Notch

Table of contents

Notch and Suppressor of Hairless

In the development of the socket cell of the mechanosensory organ, Su(H) does not co-localize with Notch and Deltex at the apicolateral membrane. Notch also colocalizes with F-actin at the apex of the socket cell. Instead Su(H) protein appears to be distributed evenly in the cytoplasm of the socket cell. This result is paradoxical since Notch is believed to regulate the cytoplamic retention of Su(H). Implied is a dynamic equilibrium between membrane-associated and cytoplasmic Su(H) that is largely in favor of the accumulation of Su(H) in the cytoplasm (Gho, 1996).

The role of the Notch signaling pathway has been examined in the transcriptional regulation of two Drosophila Enhancer of split [E(spl)] genes. Using a reporter assay system in Drosophila tissue culture cells, a significant induction of E(spl) m gamma and m delta expression is observed after cotransfection with activated Notch. Characterization of the 5' regulatory regions of these two genes led to the identification of a number of target sites for the Suppressor of Hairless [Su(H)] protein, a transcription factor activated by Notch signaling. Su(H) binding sites are present in the upstream regions of both E(spl) genes. Notch-inducible expression of E(spl) m gamma and m delta, both in cultured cells and in vivo, is dependent on functional Su(H). Although overexpression of Su(H) augments the level of induction of the reporter genes by activated Notch, Su(H) alone is insufficient to produce high levels of transcriptional activation. Despite the synergy observed between activated Notch and Su(H), the former affects neither the nuclear localization nor the DNA binding activity of the latter. The behavior of Drosophila Notch is consistent with a mechanism whereby N activates Su(H) by covalent modification. It is unlikely that N functions to sequester Su(H) in the cytoplasm, since Su(H) is nuclear. There also are no apparent differences in strength in reporter gene activation in Drosophila between nuclear and membrane bound forms of activated Notch. In the covalent modification hypothesis, N-Dl binding could result in the binding and/or activation of a modifying enzyme (such as a kinase or methylase) which could act on Notch-bound Su(H) (Eastman, 1997).

Cell-cell signaling mediated by the receptor Notch regulates the differentiation of a wide variety of cell types in invertebrate and vertebrate species, but the mechanism for signal transduction following receptor activation is unknown. A recent model proposes that ligand binding induces intracellular processing of Notch; the processed intracellular form of Notch then translocates to the nucleus and interacts with DNA-bound Suppressor of Hairless [Su(H)], a transcription factor required for target gene expression. Intracellular cleaveage has been suggested to occur within either the transmembrane domain or the first 10 amino acids of the cytoplasmic domain. Since intracellular processing of endogenous Notch has so far escaped immunodetection, a sensitive nuclear-activity assay was devised to indirectly monitor the processing of an engineered Notch in vivo. First, the non-membrane-tethered intracellular domain of Notch, fused to the DNA-binding domain of Gal4, regulates transcription in a Delta-independent manner. This transcriptional regulation requires Su(H) activity, suggesting that Su(H) may not only target the Notch intracellular domain to the DNA but may also have an additional function. For instance, Su(H) may be required to protect processed Notch from degradation, or participate in transcriptional activation together with processed Notch. Subsequently, full-length Notch, containing the Gal4 DNA-binding domain inserted 27 amino acids carboxy-terminal to the transmembrane domain, activates transcription in a Delta-dependent manner. These results provide indirect evidence for a ligand-dependent intracellular processing event in vivo, supporting the view that Su(H)-dependent Notch signaling involves intracellular cleavage, and transcriptional regulation by processed Notch (Lecourtois, 1998).

Drosophila Notch is processed in a ligand-dependent fashion to generate phosphorylated, soluble intracellular derivatives. During most of Drosophila embryogenesis, two size classes of N proteins are coimmunoprecipitated by antibodies against Su(H). These include full-length N proteins and, to a greater extent, phosphoproteins of ~114-kD, Npp114. Unlike mammalian systems in which N exists predominantly as a heterodimer, during Drosophila embryogenesis, the bulk of N exists as the full-length form. When dephosphorylated, Npp114 resolves into three proteins, each ~100 kD: Np100A, Np100B, and Np100C. Through most of embryogenesis, the most abundant of these proteins is Np100B, Np100C being found only late in development. The size difference between the two proteins might be because Np100C has been cleaved further than Np100B, or the two proteins may both have the same amino termini, but Np100C might have been additionally cleaved at the carboxyl terminus. It is also possible that there is a precursor-product relationship between the two. In any case, the occurrence of Np100C only late in embryogenesis suggests that production of these forms of N is under developmental control. Throughout most of embryogenesis, the majority of processed N proteins that are associated with Su(H) show some level of phosphorylation. Full-length N has been shown previously to be phosphorylated on serines. It is not known how the latter relates to the phosphorylation described here, although the presence of hypophosphorylated forms of N bound to Su(H) suggests that the two events are unrelated. How this phosphorylation is effected and how it influences N function is not known. There are two lines of evidence that suggest that phosphorylation is not an immediate consequence of ligand binding and cleavage. (1) Most if not all of NIntra1790 (a constructed soluble intracellular domain of Notch) is phosphorylated -- none of which has been produced as a result of ligand binding and cleavage of N. (2) Overexpression of Dl induces at least one processed form of N which is hypophosphorylated. In addition, phosphorylation of NIntra1790 is not dependent on the presence of Su(H). Because most, if not all, of NIntra1790 is phosphorylated and there is an enrichment of Npp114 in the soluble fraction, perhaps phosphorylation is related to the release of cleaved intracellular N from the membrane. Alternatively, phosphorylation may promote nuclear translocation or association with Su(H), or both. There is some salt extractable Npp114 associated with Su(H) in the membrane fraction. Finding the intracellular domain of N, which contains functional nuclear localization signals either in the membrane or cytoplasmic fractions, indicates that the cell contains mechanisms to restrain the nuclear entry of N cleavage products. Because it has been demonstrated that the cdc10 repeats of N mediate homodimerization, newly produced intracellular forms of N may be retained by full-length forms of N at the membrane. This association might be particularly favored if, as believed, the receptor is presented at the cell surface as a dimer. It is also conceivable that Npp114 is retained on the membrane by a complex of Su(H) and full-length N (Kidd, 1998).

Su(H) may regulate nuclear entry of N. With respect to cytoplasmic retention of Su(H)/Npp114 complexes, regulation may come from Su(H) itself. Whereas coexpressing high levels of NIntra along with Su(H) in S2 cells results in both proteins translocating to nuclei, when low levels of NIntra are coexpressed along with Su(H) in S2 cells, there is retention of NIntra in the cytoplasm. This suggests that excess Su(H) can promote cytoplasmic localization of soluble, intracellular forms of N. Given that there are multiple binding sites for Su(H) in the cytoplasmic domain of N, differences in subcellular localization could reflect the number of Su(H) molecules bound to N, with changes in stoichiometry resulting from increased levels of intracellular N in response to ligand. Because in vivo levels of Su(H) appear to be in excess of soluble Notch product due to sufficient Su(H) to bind to ectopically expressed NIntra and generate gain of function phenotypes, the cytoplasmic retention observed in Su(H)+ embryos is expected from the S2 cell studies. Further supporting the view that Su(H) can retain soluble N in the cytoplasm in vivo, it has been found that lowering the dose of Su(H) promotes nuclear localization of NIntra in embryos (Kidd, 1998).

