Protein Interactions : Interaction of Armadillo with Teashirt

One function of the Wingless signal cascade in Drosophila larvae is to determine the ‘naked’ cuticle cell-fate choice instead of the denticled one. Wingless stabilizes cytoplasmic Armadillo, which may act in a transcriptional activator complex with the DNA-binding protein T-cell factor (also known as Pangolin). As these components are critical for all Wingless-dependent patterning events, the problem arises as to how specific outputs are achieved. The Teashirt zinc finger protein is found to be necessary for a subset of late Wingless-dependent functions in the embryonic trunk segments where the teashirt gene is expressed. Loss of wg results in larvae with smaller segments covered with denticles and with no naked cuticle, a phenotype similar to hypomorphic loss-of-function mutations in armadillo. Loss of tsh function affects the identity of the prothorax, which is replaced by labial identity, but segment identity is not affected in the posterior thorax or in the eight abdominal segments. The size of the posterior segments in the ventral epidermis is reduced in the trunk in tsh -null mutants, as compared with wild-type larvae. In tsh -null larvae, abnormal denticle belts differentiate with smaller naked regions in-between, showing that tsh is required for patterning of both the naked and the denticled regions. Notably, denticles differentiate in the naked cuticular regions, especially in the ventral midline, of tsh-null larvae; this phenotype resembles that which arises from late loss-of-Wg signaling in the cell-specification phase (Gallet, 1998).

Armadillo and Teashirt proteins showed similar Wingless-dependent modulation patterns in homologous parts of each trunk segment in embryos, with high levels of nuclear Teashirt and intracellular Armadillo within cells destined to form naked cuticle. Overexpression of Wingless, Tsh and stabilized Arm produce a naked cuticle phenotype. It is concluded that the production of naked cuticle is temporally correlated with high levels of Tsh protein and stabilized, cytoplasmic Arm. Mutations in tsh therefore resemble those of the Wg signaling cascade, but Tsh has an independent homeotic function (Gallet, 1998).

Transcription of the wg gene depends initially on regulation from the pair-rule genes, and then is maintained differently during the early (cell-stabilization) and late (cell-specification phases of embryogenesis. During the cell-stabilization phase (stages 7-10), Wg ensures the regulation of the target gene engrailed, but thereafter, during the cell-specification phase (stage 10 onward), Wg is required to maintain wg but not en expression. Teashirt is found to be required for the maintenance of the late Wingless signaling target gene wingless but not for an earlier one, engrailed (Gallet, 1998).

The Tsh protein is detected initially in the cytoplasm soon after the blastoderm stage in a central region of the embryo. By stage 9, Tsh occupies the trunk segments and is modulated, being concentrated in the nuclei in homologous regions of each neuromere. The domains of high nuclear Tsh coincide with high cytoplasmic localization of Arm. Lower levels of intracellular Arm and nuclear Tsh separate the stronger intracellular stripes. By stage 10, Tsh retains a segmentally modulated pattern in dorsal and lateral parts of each trunk segment, with high nuclear Tsh overlapping the En-positive cells, but in the most ventral parts, Tsh becomes highly concentrated in the nuclei of all cells. Therefore, there is a correlation between high levels of Tsh and Arm inside the cells at stage 9 and the production of naked cuticle in wild-type embryos. arm function is required for the nuclear modulation of Tsh in the trunk segments during stage 9-10 (Gallet, 1998).

Teashirt has been found to associate with, and require, Armadillo in a complex for its function. Full-length Arm protein is not easily accessible to Tsh in vitro, indicating that Tsh and/or Arm needs to be modified in order to bind. Arm might act as a cytoplasmic to nuclear transporter for Tsh. It is concluded that Teashirt binds to, and requires, Armadillo for the naked cell-fate choice in the larval trunk. Teashirt is required for trunk segment identity, suggesting that Teashirt provides a region-specific output for Armadillo activity. Further modulation of Wingless is achieved in homologous parts of each trunk segment where Wingless and Teashirt are especially active. These results provide a novel, cell-intrinsic mechanism to explain the modulation of the activity of the Wingless signaling pathway (Gallet, 1998).

Wnt signaling is a key pathway for tissue patterning during animal development. In Drosophila, the Wnt protein Wingless acts inside cells to stabilize Armadillo where Arm binds to at least two DNA-binding factors that regulate specific target genes. One Armadillo-binding protein in Drosophila is the zinc finger protein Teashirt. A 23 amino acid domain (between aa 692 and 715) in Arm is necessary for the interaction with Tsh. This domain lies in the most conserved part of the C-terminal domain of Arm. Wingless signaling promotes the phosphorylation and the nuclear accumulation of Teashirt. This process requires the binding of Teashirt to the C-terminal end of Armadillo. Evidence is presented that the serine/threonine kinase Shaggy is associated with Teashirt in a complex (Gallet, 1999).

To investigate the effects of Wg signaling on Tsh phosphorylation, Western blots were performed on proteins extracted from stage 9-11 embryos mutant for different components of the Wg pathway. Mutant embryos that constitutively transduce Wg and those lacking signal transmission were selected. In wild-type embryos, different hyperphosphorylated forms of Tsh are present. In constitutive Wg signaling mutant embryos, the most hyperphosphorylated forms are predominant. Conversely, in Wg signaling loss-of-function mutants, the upper band is fainter and the 116 kDa form is more apparent. By probing the same blot with an anti-tubulin antibody and by densitometric analysis, the relative amount of Tsh in the different mutants can be correlated: there are equal amounts of Tsh in wild-type and in embryos with gain of Wg signaling function, but there is less Tsh in mutants lacking Wg signalling function. This is consistent with a decreased level of nuclear Tsh observed in the absence of Wg function. Taken together, the results indicate that the Tsh phosphorylation and the increasing nuclear level of Tsh is in part dependent on the Wg pathway. Nevertheless, even in mutants lacking signal transmission, Tsh is still phosphorylated and localized in the nucleus, indicating that other factors are acting on Tsh independently of Wg (Gallet, 1999).

Wg signal acts by inhibiting the activity of Sgg, which would otherwise promote the degradation of Arm inside the cell. Thus Arm accumulates inside the cell and can interact with its partners. Loss of Sgg activity causes the stabilization of intracellular Arm everywhere in the segment promoting the production of naked cuticle in the trunk. When Wg does not signal, Sgg is thought to promote phosphorylation of Arm on an N-terminal motif, leading to Arm degradation via the ubiquitination pathway. In order to test the interaction between Tsh and Sgg, germ-line clones of sgg were induced. The distribution of Tsh was examined in such embryos. As expected, nuclear Tsh level is high, as in embryos constitutively expressing the Wg pathway. In order to analyse the epistasis between tsh and sgg, sgg cuticles were examined with or without tsh activity. Whereas sgg germ-line clones give naked cuticle, absence of tsh gives larvae with reduced naked cuticle and a lawn of denticles. Therefore Tsh acts downstream of Sgg. Finally, mmunoprecipitations were performed to test whether Sgg and Tsh are in a complex. Using affinity-purified anti-Tsh, Sgg co-immunoprecipitates with Tsh. Together, these results show that Tsh is epistatic to Sgg; that the nuclear Tsh level is also Sgg-dependent, and that in vivo Tsh is in a protein complex with Sgg (Gallet, 1999).

How could the C-terminal domain of Arm act as transcripitional transactivator? Two models are presented for Arm function. In the first model, Arm/beta-catenin could be considered as a bridge between a general DNA-binding factor (e.g. TCF) and a specific transcription factor like Tsh, which modulates a specific function for signaling. In this model the transactivating domain of Arm could bind several different and localized factors allowing tissue specific output. In the shuttle model, on Wg signaling, Arm could interact with general (dTCF) as well as specific factors (Tsh) and translocate them into the nucleus to activate Wg target genes. Arm/beta-catenin shares homology with the importins/karyopherins, which are cytoplasmic receptors for proteins containing nuclear localization sequences (NLS) allowing their docking to the nuclear pore. In agreement with this, it has been shown that Arm is able to interact with the nuclear pore machinery. In this model Arm/beta-catenin is detected in the nucleus in cells where Wg is signaling. Furthermore, Tsh is able to produce naked cuticle in the absence of zygotic dTCF, suggesting that Tsh and dTCF act independently for Wg signaling. Further experiments are required to establish whether Tsh acts directly with dTCF and especially to determine whether or not Arm/beta-catenin is part of a transcription complex or acts simply as a nuclear shuttle. These two models can be considered together where, upon an extracellular signal (e.g. Wg/Wnt), a cytoplasmic protein (e.g. Arm/beta-catenin) recruits specific factors (e.g. Tsh) and stimulates their entry into the nucleus, where this bipartite complex could collaborate with general DNA-binding factors (e.g. TCF) to regulate specific target genes of the pathway. There are several results in favour of this model: (1) TCF is localized in the nucleus independent of Arm; (2) TCF is able to bind other specific factors, including Groucho or dCBP, to repress Wg/Wnt target genes; (3) Tsh does not contain canonical NLS signal and requires an unknown mechanism for entry into the nucleus. Nevertheless, Tsh is the first example of a protein binding Arm in its C-terminal domain and potentially is able to participate in the transactivating process (Gallet, 1999 and references).

One function of the Wingless signaling pathway is to determine the naked, cuticle cell fate choice in the trunk epidermis of Drosophila larvae. The zinc finger transcripton factor Teashirt (Tsh) binds to the transactivator domain of Armadillo to modulate Wingless signaling output in the embryonic trunk and contributes to the naked cell fate choice. The Hedgehog pathway is also necessary for the correct specification of larval epidermal cell fate, which signals via the zinc finger protein, Cubitus interruptus (Ci). Ci also has a Wingless-independent function, which is required for the specification of the naked cell fate; previously, it had been assumed that Ci induces naked cuticle exclusively by regulation of wg. Wg and Hh signaling pathways may be acting combinatorially in the same, or individually in different, cells for this process, by regulating common sets of target genes. (1) The loss of the naked cuticular phenotype in embryos lacking ci activity is very similar to that induced by a late loss of Wg function. (2) Overexpression of Ci causes the suppression of denticles (as Wg does) in absence of Wg activity in the anterior trunk. Using epistasis experiments, it has been concluded that different combinations of the three proteins Tsh, Ci, and Arm are employed for the specification of naked cuticle at distinct positions both along the antero-posterior axis and within individual trunk segments. Finally, biochemical approaches suggest the existence of protein complexes consisting of Tsh, Ci, and Arm (Angelats, 2001).

The cuticles of ci null embryos resemble those that lack wg function specifically during the cell fate specification phase. In both genotypes, the bands of naked cells are reduced though they are not totally lost and the number of denticles is increased, suggesting that both Wg and Ci are required for the patterning of the naked regions. Closer comparison of the denticle identities from these embryos reveals that the expansion of denticles belts correspond, in both cases, to an increase in the number of denticles of types 2, 3, and 4. Since the EGF pathway, and particularly rhomboid (rho), is required to specify these denticle identities, rho expression was examined in wgts embryos. When wgts embryos are shifted to the restrictive temperature at stage 10-11, rho expression is expanded posteriorly in a similar way as that observed in ci94 embryos. These observations support the idea that Ci has a function related to the late activity of Wg signaling (Angelats, 2001).

Thus examination of rhomboid expression and cuticle patterns shows the close similarity of phenotype between a late loss of wg function and the loss of function of ci. Following ectopic expression, Ci is able to promote the specification of naked cuticle in the absence of Wg signaling, showing that Ci is acting downstream or in parallel to Wg during the specification phase. This capacity of Ci to induce the naked cell fate was previously explained by ectopic expression of wg, but the experiments described here show that Ci can also act directly for the specification of the naked cell fate choice especially in the anterior trunk segments. It is believed that UASCi produces high levels of full-length Ci, resulting in the saturation of its normal negative regulation, producing naked cuticle (Angelats, 2001).

Ci, Arm, and Pangolin act in a combinatorial fashion to regulate the expression of dpp in the wing disc. Thus, in addition to the well-known regulatory effects of Hh on wg, it is proposed that downstream components of these signaling pathways may interact directly for gene regulation. Similar arguments may apply in vertebrates where Wnt signaling has been shown to be critical for the regulation of the Ci orthologs Gli2 and Gli3. These considerations and the current results support the hypothesis that Wg and Hh signaling components (Arm and Ci, respectively) have overlapping and thus common functions for patterning, at least in some cells (Angelats, 2001).

The capacity of Ci to mimic Wg activity seems to be position-specific since Ci never suppresses the denticles in the most posterior part of the abdomen (from A5 to the tail) in the absence of Wg activity. In this region of the body, the presence of another unidentified factor may modify the activity of Ci (Angelats, 2001).

Morphological examination of the wild-type trunk segments shows that a typical thoracic segment has fewer and smaller denticle belts compared to those in any abdominal segment. Consequently, thoracic segments generally possess more naked cuticle than abdominal ones. Ci and Arm exhibit differences in their ability to induce naked cuticle in different parts of the trunk. The activity of Arm is crucial for the transduction of the Wg signaling pathway and plays a pivotal role in the trunk for naked cuticular identity. Despite this, ectopic production of stabilized Arm or Wg does not replace denticles of the prothoracic beard with naked cuticle. Loss of Ci activity affects this process of patterning, suggesting that Ci activity acts with Arm signaling for the patterning of the beard. In accord with this hypothesis, ectopic Ci, with or without Wg/Arm signaling, suppresses denticles in the beard. In this context, it is interesting to note that loss of the Wg signaling component sgg induces naked cuticle in the trunk, as expected for constitutive Wg signaling, but in the prothorax no beard develops, contrary to the effects of ectopic Wg signaling (Angelats, 2001).

Tsh activity is also critical for the identity of the prothoracic segment, raising the possibility that Tsh cooperates with the Hh and Wg signaling pathways for patterning of the beard. In conclusion, it is thought that different combinations of dTcf/Arm, Ci, and Tsh proteins are acting to specify the naked cuticular choice, both in different A/P positions along the body and at distinct positions within segments, presumably by acting on common and overlapping sets of downstream target genes (Angelats, 2001).

The differential effects of ectopic Ci or ArmS10c along the A/P axis for the induction of naked cuticle may depend on the Hox proteins, which are known to act in distinct parts of the trunk for segmental identity in combination with Tsh. For example, tsh cooperates with the Sex combs reduced Hox gene for patterning of the prothorax. These results are consistent with the idea that combinations of signaling effectors, Tsh and Hox proteins determine epidermal patterning, since their binding sites are often clustered on the enhancers of target genes (Angelats, 2001).

Consistent with the idea that signaling effectors and Tsh act together during epidermal patterning, Ci, Arm, and Tsh form protein complexes in vivo. Tsh is a phosphoprotein whose phosphorylation is induced in part by Wg signaling. Additionally, hyper-phosphorylated forms of Tsh are found in the nucleus whereas hypophosphorylated forms are predominantly in the cytoplasm. By coimmunoprecipitation, only a hyperphosphorylated form of Tsh coimmunoprecipitates with Ci, suggesting that the interaction between the two proteins takes place in the nucleus. However, only hypo-phosphorylated Tsh interacts with Arm (Angelats, 2001).

These results favour the existence of bipartite complexes (Arm-Tsh and Tsh-Ci) rather than tripartite complexes in vivo. However, the existence of a complex containing these three molecules cannot be excluded (Angelats, 2001).

Protein Interactions : Interaction of Armadillo with Presenilin

Both loss of expression and overexpression of Presenilin suggested a role for this protein in the localization of Armadillo/beta-catenin. In blastoderm stage Presenilin mutants, Arm is aberrantly distributed, often in Ubiquitin-immunoreactive cytoplasmic inclusions predominantly located basally in the cell. These inclusions are not observed in loss of function Notch mutants, suggesting that failure to process Notch is not the only consequence of the loss of Presenilin function. Human presenilin 1 expressed in Drosophila produces embryonic phenotypes resembling those associated with mutations in armadillo; embryos exhibit reduced Armadillo at the plasma membrane; this is likely due to retention of Armadillo in a complex with Presenilin. The interaction between Armadillo/beta-catenin and Presenilin 1 requires a third protein, which may be delta-catenin. These results suggest that Presenilin may regulate the delivery of a multiprotein complex that regulates Armadillo trafficking between the adherens junction and the proteasome (Noll, 2000).

Arm at the cell membrane is associated with E-cadherin, and the continued expression and function of E-cadherin are dependent on the presence of functional Arm. E-cadherin is encoded by the shotgun gene, and shotgun mutant embryos develop poorly formed cuticles due to a loss of cell adhesion. It was reasoned that, if the increased level of cytoplasmic Arm observed in the presence of overexpression of hPS1 correlates with a depletion of the membrane-associated pool of Arm, then overexpression of hPS1 should result in a shotgun-like cuticle phenotype. Indeed most hPS1 embryos that survive long enough to secrete cuticle exhibit phenotypes that resemble the loss of E-cadherin function. In embryos that develop more cuticular elements, additional phenotypes include loss of head structures, variable degrees of segmental fusion, and a 'dorsal open' phenotype. In a small subset of animals (~10%) that survive to secrete cuticle, a weak/moderate wingless-like cuticle phenotype is observed, and this phenotype can be correlated with a failure to maintain Engrailed expression (Noll, 2000).

Because Drosophila PS appears to have a role in the Notch pathway, embryos expressing hPS were labeled with Fas III to assess the integrity of the ventral ectoderm and to look for any indication of hypertrophy of the nervous system. When the ectodermal cells associated with the ventral midline were examined there was evidence of some disruption in patterning as indicated by the meandering of the midline, but the epidermis was found to be intact. Further, when the Fas III-positive cells in the nervous system of these same embryos were visualized the number and location of Fas III-positive cells were found to be normal. This indicates that the nervous system hypertrophy at the expense of ventral ectodermal cell fates that is found as a result of disruption in the Notch signaling pathway does not occur when UAS-hPS1 is expressed. Since Fas III can be found at the cell membrane, the localization of Fas III was examined. Compared to wild-type, there was no obvious mislocalization of Fas III as a result of UAS-hPS1 expression. This suggests that the mislocalization of Arm as a result of UAS-hPS1 expression is not due to some nonspecific effect, but rather reflects a specific interaction been Arm and hPS1 (Noll, 2000).

The resemblance to shotgun mutations of the cuticle phenotypes associated with hPS1 expression strongly implicates dysfunction in adhesion and cytoskeletal organization. To investigate the basis of this phenotype further, phalloidin staining, used to reveal the distribution of actin, was carried out in these embryos at stages before cuticle deposition. UAS-hPS animals that make it through cellularization usually fail to initiate and/or complete dorsal closure, a phenotype resembling that observed in some Arm mutations. At the stage when an accumulation of actin in the peripheral nervous system is clearly apparent, dorsal closure should be approaching completion. However, in the hPS1 animals, the leading edge cells do not undergo the proper change in shape, and there is no accumulation of actin along the dorsal most edge of the cells. Cells all along the dorsoventral axis of the epidermis fail to stretch and take on the proper thin, cuboidal shape. Alterations in the normal distribution of actin due to expression of hPS1 can also be observed in blastoderm stage embryos, concurrent with the mislocalization of Arm to the cytoplasm. In hPS1 animals, the overall amount of actin present at the membrane is greatly reduced, resulting in a thin, spotty phalloidin staining pattern in some regions of the embryo and often in a complete degeneration of the membrane structure in other regions. In areas where hexagonal arrays are not present, large patches of intense phalloidin staining are evident (Noll, 2000).

It is unknown whether the interaction between PS1 and beta-catenin is direct or requires additional factor(s). To address this issue, different mouse beta-catenin deletion constructs were prepared and tested for interaction with PS1. PS1 failed to coimmunoprecipitate with beta-catenin antibodies and beta-catenin did not coimmunoprecipitate with PS1 antibodies. Taken together, these observations suggest that some cofactor or posttranslational modification is required for beta-catenin to bind to PS1, and this cofactor is not present in the yeast two-hybrid or coimmunoprecipitation assays. Because delta-catenin binds directly to PS1, could delta-catenin outcompete beta-catenin from an in vivo complex with PS1 in CHO cells? The minimal delta-catenin interactive fragment requires both the last four Arm repeats and a portion of the carboxy terminal sequence just beyond the Arm repeats. Expression of the delta-catenin DEco fragment (residues 828-1127) almost completely displaces beta-catenin from the PS1 complex. These experiments support the hypothesis that beta-catenin interacts with the hydrophilic loop of PS1 via a third protein, which competes with the DEco fragment of delta-catenin for binding to PS1. The most parsimonious explanation for these observations is that beta-catenin associates with the complex via full-length delta-catenin itself, but is unable to do so in the presence of the DEco fragment alone. If this were the case, then full-length delta-catenin would not be expected to compete beta-catenin from the PS1 complex, as the DEco fragment of delta-catenin did. Furthermore, it should be possible to demonstrate a direct interaction between delta-catenin and beta-catenin. Indeed, in the presence of full-length delta-catenin, beta-catenin is retained in the PS1 complex, suggesting that delta-catenin is capable of mediating beta-catenin association with PS1 (Noll, 2000).

Thus, embryos derived from presenilin germline clone females exhibit mislocalization of Armadillo. These embryos contain cytoplasmic inclusions that are both Arm and Ubiquitin immunoreactive, suggestive of a failure to target Arm to a degradative pathway. A role for PS in regulating the degradation of proteins is suggested by other PS interactions. sel-12, the C. elegans ortholog of PS, interacts with sel-10, a member of the Cdc4p family which targets proteins for Ubiquitin-mediated turnover. Furthermore, the fly ortholog of Cdc41p, Slimb, may target beta-catenin for Ubiquitin/proteasome degradation. Also, the LEF/beta-catenin complex is thought to be affected in its translocation to the nucleus by mutations in PS. A genetic relationship between Drosophila PS and Arm is also suggested by a genetic modifier screen for mutations that can suppress the armadillo mutant phenotype. Together these observations implicate PS in a complex with beta-catenin as a means to target beta-catenin and possibly its cargo for degradation or other functions at remote sites in the cell. Thus the Presenilin/ beta-catenin complex may serve as an endoplasmic reticular staging platform for complex assembly and targeting to a variety of cellular destinations including the proteosome (Noll, 2000).

