the formation of the wing margin. Dx binds to the Notch intracellular domain and its overexpression promotes Notch endocytosis and endocytic-dependent Notch activation, which in the wing results in ectopic margin. This phenotype was used to genetically screen for components required for Dx-induced Notch signaling, and a number of proteins were identified that comprise the HOPS and AP-3 complexes. In Drosophila the proteins encoded by these genes are best known for their role in biogenesis of lysosomal-related pigment granules that contribute to eye color, but they are also involved in the biogenesis of lysosomes, and autophagy (Lloyd, 1998; Pulipparacharuvil, 2005; Lindmo, 2006; Falcon-Perez, 2007). Their involvement in activating a developmental signaling pathway has not been demonstrated previously. Mutations in these components together with manipulations of early/late endosomal trafficking, and immunolocalization studies, were ised to dissect the endocytic Notch activation pathway and to show how the delivery of Notch to the late endosome/lysosome may both activate and downregulate Notch signaling (Wilkins, 2008).

The results suggest that Dx acts to promote entry of full-length Notch into the endosomal trafficking pathway, and also to direct Notch to the late endosome/lysosome limiting membrane by preventing its sorting into the internal compartments. This would allow the Notch intracellular domain to remain cytoplasmically accessible and available for signaling. It is proposed that in this location the Notch extracellular domain is subject to proteolytic degradation, being the only part of Notch that would be exposed to the internal lysosomal lumen. The resulting membrane tethered, truncated product would then be a substrate for intramembrane proteolysis by Psn, which is known to be present and active in the limiting lysosomal membrane. This would release the Notch intracellular domain for trafficking to the nucleus and signal activation. Consistent with this model, it was found that Dx induced the accumulation of extracellular domain-truncated fragments of Notch in a plane that is 3 μm below the adherens junction. The associated Notch signaling was also shown to be Psn-dependent. Dx promoted the ubiquitination of Notch and it is likely that this covalent modification controls one or both of the outcomes of Dx activity, as ubiquitination has been associated with many sorting steps in protein trafficking pathways (Wilkins, 2008).

HOPS and AP-3 gene mutations blocked Dx-dependent Notch signal activation. The HOPS complex mediates the progression of early endosomal Rab5 positive vesicles to late endosomal Rab7 positive vesicles (Rink, 2005). The AP-3 complex acts as an adaptor in the early endosome and the Golgi, which recognizes sorting signals such as di-leucine motifs and recruits integral membrane proteins for trafficking to the limiting membranes of lysosomes and related organelles (Peden, 2004; Theos, 2005). It is possible that AP-3 contributes to Notch signaling by ensuring delivery of Notch to the lysosomal limiting membrane, or by allowing the proper lysosomal localization of a membrane protein component that is required for endosomal Notch activation at that location. The data show that reducing AP-3 function leads to an accumulation of Notch in an enlarged tubular compartment associated with the early endosome, consistent with the former explanation (Wilkins, 2008).

Alternative locations for Notch endosomal activation should also be considered however. For example the apparent requirement for HOPS and AP-3 genes for Notch signaling could result from the failure of the degradative removal of nonactivated full-length Notch. In this explanation AP-3 and HOPS mutations may lead to the forced accumulation of full-length Notch in an early endocytic compartment, where it may sequester factors required for the trafficking and Psn-dependent cleavage of ligand-activated Notch. This might explain the dominant-negative effect on endogenous Notch signaling that results from the expression of Dx in an AP-3 or HOPS mutant background. However a number of the results argue against this alternative explanation. First, even though strong early endosomal accumulation of Notch is induced by Rab5 expression, there is no measurable effect on the endogenous Notch signal. Second, the coexpression of Rab7QL with Rab5 activates Notch signaling and this is associated with relocalization of Notch to late endosomal compartments. In addition car¹ mutants suppress the latter ectopic Notch signal, but this does not produce a dominant-negative effect on the endogenous Notch signal. Finally Su(dx) coexpression blocks the Dx-induced signal without causing early endosomal accumulation. Instead it redirects Notch away from the limiting membrane into the center of late endosomal compartments, consistent with the model of endosomal Notch activation (Wilkins, 2008).

The developmental role of Dx has been ambiguous because different studies have proposed that it acts either positively or negatively on Notch signaling. However the current model can now account for these diverse observations. It is possible that combinations of ubiquitin tags on Notch result in a hierarchy of control at different steps in the trafficking pathway that determine a positive or negative outcome on Notch activity. Alternatively Su(dx) might modify the activity of an as yet unknown trafficking regulator. It was not possible to detect Su(dx)-dependent Notch ubiquitination using the N^TAP pull-down assay, however this may be due to deubiquitination of Notch during its transfer to the late endosomal internal lumen (Mukhopadhyay, 2007). Further work will be required to understand the biochemical basis of this combinatorial regulation (Wilkins, 2008).