When tethered directly to DNA, the cytoplasmic domain of N can activate transcription. Conversely, a viral activator fused to Su(H) can substitute for at least some N functions during embryogenesis. It is suggested that one function of soluble forms of N is to bind to Su(H), and in the nucleus, to act directly as a transcriptional transactivator of the latter protein. The data presented here suggest that the prime function of the sequences downstream of the cdc10 repeats is to provide transactivator activity. In accord with this, the cytoplasmic domain of N has many features that are found in transcriptional activators. Although it is possible that N indirectly confers activating ability on Su(H), given the finding of appropriately processed N proteins, which contain functional nuclear localization signals preferentially associated with Su(H), the simplest interpretation of thes results is that one function of N is to bind to Su(H) and in the nucleus to directly act as its transcriptional transactivator. Recently it has been suggested that N activates transcription by disrupting the formation of a repressor complex between Su(H) and a histone deacetylase complex (SMRT/HDAC-1). The data here suggest that rather than simply disrupting the Su(H)/SMRT/HDAC-1 complex, Npp114 plays a more active role in providing transactivator activity to Su(H) (Kidd, 1998 and references).

The Notch pathway plays a crucial and universal role in the assignation of cell fates during development. In Drosophila, Notch is a transmembrane protein that acts as a receptor of two ligands, Serrate and Delta. The current model of Notch signal transduction proposes that Notch is activated upon binding its ligands and that this leads to the cleavage and release of its intracellular domain (also called Nintra). Nintra translocates to the nucleus where it forms a dimeric transcription activator with the Su(H) protein. In contrast with this activation model, experiments with the vertebrate homolog of Su(H), CBF1, suggest that, in vertebrates, Nintra converts CBF1 from a repressor into an activator. The role of Su(H) in Notch signaling during the development of the wing of Drosophila has been assessed. The results show that, during this process, Su(H) can activate the expression of some Notch target genes and that it can do so without the activation of the Notch pathway or the presence of Nintra. In contrast, the activation of other Notch target genes requires both Su(H) and Nintra, and, in the absence of Nintra, Su(H) acts as a repressor. The Hairless protein interacts with Notch signaling during wing development and inhibits the activity of Su(H). These results suggest that, in Drosophila, the activation of Su(H) by Notch involves the release of Su(H) from an inhibitory complex, which contains the Hairless protein. After its release Su(H) can activate gene expression in the absence of Nintra (Klein, 2000).

The loss of H function seems to elicit Su(H)-dependent target gene expression in the wing pouch, a region probably devoid of Notch activity. This suggests that the inactivation of H is sufficient to activate Su(H). To test further this conclusion, an examination was performed to see whether the activity of the vgBE is maintained in H mutant wing pouches if Notch is concomitantly removed. For this, Notch mutant clones were induced in H mutant wing discs. In H mutant wing pouches, weak ubiquitous expression of the vgBE is observed throughout the whole area of the wing, confirming the clonal analysis. vgBE is also active in several Notch mutant clones near the DV and anteroposterior (AP) boundary, but the activity is not maintained in all clones. One explanation for this might be again the requirement of other so far unidentified factors emanating from the two compartment boundaries. In agreement with this, the vgBE enhancer has a late expression domain along the AP boundary, suggesting an input from these areas for its proper expression. However this domain is also dependent on Notch during normal development. The removal of the Su(H)-binding site in the enhancer leads to the loss of all expression domains in the wing pouch, suggesting that Su(H) is required (Klein, 2000).

Therefore, the fact that the cells of several mutant clones do express the vgBE suggest that the vgBE can be activated in the complete absence of Notch activity and that the inactivation of H is sufficient to activate Su(H). No activation of the vgBE was ever found in Notch mutant clones induced in wild-type wing pouches, suggesting that during wild-type development, the activity of Notch is required to activate the vgBE. Hence, Notch probably activates Su(H) through inactivation of H. An examination was performed to see whether the degree of endogenous Su(H) activation that results from the removal of H is sufficient to elicit a biological effect. To assay this, it was asked whether or not removal of H activity can induce Su(H)-dependent development of the pouch in wing discs in which Notch signaling is absent, such as apterous and Presenilin mutant wing discs. Loss of H function rescues the loss of wing development of ap mutants: whereas ap mutants have no wing pouch, ap;H double mutants have large wing pouches with no margin structures. The enlarged pouch of the double mutant discs expresses spalt (sal) and the two vg reporters, vgQE and vgBE, all of which are expressed specifically in the wing pouch in a Notch/Su(H)-dependent manner and are not expressed in ap mutants. In contrast, no wg expression is induced in these double mutant discs, suggesting that the observed rescue is likely to be due to the activation of Su(H) in the double mutants. This is strongly supported by the fact that Su(H);H double mutants exhibit a small wing rudiment identical to that of Su(H) mutants. Expression of UAS-vg by dpp-Gal4 in ap mutant discs can recover the pouch-specific expression domain of sal, suggesting that the activation of vg expression by Su(H) is responsible for the recovered sal expression in the ap;H double mutant wing discs. Similar to overexpression of UAS-Su(H) in ap mutant wing discs, the pouch in ap;H mutant discs develops near the residual wg expression in the remaining hinge. As expected from the analysis of the wing discs, the pharate adult ap;H double mutants have large wing pouches, which are devoid of any margin like structure such as innervated bristles (Klein, 2000).

The effects on wing development of removing H in Psn mutants were examined. As in the case of ap, loss of function of H effects a strong rescue of the wing pouch in the Psn;H mutant discs in comparison to the Psn mutant discs. However, in this case, the morphology of the discs is more like wild type and, in contrast to ap;H mutant discs, the pouch develops at its normal place. Closer monitoring of double mutant discs reveals some expression of wg and the vgBE along the DV boundary. This suggests that, in contrast to the situation of ap mutants, in Psn mutants, there is some activation of Notch and it seems that the lack of H activity can enhance this residual signaling of Notch at the DV boundary. This is remarkable considering that the wing phenotype caused by the loss of Psn is stronger than that caused by loss of Su(H) function. Taken together, these results provide further evidence for a positive transcriptional activity of Su(H). They further show that H is an antagonist of Su(H) during early wing development and that it suppresses the activity of Su(H) in the absence of Notch signaling. The results also suggest that the inactivation of H is sufficient to activate Su(H) and that the activity of Notch is required to inactivate H during normal development (Klein, 2000).