Before beta-catenin joins alpha-catenin and arrives at the plasma membrane, it forms a 'preadhesion' complex with Cadherin that is required for ER exit and membrane delivery of the complex. The delivery of Arm requires that the cell specify a polar trafficking route to the site of the adherens junctions at the apical part of the cell during blastoderm stages. The fact that many of these inclusions are located basally suggests impaired apical trafficking in the absence of PS. Although apparently reduced, sufficient Arm does reach the adherens junctions in these embryos so they do not develop an early adhesion defect phenotype. Instead, the phenotype includes a neurogenic defect thought to be related to the role of PS in cleaving Notch to generate an active product. The reports of the close resemblance between the Notch and PS phenotypes suggested a highly restricted function for PS: enhancement of Notch function by facilitating Notch cleavage. However, loss of Notch does not produce the Arm inclusions observed with loss of PS. This finding suggests a broader function for PS that extends beyond its role in Notch processing (Noll, 2000).

One site of residence for beta-catenin is in a complex with Axin, APC, and GSK3beta where it mediates regulation of Wnt signaling. Although quantitatively less frequent than the shotgun phenotype among embryos expressing hPS1, phenotypes that resemble the loss of wingless activity were occasionally observed, suggesting that binding to hPS1 also successfully competes beta-catenin away from its signaling pool. In conjunction with the evidence that PS is involved in Notch activation by releasing its cytoplasmic domain these findings suggest another link between the Wingless and Notch pathways. Previous studies reported genetic interactions between wg and N and direct interactions between these pathways via Dishevelled, as well as isolation of wg mutations in screens for genetic modifiers of Notch and vice versa. PS is primarily localized in the ER, but cleavage of Notch occurs either at or close to the cell surface. Transit of PS to the region of the adherens junction could resolve the contradiction between previous views regarding the location of PS in the endoplasmic reticulum and the cleavage of Notch either in or near the plasma membrane. PS is associated with two proteins -- beta-catenin and delta-catenin -- whose destination is the adherens junction. Both Notch and Wingless have also been reported in the region of the adherens junction. A large regulatory complex associated with PS may cleave Notch leaving the released cytoplasmic fragment to translocate to the nucleus and activate transcription or prevent transcription by binding through its carboxy terminus to Dsh. Dishevelled may independently localize to intracellular junctions through its discs large homology (DHR) region or utilize the PS complex to direct the Notch cytoplasmic fragment toward a degradative pathway (Noll, 2000 and references therein).

Alternatively, members of the PS complex such as beta-or delta-catenin may regulate the inhibitory interaction between Notch and Dishevelled. Because both of the putative substrates for PS (Notch and the amyloid precursor protein) transit through the ER, PS-associated proteins may serve in the ER to prevent premature cleavage. If PS cleaves Notch, it is curious that the expression of hPS1 does not induce a Notch activation phenotype. Neither was a Notch activation phenotype reported when the endogenous Drosophila PS was overexpressed. Significant inhibitory controls must be present that either prevent Notch cleavage or prevent the activity of the Notch active fragment. The interactions of beta-catenin with other proteins are complex and numerous. PS1 may delimit the components of the beta-catenin complex at specific cellular locales and allow it to discriminate among potential binding partners. In the case of the beta-catenin/APC complex, GSK3beta can phosphorylate both proteins. Although a direct association of GSK3beta with beta-catenin could not be demonstrated in vitro, it has been observed that Axin simultaneously and directly binds to APC, beta-catenin, and GSK3beta. The binding of all three proteins to axin may coordinate beta-catenin down-regulation by bringing these proteins into proximity (Noll, 2000).

GSK3beta binds PS1 between residues 259 and 298 of the fragment that is N-terminal after endoproteolytic cleavage. This site differs from the delta-catenin binding site on hPS1, which spans residues 319 to 371. Thus PS may coordinate the entry of both beta-catenin and GSK3beta into the complex with APC and Axin. Alternatively, PS may coordinate the trafficking routes of beta-catenin as it assembles and shifts large multicomponent protein complexes to diverse destinations in the cell (Noll, 2000).

Protein Interactions : Interaction of Armadillo with Brahma

Wnt-induced formation of nuclear Tcf-ß-catenin complexes promotes transcriptional activation of target genes involved in cell fate decisions. Inappropriate expression of Tcf target genes resulting from mutational activation of this pathway is also implicated in tumorigenesis. The C-terminus of ß-catenin is indispensable for the transactivation function, which probably reflects the presence of binding sites for essential transcriptional coactivators such as p300/CBP. However, the precise mechanism of transactivation remains unclear. An interaction between ß-catenin and Brg-1, a component of mammalian SWI/SNF and Rsc chromatin-remodeling complexes, is demonstrated. A functional consequence of reintroduction of Brg-1 into Brg-1-deficient cells is enhanced activity of a Tcf-responsive reporter gene. Consistent with this, stable expression of inactive forms of Brg-1 in colon carcinoma cell lines specifically inhibits expression of endogenous Tcf target genes. In addition, genetic interactions are observed between the Brg-1 and ß-catenin homologues in flies. It is concluded that ß-catenin recruits Brg-1 to Tcf target gene promoters, facilitating chromatin remodeling as a prerequisite for transcriptional activation (Barker, 2001).

Development in flies was selected as a model system to gain evidence for a functional interaction between Brg-1 and ß-catenin in vivo, assuming that this interaction would be conserved between mammals and Drosophila. It was asked whether reducing the gene dosage of brahma, the founder of the Brg-1 gene family, affects the mutant phenotypes caused by activation or depletion of Armadillo. First, a strain (GMR.Arm*) was used in which a constitutively activated form of Armadillo is overexpressed in the larval eye disc. The mutation in Arm* mimics the oncogenic point mutation S45F in the putative GSK3ß phosphorylation site of ß-catenin that renders the latter constitutively active. Oncogenic forms of ß-catenin such as this are potent transcriptional coactivators of Tcf. Flies bearing GMR.Arm* show rough and slightly glazed eyes whose size is reduced compared with the wild-type, due to late onset of apoptosis in the pupal disc caused by Arm* and dTcf. This phenotype is independent of armadillo gene dosage, but is reversed considerably towards wild-type in dTcf heterozygotes, whose gene dosage is reduced by half. This rough eye phenotype was reversed even further towards wild-type in brahma heterozygotes. Finally, a similar phenotypic suppression was observed in flies heterozygous for moira, a gene encoding another component of the Brahma complex. It is concluded that the mutant eye phenotype caused by activated Armadillo is as sensitive to the levels of Brahma complex components as it is to dTcf levels, indicating that the Brahma complex is required for the activity of Arm* (Barker, 2001).

It was also asked whether heterozygosity of Brahma complex genes would affect the mutant wing phenotype caused by Armadillo depletion in the wing disc. In the wing, armadillo is required for the integrity of the margin, and sequestration of Armadillo at the membrane by overexpression of the intracellular domain of cadherin (Armunder) in the posterior wing disc causes extensive notches in the posterior wing. This phenotype is worsened by heterozygosity for activating genes of the Wingless pathway, and suppressed by heterozygosity of antagonists of this pathway. In particular, in Armunder flies heterozygous for armadillo the posterior wing margin is completely absent, and the posterior wing area is much reduced. Likewise, dTcf heterozygotes showed on average slightly narrower wings, and less residual posterior margin than Armunder controls. This modifying effect of dTcf is much milder than that observed in the eye, perhaps reflecting a dual function of dTcf in the wing margin (activating as well as repressing) similar to that observed in the embryonic cuticle. Significantly, brahma heterozygotes show considerably narrower wings than the controls. Indeed, brahma heterozygosity enhances the wing margin phenotype as strongly as armadillo heterozygosity. Finally, a slight worsening of this phenotype is also observed in moira heterozygotes. These genetic experiments in flies indicate functional interactions between Brahma complex genes and Armadillo/dTcf. Consistent with this, it has been reported that embryos derived from near-sterile brm transheterozygous mothers show reduced expression of dTcf target genes such as Ultrabithorax and engrailed (Barker, 2001).

Taken together, these fly genetic data support the conclusions from experiments in mammalian cells that the Brg-1 complex contributes to the activity of the ß-catenin-Tcf transcription factor (Barker, 2001).

Groucho corepressor proteins, which repress Tcf target gene activity in the absence of Wnt signaling, are known to recruit histone deactylases and are likely to effect repression by altering chromatin structure. Additionally, a recent study has demonstrated a role for SWI/SNF-mediated chromatin remodeling of Tcf target gene promoters in ensuring effective repression of gene activity in the absence of ß-catenin during fly development. Potentially, Groucho proteins in complex with Tcf could recruit Brahma complexes to target gene promoters through an interaction mediated by the histone deacetylase rpd3. The data support a mechanism in which ß-catenin accumulation following Wnt signaling promotes the formation of ß-catenin-SWI/SNF (or -Rsc) complexes in the nucleus, in competition with Groucho repressor complexes. In cooperation with the histone-acetylating activity of ß-catenin-bound p300/CBP, Brg-1-associated complexes would then remodel the chromatin structure of target gene promoters into a conformation more accessible to the basal transcription machinery, enhancing transactivation of target genes and leading to cellular responses. The initially paradoxical observation that chromatin-remodeling complexes are required for both the activation and repression of perhaps the same set of target genes can be resolved by the finding that in vitro, SWI/SNF and Rsc can catalyse both forward and reverse nucleosome remodeling reactions (Barker, 2001).

Protein Interactions : Interaction of Armadillo with Chibby

Inappropriate activation of downstream target genes by the oncoprotein ß-catenin is implicated in development of numerous human cancers. ß-catenin and its fruitfly counterpart Armadillo act as coactivators in the canonical Wnt/Wingless pathway by binding to Tcf/Lef transcription factors. A conserved nuclear protein, named Chibby, has been identified in a screen for proteins that directly interact with the C-terminal region of ß-catenin. In mammalian cultured cells Chibby inhibits ß-catenin-mediated transcriptional activation by competing with Lef-1 to bind to ß-catenin. Inhibition of Drosophila Chibby by RNA interference results in segment polarity defects that mimick a wingless gain-of-function phenotype, and overexpression of the wingless target genes engrailed and Ultrabithorax. In addition, epistasis experiments indicate that chibby acts downstream of wingless and upstream of armadillo (Takemaru, 2003).

ß-catenin/Armadillo (Arm) is composed of 12 Arm repeats flanked by unique N and C termini. Both the N- and C-terminal regions exhibit transcriptional activation activity, but the most potent transactivation domain resides in the C terminus. To isolate cofactors that bind to the C-terminal region of ß-catenin and that could modulate its transducing activity, a screen was carried out with the yeast Ras recruitment system (RRS) that detects protein-protein interactions at the inner surface of the plasma membrane. As bait in the screen, Arm repeat 8 to the C-terminus was used. Of 79 positive clones, 59 encoded a full-length previously unknown human protein named Chibby (Cby, meaning 'small' in Japanese, named after the RNAi phenotype in fly). Human Cby contains 126 amino acids with predicted nuclear localization signals and a coiled-coil domain, and is evolutionarily conserved from fly to human (Takemaru, 2003).

With regard to the expression of cby, Northern blot analysis detects a single 1.2-kilobase (kb) RNA that is expressed at different levels in several tissues of the adult human. Cby protein is predominantly nuclear in a punctate manner, as visualized by immunostaining of the endogenous protein in COS7 cells and other cell lines. Transfection of Wnt-1, Wnt-5a or ß-catenin did not significantly change the nuclear localization of Cby in COS7 cells (Takemaru, 2003).

The yeast RRS was then used to define the Cby binding domain of ß-catenin; Arm repeat 10 to the C-terminus is required for its interaction with Cby. Consistent with this finding, in vitro pull-down assays demonstrated that Cby directly binds to the C-terminal region of ß-catenin as well as to the full-length protein, but not to the N-terminal region. However, Cby associates with ß-catenin through its C-terminal half (Takemaru, 2003).

To test for their interaction in vivo, human embryonic kidney 293T cells were co-transfected with expression plasmids encoding Myc-tagged ß-catenin and Flag-tagged Cby, and the lysates were immunoprecipitated. As predicted, ß-catenin co-immunoprecipitates with Cby and vice versa. Finally, whether the endogenous proteins interact was investigated by immunoprecipitations from nuclear extracts of non-transfected 293T cells. Immunoprecipitation of ß-catenin pulls down Cby, but not the unrelated nuclear proteins Cyclin A or TFIIB. It is concluded that Cby is normally present in a complex with ß-catenin in vivo (Takemaru, 2003).

To evaluate whether Cby modifies ß-catenin-dependent signalling, Tcf (T-cell factor) reporter assays were performed in 293T cells. Overexpression of Cby represses the transcriptional activation of the ß-catenin-dependent Tcf reporter by Wnt-1 or stabilized ß-catenin in a dose-dependent fashion, showing minimal effects on a mutant reporter. Similar results were obtained using other cell types including COS7 and NIH3T3. Consistent with these results, the activity of the ß-catenin-dependent cyclin D1 reporter was also repressed by co-expression of Cby with stabilized ß-catenin. ß-catenin protein remains stable in the presence of high levels of Cby, indicating that Cby does not promote ß-catenin degradation (Takemaru, 2003).

To investigate possible alternative mechanisms by which Cby inhibits ß-catenin-dependent gene activation, Myc-tagged ß-catenin and Flag-tagged Cby were co-transfected with increasing amounts of hemagglutinin (HA)-tagged Lef-1 (lymphoid-enhancer factor-1), then immunoprecipitated with an anti-Myc antibody. Because the amount of Cby co-precipitating with ß-catenin decreases as the amount of Lef-1 associating with ß-catenin increases, it is concluded that Cby competes with Lef-1 for binding to ß-catenin. In contrast, Cby has no apparent effect on the interaction between ß-catenin and APC or CBP. Additional data suggest that Cby negatively regulates ß-catenin-mediated transactivation by competing with Tcf/Lef transcription factors in mammalian cells (Takemaru, 2003).

To investigate the role of Cby in Wnt signalling during development, Drosophila was examined because the fruitfly expresses a Cby ortholog that, like the human Cby described above, inhibits ß-catenin-dependent activation of reporter in mammalian cultured cells. cby double-stranded RNA was injected into wild-type embryos; this resulted in transformation of ventral denticles into naked cuticle. This phenotype is similar to embryos that overexpress wingless (wg) in all epidermal cells. In contrast, losing wg pathway activity results in transformation of naked cuticle into denticles. In addition, the head structures of cby(RNAi) embryos were missing, and the embryos were small. These phenotypes were also observed in embryos that express intron-spliced snapback RNA corresponding to cby. To confirm the specificity of the RNAi phenotype, a short interfering RNA was injected whose sequence did not overlap with either the dsRNA or snapback RNA used earlier. The siRNA duplex produced a highly similar phenotype, indicating that the phenotype is caused by specific reduction of cby activity. The cuticle phenotype suggests that cby functions as an antagonist of the wg pathway. To test this possibility further, expression of the gene engrailed (en), which is transcriptionally activated by wg signalling in the cuticle-secreting embryonic epidermis, was examined (Takemaru, 2003).

En is normally expressed in segmental stripes two cells wide, immediately behind cells expressing wg. In cby(RNAi) embryos, the En stripes expanded by another row of cells, a phenotype similar to one observed when embryos overexpress wg in the en domain. This indicates that cby normally represses expression of en. Because en is in turn required to maintain wg expression, wg activates its own expression through a paracrine feedback loop. RNAi depletion of cby results in expansion of wg messenger RNA and protein expression, further indicating that loss of cby function leads to wg pathway activation. The expanded width and intensity of the Wg stripes is greater than in embryos where wg signalling is maximally stimulated. This suggests that in addition to wg signalling, cby acts in a regulatory process at present unknown (Takemaru, 2003).

Experiments with mammalian Cby suggest that it inhibits Wnt signalling by binding to ß-catenin in the nucleus and blocking its interaction with Tcf/Lef transcription factors. If Drosophila Cby functions in a similar manner, then loss of cby should not affect the abundance or localization of Arm (ß-catenin). Arm localizes to nuclei in stripes of Wg-responding cells at stage 9 of embryogenesis (Takemaru, 2003).

Arm protein was examined in cby(RNAi) embryos; Arm abundance and localization were not detectably affected. If Cby were involved in transducing the Wg signal, then Wg would be predicted to lie upstream of Cby in an epistasis genetic pathway. To test for epistasis, the expression of a wg-responsive UbxB-lacZ reporter gene was examined in the embryonic midgut (Takemaru, 2003).

Expression is controlled by an enhancer that interacts with Drosophila Tcf and is activated in a manner dependent upon Wg signalling. Wg is expressed in parasegment (ps) 8, where it controls UbxB-lacZ expression in visceral mesoderm throughout ps7-9, as well as development of the second midgut constriction. In a wg null embryo, UbxB-lacZ expression is greatly reduced and the midgut constriction fails to form, indicating a strong dependence on wg function. In cby(RNAi) embryos, lacZ expression expanded into anterior and posterior parasegments, and the second but not the first midgut constriction formed; these phenotypes are reminiscent of moderate wg misexpression throughout the midgut. Thus, consistent with the observations of cby function in the epidermis, cby represses wg-dependent gene expression and development in visceral mesoderm. When cby was depleted by RNAi in wg null embryos, UbxB-lacZ expression was not blocked, and development of the second midgut constriction occurred. The wg;cby(RNAi) embryos resembled wild-type more than cby(RNAi) embryos; they sometimes exhibited more restricted lacZ expression and a first midgut constriction. Since embryonic RNAi usually leads to reduced levels of gene product, this implies that the residual cby product represses visceral mesoderm more effectively in the absence of wg. These results establish that wg function is mediated, at least in part, through cby (Takemaru, 2003).

The relationship was examined between cby and arm. Embryos that are arm;cby(RNAi) resemble arm embryos; they display background lacZ expression and strongly inhibit second midgut constriction. The block to midgut constriction is possibly incomplete, owing to residual arm activity supplied maternally (embryos depleted of maternal arm product exhibit a complete block to this constriction). The similarity of phenotypes of arm and arm;cby(RNAi) embryos suggests that cby functions upstream of arm in Wg signalling, and that in wild-type embryos the role of cby is to repress arm. These results suggest that cby activity is not mediated through a redistribution of ß-catenin/Arm protein, but rather from inhibiting nuclear ß-catenin/Arm activity. This interpretation is consistent with Cby's conserved ability to associate with ß-catenin and compete with Tcf/Lef for ß-catenin. Such a competitive block would account for Cby attenuating Tcf/Lef dependent gene activation in both mammalian cells and Drosophila embryos (Takemaru, 2003).

It is concluded that Cby is a nuclear protein that is conserved throughout evolution. It antagonizes Wnt/Wg signalling by inhibiting ß-catenin/Arm function in mammalian cells and in Drosophila, raising the possibility that it may be a tumor suppressor gene. In this regard, Cby expression was found to be significantly downregulated in thyroid and metastatic uterine tumors, and nuclear Cby staining is missing or considerably weaker in thyroid tumors. Dysregulation of ß-catenin signalling has been reported in these types of cancers, so the decreased levels of Cby expression might be relevant to tumor formation (Takemaru, 2003).

Distinct sites in E-cadherin regulate different steps in Drosophila tracheal tube fusion: Short stop and E-cadherin form a feedback loop in which E-cadherin, via ß-catenin, recruits Shot to new contacts between the fusion cells

How E-cadherin controls the elaboration of adherens junction-associated cytoskeletal structures crucial for assembling tubular networks was investigated. During Drosophila development, tracheal branches are joined at branch tips through lumens that traverse doughnut-shaped fusion cells. Fusion cells form E-cadherin contacts associated with a track that contains F-actin, microtubules, and Short stop (Shot), a plakin that binds F-actin and microtubules. Live imaging reveals that fusion occurs as the fusion cell apical surfaces meet after invaginating along the track. Initial track assembly requires E-cadherin binding to ß-catenin. Surprisingly, E-cadherin also controls track maturation via a juxtamembrane site in the cytoplasmic domain. Fusion cells expressing an E-cadherin mutant in this site form incomplete tracks that contain F-actin and Shot, but lack microtubules. These results indicate that E-cadherin controls track initiation and maturation using distinct, evolutionarily conserved signals to F-actin and microtubules, and employs Shot to promote adherens junction-associated cytoskeletal assembly (Lee, 2003).

Junctional contacts between cells are important for organizing the cytoskeleton and regulating cell polarity. The large size of plakins and their modular abilities to bind different cytoskeletal elements make them potentially well suited to play key organizational roles. However, except in the case of desmosomes, where the plakin desmoplakin appears to be a crucial for organizing junction-associated cytoskeleton, functional association of plakins with other cell-cell junctions has not been described (Lee, 2003).

In selected cell types, Shot localizes with proteins of the adherens junction and may play a role in adherens junction-mediated organization of the cytoskeleton. It is proposed that Shot and E-cadherin form a feedback loop in which E-cadherin, via ß-catenin, recruits Shot to new contacts between the fusion cells and Shot stabilizes the contacts. The cytoskeleton organizes around these contacts because adherens junction associated Shot promotes the assembly of an F-actin/microtubule-rich track. This track grows to span the fusion cells, extending the reach of the junctions through the cells. The recruitment mechanism may be indirect in that new adherens junctions in fusion cells are centers for cytoskeletal assembly, and Short Stop binds F-actin and microtubules. Alternatively, Shot may associate directly with E-cadherin or associated proteins. The assembly of Shot with F-actin and microtubules may stabilize E-cadherin contacts simply by bringing in cytoskeletal proteins that bind E-cadherin or associated proteins. For example, EB1, which is present in the fusion track, co-immunoprecipitates with a C-terminal fragment of Shot in cultured cells and associates with APC. APC interacts with ß-catenin to control tubulogenesis in vitro (Lee, 2003).

It is proposed that the assembly and maturation of a cytoskeletal intermediate are two E-cadherin-dependent steps in tracheal cell fusion. Imaging of fixed and live embryos suggests that fusion proceeds through the assembly and maturation of a cytoskeletal track associated with adherens junctions. The track forms after contact between the fusion cells, and persists for ~1 hour before fusion occurs (Lee, 2003).