Endocytic activation of Notch that occurs ectopically when endosomal sorting is disrupted has previously been shown to be largely independent of DSL ligands. It has also shown that signaling induced by overexpression of Dx is independent of DSL ligands (Hori, 2004). This study shows that reducing HOPS and AP-3 function does not reduce Notch activation by ectopic ligand expression, but does block the Dx-induced signal, suggesting the existence of two different activation mechanisms. The possibility that endogenous ligand-initiated signaling can, in part, involve Psn-mediated cleavage in the endosomal pathway is not, however, excluded. Once ligand promoted ectodomain shedding occurs at the cell surface, the remaining membrane-tethered Notch will become accessible for activation by metalloprotease-mediated S2 and Psn-mediated S3 cleavages. This may occur at the cell surface, but some Notch may enter the endosomal pathway and remain available for activation (Kanwar, 2008; Vaccari, 2008). It is possible that, following ectodomain shedding, the endosomal entry of membrane-tethered Notch intracellular domain is promoted by Dx. The lack of sensitivity of signaling induced by ectopically expressed ligands to carnation and ruby mutations may be because most ligand-induced Psn cleavage of Notch already occurs before Notch is trafficked to the late endosomes. A contribution cannot however be rule out of the late endosomal location in generating a proportion of the endogenous ligand-induced signal. In contrast, when Dx is overexpressed, full-length Notch is removed from the ligand-accessible pool and ectodomain shedding and Notch activation may not then occur until after the AP3- and HOPS-mediated transfer to the late endosomal/lysosomal pool making Dx-induced signaling critically dependent on this step. At present, however, the amount of signal generated through endocytosis of full-length Notch driven by endogenous Dx cannot be assessed (Wilkins, 2008).

The viability of severe or null AP-3 gene mutations (Ooi, 1997; Mullins, 1999; Mullins, 2000) and examination of mitotic clones of the null dor⁸ HOPS mutant (Sevrioukov, 1999) demonstrates that neither complex is essential for Notch signaling. However, the data suggest that several HOPS and AP-3 components are required to maintain full Notch signaling levels. This may occur through a proportion of ligand-activated, ectodomain shed Notch reaching the late endosome, or through the Dx-driven endocytosis of full-length Notch. It was observed that mutations of lt, rb, cm, and dx led to reduction in expression of sim, a Notch target gene activated during midline formation. Neurogenic phenotypes, which would be expected from a failure of Notch-mediated lateral inhibition, were also observed. Interestingly, mixed phenotypes consisting of both expansion and loss of nervous system were also observed. Although the reason for loss of neurons is not yet resolved, it is interesting to speculate whether these variable phenotypes could be accounted for by endocytic trafficking contributing both negatively and positively to Notch activation. In this model, endocytic trafficking will promote the Dx-regulated activation of Notch and also reduce the flux through the canonical DSL ligand-driven pathway by decreasing the ligand accessibility of Notch. The overall contribution of Dx may result from a balance of these opposing effects and this may act to smooth out noise in the Notch signaling levels. If such a smoothing function was not in place then fluctuations might, in some embryos, be amplified by feedback mechanisms beyond acceptable upper or lower thresholds resulting in the mixed phenotypes that were observed. Interestingly the presence of the wild-type Dx/HOPS/AP-3 pathway appears to provide a compensation mechanism that becomes more critical when development proceeds at higher temperatures (Wilkins, 2008).

Genetic interactions of Notch alleles with loss-of-function mutations in HOPS and AP-3 complex genes indicate that, like Dx, these complexes can contribute to endogenous levels of Notch activity at the wing margin. No wing margin defects were observed in HOPS or AP-3 mutants in the absence of such genetic interactions, however. Nor were wing margin phenotypes obsered in mitotic clones of dor8. It is not clear why HOPS, AP-3, and dx mutants can display similar effects in embryo development but only dx displays a wing margin phenotype. It is possible that the latter results, in part, from additional activities of Dx. Alternatively, other factors may partly substitute for AP-3 and HOPS function in some tissues, as has been proposed to explain the formation of some pigment granules in null rb¹ mutants (Mullins, 2000). Examples of such redundancy in trafficking routes have also been documented in mammalian cells. In contrast, dx wing phenotypes were enhanced by mutations in other components of the endocytic pathway. This result could be explained if entry of Notch into the endocytic pathway is not completely removed in the absence of dx, as has been previously observed. Interestingly despite both rb¹ and cm¹ being null mutations in different AP-3 complex components, the phenotypes of cm¹ were less severe. Although moderating influences of genetic background cannot be ruled out, other recent work has also shown phenotypic differences between cm¹ and rb¹ (Simonsen, 2007), suggesting that the developmental requirement of the two genes is not equivalent. Further work will now be required to establish the relative contributions for all the different components of the HOPS and AP-3 complexes and their associated proteins (Wilkins, 2008).