Overexpression of Su(H) leads to three different responses: (1) activation, as is the case for vg, some E(spl) genes, Dl and Ser; (2) inactivation, as shown for cut and E(spl)m8; or (3) no effect, as is the case for wg. This differential behavior is, at least in some cases, a consequence of direct binding of Su(H) to the promoters: the vgBE as well as the E(spl) genes contain Su(H)-binding sites to which Su(H) binds; such sites are necessary for the activation of these genes in vivo. Despite that, they react differently towards Su(H) overexpression. Since E(spl)m8, which is suppressed by Su(H) overexpression, can be activated by expression of Su(H)VP16 or Nintra, it is concluded that Nintra is required in addition to Su(H) to activate E(spl)m8 expression. The results suggest that, in this case, Nintra probably acts as an activation domain of a dimeric transcription factor containing Su(H), as has been proposed. From this, it follows that Nintra might have two function during a Notch signaling event: first it inactivates H, which leads to the release of Su(H) and then, in some instances, it provides the transactivation domain for free Su(H) to activate the expression of target genes (Klein, 2000).

Flies carrying reporter lacZ constructs with up to 12 Su(H)-binding sites do not display any activity in the wing disc. This suggests that Su(H) (even in association with Nintra) is not sufficient to activate transcription and requires other collaborating factors. It further suggests that, even in promoters that can be activated by Su(H) in the absence of Nintra, Su(H) probably interacts with other factors to promote gene expression. This is confirmed by a study of the vgBE. Although the Su(H)-binding site is absolutely necessary for its activity, other sites are equally important. So far the factors that bind to these sites are not identified. The dependence of Su(H) on these others factors is probably the reason for the differential expression of Notch target genes in H and H/N mutant clones that have been observed (Klein, 2000).

Recently it has been shown that Su(H) acts as a suppressor of single minded transcription during the formation of the midline cells in the embryonic central nervous system of Drosophila. This observation provides the first evidence that Su(H), like its mammalian counterpart CBF1, can act as a suppressor of transcription. The inactivation of the cut and E(spl)m8 expression in absence of Nintra suggests that Su(H) can act as a suppressor of gene expression also during adult development and provides further evidence for a suppressing activity of Su(H). However, this suppression is context dependent and not a general feature of Su(H). This context dependency might also exist for CBF1, since only the reaction of a small number of genes towards its activity has been tested so far and it is possible that some target genes can be activated by CBF1 in the absence of Nintra in a similar way, as has been shown for Su(H). In summary, these results suggest that the consequence of the binding of Su(H) to a promoter is dependent on its local architecture and, therefore, Su(H) can at the same time activate and suppress gene expression, like many other transcription factors. The removal of both the maternal and zygotic expression of H during embryogenesis seems to be of no consequence for the embryo. Since the overactivation of Notch/Su(H) signaling during embryogenesis has deleterious consequences, this observation contradicts the conclusion that H is required to inactivate Su(H). However, the context dependency and differential reaction of the target genes observed during wing development offer two explanations for this discrepancy, without having to postulate an unknown factor, which can functionally replace H. First, it is likely that the interacting factors, which are required for gene expression in concert with Su(H), are different during embryogenesis and this could modulate the responsiveness of the target promoters. This conclusion is supported by the observation that the genes of E(spl)C, although probably all requiring Su(H) for their expression, are all very similarly expressed in the embryo, but their expression pattern in the wing imaginal disc is very different. Another explanation is that the target promoters of binding Su(H) during embryogenesis might be all of the type that require the additional activity of Nintra. Therefore they would stay inactive even in the presence of free Su(H) until Notch is activated (Klein, 2000).

Notch signal transduction centers on a conserved DNA-binding protein called Suppressor of Hairless [Su(H)] in Drosophila species. In the absence of Notch activation, target genes are repressed by Su(H) acting in conjunction with a partner, Hairless, which contains binding motifs for two global corepressors, CtBP and Groucho (Gro). Usually these corepressors are thought to act via different mechanisms; complexed with other transcriptional regulators, they function independently and/or redundantly. This study investigated the requirement for Gro and CtBP in Hairless-mediated repression. Unexpectedly, it was found that mutations inactivating one or the other binding motif can have detrimental effects on Hairless similar to those of mutations that inactivate both motifs. These results argue that recruitment of one or the other corepressor is not sufficient to confer repression in the context of the Hairless-Su(H) complex; Gro and CtBP need to function in combination. In addition, this study demonstrates that Hairless has a second mode of repression that antagonizes Notch intracellular domain and is independent of Gro or CtBP binding (Nagel, 2005).

To test the repressive effects of Hairless in the absence of N^ICD, Hairless ability to inhibit transcription in the presence of Grainyhead (Grh) was tested. The Notch response (NRE) reporter contains binding sites for the transcriptional activator Grh that stimulate transcription fourfold in the absence of N^ICD and increase the stimulation seen in the presence of N^ICD. Addition of full-length Hairless inhibits these effects, reducing transcription in the presence of Grh alone by 50%. Furthermore, this inhibitory effect is dependent on Su(H), as indicated by a lack of repression of HDeltaS, and requires both CtBP and Gro, since Hairless proteins with either interaction domain mutated (HDeltaC, H*C, HDeltaG, H*G) lose most of their repressive activity. Again, the levels of activity with the single mutants are similar to the levels seen with the double-mutant forms of the protein (HDeltaGC, H*GC) and all resulted in >90% of the expression seen with Grh. These experiments suggest that Hairless has two modes of repression, one that operates by repressing the transcriptional machinery through its recruitment of global corepressors and a second that operates by directly antagonizing N^ICD (Nagel, 2005).

These data confirm therefore that both Gro and CtBP can function as corepressors with Hairless, and indeed both factors are necessary for full repression by Hairless on the NRE; preventing the interaction with one or the other factor severely compromises Hairless activity. This is in apparent contrast to the effects on vg^BE-LacZ, for which only Gro appears essential. Furthermore, the two cofactors appear to act together, since Hairless proteins lacking both interaction motifs retains a level of repression that is comparable to the results seen upon removing either alone (Nagel, 2005).

Previous studies of CtBP and Gro have argued that they mediate repression in qualitatively different ways, although both are thought to recruit histone deacetylases. Gro has predominantly been associated with so-called long-range repression, as it operates to dominantly silence modular enhancers. In contrast, CtBP appears to act in a local way to inhibit activators that are bound nearby. However, these models do not appear compatible with a combined requirement for Gro and CtBP in Hairless-mediated repression. Furthermore, direct fusion of a Gro interaction domain to the Su(H) protein is sufficient to convert it into a potent repressor, as described for other transcriptional regulators. Why should Gro and CtBP therefore be interdependent in the context of Hairless recruitment? One simple explanation would be that one or the other corepressor is needed to specifically counteract N^ICD activation. For example, CtBP interferes with recruitment of p300, a histone acetyltransferase that is reported to interact with mammalian N^ICD. However, the data suggest that CtBP and Gro are both needed to repress Grh even in the absence of N_ICD, arguing that each corepressor can only perform a subset of its functions in the context of Hairless. Maybe the two corepressors recruit different enzymatic activities that are needed together to promote repression. If the Hairless complex were incompatible with oligomerization of Gro, which is reported to be important for stable repression, Gro might be able to recruit histone deacetylases but not to promote spreading of the repression complex. And if CtBP, which in mammals has been found complexed with methyl transferases as well as deacetylases, could recruit only histone methyl transferases, the corepressors would each confer a critical component on the Hairless complex. A more complete understanding of the molecular functions of Gro and CtBP in the context of chromatin dynamics and transcription complexes will be needed to determine why Hairless requires their coordinate activities in many developmental scenarios, as has been shown in this study (Nagel, 2005).