In this model, the ß-catenin-binding site and the juxtamembrane site in the E-cadherin cytoplasmic domain operate sequentially and in the same E-cadherin molecule to promote fusion. In mutant embryos in which either ß-catenin or its binding site is defective, fusion cells make contact but track assembly is not observed. These data suggest that E-cadherin may initiate track assembly via ß-catenin. A mutation in the juxtamembrane site dominantly inhibits track maturation. Microtubules are generally absent from fusion tracks in these embryos, though some F-actin and Shot assembly occurs. In E-cadherin/shotgun (shg) mutant embryos, E-cadherin bearing this juxtamembrane mutation supports a low level of F-actin/Shot track formation, but the tracks do not mature. In addition, this juxtamembrane mutant E-cadherin causes progressive delocalization of the apical tracheal cytoskeleton in shg mutant embryos (Lee, 2003).

Both the ß-catenin and juxtamembrane binding sites are required for E-cadherin localization to adherens junctions, although only the juxtamembrane mutation seems to interfere with endogeneous E-cadherin localization. The results suggest that like mammalian E-cadherin, an evolutionarily conserved juxtamembrane site is required for some E-cadherin functions. Similar effects of mutations in the juxtamembrane site were observed in mammalian tissue culture cells. However, juxtamembrane site function in Drosophila E-cadherin probably does not require p120 (Lee, 2003).

Dominant effects on localization appear sensitive to expression levels, whereas effects on fusion are less so, suggesting that defects in localization are not enough to explain the defects in track maturation. Possibly, effects on localization also reflect defects in organizing the cytoskeleton, as has been observed in studies in which dominant alleles of Rho family GTPases affect cadherin localization in culture (Lee, 2003).

It is proposed that the ß-catenin-binding site and ß-catenin are required for track assembly, and that the juxtamembrane site regulates other proteins involved in a later maturation step. This later step likely requires microtubules. The microtubules or associated proteins may reinforce the initial F-actin assembly in the track, as F-actin in fusion tracks appears to be abnormally or poorly assembled in embryos expressing AAA-JXT mutant E-cadherin in tracheal cells. The microtubules appear to be also required for remodeling the fusion cell apical surfaces and also for bringing them together to fuse. In embryos expressing AAA-JXT mutant E-cadherin in tracheal cells, fusion cell apical surfaces do not develop or seal gaps at appropriate times, and fusion tracks persist substantially longer, if they resolve at all (Lee, 2003).

The microtubule regulated steps during fusion therefore likely involve effects on F-actin dynamics. Microtubule-associated factors that may regulate the F-actin cytoskeleton include Rac GTPase and exchange factors for Rho GTPase. Rac1 affects E-cadherin dependent adhesion in tracheal cells and a mutation in the juxtamembrane site in mammalian E-cadherin analogous to the one described in this study affects Rac activation. RhoA activation inhibits fusion track assembly. Downstream interactions between F-actin and microtubules, such as those mediated by Shot, may vary with cell type to produce distinct morphogenetic outcomes. Further studies of tracheal tube fusion, a genetic system in which adherens junction associated structures can be visualized in living embryos, promises to identify the regulatory molecules that allow E-cadherin to direct F-actin and microtubule assembly from the ß-catenin binding and juxtamembrane domains (Lee, 2003).

Notch modulates Wnt signalling by associating with Armadillo/ß-catenin and regulating its transcriptional activity

The establishment and stability of cell fates during development depend on the integration of multiple signals, which ultimately modulate specific patterns of gene expression. While there is ample evidence for this integration at the level of gene regulatory sequences, little is known about its operation at other levels of cellular activity. Wnt and Notch signalling are important elements of the circuitry that regulates gene expression in development and disease. Genetic analysis has suggested that in addition to convergence on the transcription of specific genes, there are modulatory cross-regulatory interactions between these signalling pathways. The nodal point of these interactions is an activity of Notch that regulates the activity and the amount of the active/oncogenic form of Armadillo/ß-catenin. This activity of Notch is independent of that induced upon cleavage of its intracellular domain and which mediates transcription through Su(H)/CBF1. The modulatory function of Notch described in this study, contributes to the establishment of a robust threshold for Wnt signalling which is likely to play important roles in both normal and pathological situations (Hayward, 2005).

A soluble form of the intracellular domain of Notch, NICD, acts as an activated Notch receptor and provides constitutive Su(H)-dependent Notch signalling. Whereas a chimera between the extracellular and transmembrane domains of the receptor tyrosine kinase (RTK) Torso and the intracellular domain of Notch (TNotch) prevents the cleavage of Notch and the translocation of its intracellular domain to the nucleus. However, this chimeric molecule is still capable of signalling, as reflected by the loss of neural precursors during neurogenesis. This signalling event is likely to be independent of Su(H) because while NICD and full length Notch are able to activate transcription of either a Su(H) reporter in vivo or the Notch target gene wingless, TNotch is unable to do so. Thus TNotch behaves as a gain-of-function allele but one specific for a particular function of Notch which might not involve Suppressor of Hairless. In agreement with this, TNotch is unable to rescue a complete loss of function of Notch (Hayward, 2005).

The inputs of Notch and Wingless signalling on the development of the wing are well characterised. Notch and Wingless signalling cooperate in the development of the wing and in the case of Notch the effects are mediated by NICD. To test if the cleavage-independent function of Notch modulates Wingless signalling, NICD and TNotch were expressed at the same time that Wingless signalling was activated either with ectopic expression of Wingless or of a constitutively active form of Armadillo, Armadillos10. This form of Armadillo lacks the Shaggy/GSK3ß phosphorylation sites and provides Wingless-independent signalling by escaping degradation by the Axin-based destruction complex. Expression of either Wingless or Armadillos10 along the AP boundary results in an expansion of the hinge region and the occasional appearance of extra wing tissue off the notum. However, the effects of the intracellular domain of Notch depend on its molecular disposition. Expression of NICD along the AP boundary induces the appearance of an ectopic wing margin and promotes the growth of the wing, while expression of TNotch leads to a slight reduction in the overall size of the wing pouch region of the disc. In the developing wing, co-expression of NICD with either Wingless or Armadillos10 leads to a synergistic effect of extra growth of the wing tissue. In contrast to NICD, TNotch is very effective in suppressing the effects of ectopic expression of Wingless and, surprisingly, also of Armadillos10 (Hayward, 2005).

Since Armadillos10 provides Wingless signalling constitutively and expression of TNotch does not affect the expression of Wingless in the third instar discs, these results argue that a Su(H)-independent Notch activity modulates Wingless signalling by targeting the activity of Armadillo. To test this further, the effects of TNotch were analyzed on the ability of Armadillos10 to induce expression of Wingless target genes, Distalless (Dll) a low threshold target of Wingless, and the proneural gene senseless (sens), which like other proneural genes, provides a high threshold target. Both are elevated and ectopic in the presence of Armadillos10, and in both cases TNotch markedly suppresses this effect (Hayward, 2005).

To test whether the effects observed are restricted to the developing wing, the effects of TNotch on the cuticle pattern of the first instar larva were monitored. In the wild-type each segment contains an anterior region decorated with denticles and a 'naked' posterior region, devoid of denticles. The extent of the 'naked' region depends on the level of Wingless signalling, and ubiquitous Wingless signalling associated with strong expression of Armadillos10 results in cuticles all devoid of denticles. By modulating the levels of expression of Armadillos10 it is possible to modulate the extent of denticle loss: weak expression leads to a patchy loss of denticles in contrast, strong expression results in ventral cuticles completely devoid of denticles. Expression of TNotch modulates the effects that Armadillos10 has on the pattern of the cuticle: while strong effects of Armadillos10 are often suppressed. This observation confirms that Notch exerts a negative modulation on Wnt signalling and suggests that this might be a general phenomenon. Altogether these observations suggest that there is an activity of Notch, independent of Su(H), which modulates the Wingless signalling pathway at or below the level of Armadillo (Hayward, 2005).

Armadillo and Notch show a high degree of co-localisation at the adherens junction of the epidermal cells of the wing disc. To test whether Notch and Armadillo are associated in the cell, Notch from developing embryos was immunoprecipitated and Armadillo was sought among the co-immunoprecipitated proteins. Two different anti-Notch antibodies were used and in both cases Armadillo protein was detected in the same protein complex as the immunoprecipitated Notch protein. Interestingly, the predominant form of Notch protein detected in these assays is unprocessed and uncleaved, suggesting that this complex is membrane associated. The reverse experiment, in which Armadillo protein is immunoprecipitated, was also undertaken; here an unprocessed and uncleaved form of Notch was found to be associated with Armadillo. Previous experiments have indicated that Dishevelled, another element of Wnt signalling, can associate with Notch in a yeast two-hybrid assay. This was confirmed and this association was shown in the same immunoprecipitates from embryos in which the complex between Notch and Armadillo was found. These results indicate that the intracellular domain of Notch and a proportion of the Armadillo protein of the cell are associated in the same protein complex. Preliminary data suggests that this association is preferentially mediated by the region C-terminal to the cdc10/ANK repeats and such an interaction might be an element in the functional interactions described in this study (Hayward, 2005).

The precise mechanism whereby Wnt proteins elicit the activity of ß-catenin is still under scrutiny but it is generally agreed that the stability and amount of cytoplasmic Armadillo/ß-catenin are rate-limiting steps in the signalling event. This pool of Armadillo/ß-catenin is under very tight control by a destruction complex assembled on Axin, which together with Shaggy/GSK3ß are the main targets of Wnt signalling. However, there is increasing evidence that high levels of cytoplasmic Armadillo/ß-catenin are not sufficient to promote Wnt signalling. Recently emphasis has been placed on the observation that Axin can regulate the activity of Armadillo/ß-catenin in a Shaggy/GSK3ß-independent manner. This has led to the conclusion that Wnt regulates the activities of Shaggy/GSK3ß and Axin co-ordinately and that there might be other factors contributing to the control of Armadillo/ß-catenin activity. Consistent with this possibility it has been reported that Wnt signalling can regulate the activity of stable oncogenic forms of ß-catenin (Hayward, 2005).

This study shows that Notch signalling provides an important input into Wnt signalling in Drosophila by associating with Armadillo and regulating its levels and activity during Wingless signalling. This activity of Notch, which is different and probably independent of that which mediates CBF1/Su(H)-dependent signalling, lies functionally downstream of Shaggy/GSK3ß and targets the concentration and activity of the hypophosphorylated form of Armadillo. It can also modulate the activity of an oncogenic form of vertebrate ß-catenin and this functional interaction between Notch and Armadillo has been shown to extend to the vertebrate system, with mNotch1 regulating the activity of ß-catenin in tissue culture cells (Hayward, 2005).

A role for Notch in the modulation of Wnt signalling has been inferred from genetic analysis. However, although these results indicate that Notch antagonises Wnt signalling, alone they do not provide insights into the mechanism of the interaction. The current study does, and it is likely that the molecular interactions that are reported in this study underpin the observed modulation of Wnt signalling by Notch. Wingless signalling can be activated in vivo in the absence of Notch and this activation does not require Dishevelled. The observations that removal of Notch in cl8 cells leads to activation of a synthetic Wnt reporter confirm this and suggest a direct regulatory effect of Notch on the mechanism of Wnt signalling. Furthermore, the effects of Notch on the activated form of Armadillo offer an explanation for why removal of Notch can bypass a requirement for Dishevelled. It may well be that even under steady state conditions there is a small amount of hypophosphorylated, active Armadillo/ß-catenin which escapes the Axin/GSK3ß mediated degradation. Given the high specific activity of this molecule, it is not surprising that there might be further mechanisms that control it. Notch appears to be an essential part of these mechanisms and in its absence this active form of Armadillo would operate even in the absence of Dishevelled. Axin is also likely to be involved in the regulation of the active form and Axin can also suppress the effects of an activated form of Armadillo. It will be of interest to explore the relationships between Notch and Axin (Hayward, 2005).

Previous studies have implicated Deltex and Dishevelled as important elements of the interaction between Notch and Wingless signalling. Both proteins bind Notch, but they do so in different places. Deltex binds to the cdc10/ANK repeats and promotes Su(H)-independent Notch signalling. Whereas, Dishevelled binds within a broad region C-terminal to this domain and reduces the Su(H)-independent activity of Notch. This study has shown that Armadillo also physically interacts with Notch, probably through the same broad region that binds Dishevelled. Mutations in Notch that impair this domain result in Notch receptors that interfere with Wnt signalling. The deletion of this region reduces the efficiency with which the intracellular domain of Notch affects the levels and activity of Armadillo. Together these observations underscore the role of this region of Notch in mediating interactions between Notch and Wnt signalling by targeting the active form of Armadillo/ß-catenin (Hayward, 2005).

The relationship between Notch and Armadillo in Drosophila extends to their vertebrate homologues, Notch1 and ß-catenin. This interaction, rather than an interaction of Dishevelled with Notch/CBF signalling, might reflect the functional relationships between the two signalling systems that have been reported during the development of the skin the immune system and in somitogenesis. In these instances Wnt and Notch drive alternative fates (skin and immune system) or act antagonistically (somites) perhaps by a combination of their individual pathways and the modulatory interaction described in this study. One consequence of this modulatory interaction might also be the observed tumour suppressor function of Notch1 in the mouse skin where removal of Notch1 results in the generation of tumours associated with an increase in the levels of active ß-catenin and Wnt signalling. While some of the elevation of ß-catenin in these cells might be a secondary consequence of activation of Wnt signalling, the current observations suggests that the loss of Notch1 can also contribute to this increase by allowing the activation of cß-catenin. In a different study carboxyl-terminal deletions in Notch1, which include the region that binds Dishevelled and Armadillo, enhanced the oncogenic effects of a chimeric E2A-PBX1 protein. It is possible that some of this effect is due to misregulation of ß-catenin in the tumours (Hayward, 2005).

In summary, Notch provides a modulatory input in the activity of Armadillo/ß-catenin. This modulation provides two functions: it establishes a threshold for Wnt signalling that is likely to play an important role in the patterning of tissues and the assignation of cell fates during development and, in addition it provides a stringent regulation of the activated form of Armadillo/ß-catenin. The second function might be crucial in pathological situations and might contribute to the understanding of Notch as a tumour suppressor (Hayward, 2005).

Notch synergizes with axin to regulate the activity of armadillo in Drosophila

Cell fate decisions require the integration of various signalling inputs at the level of transcription and signal transduction. Wnt and Notch signalling are two important signalling systems that operate in concert in a variety of systems in vertebrates and invertebrates. There is evidence that the Notch receptor can modulate Wnt signalling and that its target is the activity and levels of Armadillo/β-catenin. This function of Notch has been characterized in relation to Axin, a key element in the regulation of Wnt signalling that acts as a scaffold for the Shaggy/GSK3beta-dependent phosphorylation of Armadillo/beta-catenin. While Notch can regulate ectopic Wingless signalling caused by loss of function of Shaggy, it can only partially regulate the ectopic Wnt signalling induced by the loss of Axin function. The same interactions are observed in tissue culture cells where a synergy is observed in between Axin and Notch in the regulation of Armadillo/β-catenin. These results provide evidence for a function of Axin in the regulation of Armadillo that is different from its role as a scaffold for GSK3β (Hayward, 2006).

Requirements of genetic interactions between Src42A, armadillo and shotgun, a gene encoding E-cadherin, for normal development in Drosophila

Src42A is one of the two Src homologs in Drosophila. Src42A protein accumulates at sites of cell-cell or cell-matrix adhesion. Anti-Engrailed antibody staining of Src42A protein-null mutant embryos indicated that Src42A is essential for proper cell-cell matching during dorsal closure. Src42A, which is functionally redundant to Src64, was found to interact genetically with shotgun, a gene encoding E-cadherin, and armadillo, a Drosophila ß-catenin. Immunoprecipitation and a pull-down assay indicated that Src42A forms a ternary complex with E-cadherin and Armadillo, and that Src42A binds to Armadillo repeats via a 14 amino acid region, which contains the major autophosphorylation site. The leading edge of Src mutant embryos exhibiting the dorsal open phenotype is frequently kinked and associated with significant reduction in E-cadherin, Armadillo and F-actin accumulation. This phenotype suggests that not only Src signaling but also Src-dependent adherens-junction stabilization are essential for normal dorsal closure. Src42A and Src64 are required for Armadillo tyrosine residue phosphorylation but Src activity may not be directly involved in Armadillo tyrosine residue phosphorylation at the adherens junction (Takahashi, 2005).

In ectodermal cells, strong Src42A signals in apical or apicolateral regions were always associated with strong E-cad signals. E-cad is a core component of the adherens junction that is responsible for cell-cell adhesion and, hence, most, if not all, E-cad-associated membranous Src42A is probably related to adherens junction-dependent cell-cell adhesion (Takahashi, 2005).

A considerable fraction of ectodermal cells were also found associated with the second type of basal Src42A free of E-cad. E-cad-free Src42A is localized on the ectoderm/mesoderm interface and eliminated from ectodermal cells that have evaginated or invaginated without mesoderm association. The extracellular matrix (ECM) comprises several groups of secreted proteins such as integrin ligands. During embryogenesis, different cell layers become properly connected, most probably via cell adhesion to ECM. E-cad-free Src42A may thus be related to integrin-mediated cell-matrix adhesion. Cell-ECM adhesion may not be restricted to the interface between ectodermal and mesodermal cell layers. Strong Src42A signals have actually been found present on the interface between mesodermal and endodermal cell layers (Takahashi, 2005).

The current study shows that, as with JNK signaling genes, Src is required not only for thick F-actin accumulation at the leading edge but proper cell-cell matching along the midline seam as well. JNK signaling, which includes hemipterous (hep) and basket (bsk), is essential for dorsal closure of the embryonic epidermis in Drosophila. Based on examination of Tec29 Src42A mutant phenotypes, it has been suggested that Src42A acts upstream of bsk (Takahashi, 2005).

The adherens junction is necessary for cell-cell adhesion and thick F-actin accumulation occurs at the level of the adherens junction at the leading edge. Since E-cad and Arm signals along with actin signals are reduced significantly at the leading edge in Src42A26-1;Src64P1/+ embryos and the leading edge of the mutants is significantly kinked, the absence of Src protein from the adherens junction may possibly result in destruction of structural integrity, implying that the adherens junction is also involved in dorsal closure regulation in a structural way (Takahashi, 2005).

Dorsal closure and CNS defects similar to those in Src mutants have been observed in abl mutants. In vertebrates, Abl is tyrosine-phosphorylated with Src and is capable of interacting with delta-catenin, an E-cad-binding protein. Abl may thus function as well downstream of Src signaling in Drosophila. Germ-band retraction and possibly too, head involution, both of which require Src activity, may be regulated by the two above distinct Src functions. alpha1,2-laminin and alphaPS3ßPS integrin have clearly been shown to be essential for spreading a small group of amnioserosa epithelium cells over the tail end of the germ band during germ-band retraction. shg activity has also been shown to be essential for normal germ-band retraction and head involution (Takahashi, 2005).

Src-dependent dynamical regulation of E-cad-dependent cell-cell adhesion may also be necessary for visual system formation. E-cad overexpression or elimination of EGFR activity have been shown to render optic placode cells incapable of invaginating and prevent the separation of Bolwig's organ precursors from the optic lobe. Virtually identical phenotypes were induced by loss of Src activity, suggesting involvement of at least the adherens junction Src in larval visual system formation and that Src should function either upstream or downstream of EGFR signaling (Takahashi, 2005).

Pygopus and Legless target Armadillo/beta-catenin to the nucleus to enable its transcriptional co-activator function

Wnt signalling controls the transcription of genes that function during normal and malignant development. Stimulation by canonical Wnt ligands activates beta-catenin (or Drosophila melanogaster Armadillo) by blocking its phosphorylation, resulting in its stabilization and translocation to the nucleus. Armadillo/beta-catenin binds to TCF/LEF transcription factors and recruits chromatin-modifying and -remodelling complexes to transcribe Wnt target genes. The transcriptional activity of Armadillo/beta-catenin depends on two conserved nuclear proteins recently discovered in Drosophila, Pygopus (Pygo) and Legless/BCL-9 (Lgs). Lgs functions as an adaptor between Pygo and Armadillo/beta-catenin, but how Armadillo/beta-catenin is controlled by Pygo and Lgs is not known. This study shows that the nuclear localization of Lgs entirely depends on Pygo, which itself is constitutively localized to the nucleus; thus, Pygo functions as a nuclear anchor. Pygo is also required for high nuclear Armadillo levels during Wingless signalling, and together with Lgs increases the transcriptional activity of beta-catenin in APC mutant cancer cells. Notably, linking Armadillo to a nuclear localization sequence rescues pygo and lgs mutant fly embryos. This indicates that Pygo and Lgs function in targeting Armadillo/beta-catenin to the nucleus, thus ensuring its availability to TCF during Wnt signalling (Townsley, 2004).

Dissecting nuclear Wingless signalling: Recruitment of the transcriptional co-activator Pygopus by a chain of adaptor proteins including Arm, Pan, and Legless

Members of the Wingless (Wg)/Wnt family of secreted glycoproteins control cell fate during embryonic development and adult homeostasis. Wnt signals regulate the expression of target genes by activating a conserved signal transduction pathway. Upon receptor activation, the signal is transmitted intracellularly by stabilization of Armadillo (Arm)/beta-catenin. Arm/beta-catenin translocates to the nucleus, interacts with DNA-binding factors of the Pangolin (Pan)/TCF/LEF class and activates transcription of target genes in cooperation with the recently identified proteins Legless/BCL9 (Lgs) and Pygopus (Pygo). This study analyses the mode of action of Pan, Arm, Lgs, and Pygo in Drosophila cultured cells. Evidence is provided that together these four proteins form a chain of adaptors linking the NH2-terminal homology domain (NHD) of Pygo to the DNA-binding domain of Pan. NHD has potent transcriptional activation capacity, which differs from that of acidic activator domains and depends on a conserved NPF tripeptide. A single point mutation within this NPF motif abolishes the transcriptional activity of the Pygo NHD in vitro and strongly reduces Wg signalling in vivo. Together, these results suggest that the transcriptional output of Wg pathway activity largely relies on a chain of adaptors designed to direct the Pygo NHD to Wg target promoters in an Arm-dependent manner (Stadeli, 2005).