Given that the AP-3/HOPS activation pathway is one way in which Notch signaling can acquire ligand-independence, it should now be considered whether Notch can be ectopically activated by this route in some Notch-dependent tumors. If such tumors are identified, targeting HOPS and AP3 components may preferentially affect the tumor, while sparing normal signaling. Many other proteins have been shown to undergo regulated membrane proteolysis following ectodomain shedding. The finding that the late endosome/lysosome can activate this process for Notch may have implications for understanding the mechanisms of signaling of other developmentally and pathologically relevant membrane receptors (Wilkins, 2008).

DEVELOPMENTAL BIOLOGY

Effects of Mutation or Deletion

All deltex mutant alleles behave as recessive viable mutants with affected wing, ocellar and eye morphology. deltex mutants suppress certain Notch mutants and intereact with delta and mastermind in a similar fashion (Xu, 1990 and Gorman, 1992).

The Notch receptor signaling pathway regulates cell differentiation during the development of multicellular organisms. A number of genes are known to be either components of the pathway or regulators of the Notch signal. One candidate for a modifier of Notch function is the Drosophila Suppressor of deltex gene [Su(dx)]. Four new alleles of Su(dx) have been isolated and the gene has been mapped between 22B4 and 22C2. Loss-of-function Su(dx) mutations were found to suppress phenotypes resulting from Notch loss-of-function signaling and to enhance gain-of-function Notch mutations. Hairless, a mutation in a known negative regulator of the Notch pathway, is also enhanced by Su(dx). Phenotypes were identified for Su(dx) in wing vein development. Homozygous mutant flies for one allele [Su(dx)sp] have a wild-type vein pattern at 25 degrees C. However, when they are kept at 29 degrees, a recessive wing vein gap phenotype appears. The phenotype is manifested most often in veins L.IV and L.V, distal to the posterior cross-vein. Gaps are found frequently in L.II as well, but never in L.III. Three other alleles display wing vein gaps at 29 degrees when placed over Su(dx)sp. At 25°, the same combinations of alleles have intact longitudinal veins, but forked or incomplete cross-veins. A role was demonstrated for the gene between 20 and 30 hr after puparium formation. A temperature upshift after 28 hr after puparium formation allows normal development of the veins. This corresponds to the period when the Notch protein is involved in refining the vein competent territories (Fostier, 1998).

A number of observations indicate that the wild-type function of Su(dx) is as a negative regulator of the Notch pathway. The temperature-sensitive wing vein gap phenotype is similar to that observed for gain-of-function Abruptex alleles of Notch. Complementation tests over the deficiency have shown that the Su(dx) mutants described result in a loss of function of Su(dx). This is an important prerequisite for interpreting the wild-type function of Su(dx). The haplo-insufficient phenotype of Notch is suppressed by Su(dx) mutations, as is the mutation of Delta, the Notch ligand. In contrast, the gain-of-function AxE2 mutation of Notch is enhanced by Su(dx). This is similar to the known genetic interactions of Hairless with these Notch mutants. Hairless is a negative regulator of the Notch pathway, and it functions by binding to and inhibiting Suppressor of Hairless, a Notch-responsive transcription factor. The fact that Su(dx) enhanced the Hairless phenotype indicates that the two genes are regulating the Notch signal in the same direction. Similarly, the observed suppression of deltex is as expected. Because deltex is a positive regulator of Notch function, its mutation should be compensated by a mutant that leads to a hyperactivation of the Notch signal (Fostier, 1998).

Activation of the Notch pathway can be mimicked by ectopic E(spl)mß expression in the wing, which results in gaps in the veins. The strength of this phenotype is dependent on the dosage of the expressed E(spl)mß, and the phenotype is enhanced in a Su(dx) mutant background. It is hypothesized that the Su(dx) mutation leads to an elevation of Notch signaling and increased expression of endogenous E(spl)mß, which augments the ectopically expressed protein levels. However, the alternative possibility that the enhanced phenotype may be caused by an upregulation of the downstream response to the activity of expressed E(spl)mß cannot be ruled out. Support for a negative regulatory function for Su(dx) also comes from comparison of Su(dx) phenotypes with those resulting from ectopic expression of activated Notch and wild-type deltex proteins. It is possible to make a constitutively activated Notch receptor by expressing a truncated form that lacks the extracellular domain. The Notch pathway can also be upregulated by overexpression of wild-type deltex. When activated Notch or wild-type deltex are expressed under control of a heat shock promoter 0-24 hr APF, a wing vein gap phenotype appears. In both cases, this phenotype is strongly enhanced in a heterozygous nd (a recessive Notch allele with a wing margin loss phenotype that is similar to the loss of one copy of Notch) background, similar to the interaction between Su(dx) mutants and nd. Thus, the Su(dx) mutation mimics an elevation of the Notch signal. Taken together, these data indicate a role for Su(dx) as a negative regulator of the Notch pathway. The existence of feedback regulatory loops in the control of Notch signaling makes the position of Su(dx) protein in the Notch pathway difficult to define through genetic analysis. Su(dx) mutants were first identified through their interaction with deltex. It cannot be concluded that the corresponding proteins interact directly, however, especially as there are genetic interactions between Su(dx) and a number of Notch pathway genes. The precise function of Su(dx) will only be resolved through cloning of the gene and analysis of its function at the molecular level, which is in progress. It is likely, therefore, that the further characterization of Su(dx) and its interacting mutations will be fruitful for the understanding of Notch pathway regulation (Fostier, 1998).