Geometry and protein interactions at Su(H) binding sites

Cell-specific gene regulation is often controlled by specific combinations of DNA binding sites in target enhancers or promoters. A key question is whether these sites are randomly arranged or if there is an organizational pattern or 'architecture' within such regulatory modules. During Notch signaling in Drosophila proneural clusters, cell-specific activation of certain Notch target genes is known to require transcriptional synergy between the Notch intracellular domain (NICD) complexed with CSL proteins bound to 'S' DNA sites and proneural bHLH activator proteins bound to nearby 'A' DNA sites. Previous studies have implied that arbitrary combinations of S and A DNA binding sites (an 'S+A' transcription code) can mediate the Notch-proneural transcriptional synergy. By contrast, this study shows that the Notch-proneural transcriptional synergy critically requires a particular DNA site architecture ('SPS'), which consists of a pair of specifically-oriented S binding sites. Native and synthetic promoter analysis shows that the SPS architecture in combination with proneural A sites creates a minimal DNA regulatory code, 'SPS+A', that is both sufficient and critical for mediating the Notch-proneural synergy. Transgenic Drosophila analysis confirms the SPS orientation requirement during Notch signaling in proneural clusters. Evidence that CSL interacts directly with the proneural Daughterless protein, thus providing a molecular mechanism for this synergy. It is concluded that the SPS architecture functions to mediate or enable the Notch-proneural transcriptional synergy which drives Notch target gene activation in specific cells. Thus, SPS+A is an architectural DNA transcription code that programs a cell-specific pattern of gene expression (Cave, 2005).

The functional significance of the SPS element has not been determined, but initially, it was proposed that the arrangement of the S binding sites in the SPS may function to mediate cooperative DNA binding by CSL proteins, or it may be necessary for the recruitment of other proteins to the promoter. Subsequent studies, though, showed that CSL, NICD, and Mam "ternary complexes" can assemble on single S sites. To date, no studies have experimentally addressed whether there are significant functional differences between SPS elements and single S or other non-SPS binding site configurations, and the mechanistic function of the SPS element is not known (Cave, 2005).

In Drosophila, five of the seven bHLH repressor genes in the E(spl)-Complex contain an SPS element in their promoter regions, and four of these bHLH R genes contain both SPS and proneural bHLH A protein binding (A) sites. These four bHLH R genes (the m7, m8, mγ, and mδ genes, collectively referred to as the 'SPS+A bHLH R' genes have been shown genetically to depend upon proneural bHLH A genes for expression. In addition, transcription assays in Drosophila cells with at least two of these four genes (m8 and mγ) have shown that there is strong transcriptional synergy when NICD and proneural proteins are expressed in combination. These SPS+A bHLH R genes also have similar patterns of cell-specific expression within proneural clusters. Following determination of the neural precursor cell from within a proneural cluster of cells, Notch-mediated lateral inhibition is initiated and these SPS+A bHLH R genes are specifically upregulated in all of the nonprecursor cells but not in the precursor cell. The absence of NICD, and the presence of specific repressor proteins such as Senseless, prevent upregulation of SPS+A bHLH R genes in the precursor cells (Cave, 2005).

This study shows that there are important functional differences between the SPS architecture and non-SPS configurations of S binding sites. The SPS architecture is critical for synergistic activation of the m8 SPS+A bHLH R gene by Notch pathway and proneural proteins. Whereas previous studies have focused on which regulatory genes and proteins function combinatorially to activate SPS+A bHLH R gene expression, this study focuses on the underlying DNA transcription code that programs the Notch-proneural transcriptional synergy that drives cell-specific gene transcription. The results of previous studies have implied that an apparently arbitrary combination of S and A binding sites (S+A transcription code) is sufficient for transcriptional activation of SPS+A bHLH R genes. By contrast, this study shows that a minimal transcription code, SPS+A, is sufficient and critical for mediating Notch-proneural synergistic activation of these genes. The SPS+A code is composed of the specific SPS binding site architecture in combination with proneural A binding sites. Furthermore, evidence is presented that direct physical interactions between the Drosophila Su(H) and Daughterless protein mediate the transcriptional synergy, thus providing a molecular mechanism for the Notch-proneural synergy. Together, these studies show that the SPS architecture functions to mediate or enable the transcriptional synergy between Notch pathway and proneural proteins and that SPS+A is an architectural transcription code sufficient for cell-specific target gene activation during Notch signaling (Cave, 2005).

To test whether the SPS binding site architecture is important for Notch-proneural synergy, the ability of Drosophila NICD (dNICD) and proneural bHLH A proteins, such as Achaete and Daughterless (Ac/Da) to synergistically activate the wild-type native m8 promoter and SPS architecture variants was examined. Whereas the native m8 promoter carries the wild-type SPS architecture of S binding sites, the m8 promoter variants contain either a disrupted S site, leaving a single functional S site (S_F-X or X-S_R), or orientation variants in which the orientation of one or both S sites have been reversed (S_R-S_F, S_F- S_F, and S_R-S_R) (Cave, 2005).

The native m8 promoter is synergistically activated in transcription assays by coexpression of dNICD and Ac/Da, but it is only weakly activated by expression of dNICD or proneural Ac/Da proteins alone. However, neither promoter with a single S binding site (S_F-X or X-S_R) can mediate synergistic interactions between dNICD and proneural proteins. In fact, both single S site promoters are only weakly activated when proneural and dNICD proteins are expressed individually or together. Thus, single S sites are not sufficient to mediate Notch-proneural synergy in these contexts, even though they are in the same position as the SPS in the wild-type m8 promoter (Cave, 2005).

When the number of S binding sites are maintained, but the orientation of these sites within the SPS is varied (S_R-S_F, S_F-S_F, and S_R-S_R), only the wild-type (S_F-S_R) SPS orientation is synergistically activated by coexpression of dNICD and proneural Ac/Da proteins. Thus, the wild-type SPS architecture of S binding sites is clearly necessary for the m8 promoter to mediate transcriptional synergy between NICD and the proneural protein complexes assembled on the SPS and A sites, respectively (Cave, 2005).

The transcriptional synergy between NICD and proneural proteins mediated by the SPS element is crucial for the coactivation by the Mastermind (Mam) protein. Coexpression of Mam with both dNICD and proneural proteins provides a strong coactivation of transcription of the wild-type m8 promoter. However, this strong coactivation is not observed with any of the non-wild-type m8 SPS variants, which also cannot mediate Notch-proneural synergy. Thus, coactivation by both the NICD and Mam cofactors is strongly dependent on synergistic interactions with proneural combinatorial cofactors, and the specific SPS architecture is critical for mediating this synergy (Cave, 2005).