The identification of β-catenin as the mediator of Wnt signals has spurred investigations and discussions of how a cell adhesion molecule (relocated to the nucleus) can function as a transcriptional activator. One step towards a molecular understanding of nuclear β-catenin function was the discovery of Lgs. Genetic and biochemical experiments indicated that both proteins assist β-catenin in its transactivator role and led to the hypothesis that Lgs functions to recruit Pygo to the β-catenin/TCF complex and hence to the regulatory regions of Wnt target genes. By a series of transcriptional assays in cultured Drosophila cells, this study shows that the four dedicated nuclear components of the Wg signalling pathway, Pan/TCF, Arm/β-catenin, Lgs, and Pygo, act together in a linear manner in order to activate target gene expression. The results have led to the formulation of a 'chain of adaptors' model for nuclear Wg signalling, assigning each of these four proteins a linker function connecting a proximal and a distal component, culminating in the recruitment of the NH2-terminal homology domain of Pygo (PygoNHD) to the promoter of Wg targets. In S2 cells, PygoNHD stimulates reporter gene expression when directly tethered to DNA, indicating that this domain is capable of bestowing transcriptional activity on Arm/β-catenin.

Arm/β-catenin is composed of three domains, each of which bears transcriptional activity when tethered to DNA: the NH2-terminal region. The activity of the NH2-terminus of Arm might not be relevant in the context of the full-length protein, since only an isolated NH2-terminal fragment of Arm[wt] shows transcriptional activity when tethered to DNA. In contrast, an NH2-terminal fragment of ArmS10 or ArmN-R4[D164A] does not show transcriptional activity in this assay. It is thus proposed that Arm's co-activator capacity maps primarily to the COOH-terminal domain and to the Lgs/Pygo recruiting region located in Arm repeats 1-4. While the COOH-terminus of Arm might activate gene expression by recruiting cofactors like the histone acetyltransferase CBP/p300 or the chromatin remodelling enzyme Brg-1/Brahma, the findings indicate that the activity of ArmR1-4 strictly relies on Lgs and Pygo (Stadeli, 2005).

The role of Lgs serving as an adaptor protein to link Pygo with Arm is widely accepted. In contrast, the role of Pygo is somewhat controversial. The finding that the replacement of PygoPHD by LgsHD2 or the fusion of LgsHD2 with PygoNHD results in chimeric proteins that can substitute for both Lgs and Pygo function implies that the LgsHD1 as well as the PygoPHD are dispensable if the PygoNHD is brought to Arm by more direct means. Interestingly, it has been shown in human 293T cells that the activity of DNA-tethered Pygo depends on its PHD. When similar assays were carried out using DNA-tethered constructs in Drosophila S2 cells, it was found that PygoΔPHD is about half as active as full-length Pygo. However, deletion of the NHD leads to a more pronounced decrease in activity, while the isolated NHD is almost as active as full-length Pygo, indicating that the NHD is the key activating domain of Pygo. Consistent with this view, the above-mentioned chimeric proteins lose their rescuing activity if the PygoNHD is mutated. Collectively, these results strongly suggest that Pygo, by means of its NHD, acts as a transcriptional co-activator in Wg signalling (Stadeli, 2005).

Analysis of DNA-tethered PygoNHD in S2 cells reveals that this domain does not act as a classical acidic activator domain. Rather, the conserved tripeptide NPF plays a crucial role in the activity of PygoNHD. Pygo is one out of 791 NPF motif-containing proteins in Drosophila. In yeast, a NPF motif has been found to serve as a recognition motif for proteins bearing Eps15 homology (EH) domains. Since EH domain-containing proteins might work as integrators of signals controlling cellular pathways as diverse as endocytosis, cell proliferation, or nucleocytosolic export, a possible functional link between such proteins and Pygo was examined. However, RNAi-mediated knock-down of all four Drosophila EH domain-containing proteins (CG1099, CG6148, CG6192, CG16932) in the UAS-luc/G4DBD-PygoNHD assay did not cause a decrease in reporter activity, suggesting that the PygoNHD, despite its NPF motif, does not rely on such EH domain-containing proteins for its capacity to activate transcription (Stadeli, 2005).

As an extension of this 'chain of adaptors' model, it is tempting to assume that Pygo, through its NHD, might recruit factors with enzymatic activities, such as histone acetyl transferases (HAT) or chromatin remodelling proteins. Such interactions may depend on an intact Pygo NPF motif. It is important to mention that mutations in this motif, although almost abolishing activity of the isolated DNA-tethered PygoNHD, retain some activity in the context of the full-length protein. It is possible that these mutations do not completely prevent presumptive interactions with positive regulators of transcription; some other part of Pygo may act in a partially redundant manner together with the NHD. Confirmation of such explanations will have to await the identification of PygoNHD interacting proteins (Stadeli, 2005).

BCL9-2 binds Arm/β-catenin in a Tyr142-independent manner and requires Pygopus for its function in Wg/Wnt signaling

The Wingless signal transduction pathway controls fundamental processes during animal development. Deregulation of the Wnt pathway has been causally linked to several forms of cancer, most notably to colorectal cancer. In response to Wnt signaling, Armadillo/β-catenin associates in the nucleus with DNA bound TCF and several co-factors, among them Legless/BCL9, which provides a link to Pygopus. Recently, the second vertebrate homologue of Legless, BCL9-2 (or B9L), was characterized and proposed to mediate Wnt signaling in a Pygopus-independent manner, by binding to a Tyrosine-142-phosphorylated form of β-catenin. This study examined the role of Tyrosine-142 phosphorylation in several assays and it was find not to be important for the recruitment of BCL9-2, nor for the transcriptional activity of β-catenin in cultured mammalian cells, nor is it important in Drosophila for Wg signaling activity in vivo. Furthermore, BCL9-2 can functionally replace Lgs both in cultured cells as well as in vivo and this rescue activity depends on the ability of BCL9-2 to bind Pygo. These results do not show a significant functional difference between BCL9-2 and BCL9 but rather suggest that the two proteins represent evolutionary duplicates of Legless, which have acquired distinct expression patterns while acting in a largely redundant manner (Hoffmans, 2007).

Previous studies have ascribed unique properties and functions to BCL9 and BCL9-2, namely that BCL9-2 functions independently of Pygo and depends on phosphorylation of tyrosine 142 of β-catenin to be able to interact with it. Initially attempts were made determine if residue D164 of β-catenin, crucial for BCL9 binding, was also important for BCL9-2 binding. This study was extended to compare the signaling properties of the two proteins in other available assays. Although the repertoire of these assays has some limitations (e.g. a deficit in vertebrate genetics), these experiments did not reveal any significant functional differences between the two human homologs. Instead, it appears that BCL9 and BCL9-2 represent evolutionary duplicates of Lgs that can function in a largely redundant manner (Hoffmans, 2007).

Evidence was first provided that binding of BCL9, BCL9-2 and Lgs to Arm/β-catenin does not depend on tyrosine residue 142 of Arm/β-catenin. Single amino acid substitutions of Y142 had no effect on the transcriptional activity of Arm/β-catenin, neither in vivo nor in tissue culture cells. It was next shown that BCL9-2 can functionally replace Lgs in vivo and in cultured cells, and that this activity of BCL9-2 depends on its ability to bind Pygo. Finally, it was found that BCL9 and BCL9-2 can interact with the same partners and form similar complexes in the nucleus of stimulated cells (Hoffmans, 2007).

It was previously reported that BCL9-2 interacts efficiently with β-catenin only if the latter is phosphorylated on tyrosine 142. In the absence of tyrosine kinase activity no interaction of the two proteins was detected in a special yeast two-hybrid system. Furthermore, a mutation of β-catenin's tyrosine 142 to alanine abrogated binding to BCL9-2. In contrast, the current binding studies indicate that the binding of Arm/β-catenin to BCL9-2 does not dependent on Y142 phosphorylation. In agreement with these results, BCL9-2 was also identified as a β-catenin binding partner in a standard yeast two-hybrid system that lacks protein tyrosine kinase activity. One explanation to account for the discrepancy between the two results may be attributed to the use of different lengths of β-catenin proteins in the binding assays. While full-length β-catenin was used in the present study, a previous study used a shortened protein containing only the Armadillo repeats, and another study used a form that in addition to the Arm repeats contained small parts of the N- and C-termini (Hoffmans, 2007).

The arm2a9 allele is the strongest arm allele available, has a frameshift mutation in Arm repeat 3 and fails to provide both adhesion function as well as Wg transduction activity. A transgene encoding Arm-Y142A was able to rescue arm2a9 males, but less efficiently as the control transgene encoding wild-type Arm. This difference was attributed to a slight deficit in adhesion function, a hypothesis supported by the observations that Arm-Y142A exhibited reduced α-catenin binding while it fully rescued a signaling-defective but adhesion-competent allele of arm (armS7X-2). Furthermore, no difference could be observed in Wg signaling activity when Arm-Y142A and Arm-wt were interrogated in a quantitative signaling assay in S2 cells. Together, these experiments indicate that the transduction of the Wg signal through Arm does not depend on residue Y142 (Hoffmans, 2007).

To compare the BCL9-2 and BCL9 proteins their ability to substitute for Lgs in Wg signaling was tested. Both human homologs are able to provide Lgs signaling function. Interestingly, previous studies reported that the signaling activity of BCL9-2 does not depend on Pygo binding, but on the C-terminus of BCL9-2. However, in the current replacement assays in Drosophila, found that both human proteins depended equally on their Pygo-binding domain (HD1). At the amino acid level BCL9 and BCL9-2 share three homologous regions of 20-30 amino acids in their C-termini. These short clusters are not found in Lgs; furthermore, it was previously found that the C-terminal half of Lgs is dispensable for Lgs function in Drosophila. Future studies will have to clarify a potential contribution of these C-termini to Wnt signaling in vertebrates. The conservation of these clusters in both BCL9 and BCL9-2 suggests that whatever the function of the C-terminus it is conserved between the two proteins. One approach that might shed light on this issue is to generate mice lacking BCL9 and BCL9-2 and see if the phenotypes of these animals can be rescued by a transgene ubiquitously expressing either only BCL9, BCL9-2 or Lgs. Less stringent signaling assays based on RNAi in cultured mammalian cells were uninformative, as the simultaneous addition of two siRNAs (against both bcl9 and bcl9-2) decreased their knock-down efficiency, complicating the interpretation of the results (Hoffmans, 2007).

Phylogenetic sequence comparisons indicate that a relatively recent event of gene duplication created the two BCL9 forms in vertebrates. The current results indicate that these proteins are essentially identical, at least regarding their interaction with β-catenin as well as regarding their capacity to transduce the Wg signal and to form higher order complexes containing β-catenin and Parafibromin. It is thus suggested that the BCL9 proteins function in a largely redundant manner, as adaptor proteins for the recruitment of Pygo to the β-catenin-TCF complex (Hoffmans, 2007).

Regulatory mechanisms required for DE-cadherin function in cell migration and other types of adhesion: ß-catenin is required only to link DE-cadherin to alpha-catenin

Cadherin-mediated adhesion can be regulated at many levels, as demonstrated by detailed analysis in cell lines. This study examines the requirements for Drosophila epithelial (DE) cadherin regulation in vivo. Investigating Drosophila oogenesis as a model system allowed the dissection of DE-cadherin function in several types of adhesion: cell sorting, cell positioning, epithelial integrity, and the cadherin-dependent process of border cell migration. Multiple fusions were generated between DE-cadherin and alpha-catenin as well as point-mutated ß-catenin and their ability to support these types of adhesion were analyzed. It was found that (1) although linking DE-cadherin to alpha-catenin is essential, regulation of the link is not required in any of these types of adhesion; (2) ß-catenin is required only to link DE-cadherin to alpha-catenin, and (3) the cytoplasmic domain of DE-cadherin has an additional specific function for the invasive migration of border cells, which is conserved to other cadherins. The nature of this additional function is discussed (Pacquelet, 2005).

Classic cadherin proteins have multiple essential roles during animal development both in keeping tissues/epithelia intact and in allowing dynamic cell rearrangements. One dramatic example of the latter is the invasive migration of border cells during oogenesis, for which DE-cadherin is essential. This study investigates which features of DE-cadherin are required for migration, and these features are compared with features that are required more generally for other adhesion functions. Cadherin proteins are well conserved from fly to man; the cytoplasmic domain, in particular, is well conserved, and it interacts with the cytoskeleton. Therefore, the in vivo genetic analyses focused on dissecting the functions of DE-cadherin cytoplasmic domain. In the type of in vivo replacement experiments performed, clear conclusions could be made about what is and is not required under physiological conditions. This is the strength of the analysis, and it is thought to be important to further the understanding of the much-studied cadherin molecules. Generally speaking, the idea cannot be excluded that a type of regulation that is not genetically required does, in fact, occur under normal conditions and contribute somewhat to regulation (e.g., to make the system more robust) (Pacquelet, 2005).

Focus was initially placed on a conserved tyrosine of ß-catenin, the phosphorylation of which may induce ß-catenin to dissociate from cadherin, resulting in a decrease of adhesion. This conserved tyrosine (and, hence, its phosphorylation) is not essential even during border cell migration. The idea that phosphorylation of this tyrosine residue happens or that it may induce some dissociation of ß-catenin from DE-cadherin cannot be excluded. What these results show is that such phosphorylation is not an essential mechanism for adhesion regulation in any of the tested types of cadherin-dependent adhesion in vivo. Significant emphasis has been put in the literature on the putative regulatory role of this conserved tyrosine of ß-catenin. However, much of this emphasis is based on correlations between ß-catenin tyrosine phosphorylation and adhesion down-regulation. It is not clear whether ß-catenin phosphorylation is really the cause of adhesion down-regulation. In addition, the tyrosine kinase Src causes a decrease of adhesion in L cells expressing the fusion protein E-cadherin/alpha-catenin. Thus, Src-induced adhesion down-regulation can be independent of ß-catenin phosphorylation. Therefore, the ability to regulate adhesion without phosphorylating ß-catenin tyrosine may be more general (Pacquelet, 2005).

Next, it was found that neither the link between DE-cadherin and ß-catenin nor that between ß- and alpha-catenin need be regulated at all for DE-cadherin function in vivo. A fusion between DE-cadherin-FL and alpha-catenin fully substitutes for endogenous DE-cadherin during oogenesis even in the absence of endogenous ß-catenin. It was surprising to find that there is no need to regulate the link between DE-cadherin and alpha-catenin, since earlier studies using similar fusion proteins had concluded that regulation was required for mouse E-cadherin to support 'intercellular migration' (Nagafuchi, 1994). There are two main explainations for this discrepancy. (1) The previous study did not fuse alpha-catenin to E-cadherin-FL but fused to a truncated E-cadherin (analogous to DE-cadherinDeltaß/alpha-catenin). As was found in the current study, this not only affects the ability to regulate the link to alpha-catenin but also removes additional functionality from cadherin. It was not directly investigated in Nagafuchi (1994) whether the defects were caused by ß-catenin regulation as proposed. (2) Different cell types were analyzed; the previous study overexpressed E-cadherin in mouse fibroblasts that normally have very little of the protein, whereas this study investigated cells that normally depend on DE-cadherin for biological function (Pacquelet, 2005).

It is possible that the link between DE-cadherin and the actin cytoskeleton does need to be regulated but that it occurs downstream of alpha-catenin. More studies of alpha-catenin and of how its interactions are regulated will be of interest, in particular in a physiological context. Alternatively, regulation of adhesion may primarily occur by the turning over of DE-cadherin and/or DE-cadherin complexes via endocytosis. A Cbl-related E3 ligase called Hakai has been identified as a specific regulator of mammalian E-cadherin endocytosis (Fujita, 2002). It is recruited to specific phosphorylated tyrosines on E-cadherin. No evidence was found that the homologous D. melanogaster protein (CG10263) affects DE-cadherin or border cell migration, and the key docking tyrosines are not conserved. However, other regulators may play an analogous role. Finally, adhesive strength could be regulated by lateral clustering of cadherin complexes; for example, by the binding of additional regulatory proteins to the intracellular domain (Pacquelet, 2005).

The full functionality of DE-cadherin-FL/alpha-catenin in the absence of ß-catenin also indicates that ß-catenin has no essential adhesive function other than linking DE-cadherin to alpha-catenin. Based on the abnormal localization of various DE-cadherin mutants, it had been proposed that ß-catenin was required for proper translocation of cadherin to the plasma membrane. However, the relatively normal subcellular localization of DE-cadherin-FL/alpha-catenin that was observed in the absence of ß-catenin suggests that this is not generally the case. It remains possible that ß-catenin also contributes to modifying cadherin localization in D. melanogaster cells, but in a more subtle, nonessential way. This study suggests that parts of the cadherin tail that bind ß-catenin may also have ß-catenin-independent functions. This would complicate the interpretation of how modified cadherin molecules behave unless it is also investigated by ß-catenin loss-of-function experiments (Pacquelet, 2005).

In contrast with DE-cadherin-FL/alpha-catenin, a fusion protein between DE-cadherin and alpha-catenin lacking the DE-cadherin cytoplasmic tail (DE-cadherinDeltaCyt/alpha-catenin) could not substitute for DE-cadherin during border cell migration. It was targeted to the cell surface and was functional in all other contexts. This indicates that the DE-cadherin cytoplasmic tail has a specific function during invasive migration in addition to the basic ß-catenin/alpha-catenin linkage. The function could not be provided by an unrelated cytoplasmic linker (CD2) but could be provided by the corresponding region from mouse E-cadherin or D. melanogaster N-cadherin. Most likely, one or more interactions that are specific to cadherin tails have a critical function in this context. These results raise two questions: (1) why is DE-cadherin tail specifically important for border cell migration and (2) what is the molecular nature of the required function (Pacquelet, 2005)?

With regard to the specific requirement in border cells, the role of DE-cadherin in their migration needs to be considered. Given the absolute requirement for this particular cell-cell interaction to achieve invasive border cell movement, it is likely to be force bearing. DE-cadherin-mediated adhesion between the front of border cells and the attachment point on nurse cells needs to be strong enough to allow border cells to pull themselves into the compact germ line tissue. As the border cell cluster initiates migration using a long, slender cellular extension, the local force application at the tip may be quite high. As an illustration of the forces involved, it was found that mutant border cells with impaired cortical cytoskeleton will break apart when they attempt to invade, whereas other follicle cells (including centripetal cells) with the same defect appear to be relatively normal. It is suggested that the DE-cadherin tail may be required to allow a build-up of sufficiently strong adhesion to withstand forces that are involved in migration (Pacquelet, 2005).

Another important aspect of adhesion during cell movement is that it may need to be effectively down-regulated at the rear of the cells to allow cell translocation along the substrate. Experiments indicated that the primary defect for DE-cadherinDeltaCyt/alpha-catenin in border cells is not a lack of down-regulation; in other words, it is not caused by an excess of adhesion. However, an inability of DE-cadherinDeltaCyt/alpha-catenin to provide sufficient adhesion for migration as discussed above could mask possible additional (migration specific) defects of the fusion protein such as the ability to be down-regulated (Pacquelet, 2005).

The molecular nature of the DE-cadherin tail requirement in migration is in need of further investigation. The function does not simply map to any previously known signal or interaction, suggesting involvement of a novel interaction and/or a redundancy of interactions. The DE-cadherinDeltaß/alpha-catenin fusion results indicate that the most COOH-terminal domain contributes to DE-cadherin function in border cells independently of ß-catenin binding. However, this domain is not essential on its own nor when coexpressed with p120 catenin RNA interference constructs, indicating that additional important signals are located in the more proximal region of the DE-cadherin cytoplasmic domain. A mutant form of Xenopus laevis C-cadherin lacking the 94 proximal amino acids of its cytoplasmic domain can mediate some adhesion but is unable to support strong adhesion. This seems to be caused by its inability to form lateral clusters. Similarly, an absence of the proximal region in DE-cadherinDelta-Cyt/alpha-catenin could prevent its clustering and, thereby, prevent adhesion strengthening (Pacquelet, 2005).

In conclusion, this structure/function analysis of DE-cadherin in different types of cell adhesion has given new information about cadherin regulation in vivo. Several previously defined potential points of regulation that were established through detailed work in tissue culture were found not to be essential for functionality in vivo. The cytoplasmic tail of cadherin was found to have a unique role in the demanding process of invasive cell migration, possibly through a novel interaction (Pacquelet, 2005).

Rac function in epithelial tube morphogenesis: Rac regulation of salivary gland morphogenesis occurs through modulation of cell-cell adhesion mediated by the E-cadherin/ß-catenin complex

Epithelial cell migration and morphogenesis require dynamic remodeling of the actin cytoskeleton and cell-cell adhesion complexes. Numerous studies in cell culture and in model organisms have demonstrated the small GTPase Rac to be a critical regulator of these processes; however, little is known about Rac function in the morphogenic movements that drive epithelial tube formation. This study used the embryonic salivary glands of Drosophila to understand the role of Rac in epithelial tube morphogenesis. Inhibition of Rac function, either through loss of function mutations or dominant-negative mutations, disrupts salivary gland invagination and posterior migration. In contrast, constitutive activation of Rac induces motile behavior and subsequent cell death. Rac regulation of salivary gland morphogenesis occurs through modulation of cell-cell adhesion mediated by the E-cadherin/ß-catenin complex, and shibire, the Drosophila homolog of dynamin, functions downstream of Rac in regulating ß-catenin localization during gland morphogenesis. These results demonstrate that regulation of cadherin-based adherens junctions by Rac is critical for salivary gland morphogenesis and that this regulation occurs through dynamin-mediated endocytosis (Pirraglia, 2006).

This study shows that the Rac GTPases regulate salivary gland morphogenesis through modulation of cadherin/catenin-based cell–cell adhesion, likely by dynamin-mediated endocytosis. The characterization of the Rac mutant phenotypes suggests a model where Rac normally regulates cadherin-mediated cell–cell adhesion in salivary gland cells to allow enough plasticity for its invagination and migration yet keep the cells of the tube adhered to one another so that the gland can migrate as a cohesive tube. One mechanism by which cell surface cadherin levels are regulated is through selective endocytosis of E-cadherin from the apical–lateral membrane in a dynamin-mediated process. When Rac function is compromised through loss-of-function mutations or expression of dominant-negative mutations, the balance between E-cadherin at the plasma membrane and internalized E-cadherin appears to be abrogated so that more E-cadherin remains at the plasma membrane resulting in increased cell–cell adhesion and causing the gland to sever. These studies reveal the importance of precise regulation of adherens junction remodeling during cell migration in the context of a developing organ (Pirraglia, 2006).