Formation of mechano-sensory organs in Drosophila involves the selection of neural precursor cells (SOPs) mediated by the classical Notch pathway in the process of lateral inhibition. The subsequent cell type specifications rely on distinct subsets of Notch signaling components. Whereas E(spl) bHLH genes implement SOP selection, they are not required for later decisions. Most remarkably, the Notch signal transducer Su(H) is essential to determine outer but not inner cell fates. In contrast, the Notch antagonist Hairless, thought to act upon Su(H), influences strongly the entire cell lineage, demonstrating that it functions through targets other than Su(H) within the inner lineage. Thereby, Hairless and Numb may have partly redundant activities. This suggests that Notch-dependent binary cell fate specifications involve different sets of mediators depending on the cell type considered (Nagel, 2000).

The decision between the tormogen (socket) versus trichogen (shaft) fate of the pIIa progeny seems to depend strictly on the balanced doses of H and Su(H). Changes in the dose of either one pushes the equilibrium completely towards the opposite fate. Accordingly, Su(H) protein accumulates to very high levels in the future tormogen, and can thus override the elevated levels of H protein within this cell. The epistasis of H over dx regarding outer bristle cell fates can be easily explained by the dominating activity of Su(H) within the pIIa progeny. The choice between neuron and thecogen (sheath) fate is based on a quite different mechanism, because unlike H, Su(H) is not necessary for the emergence of the two opposing cell types. The default state of pIIIb descendants is neuronal. The Notch signal redirects one of these cells into thecogen fate. Although both Su(H) and dx, positively influence Notch signaling in the presumptive thecogen, none of the two is required for the generation of this cell type. Thus, the Notch signal in the thecogen might be transduced by a molecular mechanism independent of Su(H) or dx involving as yet unknown factor(s). In the absence of H, the presumptive neuron gains thecogen fate. Therefore, H has an important role in protecting the neuron from the Notch signal. Since this signal does not emanate from Su(H), H must act through unknown component(s). This is the first unambiguous example of a Su(H)-independent function of H (Nagel, 2000).

In Drosophila, Suppressor of deltex [Su(dx)] mutations display a wing vein gap phenotype resembling that of Notch gain of function alleles. The Su(dx) protein may therefore act as a negative regulator of Notch but its activity on actual Notch signalling levels has not been previously demonstrated. Su(dx) is shown to regulate the level of Notch signalling in vivo, upstream of Notch target genes and in different developmental contexts, including a previously unknown role in leg joint formation. Overexpression of Su(dx) is capable of blocking both the endogenous activity of Notch and the ectopic Notch signalling induced by the overexpression of Deltex, an intracellular Notch binding protein. In addition, using the conditional phenotype of the Su(dx)^sp allele, it has been shown that loss of Su(dx) activity is rapidly followed by an up-regulation of E(spl)mß expression, the immediate target of Notch signal activation during wing vein development. While Su(dx) adult wing vein phenotypes are quite mild, only affecting the distal tips of the veins, the initial consequence of loss of Su(dx) activity is more severe than previously thought. Using a time-course experiment it has been shown that the phenotype is buffered by feedback regulation illustrating how signalling networks can make development robust to perturbation (Mazaleyrat, 2003).