The native m8 promoter studies tested whether the organization of the S binding sites in the SPS are necessary to mediate the Notch-proneural synergy. In order to test which of these architectural features are sufficient to mediate that synergy, a set of synthetic promoters was created carrying the same SPS variants mentioned above in combination with A sites (SPS-4A reporter). These synthetic promoters thus contain the sites predicted to mediate the synergy but lack the other sites present in the native m8 promoter, which might also be necessary. This reductionist approach allows for the identification of a minimal promoter that contains only those sites that are necessary and sufficient to mediate the Notch-proneural synergy. All of these synthetic reporters are modestly activated by expression of proneural proteins alone, but expression of dNICD alone gives no activation. By contrast, only the SPS-4A reporter containing the wild-type SPS (S_F-S_R) mediates clear synergistic activation when dNICD and proneural proteins are coexpressed, and none of the SPS variants do so (Cave, 2005).

Given that functional CSL/NICD/Mam ternary complexes have been shown to assemble on single S sites and activate transcription, it was expected that promoters with single S sites could be activated at low levels by expression of dNICD in the absence of the proneural proteins and that promoters with two S sites might have more activity than single S sites. However, it was surprising to observe that all of the m8 and synthetic promoters, even with the wild-type SPS element, have very low or no activity when dNICD is expressed alone. Thus, the SPS binding site architecture does not appear to facilitate recruitment of functional NICD coactivator. This argues against previous proposals that suggested that the SPS architecture might function to recruit other proteins to the promoter. Thus, given that the wild-type SPS architecture is necessary and sufficient for Notch-proneural synergy, these results indicate that the function of the SPS element is to enable synergistic interactions with proneural proteins (Cave, 2005).

The synthetic promoters do not carry bHLH R sites, which are present in all E(spl)-C gene promoters. Thus, these sites clearly are not necessary for Notch-proneural synergy, although they may modulate it in vivo. It has been proposed that other repressor proteins bind the mγ and mδ SPS+A bHLH R gene promoters to restrict their expression to a subset of proneural clusters. Although these hypothetical repressor binding sites may be necessary to program the full mγ and mδ gene expression pattern, the current results indicate that they are not necessary for the Notch-proneural synergy that drives nonprecursor cell-specific upregulation (Cave, 2005).

Both the m8 and SPS-4A synthetic reporter contain a hexamer sequence that has been coconserved with the SPS element. Elimination of that hexamer site in a synthetic promoter does not disrupt Notch-proneural, suggesting that Notch-proneural synergy in vivo is not dependent on the hexamer site (Cave, 2005).

Together, the synthetic and m8 promoter results indicate that SPS+A is a minimal transcription code that is both necessary and sufficient for Notch-proneural synergy in Drosophila. The results with the promoters that were tested show that Notch-proneural transcriptional synergy requires the specific organization or architecture of the SPS element, in addition to its combination with proneural A binding sites. All of the promoters with SPS variants failed to mediate this synergy. This clearly indicates that arbitrary combinations of S and A binding sites are not sufficient to mediate Notch-proneural synergy (Cave, 2005).

An important question is whether there are other DNA binding transcription factors that can combinatorially synergize with CSL/NICD transcription complexes. Previous studies have shown that Notch pathway factors can synergize with a nonproneural transcription factor, Grainyhead, suggesting that synergy with the CSL/NICD transcription complexes could be very general or nonspecific. To test whether a general coactivator, the VP16 transcription activation domain, can synergistically interact with dNICD, an essentially identical wild-type SPS-containing synthetic promoter was created in which the A sites were replaced by UAS binding sites for the yeast Gal4 transcription factor (SPS-5U). Expression of a fusion protein containing the Gal4 DNA binding domain and the constitutively active VP16 activation domain can activate the synthetic SPS-5U promoter. However, the Gal4-VP16 fusion protein does not synergize with NICD. Thus, CSL/NICD complexes do not synergize with every nearby DNA bound transcription factor, and there is at least some specificity to the synergy with bHLH A proteins. This interaction specificity could contribute significantly to selective activation of Notch target genes. Further studies will be required to determine whether other DNA binding transcription factors can combinatorially synergize with Notch signaling and whether such factors fall into distinct classes (Cave, 2005).

Given that Notch signaling and neural bHLH A proteins have been conserved between Drosophila and mammals, it was next asked whether the transcriptional synergy between these proteins is also conserved in mammalian cells. Using the same set of synthetic promoters as mentioned above, activation following expression of the mammalian NICD and neural bHLH A protein homologs (Notch-1 ICD [mNICD] and MASH1/E47, respectively) was tested in murine NIH 3T3 cells. As in the Drosophila system, expression of MASH1/E47 proteins alone produces modest activation of the wild-type (S_F-S_R) SPS-4A promoter, and mNICD alone does not produce any significant activation of the promoter. However, clear transcriptional synergy is observed with the wild-type SPS promoter when both mNICD and neural bHLH A proteins are coexpressed. Moreover, SPS-mediated synergy requires nearly the same organizational features of S binding sites as observed in Drosophila. Neither of the single S site promoters can mediate that synergy, nor can most of the orientation variants. Although the S_R-S_R promoter is activated following coexpression of both the mNICD and bHLH A proteins, it is not activated by mNICD alone (Cave, 2005).

These results indicate that the potential for transcriptional synergy between NICD and neural bHLH A proteins has been conserved in a mammalian cell system and that the SPS+A code is sufficient and critical for mediating that transcriptional synergy. This raises the possibility that there may be mammalian genes that are regulated by neural bHLH A proteins and Notch signaling via this code. Although there is an SPS element conserved in the HES-1 promoter, HES-1 does not have an A site in its proximal promoter region, and HES-1 is not activated by expression of bHLH A genes. Thus, HES-1 appears to be similar to the Drosophila E(spl)-C m3 bHLH R gene, which also has an SPS but no obvious nearby A site. Whole-genome searches are being performed for genes in mammalian systems that may be regulated by the SPS+A code (Cave, 2005).

It has been proposed that the architecture of the SPS element may mediate cooperative binding of a second CSL protein once an initial CSL protein binds the DNA. Using electromobility gel shift assays to test for cooperative binding, the ability was compared of bacterially expressed and partially purified Drosophila Su(H) protein to bind DNA probes containing either the wild-type m8 SPS or an m8 SPS with one S site mutated. If there is cooperativity, one would expect to observe the band corresponding to two DNA bound CSL proteins to be as strong or stronger than the band corresponding to a single CSL protein bound to DNA. The single S site probe serves as a control because it cannot be cooperatively bound by two Su(H) proteins, and it also serves to identify the band corresponding to a single Su(H) protein bound to the wild-type SPS probe. Similar amounts of Su(H) protein bind strongly to the wild-type probe and to the single-site probe. In particular, because single protein binding to the wild-type DNA probe did not facilitate or stabilize simultaneous binding of two S proteins, Su(H) does not appear to bind cooperatively to the two S sites in the wild-type probe. These results suggest that CSL proteins do not bind cooperatively to the SPS in vivo, although posttranslational modifications in vivo could affect these binding properties Cave, 2005).

In addition, the protein binding affinity for the S_F-S_R and S_R-S_F probes appears to be comparable, although the reversed orientation of the two S sites would have likely disrupted cooperative binding if it were present. This result strongly suggests that the complete lack of activation by S_R-S_F sites in all of the promoters tested is not due simply to decreased ability of Su(H) protein to bind to the S_R-S_F orientation variant Cave, 2005).