In all stage 14 Rac1L89 mutant embryos examined, the salivary gland broke apart close to its approximate mid-point. Reduction in cadherin levels rescues the mutant Rac severing phenotype, suggesting that severing occurs because loss of Rac leads to an increase in cadherin-mediated cell–cell adhesion. At least two possible explanations for the midpoint severing phenotype are envisioned. In the first scenario, levels of cadherin remodeling may differ throughout the gland such that in Rac1L89 embryos the cells in the distal tip are least affected and cells in the mid-region of the gland are most affected by the increase in cadherin function. In this situation, when the distal cells begin to migrate posteriorly, the increased adhesivity of the mid-region cells prevents their migration and causes the gland to sever in the middle. In the second scenario, movement of the mid-region and the distal region of the gland may occur through different mechanisms. It is possible that while the distal most cells migrate by undergoing cell shape change and extending prominent protrusions in the direction of migration, cells in the middle of the gland may follow the distal cells by rearranging their positions along the gland, such as occurs during the convergence extension movements observed in epithelial morphogenesis. Dynamic remodeling of E-cadherin may be particularly important for proper rearrangement of the mid-region cells and an inability to rearrange when E-cadherin adhesion is increased may cause severing of the gland and subsequent separation of the migrating distal portion from the rest of the gland. Alternatively, it is possible that both of these scenarios are at play during normal salivary gland migration. Currently it is not possible to distinguish between these possibilities. In the developing tracheal tubes, Rac1 is required for cell rearrangements; in tracheal cells expressing a dominant-negative Rac1 mutation, the dorsal branch was shorter than that of wild-type embryos. Therefore, it will be important to determine whether cell rearrangement plays a role during salivary gland migration and to further elucidate the role of the Rac genes in this process (Pirraglia, 2006).

When Rac1 function is over-activate, dynamin-mediated endocytosis of E-cadherin may be increased, resulting in decreased cadherin at the plasma membrane, and decreased cell–cell adhesion. The loss of adhesion leads to the dispersal of salivary gland cells and ultimately cell death. Preventing Rac1V12-induced cell death led to the formation of abnormally shaped glands demonstrating that the Rac1V12 salivary gland phenotype is primarily due to abrogation of gland morphogenesis and not to activation of the apoptotic pathway. Moreover, since wild-type full-length E-cadherin is sufficient to rescue the Rac1V12 salivary gland phenotype, loss of cadherin function appears to be the primary cause for salivary gland defects. Thus, the Rac genes function in salivary gland cells to regulate E-cadherin-mediated cell–cell adhesion during tube morphogenesis (Pirraglia, 2006).

During salivary gland morphogenesis, gland integrity is kept intact while cells perform extensive cell shape changes and movements. Rac-regulated endocytosis of E-cadherin is one mechanism by which cell–cell adhesion is likely to be downregulated temporarily. After E-cadherin is endocytosed, it can be recycled back to the cell surface, sequestered transiently inside the cell or routed to late endosomes and lysosomes for degradation. Once salivary gland migration is complete and the gland has reached its final position, cell–cell adhesion may then need to be strengthened again in the mature gland and Rac activity may be downregulated to promote increase in surface cadherins (Pirraglia, 2006).

In addition to endocytosis, studies in mammalian cultured cells have shown that Rac can regulate levels of cell surface E-cadherin by other mechanisms, such as cleavage by presenilins and metalloproteinases, or tyrosine phosphorylation of the cadherin adhesion complex in a process involving reactive oxygen species. Thus, it will be interesting to determine whether additional mechanisms of E-cadherin regulation exist in salivary gland cells during gland morphogenesis (Pirraglia, 2006).

Numerous studies in cell culture have demonstrated that recycling of E-cadherin occurs in both a clathrin-dependent and caveolin-dependent manner. Since dynamin mediates both clathrin- and caveolin-dependent endocytosis, these studies do not allow distinguishing which type is involved in cadherin endocytosis during salivary gland migration. Alternatively, both types of endocytosis may mediate Rac1 regulation of E-cadherin in salivary gland cells (Pirraglia, 2006).

Expression of the Rac1V12 mutation in salivary gland cells leads to loss of expression of salivary gland specific proteins, apical–basal polarity proteins and E-cadherin/β-catenin. Concomitant with changes in gene expression, Rac1V12 mutant salivary gland cells lose adhesion to each other and subsequently migrate away or die by apoptosis. The data suggest that overactivation of Rac1 primarily affects E-cadherin/β-catenin-mediated adhesion and salivary gland cell fate and that the observed cell death is a secondary consequence of these earlier changes. When cell death was prevented in Rac1V12 embryos by expressing p35, more cells expressed the salivary gland specific protein PH4αSG1 than in Rac1V12 embryos; however, the expression level was drastically reduced compared to wild-type, suggesting that even in the Rac1V12p35 cells, cell differentiation was still mostly altered. Moreover, Rac1V12p35 salivary gland cells did not form a normal gland, demonstrating a role for Rac1 in gland morphogenesis. It is possible that apoptosis of Rac1V12 cells is brought about by the loss or reduction of Forkhead (Fkh) function. Fkh is expressed early in the salivary gland placode and its expression is maintained throughout embryogenesis. In the absence of fkh function, salivary gland cells die by apoptosis during the invagination stage. Since expression of dCREB-A and PH4αSG1 is reduced in Rac1V12 mutant salivary gland cells, it is possible that Fkh expression is also similarly reduced, thereby, causing the cells to undergo apoptotic cell death (Pirraglia, 2006).

Many human cancers are due to epithelial-derived tumors. When epithelial cells metastasize, they first undergo an epithelial to mesenchymal transition (EMT) before migrating away from the primary tumor to invade surrounding tissues. EMT is characterized by the loss of epithelial polarity and cell–cell adhesion. When Rac1V12 was expressed in salivary gland cells, expression of apical membrane proteins, Crumbs and aPKC and adherens junction proteins E-cadherin and β-catenin, was either lost or mislocalized. Based on these criteria, activation of Rac1 function induces features characteristic of early changes in EMT and metastasis. Interestingly, the expression levels of Rho GTPases are found to be elevated in a number of human cancers. For example, increased Rac protein levels and fast-cycling Rac mutations have been correlated with colorectal and breast tumors. Expression of constitutively active Rac1 causes some salivary gland cells to lose polarity and adhesion to neighboring cells and migrate away in a manner similar to EMT. These findings suggest that Rac1-regulated endocytosis of E-cadherin in the Drosophila salivary glands may be critical in maintaining epithelial character and preventing the loss of cell–cell adhesion and cell polarity. The Drosophila salivary gland might thus be powerful as a simple system to identify and characterize mechanisms that regulate cadherin-based cell–cell adhesion and certain aspects of EMT (Pirraglia, 2006).

The Fes/Fer non-receptor tyrosine kinase cooperates with Src42A to regulate dorsal closure in Drosophila: DFer localises to AJs and regulates ß-catenin phosphorylation

Fes/Fer non-receptor tyrosine kinases regulate cell adhesion and cytoskeletal reorganisation through the modification of adherens junctions. Unregulated mammalian Fes/Fer kinase activity has been shown to lead to tumours in vivo. Drosophila Fer localises to adherens junctions in the dorsal epidermis and regulates a major morphological event, dorsal closure. Mutations in Src42A cause defects in dorsal closure similar to those seen in dfer mutant embryos. Furthermore, Src42A mutations enhance the dfer mutant phenotype, suggesting that Src42A and DFer act in the same cellular process. DFer is required for the formation of the actin cable in leading edge cells and for normal rates of dorsal closure. A gain-of-function mutation in dfer (dfergof) expresses an N-terminally fused form of the protein, similar to oncogenic forms of vertebrate Fer. dfergof blocks dorsal closure and causes axon misrouting. In dfer loss-of-function mutants ß-catenin is hypophosphorylated, whereas in dfergof ß-catenin is hyperphosphorylated. Phosphorylated ß-catenin is removed from adherens junctions and degraded, thus implicating DFer in the regulation of adherens junctions (Murray, 2006),

DFer localises to adherens junctions during Drosophila embryogenesis, where it regulates leading edge morphology and dorsal closure. In dfer null mutants, P-Tyr staining is reduced at the leading edge, in particular at the actin-nucleating centers. Two potential substrates of DFer, p120ctn and ß-catenin, are localised to adherens junctions. It was found that in dferΔ1 mutants ß-catenin phosphorylation is reduced. Conversely, ß-catenin is more highly phosphorylated in dfergof mutants, demonstrating that the role of Fer in the phosphorylation of ß-catenin is conserved in Drosophila. Interestingly, the overall level of ß-catenin at cell-cell junctions is lower in dfergof mutants, suggesting that phosphorylated ß-catenin is lost from AJs and subsequently degraded (Murray, 2006),

dfer mutants also exhibit a disorganised and reduced F-actin cable at the leading edge. Formation of the F-actin cable appears to depend on adherens junctions, as F-actin nucleation begins at the level of the AJs and the F-actin cable is disrupted in DE-Cadherin mutants. It has been suggested that elevated levels of cytoplasmic α-catenin near stable AJs could favour the formation of F-actin bundles. DFer may contribute to the formation of the F-actin cable by phosphorylating ß-catenin, reducing its affinity for α-catenin, and thereby increasing the local levels of cytoplasmic α-catenin. If DFer promotes stable F-actin bundles then the regulated loss of DFer from the leading edge at stage 14 may enable the more motile Arp2/3 regulated F-actin filopodia to form and complete dorsal closure by 'zipping up' (Murray, 2006),

DFer and Src42A cooperate during dorsal closure. DFer localises to AJs and regulates ß-catenin phosphorylation. In Drosophila, Src42A binds and phosphorylates ß-catenin, although this may not be direct. Consequently, the more severe phenotypes seen in the dfer;Src42A loss-of-function mutants are most likely due to a combined loss of phosphorylation on at least two different tyrosine residues of ß-catenin (Murray, 2006),

dfer mRNA is upregulated in leading edge cells. This, together with reports that vertebrate v-Fps and Fes mediate JNK pathway activation (Li, 1998), suggested that dfer might activate the JNK pathway during dorsal closure. Although DFer itself cannot induce dpp expression, it does play a supporting role in the maintenance of dpp levels, as revealed in the Src42A mutant background. A similar failure in the maintenance of dpp, as opposed to its induction, is seen in mutants of the Wnt pathway. Given the comparable phenotypes, and the fact that phosphorylated ß-catenin is reduced in dfer mutants, it is possible that DFer contributes to the maintenance of dpp via the Wnt pathway (Murray, 2006),

This study isolated a novel, gain-of-function mutation, dfergof, in which a fragment of the White protein is fused to the N terminus of Dfer. This protein, Wex1-DFerRB, is analogous to oncogenic forms of Fps in which part of the viral GAG protein is fused to the N terminus of the endogenous proto-oncogene, generating an activated kinase. Although dfergof mutants express DFer at higher levels, this alone seems unlikely to account for the observed defects, as overexpression of DFerRB gives no obvious embryonic phenotype (Murray, 2006),

In dfergof mutants, the leading edge cells fail to elongate and the F-actin-rich filopodia are greatly reduced. The overall levels of the AJ junction components DE-Cadherin and ß-catenin are reduced, and ß-catenin is hyperphosphorylated. This suggests that AJs are disrupted in dfergof mutants. By contrast, it is interesting that the morphology of amnioserosal cells is shifted to a more motile appearance: F-actin is reduced at the cortex and there is an increase in the number of filopodia, perhaps because of a disruption of cell-cell junctions. In vertebrates, Fer has the capacity to both positively and negatively regulate cadherin-complex stability. This dual function may reflect a difference in binding partners present at AJs in different tissues (Murray, 2006),

Although loss of dfer does not appear to affect axon guidance, dfergof mutants have a clear CNS phenotype in which axons aberrantly cross the midline. A similar phenotype is seen with overexpression of the Abelson tyrosine kinase, which antagonises the receptor Robo. dfergof mutants also disrupt axon guidance in the PNS, with some general misrouting of motor nerves and some overly large synapses. In vertebrates, Fer associates with N-Cadherin in elongating neurites, where it can coordinately regulate N-Cadherin and integrin adhesion (Arregui, 2000). Fer has been shown to be concentrated in growth cones of stage 2 hippocampal neurons and is required for neuronal polarisation and neurite development (Lee, 2005). Similar to the leading edge, DFer may be required at growth cones to regulate filopodial extensions. In chick retinal cells, the phosphatase PTP1B when phosphorylated by Fer, localises to the catenin-binding domain of N-Cadherin (Xu, 2004). Interestingly, the Drosophila homologue of PTP1B, DPTP61F, is expressed in the CNS and binds to the axon guidance molecule Dock (Murray, 2006),

Strikingly, all of the phenotypes associated with dfergof mutants are rescued by expression of the Puckered tyrosine phosphatase. Given that JNK pathway activity appears normal in dfergof mutants, Puckered may target DFer itself, or its substrates, at least one of which is hyperphosphorylated in dfergof mutants. A role for DFer during Drosophila embryonic development in the regulation of AJ stability, in the formation of the contractile leading edge during dorsal closure, and in axon guidance. It cooperates with Src42A to regulate ß-catenin phosphorylation at AJs. A gain-of-function mutant with structural similarity to oncogenic forms of vertebrate Fer was isolated. Unregulated Fer activity leads to oncogenesis, possibly through unregulated epidermal to mesenchymal transition. This study has shown that activated DFer, or loss of DFer together with Src42A, disrupts AJs. This may provide a model for studying oncogenesis in the whole organism (Murray, 2006),

Parafibromin/Hyrax activates Wnt/Wg target gene transcription by direct association with β-catenin/Armadillo

The Wnt pathway controls cell fates, tissue homeostasis, and cancer. Its activation entails the association of β-catenin with nuclear TCF/LEF proteins and results in transcriptional activation of target genes. The mechanism by which nuclear β-catenin controls transcription is largely unknown. A novel Wnt/Wg pathway component has been genetically identify that mediates the transcriptional outputs of β-catenin/Armadillo. Drosophila Hyrax and its human ortholog, Parafibromin, components of the Polymerase-Associated Factor 1 (PAF1) complex, are required for nuclear transduction of the Wnt/Wg signal and bind directly to the C-terminal region of β-catenin/Armadillo. Moreover, the transactivation potential of Parafibromin/Hyrax depends on the recruitment of Pygopus to β-catenin/Armadillo. These results assign to the tumor suppressor Parafibromin an unexpected role in Wnt signaling and provide a molecular mechanism for Wnt target gene control, in which the nuclear Wnt signaling complex directly engages the PAF1 complex, thereby controlling transcriptional initiation and elongation by RNA Polymerase II (Mosimann, 2006).

Three lines of evidence argue for the notion that Hyx represents a component of the Wg pathway. (1) The initial observation that increased expression of hyx can overcome the dominant-negative effect of overexpressed lgs17E provides a first indication that Hyx positively influences Wg signaling outputs in vivo. lgs17E encodes an altered form of Lgs which contains a mutation in its Arm-interacting domain that severely decreases binding of Lgs to Arm and consequently the recruitment of Pygo to Arm. When provided in excess, Lgs17E protein likely impairs the function of nuclear Arm by outcompeting endogenous Lgs and thus disturbs the sensitive balance and/or sequence of factors normally recruited at Wg-responsive enhancers. Elevating the levels of a positively acting nuclear factor involved in Wg signaling, in this case Hyx, could readily explain the reversion of the Lgs17E phenotype in genetic assays. (2) The subsequent observation that genetic reduction of hyx function in imaginal discs as well as the RNAi-mediated knock-down of hyx expression in S2 cells caused a severe decrease in Wg pathway activity is a strong argument for a requirement of Hyx in Wg signaling. (3) Ultimate confirmation of the above genetic claims was the discovery of Hyx as a direct binding partner of Arm. Together these observations provide a solid basis for a model in which Hyx plays a key role in mediating the transcriptional output of Arm in response to Wg pathway activation. In contrast to the Arm partners Lgs and Pygo, Hyx is most likely not a component dedicated solely to the Wg pathway. The phenotypes associated with hyx loss-of-function mutations indicate that Hyx is involved in other developmental processes, possibly in the transcriptional output of some other signal transduction pathway(s) (Mosimann, 2006).

The high degree of homology between Hyx and its single human ortholog suggested that Parafibromin serves the same function in Wnt signaling as Hyx in Wg signaling. Indeed, with the exception of genetic evidence for an in vivo requirement, equivalent lines of reasoning as those arrived at for Hyx argue for an important role of Parafibromin in human β-catenin signaling. What could this role be? It has recently been shown that Parafibromin/Hyx represents the Cdc73 subunit of a metazoan PAF1 complex (Rozenblatt-Rosen, 2005; Yart, 2005; Adelman, 2006). The yeast PAF1 complex has originally been found associated with initiating and elongating forms of RNAPII. Moreover, the PAF1 complex interacts genetically and physically with the histone H2B ubiquitination complex, the Set1 methylase-containing COMPASS complex, and Set2, thus conferring control over a number of distinct histone modifications on RNAPII. Together, these findings suggest important conserved functions of the PAF1 complex in coordinating histone modifications 'downstream' of chromatin preparation on target promoters to ensure proper initiation, elongation, and memory of transcription (Mosimann, 2006).

To date, Cdc73p has not been reported to interact directly or indirectly with a sequence-specific DNA binding transcription factor, and it is not clear how the PAF1 complex is recruited to its target genes. However, the metazoan homologs Parafibromin and Hyx share an extended N-terminal region, not present in Cdc73p, which was found to physically interact with the core Wnt/Wg component β-catenin/Arm. It is thus tempting to speculate that during metazoan evolution, Cdc73 homology proteins evolved in their N-terminal sequences interaction domains for certain signal transduction pathways, such as the Wnt/Wg pathway, while conserving C-terminal sequences for PAF1 complex and/or RNAPII association (Mosimann, 2006).

β-catenin/Arm has two “branches” of transcriptional output, an N-terminal and a C-terminal branch, which can be separated experimentally. The N-terminal activity maps to Arm repeat 1 and can be attributed to the recruitment of Lgs and Pygo. The current results suggest that Parafibromin/Hyx mediates an important aspect of the C-terminal output of β-catenin/Arm. The significance of any transcriptional activity mapping to C-terminal sequences of β-catenin/Arm is seemingly undermined by the finding that C-terminally truncated forms of Arm (such as the product of the allele armXM19) are able to drive Wg target gene expression under certain experimental conditions. However, the armXM19 allele exhibits robust signaling activity only when its product is 'forced' into the nucleus by overexpression of a membrane-tethered form of Arm and most likely uses the N-terminal Lgs/Pygo-dependent branch for this activity. Under physiological conditions, ArmXM19 is severely impaired for Wg signaling. ArmH8.6, which lacks only a distal portion of the CTD, retains residual transactivation potential at 18°C. This apparent correlation between signaling activity and the extent of C-terminal integrity of Arm might reflect the capacity of Arm to recruit Hyx, a view consistent with protein–protein interaction results (Mosimann, 2006).

Recent advances in the understanding of how transcriptional activators modulate gene transcription suggest a sequential recruitment of histone acetylases (such as CBP/p300) and chromatin-remodeling complexes (like SWI/SNF) to target genes before RNAPII is contacted to initiate transcription on the prepared chromatin. The β-catenin region encompassing Arm repeat 11 to the C terminus has been implicated in being necessary for chromatin remodeling using in vitro assays. Parafibromin/Hyx interacts with a region of β-catenin/Arm (repeat 12-C) that overlaps with the CBP/p300 binding site (repeat 10/11-C) and the Brg-1/Brm binding region (repeat 7-12). This raises the intriguing possibility of a concerted or sequential recruitment of chromatin remodeling factors during the control of Wnt/Wg-responsive genes to the C-terminal portion of β-catenin/Arm, as is being reported for other transcription factors. In such a scenario, CBP/p300 and Brg-1/Brm would, in sequential or arbitrary order, mediate chromatin remodeling steps at β-catenin/Arm-dependent target genes before the Parafibromin/Hyx-mediated recruitment of a PAF1-like complex orchestrates later transactivation steps involving the preparation of RNAPII with histone methylase complexes. In a final step, the PAF1 complex, including Parafibromin/Hyx, may be transferred from β-catenin/Arm to RNAPII to travel with it through the actively transcribed gene (Mosimann, 2006).

What role does the Wnt/Wg pathway component Pygo play in such a model? In several readouts it was found that the Parafibromin/Hyx-enhanced transactivation activity of β-catenin is dependent on Pygo. The Pygo-Parafibromin/Hyx dependence is interpreted as an indication for a more general cross talk between Pygo and proteins interacting with the C-terminal region of β-catenin/Arm. Thus, Pygo could act as a flexible recruitment module to facilitate the exchange or stabilization of transactivating complexes that sequentially bind to the β-catenin C terminus. It is therefore proposed that Wnt/Wg target gene activation might be a concerted, Pygo-guided process, which dynamically coordinates the sequential action of transcriptional modulators at the central scaffold protein β-catenin/Arm (Mosimann, 2006).

The yeast PAF1 complex shows cotranscriptional association with a wide range of genes and has therefore been considered a general transcription cofactor complex. However, deletion of individual components of this complex does not have a global effect on mRNA transcription but instead has a more selective impact on the transcription of only a subset of genes. Currently, aside from findings of an involvement in Wnt signaling, little is known about the target gene spectrum of metazoan PAF1-like complexes. Recently published data indicate that, as in yeast, the Drosophila PAF1-like complex is broadly associated with active genes but, functionally, Cdc73/Hyx seems only necessary for a subset of PAF1 complex targets. This would be consistent with a view that Parafibromin and Hyx provide an adaptor function only to certain transcription factors, such as, for example, β-catenin and Arm. Indeed, in vivo and in vitro assays indicate that in contrast to a cohort of other genes, whose expression is constitutive or controlled by other pathways, Wg targets are remarkably sensitive to reduction of Hyx levels. However, since the assays severely reduced but never abolished hyx expression, currently it is not possible to evaluate the extent to which Hyx activity is also required for the transcription of targets of other pathways, which potentially are more resilient to reductions in Hyx levels (Mosimann, 2006).