To begin to unravel the mechanism of action of Su(dx), it is an important prerequisite to establish whether Su(dx) acts on the Notch pathway itself, or whether the genetic interactions observed reflect an indirect, parallel, or downstream activity. The data argue that Su(dx) can indeed negatively regulate Notch signalling, upstream of the immediate Notch target genes. (1) It has been shown, using the temperature sensitivity of the Su(dx)^sp wing vein gap phenotype, that Su(dx) loss of function is rapidly followed by the up-regulation of E(spl)mß expression in the pupal wing. (2) In third instar wing imaginal discs, it has been shown that in two enhancing genetic backgrounds, Su(dx) loss of function results in the up-regulation of wingless, another Notch target gene at the D-V boundary. (3) Su(dx) overexpression in the wing imaginal disc is capable of down-regulating the Notch-dependent expression of three genes, wingless and cut at the D-V boundary, and the vg^BE-LacZ element at both the D-V and the A-P boundaries. These data show that Su(dx) is capable of downregulating Notch in different developmental contexts and that its activity on Notch is not limited to the particular situation of wing vein development (Mazaleyrat, 2003).

Su(dx) is capable of blocking the stimulation of Notch signalling, which is induced by the overexpression of Deltex, a regulatory protein which binds to the Notch intracellular domain. Thus these data suggest that the activity of Su(dx) lies upstream of the regulation of Notch target gene expression but downstream of, or at the level of, Deltex. This, together with the rapidity of the response of increased Notch signalling that is observed following Su(dx) loss of function, supports the hypothesis that Su(dx) acts directly on the Notch pathway. In vivo data are thus consistent with the in vitro observation that a related mammalian Nedd4 family protein, Itch, can promote the ubiquitination of the Notch1 intracellular domain (Mazaleyrat, 2003).

Interestingly while Deltex expression does not block the Notch down-regulatory activity of Su(dx), it does inhibit the latter’s wing overgrowth phenotype. This uncoupling of phenotypes suggests that Su(dx) has multiple activities. One activity down-regulates the Notch signal and thus blocks the ectopic wing margin and wing growth phenotype induced by Deltex overexpression. The overexpression of Deltex may in turn titrate Su(dx) away from a second activity responsible for a distinct wing overgrowth phenotype. This could explain how the coexpression of these two proteins fails to produce a wing overgrowth when the expression of each singly does result in an overgrowth phenotype (Mazaleyrat, 2003).

Regulation of Notch signalling by non-visual ß-arrestin: a trimolecular interaction between Notch, Deltex, and Kurtz

Signalling activity of the Notch receptor, which plays a fundamental role in metazoan cell fate determination, is controlled at multiple levels. A Notch signal-controlling mechanism was uncovered that depends on the ability of the non-visual ß-arrestin, Kurtz (Krz), to influence the degradation and, consequently, the function of the Notch receptor. Krz was identified as a binding partner of a known Notch-pathway modulator, Deltex (Dx), and the existence was demonstrated of a trimeric Notch-Dx-Krz protein complex. This complex mediates the degradation of the Notch receptor through a ubiquitination-dependent pathway. These results establish a novel mode of regulation of Notch signalling and define a new function for non-visual ß-arrestins (Mukherjee, 2005).

In an effort to identify elements that are integrated into the molecular circuitry affecting Notch signalling, two independent protein-interaction screens were carried out: one based on the yeast two-hybrid system, and the other based on the identification of cellular protein complexes using the tandem affinity purification (TAP)-liquid chromatography (LC)-mass spectrometry (MS)/MS approach. Both methods identified Krz as an interacting partner of Dx (Mukherjee, 2005).

A yeast two-hybrid screen was carried out using full-length Dx as bait. Eight positive clones were isolated and found to encode overlapping krz cDNAs. Sequence analysis revealed that the amino-terminal half of Krz (amino acids 10-251) is necessary and sufficient for binding Dx. The corresponding domain of mammalian non-visual ß-arrestins, which consists of the amino-terminal half of the protein, has been shown to interact with activated GPCRs (Mukherjee, 2005).

Four Krz peptides (VGEQPSIEVSK, VFELCPLLANNK, HEDTNLASSTLITNPAQR and ESLGIMVHYK) were also identified, using LC-MS/MS, among proteins in the 50,000-55,000 relative molecular mass range that co-purified with full-length amino-terminally TAP-tagged Dx (NTAP-Dx) that was isolated from stably transfected Kc167 cells. These peptides correspond to endogenous Krz protein (with a predicted relative molecular mass of 51,200) that is expressed at normal levels. Krz was also identified as a Dx-interacting partner in an independent experiment involving another cell line (S2) that was stably transfected with the NTAP-Dx transgene (Mukherjee, 2005).

To address the functional implications of the association between the Krz and Dx proteins, an investigation was carried out to see whether mutations in krz and dx display genetic interactions. Two independent loss-of-function dx alleles, dx and dx^SM were used, and two independent krz loss-of-function alleles, krz¹ and krz². A transheterozygous combination of dx and krz alleles (dx/+; krz/+ females) resulted in wings that were indistinguishable from the wild type. However, reducing the dose of krz in a genetic background that further reduces or eliminates dx (in dx/Y; krz/+ males) elicited enhanced wing notching and vein thickening, compared with dx hemizygotes in a krz wild-type background. Similar results were obtained using two other dx alleles, dx^ENU and, importantly, a recently identified dx null allele, dx¹⁵². Given that the genetic interaction between dx and krz was observed in the absence of all dx functions, it is clear that a complete absence of dx creates a sensitized genetic background that makes development of the wing margin sensitive to a decrease in the dosage of krz (Mukherjee, 2005).