To test the in vivo relevance of the conserved S binding site orientation in SPS elements, transgenic flies were created carrying β-galactosidase reporter genes driven by native m8 promoters containing either the wild-type (S_F-S_R) or S_R-S_F variant SPS elements. Wing and eye imaginal discs containing m8 promoters with the wild-type SPS element produced strong expression in proneural cluster regions, similar to the pattern described for endogenous m8. By contrast, comparably stained wing and eye discs carrying the m8 promoter reporters with the S_R-S_F SPS variant showed no expression or very low levels of expression, respectively. Extended staining of discs containing the S_R-S_F element revealed clear but weak expression in a pattern of single cells that resembles the distribution of neural precursors in the wing discs and eye discs. This is likely due to activation via the A site by proneural proteins because proneural levels are highest in the precursor cells. However, there was no expression in the surrounding nonprecursor cells within the proneural clusters even though Notch signaling is activated in these cells. Similar neural precursor-specific m8 reporter expression patterns have been observed when the S binding sites are eliminated, indicating that reversal of the S binding site orientations is functionally equivalent to eliminating them for this aspect of Notch target gene expression. These in vivo results confirm that the conserved orientation of the S binding sites in the wild-type SPS element is essential for nonprecursor cell specific upregulation of the SPS+A bHLH R m8 genes in response to Notch signaling in proneural clusters (Cave, 2005).

To gain an insight into the molecular mechanism underlying the strong transcriptional synergy between Notch signaling and bHLH A proteins on the m8 and SPS-4A promoters, whether this synergy involves a direct physical interaction was tested by using yeast two-hybrid assays with the Drosophila proteins. These experiments revealed that the Daughterless N-terminal domain directly and specifically interacts with the Su(H) protein in the absence of the bHLH domain and C terminus (Cave, 2005).

Using transcription assays in Drosophila cells, whether the Da N terminus (DaN construct), which contains a transcription activation domain, can synergistically activate the m8 promoter was tested in the absence of both its bHLH DNA binding domain and a heterodimerization partner, like Ac. The Da N-terminal protein synergistically activates the m8 promoter when dNICD is coexpressed, apparently by direct binding of the DaN protein to endogenous CSL bound to the SPS element. These results indicate that the Notch-proneural transcriptional synergy is not mediated by cooperative DNA binding interactions between the Su(H) and proneural proteins, although such cooperative binding may mediate transcriptional synergy between some combinatorial cofactors. These results suggest that a direct interaction between Su(H) and the Da N-terminal fragment, which can occur independent of NICD, facilitates the formation of an active transcription complex when NICD is also present during Notch signaling (Cave, 2005).

These results suggest that the SPS architecture functions to enable a direct physical interaction between Su(H) and Da proteins, thus providing a molecular mechanism for the observed Notch-proneural synergy that is mediated by the SPS element. This interaction could stabilize the recruitment or functional activity of NICD, which then recruits Mam, and could explain the strong dependence of both NICD and Mam coactivation functions on the presence of proneural proteins (Cave, 2005).

In previous studies, it has been proposed that neither the synergistic activation nor the transcriptional repression mediated by CSL protein complexes imply direct interactions between CSL and DNA bound combinatorial cofactors; rather, it is likely that CSL proteins exert their effects through the recruitment of non-DNA binding cofactors, such as chromatin modifying enzymes. While this might be the case for some Notch target gene promoters, in the case of m8, the results indicate that the mechanism underlying the synergistic interactions between CSL/NICD and bHLH A proteins does involve direct physical interactions (Cave, 2005).

A mechanistic model is proposed for programming Notch-proneural synergy with the SPS+A transcription code. These studies demonstrate that there are important functional differences between SPS and non-SPS organizations of S binding sites. The critical role of the SPS binding site architecture is not predicted or explained by the previous models for Notch target gene transcription. Previous models suggest that transcription is promoted by the binding of NICD to CSL, which displaces CSL bound corepressors, thus allowing transcriptional synergy with other DNA bound combinatorial cofactors. These models have not distinguished between Notch target genes with regulatory modules that contain SPS or non-SPS configurations of S binding sites, nor do they explain or predict the critical function of the SPS binding site architecture in mediating Notch-proneural transcriptional synergy (Cave, 2005).

A revised model is proposed that incorporates the essential requirement for the specific SPS binding site architecture in combination with the proneural A binding sites for transcriptional activation of m8 and the other SPS+A bHLH R genes. These genes each contain an SPS+A module and exhibit similar cell-specific upregulation in nonprecursor cells in proneural clusters. In this new model, the specific architecture of the S sites in the SPS element directs the oriented binding of Su(H) so that it is in the proper orientation and/or conformation to enable a direct interaction with Da. This interaction is an essential prerequisite for subsequent recruitment and/or functional coactivation by NICD during Notch signaling. This Notch-proneural complex is then further activated by subsequent recruitment of Mam (Cave, 2005).

It is interesting to note that the mammalian homologs of each of the Su(H), NICD, and Da proteins have been shown to interact with the p300 coactivator; thus, when complexed together, these proteins could potentially function combinatorially to recruit p300 or a related coactivator (Cave, 2005).

In Drosophila and mammals, Notch signaling is used throughout development to activate many different target genes, and in multiple developmental pathways. Thus, it is of paramount importance that the proper target genes are selectively activated in the proper cell-specific patterns. It is known that Notch signaling can activate genes through non-SPS configurations of S sites in certain other target genes. For example, expression of the Drosophila genes single minded, Su(H), and vestigal have all been shown to be regulated by Notch signaling, and all have single S sites or multiple unpaired S sites but no SPS elements in their promoter and/or enhancer regions (Cave, 2005).

The results show that for essentially every promoter tested, NICD cannot activate in the absence of neural bHLH A combinatorial cofactors, suggesting that NICD may always require a combinatorial cofactor to activate target genes. If so, the non-SPS Notch target genes are likely also to have specific combinatorial cofactors. The results also clearly show that the Notch-proneural combinatorial synergy requires a specific configuration of S sites, the SPS. There may be other specific configurations of S binding sites that mediate synergy for different classes of combinatorial cofactors for Notch signaling (Cave, 2005).

Together, these observations suggest that specific, but unknown, non-SPS configurations of sites may program the interactions between Notch complexes and the proper combinatorial cofactors. It is speculated that these non-SPS configurations might be unique to each target gene, or it is possible that there are specific patterns or classes of S binding site configurations -- an 'S binding site subcode' -- that determine cofactor specificity. Thus, the results suggest that selective Notch target gene activation may be programmed by distinct Notch transcription codes in which specific configurations of S binding sites mediate selective interactions with specific combinatorial cofactors (Cave, 2005).

Elucidating the various transcription codes controlling target gene activation during Notch signaling will be an important goal for future studies. The results have clearly shown that the architecture of transcription factor binding sites can be crucial for control of cell-specific Notch target gene activation. The studies presented here give a glimpse into the molecular mechanisms by which a one dimensional pattern of DNA binding sites can program cell-specific patterns of gene expression (Cave, 2005).