Trabid, a new positive regulator of Wnt-induced transcription with preference for binding and cleaving K63-linked ubiquitin chains

A key effector of the canonical Wnt pathway is β-catenin, which binds to TCF/LEF factors to promote the transcription of Wnt target genes. In the absence of Wnt stimulation, β-catenin is phosphorylated constitutively, and modified with K48-linked ubiquitin for subsequent proteasomal degradation. Trabid is a positive regulator of Wnt signaling in mammalian and Drosophila cells. Trabid shows a remarkable preference for binding to K63-linked ubiquitin chains with its three tandem NZF fingers (Npl4 zinc finger), and it cleaves these chains with its OTU (ovarian tumor) domain. These activities of Trabid are required for efficient TCF-mediated transcription in cells with high Wnt pathway activity, including colorectal cancer cell lines. Trabid can bind to and deubiquitylate the APC tumor suppressor protein, a negative regulator of Wnt-mediated transcription. Epistasis experiments indicate that Trabid acts below the stabilization of β-catenin, and that it may affect the association or activity of the TCF-β-catenin transcription complex. These results indicate a role of K63-linked ubiquitin chains during Wnt-induced transcription (Tran, 2008).

In the absence of Wnt stimulation, an active mechanism operates to down-regulate β-catenin, the key effector of the canonical Wnt pathway. This involves the constitutive phosphorylation of β-catenin by the Axin complex that also includes APC, glycogen synthase kinase 3β (GSK3β), and casein kinase I. Phosphorylated β-catenin is recognized by β-TrCP, an F-box-containing adaptor, which binds to an SCF ubiquitin ligase. The latter conjugates a K48-linked ubiquitin chain to phosphorylated β-catenin, which earmarks it for subsequent proteasomal degradation. Binding of Wnt ligand to its transmembrane receptor leads to Dishevelled-mediated inhibition of the Axin complex; as a consequence, unphosphorylated β-catenin accumulates and associates with TCF/LEF factors in the nucleus, to stimulate the transcription of Wnt target genes. This involves the recruitment of a wide range of transcriptional cofactors to the C terminus of β-catenin, including the chromatin modifier and remodeling factors CBP, Brg-1, and SET1; TATA-binding and associated factors; and the transcriptional elongation factor Parafibromin. Through a more N-terminal segment, β-catenin binds to the BCL9-Pygopus complex, to recruit an unknown transcriptional coactivator and/or to facilitate efficient recruitment of β-catenin to TCF target genes. Thus, the key output of canonical Wnt signaling is a switch in transcription (Tran, 2008 and references therein).

The same SCF ubiquitin ligase complex that regulates β-catenin also controls the NF-kappaB pathway: Following TNF-α stimulation, this complex conjugates a K48-linked ubiquitin chain to phosphorylated IkappaB, an inhibitor of NF-kappaB. The subsequent proteasomal degradation of ubiquitylated IkappaB allows NF-kappaB to accumulate in the nucleus and to stimulate the transcription of NF-kappaB target genes. Notably, several components of the NF-kappaB pathway are also regulated by K63-linked polyubiquitylation. The TNF receptor-associated factors TRAF2, TRAF6, RIP, and the NEMO subunit of the IkappaB kinase are modified by K63-linked ubiquitin chains, which are thought to mediate the recruitment and activation of their effectors. For example, the binding of TAB2 to K63-polyubiquitylated TRAF6, NEMO, and RIP appears to be required for activation of NFkappaB. TAB2 binds to its polyubiquitylated substrates via its NZF motif (Npl4 zinc finger), one of many different ubiquitin-binding domains that bind to the commonly recognized hydrophobic "Ile44 patch" of the ubiquitin surface (Tran, 2008 and references therein).

The deubiquitylating (DUB) enzyme A20 antagonizes the NF-kappaB pathway, by removing the K63-linked polyubiquitylation of RIP, which appears to be necessary for its ability to down-regulate TNF-alpha-induced NF-kappaB activity. A20 belongs to the OTU (ovarian tumor) family of DUBs, which includes the largely uncharacterized proteins Cezanne and Trabid. The OTU domain is a conserved cysteine protease that possesses DUB activity (Balakirev, 2003). Overexpressed Cezanne can also down-regulate NF-kappaB signaling, whereas overexpressed Trabid had negligible effects on this pathway (Tran, 2008).

This study reports the discovery of Trabid as a new APC-interacting protein and a positive regulator of Wnt responses in mammalian cells and Drosophila. Its variant OTU domain exhibits DUB activity, and it deubiquitylates APC. Its remarkable preference for binding to, and cleaving, K63-linked ubiquitin chains are critical for Trabid's function in TCF-mediated transcription. Epistasis analysis suggests that the TCF-β-catenin complex is the functional target of Trabid (Tran, 2008).

A yeast two-hybrid (Y2H) screen was conducted with the armadillo repeat domain (ARD) of mouse Apc, and 13 independent positives were counterscreened with a mutant ARD bearing a point mutation in its putative surface that, in Drosophila, causes inactivation of E-APC. This revealed two isolates that were sensitive to this mutation. One of these encodes an internal fragment of mouse Cezanne overlapping its OTU domain (residues 252-403), which shows high sequence similarity to Trabid, and also to dTrbd, the Trabid ortholog in Drosophila and only representative of the A20 OTU family in flies. The Cezanne Y2H fragment, and a similar OTU-spanning fragment from human Trabid, both coimmunoprecipitate preferentially with wild type rather than mutant ARD in cotransfected 293 cells. The same selective association is also seen with full-length Flag-dTrbd. Furthermore, Flag-Trabid coimmunoprecipitates with coexpressed GFP-APC(1-1447), an ARD-spanning APC fragment similar to APC type 1 truncations typically found in colorectal cancer cells, but not with coexpressed GFP-APC(918-1698) that lacks the ARD. These sequence-specific interactions in yeast and mammalian cells indicate that Trabid, Cezanne, and dTrbd may be bona fide direct ARD ligands (Tran, 2008).

To test the function of the single Trabid ortholog of Drosophila in its Wingless response, the trbd locus (CG9448) was deleted by homologous recombination. Surprisingly, trbd-null mutants were viable and fertile, suggesting that trbd may be redundant with another gene. trbd function was tested in a more sensitive assay by asking whether lowering the dose of Trabid by half (in trbd heterozygotes) would affect the rough eye phenotype due to ectopic Wingless in the eye imaginal disc. Indeed, trbd heterozygosity suppresses this phenotype, similarly to dTCF heterozygosity. trbd heterozygosity also suppressed the rough eye phenotype due to overexpressed Armadillo (the β-catenin of Drosophila) to some extent. In contrast, no genetic interactions was observed between trbd and components of EGF receptor signaling, a pathway that controls differentiation in the developing fly eye: The rough eye phenotype due to activation or inhibition of this pathway remained unchanged in trbd heterozygotes. Similarly, there was no interaction between trbd and the Notch pathway, whose stimulation also causes rough eyes. These results suggest that dTrbd is required for an efficient Wingless response, but not for other signaling responses in the eye, consistent with results in human cell lines (Tran, 2008).

To date, the only known function of ubiquitylation in the Wnt pathway relates to protein turnover, especially that of its key effector β-catenin, typically conferred by K48-linked ubiquitin chains. The discovery of Trabid, a DUB enzyme that exhibits a remarkable preference for K63-linked ubiquitin, and its functional link to APC and the Wnt response of mammalian and Drosophila cells implies a distinct role of ubiquitylation in this pathway, namely, one mediated by K63-linked ubiquitin chains. These chains are likely to affect the activity rather than the stability of proteins, and Trabid may affect the transcriptional response to Wnt signaling by antagonizing the ubiquitylation of APC (Tran, 2008).

This study showed that Trabid is a bona fide DUB and that it cleaves preferentially K63-linked ubiquitin. This activity resides in its OTU domain, which possesses intrinsic DUB activity, as has been shown previously for other members of the OTU family (Tran, 2008).

Trabid exhibits an unprecedented selectivity for binding to K63- versus K48-linked ubiquitin chains, which is conferred by a minimal module of at least two linked NZF motifs. In contrast, an individual NZF motif (e.g., that of TAB3) binds indiscriminately to K48- and K63-linked ubiquitin chains, consistent with structural analysis that revealed the commonly used surface of ubiquitin for NZF binding. Notably, the three specificity-conferring NZF motifs of Trabid are not only a defining feature of Trabid orthologs, but also a fairly unique one: Although NZF domains with ubiquitin-binding signatures are found in a number of proteins, arrays of such NZF fingers are rare. It is conceivable that tandem NZF arrays confer specificity for K63-linked ubiquitin by topological restriction: K48-linked chains are known to form relatively compact structures, whereas K63-linked chains are likely to be extended (Tran, 2008).

Evidence suggests that the NZF motifs of Trabid contribute to its function in TCF-mediated transcription, although they are not required for its intrinsic DUB activity in vitro. These motifs may have a targeting function in vivo, similarly to the ubiquitin binding by the TAB adaptors, which is thought to facilitate the recruitment of ubiquitylated substrates to the TAB-associated TAK1 kinase (Tran, 2008).

Epistasis analysis indicated that Trabid acts below the stabilization of β-catenin. Consistent with this, only minor effects of Trabid depletion were detected on the ubiquitylation of β-catenin (by K48-linked ubiquitin) and on its steady-state levels. These results argue against a direct function of Trabid in proteasomal degradation (Tran, 2008).

In contrast, it was discovered that APC becomes hyperubiquitylated in Trabid-depleted cells, indicating that APC is not only a binding partner of Trabid, but also a substrate of its DUB activity. Thus, at least one function of Trabid is to antagonize the ubiquitylation of APC. This is likely to involve K63-linked ubiquitin chains, given Trabid’s preference for cleaving these chains. Also, the hyperubiquitylation of APC in Trabid-depleted cells, despite being more pronounced after MG132 treatment, neither seems to promote the degradation of APC itself, nor that of its key target, β-catenin. It thus appears that the deubiquitylation of APC by Trabid has a regulatory output (rather than antagonizing the proteasomal turnover of APC mediated by Axin in the absence of Wnt stimulation. For example, it might regulate the activity of APC in transcription. Further work will be required to clarify the molecular nature of these APC ubiquitylations, and their effects on APC’s stability and/or activity (Tran, 2008).

The results from the epistasis analysis and LEF1 chimeras suggest a role of Trabid in TCF-mediated transcription. Consistent with this, preliminary results from chromatin immunoprecipitation experiments indicate that Trabid is associated with the c-myc promoter and upstream sequences. Trabid's prime physiologically relevant substrate could be APC itself, which is recruited (together with β-TrCP) to TCF target genes to repress their transcription during sustained Wnt signaling. If so, this would imply that Trabid inactivates APC; hyperubiquitylation of APC in Trabid-depleted cells would result in its hyperactivation, possibly leading to hyperrepression (or precocious repression) of Wnt target genes, which would explain why the Wnt response is attenuated in these cells. Alternatively, the prime functional target of Trabid might be an unknown nuclear protein, a negative regulator like APC whose deubiquitylation results in its inactivation or a positive regulator of Wnt-induced transcription that becomes activated as a result of Trabid's DUB action; in the latter case, hyperubiquitylation would be expected to result in inactivation (Tran, 2008).

How does Trabid affect the TCF-mediated transcription? No change was detected in the steady-state association between β-catenin and TCF in Trabid-depleted cells. Nevertheless, the results from the LEF chimeras suggest that Trabid may affect the dynamic association or dissociation of the TCF-β-catenin complex: These chimeras might bypass the requirement for Trabid because of their direct linkage to TADs, which allows them to recruit (or retain) coactivators efficiently -- possibly more efficiently than normal TCF factors whose transcriptional activity during Wnt signaling relies on transient, and perhaps labile, association with limited amounts of nuclear β-catenin. It is therefore conceivable that Trabid increases, for example, the rate of TCF-β-catenin complex formation or the lifetime of its activity (Tran, 2008).

Ubiquitylation is beginning to emerge as a versatile controlling factor in transcriptional switches, including regulatory ubiquitylation events such as monoubiquitylation of histones. There are as yet no known regulatory transcription events that rely on K63-linked ubiquitin, although DNA repair can be regulated by K63-linked ubiquitylation (of PCNA). The turnover of regulatory ubiquitylations via DUBs may be as important as the ubiquitylations themselves, one example being the negative regulation of H2B monoubiquitylation by Ubp8, a DUB associated with the yeast histone acetylation SAGA complex. Interestingly, in vitro transcription from a β-catenin-dependent chromatin template was attenuated by a DUB inhibitor (Tran, 2008).

Given its requirement for efficient TCF-mediated transcription in colorectal cancer cell lines, Trabid has potential as a molecular target for inhibitory drugs in colorectal cancer. Obviously, more work is required to validate this target, but Trabid appears attractive because of (1) its apparent specificity for Wnt responses, (2) its enzymatic activity as a protease, and (3) its catalytic pocket that is likely to be unique among OTU domain proteases (Tran, 2008).

Wnt signaling has a key role in maintaining intestinal stem cell compartment. Furthermore, a recent compilation of gene expression data sets has identified relatively few genes (<40) commonly up-regulated in a variety of stem cell types (including skin bulge, hematopoietic, embryonic, and neural stem cells), which include TCF3 and TCF4 as well as Trabid. Notably, TCF3 plays a role in maintaining skin stem cells in an undifferentiated state, and TCF4 is required for maintaining crypts in the intestinal epithelium. Given the high Trabid levels in these stem cell compartments and Trabid's role in TCF-mediated transcription, it is conceivable that Trabid may have a function in stem cell self-renewal or differentiation (Tran, 2008).

Homeodomain-interacting protein kinases (Hipks) promote Wnt/Wg signaling through stabilization of β-catenin/Arm and stimulation of target gene expression

The Wnt/Wingless (Wg) pathway represents a conserved signaling cascade involved in diverse biological processes. Misregulation of Wnt/Wg signal transduction has profound effects on development. Homeodomain-interacting protein kinases (Hipks) represent a novel family of serine/threonine kinases. Members of this group (in particular Hipk2) are implicated as important factors in transcriptional regulation to control cell growth, apoptosis and development. This study provides genetic and phenotypic evidence that the sole Drosophila member of this family, Hipk, functions as a positive regulator in the Wg pathway. Expression of hipk in the wing rescues loss of the Wg signal, whereas loss of hipk can enhance decreased wg signaling phenotypes. Furthermore, loss of hipk leads to diminished Arm protein levels, whereas overexpression of hipk promotes the Wg signal by stabilizing Arm, resulting in activation of Wg responsive targets. In Wg transcriptional assays, Hipk enhances Tcf/Arm-mediated gene expression in a kinase-dependent manner. In addition, Hipk can bind to Arm and Drosophila Tcf, and phosphorylate Arm. Using both in vitro and in vivo assays, Hipk was found to promote the stabilization of Arm. Similar molecular interactions were observed between Lef1/β-catenin and vertebrate Hipk2, suggesting a direct and conserved role for Hipk proteins in promoting Wnt signaling (Lee, 2009).

Metazoan development is a highly dynamic and complex process that requires the action of several key signal transduction pathways. Their activity must be tightly regulated to ensure the proper patterning and growth of tissues. Regulation of a signaling pathway can occur at any level within the pathway, from perturbation of ligand-receptor interactions to regulation of the activity of transcription factors in the nucleus. Some regulators affect the on or off state of pathways, whereas others are involved in fine-tuning, an essential aspect that ensures that accurate and physiologically necessary levels of signaling are achieved without excessive signaling, which can have deleterious effects (Lee, 2009).

The ability of the Wnt pathway to control different developmental events in a temporally and spatially specific manner requires coordination between numerous regulators. Canonical Wnt signaling controls cell fate by regulating transcription of target genes. Wingless (Wg), a secreted glycoprotein, is the best characterized of the seven Drosophila Wnt ligands, and initiates the canonical pathway by binding to the Frizzled2 (Fz2) and LRP5/6/Arrow co-receptors. This leads to the activation of Dishevelled, which then inhibits the activity of the destruction complex composed of Axin, glycogen synthase kinase 3β (GSK-3β)/Zw3 and adenomatous polyposis coli (APC). As a result, cytosolic Drosophila β-catenin called Armadillo (Arm) accumulates and enters the nucleus to interact with a Tcf/Lef (Drosophila Tcf) family transcription factor to promote target gene expression. In the absence of Wg signaling, the Axin/GSK-3β/APC complex promotes the proteolytic degradation of Arm, whereas transcriptional co-repressors bind to Tcf and repress transcription (Lee, 2009).

The Nemo-like kinase family (Nlk) of protein kinases can regulate activation of Tcf/Lef target genes. In Drosophila, Nemo (Nmo) inhibits Drosophila Tcf activity, and is itself a transcriptional target of the Wg pathway. Recently Homeodomain-interacting protein kinase 2 (Hipk2) was proposed to participate in a kinase cascade to activate Nlk during the regulation of the Myb transcription factor. Therefore, attempts were made in this study to identify whether this regulation was perhaps more general and whether Drosophila Hipk played a role in regulating Nmo, and thus also Wg signaling. It was rapidly learned that Hipk exerts a positive effect on Wg signaling, distinct from Nmo, which has been more fully characterized using the developing wing as a model system (Lee, 2009).

The patterning of the adult wing blade is a tightly regulated process involving numerous essential signaling pathways, including Wg, Notch, EGFR and TGFβ, making it an excellent tissue in which to examine regulatory and epistatic relationships between many genes involved in patterning. The adult wing blade possesses five longitudinal veins (LI-LV) that extend proximally to distally. These are connected by the anterior cross vein (ACV) and posterior crossvein (PCV). The Wg pathway acts at several stages of wing patterning and growth. Wg is expressed along the dorsal/ventral boundary, which in imaginal discs is a stripe bisecting the wing imaginal disc, and in adult wings gives rise to the wing margin and bristles that surround the edge of the wing blade. Loss of wg can lead to loss of the entire wing, to wing to notum transformations, to wing notching or to loss of bristles along the entire wing margin. Wg also promotes proliferation in the wing disc and ectopic Wg can induce outgrowths from the ventral surface of the wing (Lee, 2009).

Hipk2 is a member of a conserved family of serine/threonine kinases. Vertebrate species possess four Hipk proteins (Hipk1-4) that have evolved distinct functions. Singly mutant Hipk1 and Hipk2 mice are viable, whereas double mutant mice die before birth. Drosophila possesses a single Hipk ortholog (which has been referred to as both Hipk and Hipk2) that shares extensive sequence homology within the kinase domain with members of the vertebrate family (Lee, 2009).

Vertebrate Hipk2 has been the most extensively studied member of the family. Biochemical studies have identified a growing list of Hipk2 interactors, including proteins involved in transcriptional regulation, chromatin remodeling and key components of evolutionarily conserved signaling pathways. However, the biological investigation of these interactions in multicellular organisms has been minimal. In vivo examination of murine Hipk2 protein function has thus far revealed a role in neurogenesis and homeotic transformation of the skeleton. Studies in Drosophila using transgenic flies expressing Hipk transgenes have uncovered a role for Hipk in regulating the global corepressor Groucho. Using loss-of-function mutant analyses, a role has been identified for Hipk in promoting Notch signaling during Drosophila eye development (Lee, 2009 and references therein).

This study presents an analysis of the function of Hipk in Drosophila canonical Wg signaling. Genetic studies show that ectopic hipk can rescue phenotypes owing to loss-of-function wg alleles or inhibition of the pathway with a dominant-negative Fz2 receptor. Immunohistochemical studies show that hipk positively regulates expression of Wg targets, and that Hipk can act to stabilize cellular levels of the Arm protein in wing discs. Wnt reporter assays show that both Drosophila Hipk and mouse Hipk2 can promote the Wnt-responsive Topflash reporter. In addition, Hipk/Hipk2 can promote the stabilization of Arm/β-catenin in cell culture and in vivo. These results suggest that Hipk is a positive regulator of the Wg pathway that refines Wg activity during wing development. The findings suggest that these roles may be conserved across species (Lee, 2009).

Loss of zygotic hipk results in pupal or larval lethality. Whole-mount in situ hybridization reveals that hipk is expressed broadly in a non-uniform pattern in multiple stages of development, including all imaginal discs. Removal of the maternal contribution caused embryonic lethality characterized by twisted embryos and head holes, showing that hipk is an essential gene for Drosophila development. These analyses have focused on the most severe allele, hipk4, which causes early larval lethality, or pupal lethality in trans to hipk3. Given the embryonic lethality caused by loss of maternal Hipk, it is speculated that maternally contributed Hipk perdures and obscures its requirement at later stages and impacts the severity of mutant phenotypes. The FLP/FRT technique was used to generate mutant somatic clones to examine the requirements for Hipk in patterning adult structures (Lee, 2009).

This study focused on the role of hipk in the development of the wing. Clones of cells mutant for hipk4 show ectopic veins in the anterior region of the wing blade along LII, loss of the PCV and occasional notches in the wing margin. Reducing hipk function by expression of two independent Gal4-responsive hipk-RNAi constructs in the wing pouch with sd-gal4 or vg-Gal4 also caused a wing notching phenotype reminiscent of those seen upon decreased Wg signaling (Lee, 2009).

Next, the effects of ectopic expression of hipk was studied using the Gal4-UAS system. Phenotypes were observed that suggested that hipk is involved in promoting Wg signaling. Expression of one copy of hipk in the central domain of the imaginal wing discs with omb-Gal4 induced the formation of an additional wing margin and outgrowths emanating from the distal most tip of the ventral surface of the wing. Expression of two copies of UAS-hipk enhanced this phenotype and caused the outgrowths to extend further distally from the ventral plane of the wing. These effects phenocopied the effects observed in ectopic wg-expressing clones. Similarly, expression of UAS-hipk in the wing phenocopies the ectopic venation pattern seen both upon ectopic expression of activated Arm (ArmS10) with bs-Gal4 and in nmoDB24/nmoadk2 mutants. Although control of wing vein patterning is not generally attributed to Wg signaling, ectopic venation has been observed upon elevated Wg signaling. For example, ectopic expression of constitutively active Arm by en-Gal4 in the posterior region of the wing, or ubiquitously with 69B-Gal4 or MS1096-Gal4, leads to disturbed and ectopic venation. Moreover, loss-of-function clones of sgg/zw3 (encoding the fly homolog of GSK3β, a component of the destruction complex) induce the formation of ectopic veins. The results of these phenotypic analyses of hipk are surprising because they demonstrated that Nmo and Hipk did not act in concert to inhibit Wg signaling. Rather, hipk mutant and gain-of-function phenotypes suggest a role in promoting the Wg pathway (Lee, 2009).