To extend the analysis of the interactions between Krz, Dx and Notch, the relative subcellular localization of these proteins was tested when they were co-expressed in cultured cells. Immunocytochemical analysis revealed that the expression of either HA-Krz or Flag-Dx alone resulted in a diffuse distribution throughout the cytoplasm. By contrast, co-expression of both proteins led to a redistribution of Krz and Dx into intracellular vesicles, where they co-localized. Colocalization of co-expressed Krz and Dx was also observed in vivo in the wing imaginal discs. The nature of these vesicles remains to be determined, but several known intracellular trafficking markers (which label early and late endosome compartments, the Golgi apparatus and the endoplasmic reticulum) did not seem to co-localize with the Krz and Dx proteins (Mukherjee, 2005).

To probe the functional significance of an interaction between Krz, Dx and Notch in vivo, the effects of krz loss of function on the endogenous Notch receptor were examined. To this end, krz loss-of-function clones were generated in two different tissues, the wing and eye-antennal imaginal discs, using the krz¹-null mutant and the FLP/FRT system. It was found that the levels of the Notch protein, normally expressed throughout these discs, were substantially elevated in krz mutant cells compared with the surrounding wild-type cells. This increased level of Notch was observed in both the wing and eye-antennal discs. It is noted, however, that in the eye discs, this elevated level of Notch was more prominent in krz clones that were located anterior to the morphogenetic furrow. In contrast with the upregulation of Notch, the levels of Dx were unaltered in krz mutant clones (Mukherjee, 2005).

The present study has revealed the existence of a hitherto unknown Notch-signal controlling mechanism that relies on modulating Notch-receptor levels through the activity of the krz gene that encodes the single non-visual ß-arrestin in Drosophila. Consequently, this analysis unveils a new role of ß-arrestins as regulators of Notch signalling. Mammalian non-visual ß-arrestins were originally thought to function exclusively in the desensitization and clathrin-mediated internalization of GPCRs. The range of ß-arrestin activity has been recently extended by uncovering their involvement in the regulation of other receptor systems. The data presented in this study further extend the spectrum of ß-arrestin functions, given the demonstration that the Drosophila non-visual ß-arrestin, Krz, can modulate the protein levels of the Notch receptor and, consequently, Notch signalling. This analysis indicates that the interaction between Krz and Notch is mediated by Dx (Mukherjee, 2005).

The biochemical nature of Dx and its full spectrum of activities are not yet fully understood. dx was first implicated in Notch signalling as a modifier of Notch phenotypes. Indirect evidence implied that Dx may have a role in the transcriptional regulation of Notch targets. Additional studies postulated that dx may define a node in the Notch-signalling pathway that is independent of Suppressor of Hairless (CBF1 in mammals), the classical effector of Notch signals. In mammals, Deltex seems to be an antagonist of Notch signals. However, overexpression of Dx in Drosophila can mimic the phenotypes that are associated with Notch gain-of-function mutations, and loss of dx function results in wing-margin phenotypes that are reminiscent of loss of Notch function, indicating a positive rather than a negative role in Notch signalling. Although the current data do not exclude the possibility that Dx may have a positive role in Notch signalling in certain cellular contexts, the evidence presented in this study unambiguously demonstrates that Dx, in combination with Krz, functions as a negative regulator of Notch. The results of the present analysis, together with the previously published genetic studies, indicate that Dx may behave both as an agonist and as an antagonist of Notch signalling, depending on the specific cellular context (Mukherjee, 2005).

Notwithstanding the lack of direct evidence regarding the biochemical nature of Dx, the fact that Dx contains a RING-H2 and two WWE domains indicates that Dx may function as an E3 ubiquitin ligase. In fact, E3 ubiquitin-ligase activity has been shown to exist for mammalian homologues of Drosophila Dx. Mammalian ß-arrestins have also been implicated in receptor ubiquitination events. A stable association between ß-arrestins and Class B GPCRs was shown to promote receptor ubiquitination and degradation by recruiting E3 ubiquitin ligases, such as Mdm2, to the receptor (Mukherjee, 2005).