Notch and Numb

Numb has been shown to physically interact with the intracellular domain of Notch. To assess the possibility of a direct physical interaction between Notch and Numb, a yeast two hybrid interaction assay has been carried out. In this experiment genes coding for fragments of each protein are placed in yeast cells, one attached to a coding sequence specifying a DNA binding protein, and the other attached to a coding sequence specifying a transcriptional activator domain. The binding site for the DNA binding domain is placed next to a ß-galactosidase promoter. The fragment attached to the DNA binding domain is termed the bait. If the bait interacts with the other protein fragment attached to the transcriptional activator, then ß-galactosidase is transcribed. The Notch intracellular domain consists of an N-terminal RAM23 domain, an ankyrin repeat region (serving as a protein interaction domain which interacts with Deltex, a C-terminal PEST sequence (serving to promote protein instablity), and a central domain. The N-terminal RAM23 domain does interact with Numb in the yeast two hybrid experiment. The N-terminal phosphotyrosine binding domain of Numb interacts with the N-terminal area of the intracellular region of Notch. The physical interaction of Notch and Numb has confirmed using an in vitro physical interaction assay (Guo, 1996).

Numb influences cell fate by downregulating Notch through polarized receptor-mediated endocytosis. Numb functions as a linker between a-Adaptin and Notch. a-Adaptin facilitates the endycytosis of Notch. a-Adaptin acts downstream of Numb in the determination of alternative cell fates in asymmetric cell division. During asymmetric cell division in sensory organ precursor cells, Numb protein localizes asymmetrically and segregates into one daughter cell, where it influences cell fate by repressing signal transduction via the Notch receptor. Numb acts by polarizing the distribution of a-Adaptin, a protein involved in receptor-mediated endocytosis. a-Adaptin binds to Numb and localizes asymmetrically in a Numb-dependent fashion. Mutant forms of a-Adaptin that no longer bind to Numb fail to localize asymmetrically and cause numb-like defects in asymmetric cell division. These results suggest a model in which Numb influences cell fate by downregulating Notch through polarized receptor-mediated endocytosis, since Numb also binds to the intracellular domain of Notch (Berdnik, 2002).

Drosophila a-Adaptin binds to Numb and the ear domain of a-Adaptin is critical for this interaction. Like Numb, a-Adaptin localizes asymmetrically in dividing SOP cells and preferentially segregates into the pIIb cell. a-Adaptin mutations that affect binding to Numb and abolish asymmetric localization cause cell fate transformations similar to those observed in numb. Epistasis experiments place a-Adaptin downstream of numb and upstream of Notch, suggesting that a-Adaptin is involved in the suppression of Notch signaling by Numb. These results suggest that Numb regulates cell fate by polarizing the distribution of the endocytic protein a-Adaptin which in turn is involved in the endocytosis and consequent inactivation of Notch (Berdnik, 2002).

To test whether a-Adaptin acts upstream or downstream of Notch, the Notch, a-Adaptin double mutant phenotype was examined. Inactivation of Notch during SOP division causes transformations of hair and socket cells into inner cells and leads to an apparent loss of bristles. Notch, a-Adaptin double mutants should have the Notch phenotype if a-Adaptin is upstream, but the a-Adaptin phenotype if it is downstream, of Notch. A temperature-sensitive allele of Notch (Notch^ts) was used that has no bristle phenotype at 18°C but causes essentially a complete loss of postorbital bristles when shifted to 29°C during the time of asymmetric cell divisions in the bristle lineage. When ada^ear4 mutant clones are generated in a Notch^ts background, outer cell fate transformations are observed at the permissive temperature, but not at the restrictive temperature. Thus, Notch^ts, ada^ear4 double mutant SOP cells have the Notch mutant phenotype, indicating that a-Adaptin acts upstream of, or in parallel to, Notch (Berdnik, 2002).

Numb protein, known to interact with the cytoplasmic domain of Notch, interfers with the ability of Notch to cause the nuclear translocation of Suppressor of hairless. Both the C-terminal half of the highly conserved phosphotyrosine binding domain of Numb and the C-terminus of Numb are required to inhibit Notch. Numb prevents the colocalization to the nucleus of cells of the Notch intracellular domain with Su(H) resulting in a cytoplasmic localization. Overexpression of Numb during wing development, which is sensitive to Notch dosage, reveals that Numb is also able to inhibit the Notch receptor in vivo. In the external sense organ lineage, the phosphotyrosine binding domain of Numb is found to be essential for the function but not for asymmetric localization of Numb. These results suggest that Numb determines daughter cell fates in the external sense organ lineage by inhibiting Notch signaling (Frise, 1996).

Mammalian Numb (mNumb) has multiple functions and plays important roles in the regulation of neural development, including maintenance of neural progenitor cells and promotion of neuronal differentiation in the central nervous system (CNS). However, the molecular bases underlying the distinct functions of Numb have not yet been elucidated. mNumb, which has four splicing isoforms, can be divided into two types based on the presence or absence of an amino acid insert in the proline-rich region (PRR) in the C-terminus. It has been proposed that the distinct functions of mNumb may be attributable to these two different types of isoforms. In this study, the outer optic anlage (OOA) of the Drosophila larval brain was used as an assay system to analyze the functions of these two types of isoforms in the neural stem cells, since the proliferation pattern of neuroepithelial (NE) stem cells in the OOA closely resembles that of the vertebrate neural stem/progenitor cells. They divide to expand the progenitor cell pool during early neurogenesis and to produce neural precursors/neurons during late neurogenesis. Clonal analysis in the OOA allows one to discriminate between the NE stem cells, which divide symmetrically to expand the progenitor pool, and the postembryonic neuroblasts (pNBs), which divide asymmetrically to produce neural precursors (ganglion mother cells), each of which divides once to produce two neurons. In the OOA, the human Numb isoform with a long PRR domain (hNumb-PRRL), which is mainly expressed during early neurogenesis in the mouse CNS, promotes proliferation of both NE cells and pNBs without affecting neuronal differentiation, while the other type of hNumb isoform with a short PRR domain (hNumb-PRRS), which is expressed throughout neurogenesis in the mouse embryonic CNS, inhibits proliferation of the stem cells and promotes neuronal differentiation. It was also found that hNumb-PRRS, a functional homologue of Drosophila Numb, more strongly decreases the amount of nuclear Notch than hNumb-PRRL, and can antagonize Notch functions probably through endocytic degradation, suggesting that the two distinct types of hNumb isoforms contribute to different phases of neurogenesis in the mouse embryonic CNS (Toriya, 2006).

Notch and Sanpodo

Cellular diversity is a fundamental characteristic of complex organisms, and the Drosophila CNS has proved an informative paradigm for understanding the mechanisms that create cellular diversity. One such mechanism is the asymmetric localization of Numb to ensure that sibling cells respond differently to the extrinsic Notch signal and, thus, adopt distinct fates (A and B). This study focusses on the only genes known to function specifically to regulate Notch-dependent asymmetric divisions: sanpodo and numb. sanpodo, which specifies the Notch-dependent fate (A), encodes a four-pass transmembrane protein that localizes to the cell membrane in the A cell and physically interacts with the Notch receptor. Numb, which inhibits Notch signaling to specify the default fate (B), physically associates with Sanpodo and inhibits Sanpodo membrane localization in the B cell. These findings suggest a model in which Numb inhibits Notch signaling through the regulation of Sanpodo membrane localization (O'Connor-Giles, 2003).