This study has revealed that Hipk possesses an intrinsic ability to promote Wg pathway activity and this regulatory function for Hipk is conserved in both Drosophila and mammalian cells. Through a combination of genetic and biochemical analyses, the data reveal that Hipk proteins promote Tcf/Lef1-mediated transcription. Additionally, Hipks enhances the stabilization of Arm/β-catenin in several cell lines and hipk mutant clones in the wing disc have diminished Arm protein levels. Overexpression of Hipk induces a broader domain of stabilized Arm, suggesting Hipk is required to maintain the signaling pool of cytosolic Arm. A model is proposed in which Hipk promotes the Wnt/Wg signal via its regulation of Arm stabilization (Lee, 2009).

Nlk is a conserved antagonist of the Lef1/β-catenin transcriptional complex. This research has shown that Nmo is also an inducible antagonist of the Wg signal in the developing Drosophila wing. It was previously reported that Wnt1 induces the activation of a putative Tak1-Hipk2-Nlk kinase cascade to promote the degradation of the Myb transcription factor. The current study sought to delineate the physiological relevance of this potential kinase cascade; specifically, it was determined whether these interactions played a role in the regulation of the Wg pathway. The data reveal that in Drosophila Nmo and Hipk do not form a kinase cascade in this context, rather they exert opposing effects on the same pathway, probably through distinct mechanisms (Lee, 2009).

The Wg morphogen can bring forth a spectrum of biological processes. Sustaining maximal levels of signaling could be accomplished through the amplification and enhancement of the signal in the wing margin. In support of this model, it was found that overexpression of Hipk expands the expression of Wg targets such as Dll, Sens and Ac. Transcriptional assays reveal that both Drosophila Hipk and mouse Hipk2 enhance the transcriptional activity of Tcf and Lef1, respectively, in a kinase-dependent manner. These findings strongly suggest that Hipk and Hipk2 function to enhance the activity of the transcriptional complex to promote the Wg/Wnt signal (Lee, 2009).

Accumulation of stabilized Arm is paramount to effective Wg signaling. Failure to escape the destruction complex results in Arm degradation and inhibition of Tcf-mediated gene activation. Thus understanding the regulation of Arm is central to the global understanding of how the Wg signal is modulated. In these studies, a role was revealed for Hipk in Arm stabilization. This feature is highlighted by the loss of stabilized Arm in hipk mutant clones. Additionally, in hipk mutant discs, overexpressed wild-type Arm fails to accumulate, despite its expression in domains of high Wg signaling. These findings demonstrate that Hipk plays an important role in Arm stabilization. Hipk may reduce the ability of Arm to interact with destruction complex components or may increase the nuclear retention of Arm. In the absence of Hipk, either of these scenarios would give the destruction complex more access to Arm. In agreement with such a model, it was found that increasing Hipk activity in the wing surpasses the inhibitory effects of the degradation machinery and expands the perimeter of stabilized Arm. Furthermore, it was found that the presence of Hipk or Hipk2 in cell culture stabilizes Arm/β-catenin. Thus, the enhanced transcriptional activity is probably due to the elevated availability of Arm protein (Lee, 2009).

It is crucial for normal development to maintain the proper amounts of β-catenin, since elevated levels of β-catenin can lead to cancer. Elaborate regulatory networks in the cytoplasm and nucleus are vital to maintaining appropriate levels of β-catenin. It is well documented that phosphorylation in the N terminus of β-catenin is crucial for its negative regulation. A chain of phosphorylation events begins when Casein Kinase I (CKI) primes β-catenin for successive modifications by GSK-3β. Central to this event is Axin, which provides a scaffold for APC, CK1, GSK-3β and β-catenin. N-terminally phosphorylated β-catenin is ubiquitinated by βTrCP ubiquitin ligase and targeted for degradation via the proteasome (Lee, 2009).

Wnt signaling promotes the accumulation of β-catenin; however, some of the mechanisms governing this process remain enigmatic. Although overexpression of wild-type β-catenin/Arm is unable to overcome the effects of the degradation machinery, Wnt-stimulated β-catenin can resist the activity of the destruction complex. Although achieving stabilized pools of β-catenin represents the core goal of the Wnt pathway, high levels of β-catenin are not always coupled with elevated transcription. For example, in Xenopus, alanine substitution of one of the GSK3 target residues leads to elevated β-catenin levels, without causing an increase in Tcf-mediated transcription. Thus, further posttranslational modifications of β-catenin are necessary to potentiate its signaling activity (Lee, 2009).

Furthermore, phosphorylation can affect β-catenin stability by affecting protein-protein interactions that regulate protein turnover and activity. Phosphorylation of β-catenin by Cdk5 inhibits APC binding to β-catenin, whereas phosphorylation by CK2 promotes β-catenin stability and transcriptional activity (Lee, 2009).

Recent advances have begun to unravel the molecular complexity that controls β-catenin-mediated transcription within the nucleus. Upon pathway activation, Tcf recruits Arm to the enhancers of Wg-responsive genes where Arm forms multiple transcriptional complexes along its length. Formation of these transcriptional units is needed for the transmission of the Wg/Wnt signal. Recent studies have shown that phosphorylation (distinct from the N-terminal phosphorylation that triggers β-catenin destruction) may modulate its ability to recruit these co-factors. This study found that the Hipk-dependent stabilized form of Arm is transcriptionally active and induces the expression of Wg targets, suggesting modification by Hipk may promote protein interactions (Lee, 2009).

APC and the cell-adhesion molecule E-cadherin compete with Tcf for overlapping binding sites on β-catenin. Competition between proteins may play an important role in the regulation of the Wnt signaling pathway. It is proposed that Hipks may promote the stability of Arm/β-catenin by excluding further interactions with other proteins, including those that antagonize Arm/β-catenin. Given that Hipks can also bind to Tcf/Lef1, it is predicted that these proteins may act synergistically to displace the inhibitory partners of β-catenin. In agreement with such a role, it is observed that Lef1 enhances the interaction between Hipk2 and β-catenin, and these interactions may insulate β-catenin from components of the degradation machinery (Lee, 2009).

Although Hipk phosphorylates Arm, the functional significance of this modification has yet to be determined. Hipk might facilitate the interactions between Arm and its transcriptional co-factors, as Hipk2 phosphorylation has been shown to affect gene regulation by modifying the composition of various transcriptional complexes. Hipk may also enhance the formation of the β-catenin/Tcf transcriptional complex by inducing a conformational change and/or reducing the affinity of possible inhibitors for β-catenin through the phosphorylation of β-catenin. Recently, it has been reported that Hipk2 could antagonize β-catenin/Tcf-mediated transcription in a kinase-independent manner. Although these data appear in conflict with the current findings, it was observed that the effect of Hipk2 on transcription is very cell type- and target gene-dependent, suggesting Hipk2 function is affected by its cellular context, most probably owing to the availability of targets and co-factors (Lee, 2009).

The dynamic localization of Hipk2 in the nucleus, nucleoplasm and in cytosolic speckles suggests that the protein may carry out distinct roles in each site. Given the growing list of interacting proteins, it is tempting to speculate that specific Hipk function is determined in part through its particular localization. It is also possible that Hipk/Hipk2 may act as a scaffolding protein, bringing together multiple binding partners. Ongoing biochemical studies will further uncover the molecular significance of these interactions. Hipk proteins are emerging as important components of multiple signaling networks. The current study describes the roles of Hipk and Hipk2 as Wnt/Wg regulators and sheds light on the regulatory mechanisms governing this conserved pathway (Lee, 2009).

Coop functions as a corepressor of Pangolin and antagonizes Wingless signaling

Wingless (Wg) signaling regulates expression of its target genes via Pangolin and Armadillo, and their interacting cofactors. In the absence of Wg, Pangolin mediates transcriptional repression. In the presence of Wg, Pangolin, Armadillo, and a cohort of coactivators mediate transcriptional activation. This study uncovered Coop (Corepressor of Pan) as a Pangolin-interacting protein. Coop and Pangolin form a complex on DNA containing a Pangolin/TCF-binding motif. Overexpression of Coop specifically represses Wg target genes, while loss of Coop function causes derepression. Coop is shown to antagonize the binding of Armadillo to Pangolin, providing a mechanism for Coop-mediated repression of Wg target gene transcription (Song, 2010).

Coop (CG1621) was found in an attempt to identify novel Pan-interacting proteins by a mass spectrometry-based proteomic approach. It contains an N-terminal MADF domain and a C-terminal BESS domain. This architecture is conserved in 16 Drosophila proteins, one C. elegans protein, and one zebrafish protein. The putative dimeric BESS domain does not exist in mammals, and there is one uncharacterized MADF-containing protein in humans (ZSCAN29/ZNF690). Earlier studies on other members of this family indicated that they might be involved in transcriptional regulation. The interaction between Coop and Pan was confirmed in a series of pull-down experiments. In lysates of Drosophila Kc cells, Pan was coimmunoprecipitated by Coop. Consistent with a role in transcription, Coop was localized in the nucleus in these cells. To determine if this interaction was direct, GST pull-down experiments were performed. A GST fusion of Coop could pull down in vitro translated Pan. In the reverse experiment, Coop was pulled down by a Pan central fragment (247-362) harboring its DNA-binding domain, but not by a Pan N-terminal fragment (1-160). This interaction was further mapped to the C-terminal part of Coop (161-358), which contains its BESS domain (Song, 2010).

Since the DNA-binding domain of Pan may be involved in the interaction with Coop, it was asked whether Coop affected the DNA affinity of Pan. This question was addressed in a gel shift assay. Pan (247-362) induced the mobility shift of a DNA oligonucleotide containing a consensus Pan/TCF-binding site, but did not affect that of a control oligonucleotide in which the binding site was mutated. Coop alone did not bind to this DNA probe. However, in the presence of Pan, Coop induced a supershift of this oligonucleotide. This suggests that the interaction between Coop and Pan does not interfere with Pan binding to DNA. Instead, it suggests that Coop and Pan form a complex on Pan targets (Song, 2010).

To test whether the interaction between Coop and Pan affects Wg signaling in vivo, Coop was overexpressed in wing imaginal discs and expression of Wg targets was monitored, including Distal-less (Dll), senseless (sens), and Notum/wingful (wf). The expression of Coop in the dorsal compartment of wing discs by apterous-Gal4 (apGal4) resulted in the loss of Dll protein in this domain. This repression occurred at the transcriptional level, as the expression of a Dll-lacZ (DllZ) reporter transgene was similarly repressed in the same assay. The expression of Coop in the posterior compartment of wing discs by engrailed-Gal4 (enGal4) also repressed Dll expression, but in a different pattern: Dll staining was lost in the domain away from the dorsal-ventral (D/V) boundary, and was only weakened in the domain close to the D/V boundary. The Wg morphogen is expressed at the D/V boundary and forms a concentration gradient in the wing disc. It is possible that the observations reflected a dosage-dependent effect of Coop on different levels of Wg signaling. To test this idea, Coop was also expressed at lower levels, either in clones or in the center of wing discs by spalt enhancer-Gal4 (salEGal4). Indeed, the repression of Dll expression was observed preferentially in the domain with lower levels of Wg signaling. These results implied that Coop functions to antagonize incoming Wg signaling (Song, 2010).

sens is a high-threshold target of Wg in wing discs and is expressed in two to three rows of cells flanking the D/V boundary. The expression of sens starts at the center of the wing disc and extends to the periphery at late larval stage. The expression of Coop by enGal4 resulted in the repression of sens in the posterior compartment of wing discs, while the expression of Coop by apGal4 prevented Sens from extending to the periphery in the dorsal compartment. Consistent with observations with Dll, lower levels of Coop expression by act>CD2>Gal4 or salEGal4 did not affect sens expression (Song, 2010).

wf is another known Wg target gene. A 4-kb upstream element has been isolated from the wf locus, which responded to Wg signaling in cell culture. A lacZ transgene under its control (wfZ) mimicked wf expression in wing discs. The expression of Coop repressed this wfZ in wing discs and wf reporter in cultured Drosophila cells. In addition to Wg targets in the wing disc, the expression of Coop also repressed H15, a Wg target gene in the leg disc. Thus, in the case of the four Wg target genes analyzed, Coop functioned consistently as a negative transcriptional regulator (Song, 2010).

Whether Coop generally affects transcription, or specifically affects Wg signaling was tested. The effect of Coop on the expression of Notch targets wg and cut was examined. The expression of Coop by enGal4 or other drivers did not affect expression of either gene, confirming that the effect observed on Wg targets was not due to the interference in the upstream level of Wg signaling. Then Coop was expressed by apGal4, which strongly repressed Wg targets, and the expression of other non-Wg targets was monitored. Lgs plays a key role in transducing Wg signal. No effect on lgs expression by Coop was observed. Similarly, the expression of Coop had no effect on the Hedgehog (Hh) target gene decapentaplegic (dpp), or the Dpp targets optomotor blind (omb) and spalt. Similarly, hh, patched (ptc), and en expression were unaffected. In this study it was noticed that overexpression of Coop had a weak negative effect on the Gal4/UAS system. It is therefore possible that Coop expression was reduced, leading to an underestimation of the strength of Coop's activity. Whatever the case, the effect does not compromise any of these conclusions, as none of the targets monitored in this study was driven by the Gal4/UAS system. Taken together, these results suggest that the repressive effect of Coop is probably mediated by its interaction with Pan, and not an indirect effect on wg expression or components of the Wg signaling cascade. Importantly, Coop does not appear to affect Hh or Dpp signaling, suggesting its effect on the Wg pathway is fairly specific (Song, 2010).

In the next step, it was asked whether Coop was required for the proper regulation of Wg target gene expression. The effect of Coop RNAi on the basal transcriptional levels of Wg target genes was examined in Drosophila Kc cells. For comparison, RNAi against Groucho or CtBP, two known corepressors of the Wg pathway, was also performed. Surprisingly, the knockdown of Coop mRNA in Kc cells by dsRNA treatment was very inefficient. The levels of most mRNAs could be knocked down to 10%-15% after 4 d; however, the levels of Coop mRNA remained at 60%-70% after 4 d and 40%-50% after 7 d. Even so, under this condition, the knockdown of Coop mRNA caused a twofold to threefold increase in Arm-independent basal expression of Wg target gene nkd and CG6234, similar to the effect observed with Groucho RNAi, but slightly weaker than the effect seen with CtBP RNAi. In addition, the combination of Coop and CtBP RNAi showed an additive effect (Song, 2010).

Whether reducing Coop function affected the activation of Wg target genes was examined in vivo. This was tested by expressing dsRNA against Coop in the wing disc and monitoring expression of Wg targets. Coop is expressed ubiquitously in imaginal discs. The knockdown of Coop enhanced expression of Dll and wf, suggesting that endogenous Coop also affects Wg-mediated target activation. Starting from an enhancer P element (EP) line, two Coop alleles (hereafter referred to as coop) were generated that encode truncated proteins. coop mutant flies seemed normal, but showed enhanced Wg signaling in a sensitized background: Loss of Coop enhanced a rough eye phenotype caused by ectopic Wg signaling (sev-Wg). In coop mutant clones, moderately enhanced expression of Dll was also observed in the wing disc. This is consistent with in vivo RNAi results, indicating that Coop is a repressor of Wg target genes. Although coop clones had a weaker effect than RNAi, this difference might be due to the perdurance of Coop protein. The RNAi was induced earlier than the clones of coop, thus probably eliminating Coop more thoroughly (Song, 2010).

There are 15 sequence homologs of Coop in Drosophila, and it is possible that one or several of them also had similar roles in Wg signaling. Some Coop family members were examined, and overexpression of CG6854 and Adf1 were found to also repressed Wg signaling in Drosophila cultured cells. As a next step, the possibility that CG6854 or Adf1 acts like Coop in vivo was examined. Overexpression of CG6854 strongly repressed expression of Dll and sens. However, in contrast to Coop, it also repressed expression of wg and targets of other pathways, suggesting the repressive effect of CG6854 may be less specific than that of Coop. Adf1 behaved like CG6854. Taking these results together, it is proposed that, unlike Coop, Adf1 and CG6854 are not specific repressors of the Wg pathway (Song, 2010).

Having established that Coop has a defined role in Wg signaling, in contrast to CG6854 and Adf1, the mechanism by which Coop represses Wg target genes was examined. As the interaction between Arm and Pan is essential for activation of Wg target genes, whether Coop functions by preventing this process was tested. In cultured Drosophila Kc cells, Pan and Arm were overexpressed in the absence or presence of overexpressed Coop, and Pan was coimmunoprecipitated via Arm. The presence of Coop greatly reduced the amount of Pan coimmunoprecipitated. Similarly, when Pan was coimmunoprecipitated via Coop, the presence of overexpressed Arm also prevented this interaction. These results suggest that the binding of Coop to Pan and the binding of Arm to Pan are mutually exclusive (Song, 2010).

As Coop can interact with a domain in Pan that is conserved in other TCFs, whether Coop could also affect Wnt signaling was examined. Interestingly, activation of a Wnt signaling reporter was repressed by ectopic expression of Coop in HEK293T cells. It is likely that Coop achieved this by interfering with the conserved interaction between β-catenin and TCF. These results indicate a way in which Wnt signaling could be additionally regulated by a functional homolog of Coop. Since BESS domain proteins apparently do not exist in mammals, it is postulated that a Coop-like repressor function is carried out by a TCF-interacting protein that is not necessarily structurally related to Coop. Proteomic analysis of TCF interaction partners may help to identify functional homologs of Coop in mammals (Song, 2010).

It has been shown in vitro that β-catenin and TLE1 compete for binding to Lef1. By competing for an overlapping binding site adjacent to the DNA-binding domain of Lef1, TLE1 prevents the recruitment of β-catenin. This study also shows that Coop and Arm also compete to bind Pan. Thus, the levels of nuclear β-catenin/Arm, determined by the levels of Wnt/Wg signaling, decide the transcriptional activity of TCF/Lef/Pan. This dosage-dependent, reversible mechanism helps to shape Wnt/Wg gradient-induced expression of downstream targets (Song, 2010).

Ectopic Wnt signaling, transduced via interaction between β-catenin and TCF, is often detected in human cancers. Several β-catenin-binding proteins, including ICAT and Chibby, can interfere with the interaction of these two proteins. This study reports the identification of Coop as another potential blocker of the β-catenin-TCF interaction. As β-catenin has divergent functions in more than Wnt signaling, TCF-binding proteins may help to specifically decrease the transcriptional outputs of ectopic Wnt signaling. Given the specific effect of Coop in the Wg pathway, it is believed Coop may function as a specific inhibitor of Arm-Pan interaction in Drosophila. Further studies to map the Coop-Pan interaction may uncover novel ways to prevent the interaction between β-catenin and TCF (Song, 2010).

Linking H3K79 trimethylation to Wnt signaling through a novel Dot1-containing complex

Epigenetic modifications of chromatin play an important role in the regulation of gene expression. KMT4/Dot1 is a conserved histone methyltransferase capable of methylating chromatin on Lys79 of histone H3 (H3K79). This study reports the identification of a multisubunit Dot1 complex (DotCom), which includes several of the mixed lineage leukemia (MLL) partners in leukemia such as ENL, AF9/MLLT3, AF17/MLLT6, and AF10/MLLT10, as well as the known Wnt pathway modifiers TRRAP, Skp1, and β-catenin. The human DotCom is indeed capable of trimethylating H3K79 and, given the association of β-catenin, Skp1, and TRRAP, a role was sought for Dot1 in Wnt/Wingless signaling in an in vivo model system. Knockdown of Dot1 in Drosophila (Grappa) results in decreased expression of a subset of Wingless target genes. Furthermore, the loss of expression for the Drosophila homologs of the Dot1-associated proteins involved in the regulation of H3K79 shows a similar reduction in expression of these Wingless targets. From yeast to human, specific trimethylation of H3K79 by Dot1 requires the monoubiquitination of histone H2B by the Rad6/Bre1 complex. This study demonstrates that depletion of Bre1, the E3 ligase required for H2B monoubiquitination, leads specifically to reduced bulk H3K79 trimethylation levels and a reduction in expression of many Wingless targets. Overall, this study describes for the first time the components of DotCom and links the specific regulation of H3K79 trimethylation by Dot1 and its associated factors to the Wnt/Wingless signaling pathway (Mohan, 2010).

In eukaryotic organisms, gene expression patterns are spatiotemporally regulated in a manner that allows for specification of diverse cell types and their differentiation. This spatiotemporal expression is coordinated in part by transcription factors and chromatin modifiers, and by the activity of several signaling pathways, which contribute to gene expression by regulating the transcription factors. Understanding the relationship between chromatin events and signaling pathways is crucial to understanding gene regulation, development of the organism, and disease pathogenesis (Mohan, 2010).

The nucleosome, the basic unit of chromatin, consists of histones H2A, H2B, H3, and H4, and 146 base pairs (bp) of DNA. Crystal structure studies have demonstrated that the N-terminal tails of each histone protrude outward from the core of the nucleosome. These histone tails are subject to various post-translational modifications, including methylation, ubiquitination, ADP ribosylation, acetylation, phosphorylation, and sumoylation, and such modifications are involved in many biological processes involving chromatin such as transcription, genome stability, replication, and repair (Mohan, 2010).

Histones are methylated on either the lysine and/or arginine residues by different histone methyltransferases (HMTases). Histone lysine methylation can occur as mono-, di-, or trimethylated forms, and several lysine residues of histones have been shown to be multiply methylated. This includes methylation on Lys4, Lys9, Lys27, Lys36, and Lys79 of histone H3, and Lys20 of histone H4. Almost all of the lysine HMTases characterized to date contain a SET domain, named after Drosophila Su(var)3-9, Enhancer of zeste [E(z)], and trithorax (trx). SET domain-containing enzymes can catalyze the methylation of specific lysines on histones H3 and H4, and many SET domain-containing enzymes, such as Trithorax and Enhancer of zeste, are central players in epigenetic regulation and development (Mohan, 2010).