This study reproducibly observed a small increase of Notch ubiquitination in the presence of Dx, which was further enhanced following addition of Krz. Previous studies implicated Notch in both poly- and monoubiquitination events. No increase was detected in Notch monoubiquitination following addition of Dx, Krz or both, so an increase in ubiquitination is attributed to polyubiquitination of Notch. This study, therefore, associated the formation of the Notch-Dx-Krz complex with polyubiquitination of the Notch receptor and a subsequent reduction of Notch levels, apparently via proteasomal degradation. The underlying mechanism is unknown at this point, but it is possible that the incorporation of Krz into the Notch-Dx-Krz complex may promote polyubiquitination of Notch by facilitating the ubiquitin-ligase activity of Dx, by recruiting additional E3 ligases or perhaps by inducing an altered conformation of the Notch receptor (Mukherjee, 2005).

It is worth mentioning that additional E3 ligases, such as Suppressor of deltex [Su(dx)] and Nedd4, have been associated with Notch signalling. However, no co-localization of Su(dx) with vesicles containing Krz and Dx was observed following co-transfection of these three proteins in S2 cells. The data support a connection between the formation of the Notch-Dx-Krz complex and the proteasomal rather than the lysosomal degradative pathway. However, an involvement of the Krz-Dx vesicles in the intracellular trafficking of the Notch receptor cannot be excluded, despite the fact that marker analysis has not revealed the identity of these vesicles (Mukherjee, 2005).

It has been documented that non-visual ß-arrestins are involved in trafficking of GPCRs and other types of receptors. Given that krz seems to be the only ß-arrestin in the Drosophila genome, the question is raised as to whether other, non-seven-transmembrane-receptor systems are affected in krz mutant cells. It was asked whether, similar to the Notch receptor, the levels of Frizzled or the epidermal growth factor receptor (EGFR) are affected in loss-of-function krz clones in the wing or the eye imaginal discs, and no change was found in their levels or localization. However, these observations do not exclude the possibility that krz is still involved in the regulation of these and other receptors, as is the case in mammals. If, in mammalian systems, Notch is regulated by a similar mechanism, then loss-of-function mutations in ß-arrestins may result in the upregulation of Notch signals in certain tissues. This would be particularly significant in tissues in which Notch activation has a role in tumorigenesis (Mukherjee, 2005).

Together, the loss-of-function and the complementary gain-of-function analyses indicate that Krz in involved in the regulation of Notch signalling. It is proposed that one of the biological functions of Krz is to modulate the level of the Notch receptor in the cell and thereby to optimize the amount of Notch that can participate in signalling. Such regulation of Notch by Krz is likely to be, at least in part, constitutive and may not require its interaction with a ligand (Mukherjee, 2005).

It seems unlikely that the action of the Drosophila ß-arrestin Krz is confined to the Notch signalling pathway, but further studies will be necessary to establish the spectrum of Krz function. An association of other signalling receptors with Krz would not only link them to non-visual ß-arrestin function, but it would also provide a potential mode of cross-talk with Notch. These links may be important for defining the cellular framework within which controlling mechanisms have evolved to act on evolutionarily conserved signalling pathways such as Notch (Mukherjee, 2005).

Effects of Mutation

Notch is a single-pass transmembrane receptor. The N signaling pathway is an evolutionarily conserved mechanism that controls various cell-specification processes. Drosophila Deltex (Dx), a RING-domain E3 ubiquitin ligase, binds to the N intracellular domain, promotes N’s endocytic trafficking to late endosomes, and has been proposed to activate Suppressor of Hairless [Su(H)]-independent N signaling. However, it has been difficult to evaluate the importance of dx, because no null mutant of a dx family gene has been available in any organism. This study reports the first null mutant allele of Drosophila dx. dx is involved only in the subsets of N signaling, but is not essential for it in any developmental context. A strong genetic interaction exists between dx and Su(H); this suggests that dx might function in Su(H)-dependent N signaling. These epistatic analyses suggested that dx functions downstream of the ligands and upstream of activated Su(H). A novel dx activity has been uncovered that suppresses N signaling downstream of N (Fuwa, 2006).

A null allele of dx, dx¹⁵² was generated to elucidate the functions of the dx gene during development. Homo- and hemi-zygotes of dx¹⁵² are viable and fertile, although the recessive phenotypes coincide with a subset of weak phenotypes observed in N mutants. Thus, it is concluded that dx function is not required for N signaling in any context. These results also show that the involvement of dx in N signaling is tissue-specific. In contrast, it is noted that dx seems to play a role in tissues that are not affected in the dx null mutant. For instance, dx¹⁵² /Y ;; Dl^rev10 /+ shows fusion of the second and third tarsal segments of the foreleg, while neither dx¹⁵² /Y nor Dl^rev10 /+ has this defect. Thus, dx is involved in the development of the tarsal segment. However, even in the null dx background, N signaling activity is normally above the threshold required for wildtype leg development. In the Drosophila genome, no other genes homologous to dx are found. Nevertheless, it is possible that some protein functionally related to Dx compensates for the absence of the dx gene functions. It was also found that the expressivity of dx¹⁵² phenotypes varies greatly among tissues. Compensation by another protein, which could have some tissue specificity, may also account for the variation in the expressivity of dx¹⁵² phenotypes. It is noted that, to the extent they have been tested; the genetic behavior of dx¹⁵² is similar to that of dx²⁴, which has been used in previous studies (Fuwa, 2006).