Spdo was initially identified as the homolog of the actin-associated protein Tropomodulin (Tmod), a protein that regulates actin filament length. This study finds that spdo does not encode tmod, but rather a four-pass transmembrane protein that acts upstream of Notch and downstream of Delta to specify the A cell fate. Spdo colocalizes and physically associates with the Notch receptor in vivo. Spdo also exhibits differential subcellular localization between A and B cells during asymmetric divisions, localizing primarily to the cell membrane of the A cell and to the cytoplasm of the B cell. Numb inhibits the cell membrane localization of Spdo in the B cell and Numb and Spdo physically associate in vivo. These findings support a model in which Numb acts in the B cell to block Notch activity by preventing Spdo from localizing to the cell membrane, likely through its link to the endocytic machinery. In the A cell, the absence of Numb allows Spdo to localize to the cell membrane, where it promotes Notch signaling and the A cell fate, likely through a direct association with Notch (O'Connor-Giles, 2003).

Prior studies suggest that spdo acts in the Notch pathway to mediate asymmetric divisions. However, as these studies did not order spdo function relative to members of the Notch pathway, the placement of spdo within the pathway remains uncertain. To order the action of spdo relative to the intramembranous S3 cleavage event that releases the Notch intracellular domain (NICD) from the membrane, two distinct constitutively active forms of Notch, Notch^Intra and Notch^ECN were used. While both Notch constructs function in a ligand-independent manner, Notch^ECN contains the NICD and the Notch transmembrane domain and requires proper execution of the S3 cleavage to activate transcription of Notch target genes. Notch^Intra, which comprises only the NICD, functions independently of the S3 cleavage (O'Connor-Giles, 2003).

In these experiments focus was placed on the development of eight pairs of sibling neurons that arise from spdo/Notch/numb-dependent asymmetric divisions: RP2/RP2sib, dMP2/vMP2, aCC/pCC, and five pairs of U/Usib neurons. Molecular markers can distinguish unambiguously the fate of each of these sibling neurons from their sisters. RP2/RP2sib develop from the Even-skipped (Eve)-expressing GMC4-2a. After division, RP2 retains, while RP2sib extinguishes, Eve expression. Similarly, the U/Usib neurons develop from five Eve-positive GMCs; each GMC divides to produce two initially Eve-positive neurons. The five U neurons retain Eve expression, while the five Usib neurons extinguish Eve. The dMP2/vMP2 interneurons develop from the Odd-skipped (Odd)-positive MP2 precursor. After MP2 division, dMP2 retains Odd expression and extends an axon posteriorly, while vMP2 extinguishes Odd and extends an axon anteriorly. aCC/pCC develop from the Eve-positive GMC1-1a. Both aCC and pCC retain Eve expression; however, aCC expresses 22C10 and extends a motor axon out the intersegmental nerve, while pCC is an interneuron that extends a 22C10-negative axon anteriorly. The RP2sib, pCC, vMP2, and U neurons (A fates) require spdo/Notch function for their specification, while their siblings (B fates) require numb-mediated inhibition of spdo/Notch activity for their development (O'Connor-Giles, 2003).

The two constitutively active Notch constructs were expressed throughout the CNS of wild-type and spdo mutant embryos using the Gal4/UAS system and the development of the RP2/RP2sib, dMP2/vMP2, and U/Usib neurons was followed. It was reasoned that, if spdo acts upstream of Notch, the Notch gain-of-function phenotype (A/A) should be observed. Conversely, if spdo acts downstream of Notch, the spdo phenotype (B/B) would be seen. The placement of spdo function upstream of Notch^Intra, but downstream of Notch^ECN, would indicate a requirement for spdo in the S3 cleavage of the Notch receptor. In a wild-type background, misexpression of either Notch construct is found to be sufficient to induce cells that would normally acquire the numb-dependent B fate to adopt the A fate at a moderate to high frequency depending upon the sibling pair examined. Misexpression of each Notch construct in spdo embryos is found to yield identical cell fate transformations at frequencies essentially equal to those observed in wild-type embryos misexpressing each construct. These results indicate that spdo functions genetically upstream of the S3 cleavage of Notch during asymmetric divisions (O'Connor-Giles, 2003).

Next, the placement of spdo function relative to Delta was assayed. To do this, Delta was misexpressed throughout the CNS of spdo embryos and U/Usib and RP2/RP2sib neuron development was assayed. It was reasoned that, if spdo acts downstream of, or in parallel to, Delta, then misexpression of Delta would not rescue the spdo phenotype. However, if spdo acts upstream of Delta, rescue of the spdo CNS phenotype would be observed. Misexpressing Delta was found not to rescue the spdo phenotype, indicating that spdo acts genetically downstream of, or in parallel to, Delta to promote asymmetric divisions. Together with the placement of spdo function upstream of the S3 cleavage of Notch, this result suggests that spdo functions at or near the membrane to promote Notch signaling during asymmetric divisions (O'Connor-Giles, 2003).

Genetic, molecular, and expression data suggest that Spdo promotes productive Notch signaling through a close association with Notch. To determine whether Spdo physically associates with the Notch receptor, Notch was immunoprecipitated and assayed for the coprecipitation of Spdo. Spdo was found to coprecipitate at roughly equivalent efficiencies with antibodies specific to either the intracellular or extracellular domain of Notch, suggesting that Spdo associates with the full-length Notch receptor. These data indicate that Spdo associates with the Notch receptor in vivo and suggest that Spdo promotes Notch signaling during asymmetric divisions through a physical association with the Notch receptor (O'Connor-Giles, 2003).

Notch and Dishevelled

The dishevelled gene interacts antagonistically with Notch and its ligand Delta. A direct physical interaction between Dishevelled and the Notch carboxyl terminus, distal to the cdc10/ankyrin repeats, suggests a mechanism for this interaction. It is proposed that Dishevelled, in addition to transducing the Wingless signal, blocks Notch signaling directly, thus providing a molecular mechanism for the inhibitory cross talk observed between these pathways (Axelrod, 1996). It therefore appears that Wingless and Notch talk to each other through Dishevelled.

Notch and Canoe

Besides Dishevelled and Discs large, a third Drosophila protein, Canoe, contains the GLGF/DHR motif. canoe interacts genetically with Notch and scabrous in eye, bristle and wing development, suggesting that cno has a common role with sca and N in the morphogenesis of these tissues. As there appears to be a direct physical interaction between Notch receptor and Dishevelled (Axelrod, 1996), providing a link between Notch and wingless signaling, perhaps Canoe plays a role in modifying this interaction (Miyamoto, 1995).

Notch and Shaggy

A study of epistatic relationships between shaggy/zeste white 3 and gain- and loss-of-function alleles of Notch indicates that shaggy/GSK-3 is part of a signaling pathway downstream of Notch. Vertebrate GSK-3 beta can substitute for shaggy in this function (Ruel, 1993).

Notch and Deltex

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).

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).

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 an 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).

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).

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).

Other Notch interactions

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