This study shows that the ability of Dl/Ser ligands to activate N signaling is partially reduced in the wing discs of the dx¹⁵² mutant. This result suggests that dx acts downstream of the Dl/Ser ligands to activate N signaling. When Dx is overexpressed, N signaling is induced independent of the presence of the Dl/Ser ligands. It is therefore possible that artificially elevated levels of Dx somehow overcome the Ser/Dl requirement for N activation. However, it is notable that both Dl and Ser showed a substantial ability to stimulate N signaling in the dx¹⁵² mutant disc, but they failed to activate N signaling in Su(H) mutant clones. Therefore, both dx and Su(H) play roles in ectopic vgBE activation, but while Su(H) is indispensable, Dx is required only for strong signal induction (Fuwa, 2006).

Genetically, dx has been considered a positive regulator of N signaling in Drosophila. Furthermore, mammalian homologues of dx activate the reporter genes of N signaling target genes. Also, it was shown that Dx and a dominant-negative form of Nedd4 activate the E(spl)mγ promoter in Drosophila cultured cells. However, in contrast, a human Dx homolog antagonizes N signaling in cortical neurons. This discrepancy could be explained by the finding that dx both activates and suppresses N signaling (Fuwa, 2006).

In this study, a novel genetic interaction involving dx was uncovered. It has been reported that N shows a dominant lethal interaction with hypomorphic alleles of dx. Here, it was found that Ser^94c shows a dominant lethal interaction with dx¹⁵². This result was unexpected, because Dl, which functions more broadly than does Ser, did not show a lethal interaction. Thus, it is possible that dx and Ser have common tissue specificity. However, the developmental and molecular basis of this interaction between dx and Ser remains to be addressed (Fuwa, 2006).

Another curious genetic interaction was found between dx and Su(H). The wing-vein phenotype of dx¹⁵² was suppressed dominantly by Su(H)^Δ47, a null mutation of Su(H). This dx¹⁵² phenotype is suppressed by a hypomorphic allele of Su(H), Su(H)^SF8. A similar suppression was also reported previously with the combined hypomorphic alleles of dx and Su(H). It is known that Su(H) acts both as a repressor and activator of N target genes. Thus, the lack of this repressor function in Su(H) mutants probably explains the suppression of dx¹⁵² phenotypes in combination with Su(H)/+. It has been demonstrated that Dx ectopically activates vgBE in Su(H) null mutant clones, which suggests Dx is involved in Su(H)-independent N signaling. This study showed that vgBE is activated along the A/P boundary during the late third-instar in a Su(H)- and Dx-independent manner, indicating that this activation is irrelevant to N signaling. However, this activation is not detected during the middle third-instar. Thus, previous experiments, which were carried out in middle third-instar larvae, were not affected by this background activation of vgBE (Fuwa, 2006).

It has been shown that dx modulates the endocytic trafficking of N. However, most experiments in these studies rely on the overexpression of dx. Therefore, the dx null mutant should offer the opportunity to study the requirement for dx in N transportation (Fuwa, 2006).

TRAF6 is a novel regulator of Notch signaling in Drosophila melanogaster

Notch signaling pathway unravels a fundamental cellular communication system that plays an elemental role in development. It is evident from different studies that the outcome of Notch signaling depends on signal strength, timing, cell type, and cellular context. Since Notch signaling affects a spectrum of cellular activity at various developmental stages by reorganizing itself in more than one way to produce different intensities in the signaling output, it is important to understand the context dependent complexity of Notch signaling and different routes of its regulation. This study identified TRAF6 (Drosophila homolog of mammalian TRAF6) as an interacting partner of Notch intracellular domain (Notch-ICD). TRAF6 genetically interacts with Notch pathway components in trans-heterozygous combinations. Immunocytochemical analysis shows that TRAF6 co-localizes with Notch in Drosophila third instar larval tissues. The genetic interaction data suggests that the loss-of-function of TRAF6 leads to the rescue of previously identified Kurtz-Deltex mediated wing notching phenotype and enhances Notch protein survival. Co-expression of TRAF6 and Deltex results in depletion of Notch in the larval wing discs and down-regulates Notch targets, Wingless and Cut. Taken together, these results suggest that TRAF6 may function as a negative regulator of Notch signaling (Mishra, 2014).

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date revised: 15 August 2023

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