Histone H3 at Lys79 (H3K79) can be mono-, di-, and trimethylated by Dot1, which to date is the only characterized non-SET domain-containing lysine HMTase. Dot1 is conserved from yeast to humans. In yeast, telomeric silencing is lost when Dot1 is overexpressed or inactivated, as well as when H3K79 is mutated. Unlike other histone methylation patterns, the pattern of di- and trimethylation of H3K79 in yeast appears to be nonoverlapping. It was also first discovered in yeast that monoubiquitination of histone H2B on Lys123 (H2BK123) by the Rad6/Bre1 complex is required for proper H3K79 trimethylation by Dot1. In vivo analysis of the pattern of H2B monoubiquitination in yeast demonstrated that the H3K79 trimethylation pattern overlaps with that of H2B monoubiquitination, and that the H3K79 dimethylation pattern and H2B monoubiquitination appear to be nonoverlapping. This observation resulted in the proposal that the recruitment of the Rad6/Bre1 complex and the subsequent H2B monoubiquitination could dictate diversity between H3K79 di- and trimethylation on chromatin on certain loci within the genome. In addition to a role in the regulation of telomeric silencing in yeast, Dot1 has also been shown to be involved in meiotic checkpoint control and in double-strand break repair via sister chromatid recombination. A relationship has been found between cell cycle progression and H3K79 dimethylation, but not trimethylation, by Dot1. Consequently, to date, very little is known about a specific biological role of histone H3K79 trimethylation (Mohan, 2010).

In Drosophila, H3K79 methylation levels correlate with gene activity. Mutations in grappa, the Dot1 ortholog in Drosophila, show not only the loss of silencing, but also Polycomb and Trithorax-group phenotypes, indicating a key role for H3K79 methylation in the regulation of gene activity during development. Similarly, Dot1 in mammals has been implicated in the embryonic development of mice, including a role in the structural integrity of heterochromatin. Genome-wide profiling studies in various mammalian cell lines have suggested that Dot1 as well as H3K79me2 and H3K79me3 localize to the promoter-proximal regions of actively transcribed genes, and correlate well with high levels of gene transcription (Steger, 2008). It has also been proposed that Dot1 HMTase activity is required for leukemia pathogenesis (Mohan, 2010 and references therein).

The highly conserved Wnt/Wingless (Wnt/Wg) signaling pathway is essential for regulating developmental processes, including cell proliferation, organogenesis, and body axis formation. Deregulation or ectopic expression of members of the Wnt pathway has been associated with the development of various types of cancers, including acute myeloid and B-cell leukemias. In the canonical Wnt/Wg pathway, a cytoplasmic multiprotein scaffold consisting of Glycogen synthase kinase 3-β (GSK3-β), Adenomatous polyposis coli (APC), Casein kinase 1 (CK1), Protein phosphatase 2A, and Axin constitutively marks newly synthesized β-catenin/Armadillo for degradation by phosphorylation at the key N-terminal Ser and Thr residues. Binding of the Wnt ligands to the seven-transmembrane domain receptor Frizzled (Fz) leads to recruitment of an adaptor protein, Disheveled (Dvl), from the cytoplasm to the plasma membrane. Axin is then sequestered away from the multiprotein Axin complex, resulting in inhibition of GSK3-β and subsequent stabilization of hypophosphorylated β-catenin levels in the cytoplasm. Stabilized β-catenin translocates into the nucleus and binds to members of the DNA-binding T-cell factor/lymphoid enhancer factor (TCF/LEF) family, resulting in the recruitment of several chromatin-modifying complexes, including transformation/transcription domain-associated protein (TRRAP)/HIV Tat-interacting 60-kDa protein complex (TIP60) histone acetyltransferase (HAT), ISWI-containing complexes, and the SET1-type HMTase mixed lineage leukemia 1/2 (MLL1/MLL2) complexes, thereby activating the expression of Wnt/Wg target genes (Mohan, 2010 and references therein).

Although much is known about Dot1 as an H3K79 HMTase, biochemical studies isolating to homogeneity a Dot1-containing complex have not been successful during the past decade. This study reports the first biochemical isolation of a multisubunit complex associated with Dot1, which has been called DotCom. DotCom is comprised of Dot1, AF10, AF17, AF9, ENL, Skp1, TRRAP, and β-catenin. This complex is enzymatically active and can catalyze H3K79 dimethylation and trimethylation. Indeed, nucleosomes containing monoubiquitinated H2B are a better substrate for DotCom in the generation of trimethylated H3K79. Given the association of Skp1, TRRAP, and β-catenin with DotCom, and the fact that these factors have been linked to the Wnt signaling pathway in previous studies, this study investigated the role of the Drosophila homolog of Dot1, dDot1 (Grappa), for the regulation of Wg target genes. RNAi of dDot1 leads to a reduced expression of a subset of Wg target genes, including senseless, a high-threshold Wingless target gene. Furthermore, reduction by RNAi in the levels of the Drosophila homologs of other components of DotCom that regulate the pattern of H3K79 methylation in humans also showed a similar reduction in senseless expression and other Wg target genes. Importantly, DotCom requires monoubiquitination of H2B for H3K79 trimethylation, and, in Drosophila, the loss of Bre1, the E3 ubiquitin ligase, leads to reduction of H3K79 trimethylation and decreased expression of the senseless gene. Taken together, these data support a model in which monoubiquitinated H2B provides a regulatory platform for a novel Dot1 complex to mediate H3K79 trimethylation, which is required for the proper transcriptional control of Wnt/Wg target genes (Mohan, 2010).

Although H3K79 methylation is a ubiquitous mark associated with actively transcribed genes, and its presence is a clear indicator for the elongating form of RNA polymerase II, Dot1 itself has a very low abundance and is very hard to detect in cells. This indicates that Dot1 is an active enzyme with a very high specific activity toward its substrate, H3K79. Due to the low abundance of Dot1 in cells, its molecular isolation and biochemical purification have been hindered for the past decade. This study reports the biochemical isolation of a Dot1-containing complex (DotCom) and demonstrate a specific link between H3K79 trimethylation by DotCom and the Wnt signaling pathway. The study reports (1) the identification and biochemical isolation of a large macromolecular complex (~2 MDa) containing human Dot1, in association with human AF10, AF17, AF9, ENL, Skp1, TRRAP, and β-catenin; (2) the biochemical demonstration that the human DotCom is capable of trimethylating H3K79, and the analysis of the role of histone H2B monoubiquitination in the enhancement of this H3K79 trimethylase activity of the human DotCom; (3) identification of the role of the components of DotCom in the regulation of its H3K79 methylase activity; (4) demonstration of a role for the Drosophila homolog of Dot1 and its associated factors in the Wnt signaling pathway; and, finally, (5) the identification of a specific requirement of H3K79 trimethylation, but not mono- or dimethylation, in the regulation of Wnt target transcription, thereby linking H3K79 trimethylation to Wnt signaling (Mohan, 2010).

Dot1 was initially isolated from yeast, and these studies demonstrated that the enzyme is capable of mono-, di-, and trimethylating H3K79. Subsequent molecular and biochemical studies demonstrated that prior H2B monoubiquitination by the Rad6/Bre1 complex is required for proper H3K79 trimethylation by yeast Dot1. A recent analysis of the human homolog of Dot1 suggested that its HMTase domain is not capable of trimethylating H3K79, and that this enzyme can only dimethylate its substrate. It has also been demonstrated that reconstitution of monoubiquitinated H2B into chemically defined nucleosomes, followed by enzymatic treatment with Dot1, resulted only in dimethylation of H3K79. Since these observations are in contrast with the published studies in yeast, this study tested the enzymatic activity of purified human DotCom toward monoubiquitinated and nonmonoubiquitinated nucleosomes. The studies demonstrate that the human DotCom can indeed trimethylate H3K79, and that monoubiquitination of histone H2B enhances this enzymatic property of the human DotCom. Since the enzymatic studies employ antibodies generated toward mono-, di-, and trimethylated H3K79 to identify the products of the enzymatic reactions containing human Dot1, it was important to make certain that the observations are not the result of cross-reactivity between these antibodies. Therefore recombinant nucleosomes were generated and treated with human Dot1 in the presence and absence of SAM, and the products were analyzed by MS. The chemical analysis of the products from this enzymatic reaction confirmed that human Dot1 is capable of trimethylating H3K79. The hDot1-treated nucleosome samples were digested with Endoproteinase Arg-C because previous unpublished work on analyzing yeast histone modifications by MudPIT had shown that the trimethylated peptide containing H3K79 was not detected when digesting with trypsin. Notably, McGinty (2008) performed their digestions with trypsin, which might explain their failure to detect this modification by MS (Mohan, 2010).

These studies identified several factors—including ENL, AF9, AF17, AF10, SKP1, TRA1/TRAPP, and β-catenin—as components of the human DotCom. To test the role of these factors in regulating Dot1’s catalytic activity, their levels were reduced via RNAi. These studies demonstrated that AF10 functions with Dot1 to regulate its catalytic properties in vivo. Significant differences in Dot1’s H3K79 HMTase activity were not detected in vivo when reducing the levels of ENL, AF9, and AF17. Factors that significantly alter the H3K79 methylation pattern by Dot1 are also linked to its transcriptional regulatory functions at Wnt target genes (Mohan, 2010).

Since Dot1 also appears to interact with β-catenin, and given the known role for β-catenin, Skp1, and TRRAP in the Wnt signaling pathway, the role for Dot1 and the components of its complex were tested in Wnt signaling. Drosophila is an outstanding model system for the study of the Wnt signaling pathway. Given the power of genetics and biochemistry in Drosophila, the role of dDot1 and the members of its complex in wingless signaling were tested. From this study, it was learned that down-regulation of Drosophila Dot1 and Drosophila AF10 had the most significant effects in the regulation in the expression of the Wg target senseless. Given the fact that the molecular studies demonstrated that Dot1 and AF10 have the strongest effect in the regulation of H3K79 methylation in vivowe wanted to determine whether a specific form of H3K79 methylation is required for Wnt target gene expression was tested (Mohan, 2010).

Histone H2B monoubiquitination is required for proper H3K79 trimethylation. The E2/E3 complex Rad6/Bre1 is required for the proper implementation of H2B monoubiquitination on chromatin, and this complex is highly conserved from yeast to humans. Deletion of the Drosophila homolog of Bre1 results in the loss of H2B monoubiquitination and the specific loss of H3K79 trimethylation. Interestingly, reduction in the levels of H3K79 trimethylation results in a defect in expression of one of the Wnt target genes, senseless, although the H3K79 mono- and dimethylation in this mutant background appear to be normal. In addition to senseless, the role of H3K79 methylation at other Wnt targets was tested, and the same effect was observed for Notum and CG6234. Overall, these studies demonstrate a link between H3K79 trimethylation by the DotCom and the Wnt signaling pathway (Mohan, 2010).

Wnt/Wg signaling serves a critical role in tissue development, proliferation of progenitor cells, and many human cancers. The key player in the Wnt pathway is β-catenin, which is shuttled into the nucleus at the onset of activation of the pathway. Various proteins that interact with β-catenin in the nucleus—such as CBP/p300, TRRAP, MLL1/MLL2, Brg1, telomerase, Hyrax, Pygopus, and CDK8—modulate the transcriptional output of Wg/Wnt target genes. These proteins probably provide the context specificity to Wnt response directing proliferation or differentiation effects of Wnt signaling. The finding that dDotCom is required for expression of a subset of Wg targets suggests that dDotCom might also facilitate Wg-regulated programs of transcriptional regulation in specific contexts. As most human cancers have elevated levels of Wnt signaling and require Wnt signaling for continued proliferation, DotCom might play a role in supporting the high rate of expression of Wnt target genes in such cancers (Mohan, 2010).

Several studies have found interactions between Dot1 and many translocation partners of MLL. While these associations suggest a link between Dot1 methylation and leukemogenesis, it was not clear how Dot1 methylation would participate in this process. Recently, GSK3, a regulator of β-catenin and Wnt signaling, was found to be essential for proliferation of MLL-transformed cells and for progression of a mouse model of MLL-based leukemia (Wang, 2008). These studies linking Dot1 H3K79me3 with Wnt signaling provide insight into the role of Wnt signaling and Dot1 methylation in MLL translocation-based leukemia (Mohan, 2010).

Sunspot, a link between Wingless signaling and endoreplication in Drosophila

The Wingless (Wg)/Wnt signaling pathway is highly conserved throughout many multicellular organisms. It directs the development of diverse tissues and organs by regulating important processes such as proliferation, polarity and the specification of cell fates. Upon activation of the Wg/Wnt signaling pathway, Armadillo (Arm)/beta-catenin is stabilized and interacts with the TCF family of transcription factors, which in turn activate Wnt target genes. This study shows that Arm interacts with a novel BED (BEAF and Dref) finger protein that has been termed Sunspot (Ssp). Ssp transactivates Drosophila E2F-1 (dE2F-1) and PCNA expression, and positively regulates the proliferation of imaginal disc cells and the endoreplication of salivary gland cells. Wg negatively regulates the function of Ssp by changing its subcellular localization in the salivary gland. In addition, Ssp was found not to be involved in the signaling pathway mediated by Arm associated with dTCF. These findings indicate that Arm controls development in part by regulating the function of Ssp (Taniue, 2010).

Arm is composed of 12 imperfect protein interaction repeats (Armadillo repeat domain) flanked by unique N and C termini. In an attempt to identify novel Arm-binding proteins, a yeast two-hybrid screen of a Drosophila embryo cDNA library was performed using the Armadillo repeat domain of Arm as bait. Positive clones containing the same insert of a novel gene (CG17153) were isolated that were named sunspot (ssp; named after the phenotype of mutant flies). Sequence analysis of the full-length cDNA revealed that it encodes a protein of 368 amino acids. A region near its N terminus (amino acids 34 to 98) shows similarity to the BED (BEAF and Dref) finger domain, which is predicted to form a zinc finger and to bind DNA (Taniue, 2010).

To confirm the interaction between Ssp and Arm, whether Ssp produced by in vitro translation could interact with the Armadillo repeat domain of Arm fused to glutathione S-transferase (GST) was tested. Ssp specifically interacted with the Armadillo repeat domain of Arm (amino acids 140 to 713), but failed to interact with Pendulin (Pen), a Drosophila homolog of importin α, which also possesses the Armadillo repeat domain. Pull-down assays with a series of deletion fragments of Ssp showed that a fragment of Ssp containing amino acids 235 to 307 (termed the ABR, the Arm-binding region) binds to Arm in vitro. Also, it was found that Armadillo repeats 2-8 of Arm are responsible for binding to Ssp. Although TCF is known to bind to Armadillo repeats 3-10 of Arm, Ssp did not compete with TCF for binding to Arm (Taniue, 2010).

Next, whether Ssp is associated with Arm in living cells was examined. Drosophila Schneider-2 (S2) cells were transfected with Arm along with GFP-Ssp, GFP-SspδC (amino acids 1 to 217; a mutant lacking the ABR) or GFP-SspABR (amino acids 235 to 342; a fragment containing the ABR). GFP-fusion proteins were immunoprecipitated from S2 cell lysates and subjected to immunoblotting with anti-GFP and anti-Arm antibodies. For immunoprecipitation of GFP-fusion proteins, a 13-kDa GFP-binding fragment was used derived from a llama single chain antibody, which was covalently immobilized to magnetic beads (GFP-Trap-M), as the molecular weight of GFP-SspδC is the same as that of IgG. It was found that Arm is associated with GFP-Ssp and GFP-SspABR. By contrast, Arm barely co-immunoprecipitated with GFP-SspδC. In addition, pull-down assays were also performed with a mixture of lysates of S2 cells transfected with Arm alone and GFP-Ssp alone, respectively. It was found that Ssp and Arm co-precipitate only when both proteins are co-expressed in S2 cells, excluding the possibility that Ssp and Arm associate after cells are lysed. Taken together, these results suggest that Ssp interacts via its ABR with Arm not only in vitro but also in vivo (Taniue, 2010).

One lethal P-element insertion line, l(3)j2D3j2D3, was found in which a P-element had been inserted into the gene adjacent to ssp, CG6801, which is located about 250 bp upstream of the 5' end of ssp. RT-PCR analysis revealed that the expression level and size of the CG6801 transcript were not changed compared with in wild-type larvae, which is consistent with the P-element being inserted into an intron in CG6801. To generate mutants that have a deletion in ssp but have intact CG6801, a local hop and imprecise excision approach was used. l(3)j2D3j2D3 was used in a local hop to generate a P-element insertion line, sunspotP, that completely complemented the lethality of l(3)j2D3j2D3. Then ssp mutants were generated by imprecise excision of the P-element from sunspotP. One allele was found that has a deletion of about 600 bp, and this was designated as ssp598. Sequence analysis showed that the deletion extends from a position 60 bp downstream of the presumptive ssp transcription start site to the ssp gene locus. Because this deletion removes the start codon and the BED finger domain of ssp, it is presumed that ssp598 represents a null allele for ssp. RT-PCR analysis revealed that ssp598 generates a truncated transcript. The truncated transcript encodes a peptide consisting of 13 amino acids, which is unrelated to Ssp. By contrast, RT-PCR analysis revealed that the intact CG6801 transcript is expressed in ssp598 mutant larvae, and that the expression level of CG6801 is unchanged in ssp598 mutant larvae compared with that in wild-type larvae. Furthermore, ssp598 fully complemented the phenotype of l(3)j2D3j2D3, indicating that this mutant contains intact CG6801 (Taniue, 2010).

The imaginal discs, salivary glands and central nervous system of larvae homozygous for ssp598 were smaller than those of their normal counterparts. ssp598 homozygotes reached the third instar stage, but failed to reach the pupal stage and died between 10 and 20 days after egg laying (AEL). Furthermore, melanotic pseudotumors were formed in ssp598 mutant larvae. Melanotic pseudotumors are groups of cells within the larvae that are recognized by the immune system and encapsulated within a melanized cuticle. One or more small melanotic pseudotumors first appeared in the ssp mutants at 6 days AEL, and the number and size of these melanotic pseudotumors increased during the development of the larvae. Similar phenotypes were observed with hemizygotes for ssp598 and Df(3L)BK9, which has a deletion larger than that of ssp598 and lacks ssp. In situ hybridization analysis of imaginal discs using the coding region of the ssp cDNA as a probe revealed that ssp transcripts are expressed ubiquitously. Therefore whether ubiquitous expression of ssp restores the phenotypes of ssp598 homozygous animals was examined. It was found that ubiquitous expression of the full-length ssp cDNA with the Gal4-UAS system rescued the lethality and other phenotypes associated with ssp598 homozygous animals. Taken together, these results suggest that the phenotypes of ssp598 homozygotes are caused by the loss of ssp function, and that ssp is required for cell proliferation and morphogenesis of the imaginal disc and central nervous system (Taniue, 2010).

Arm is a key transducer of Wg signaling and many of the Arm-binding proteins are known to function as a component of the Wg signal transduction pathway. To explore the possibility that Ssp is related to the Wg signal transduction pathway, the effect of Wg on the distribution of GFP-Ssp was examined. Because imaginal disc cells are too small for detailed study, focus for this analysis was placed on the third instar salivary glands, and whether the subcellular localization of GFP-Ssp is linked to Wg signaling was studied. The larval salivary gland mainly consists of secretory gland cells and imaginal ring cells. Gland cells are large polyploid epithelial cells. Small imaginal ring cells reside at the proximal end of the secretory gland. Immunostaining with anti-Wg antibody revealed that Wg is expressed in imaginal ring cells. Furthermore, Drosophila frizzled 3 (dfz3)-lacZ, a target gene of Wg signaling, was found to be expressed in imaginal ring cells and proximal gland cells, which reside within several cell diameters of the Wg-expressing cells. These results suggest that Wg signaling is active in the proximal region in the third instar salivary gland. When GFP-Ssp was expressed ubiquitously under the control of dpp-Gal4 in the larval salivary gland, GFP-Ssp was found to be localized predominantly at the nuclear envelope in proximal gland cells. In addition, GFP-Ssp was detected as aggregates in the nucleus in the distal region of the salivary gland. To examine whether this region-specific subcellular localization of GFP-Ssp is related to Wg signaling, Wg or Axin, a negative regulator of Wg signaling, was overexpressed in the salivary gland under the control of dpp-Gal4. It was found that expression of Wg along with GFP-Ssp resulted in the accumulation of a certain population of GFP-Ssp at the nuclear envelope in both the distal and proximal regions. Again, a significant amount of GFP-Ssp was localized in nuclear aggregates in both distal and proximal cells, suggesting that ectopic expression of Wg can also change the subnuclear localization of Ssp in proximal cells, from the nuclear periphery to nuclear aggregates. This result also suggests that ectopic expression of Wg in distal cells is not sufficient to change the subnuclear localization of all GFP-Ssp protein, from nuclear foci to the nuclear periphery. By contrast, when Axin was expressed along with GFP-Ssp, GFP-Ssp was detected as nuclear aggregates, not only in the distal region but also in the proximal region, but was no longer detected at the nuclear envelope. These results suggest that the subcellular localization of Ssp is regulated at least in part by Wg signaling in the third instar salivary gland (Taniue, 2010).

To examine whether the effect of Wg signaling on Ssp localization is mediated by the direct interaction between Arm and Ssp, Ssp localization was studied in larvae expressing an RNAi targeting Arm. It was found that Ssp was localized in nuclear aggregates and that Wg overexpression did not alter its localization when the expression of Arm was suppressed by RNAi. Thus, Arm is required for Wg-induced Ssp relocalization. Ssp localization was also examined in cells expressing δArm, a mutant of Arm that localizes at the plasma membrane. It was found that overexpression of δArm under the control of dpp-Gal4 results in the localization of GFP-Ssp at the plasma membrane throughout the salivary gland. Next the subcellular localization of SspδC, a mutant that lacks the ABR and is unable to interact with Arm, was examined. When GFP-SspδC was expressed ubiquitously, it was found to localize homogenously in the nucleus of both distal and proximal cells. This result indicates that the localization of Ssp to nuclear aggregates requires the ABR and suggests that Ssp requires a direct interaction with Arm to localize to its target sites in the nucleus. Furthermore, it was found that the localization of GFP-SspδC was not changed by coexpression with Wg, or δArm. Taken together, these results suggest that the direct interaction between Arm and Ssp is required for the regulation of Ssp localization by Wg signaling (Taniue, 2010).

The N-terminal region of Ssp contains a BED finger domain. This presumptive DNA-binding domain is known to be contained in several Drosophila proteins, such as Dref and BEAF-32. Dref regulates the transcription of genes involved in DNA replication and cell proliferation, including dE2F-1 and PCNA, the promoters of which contain BED finger-binding elements (BBEs). To clarify whether Ssp regulates the transcription of these genes, the expression levels of dE2F-1 and PCNA were examined. For this purpose, the P-element (lacZ) insertion lines E2F07172 and PCNA02248 were used. dE2F-1-lacZ and PCNA-lacZ expression were found to be high in distal cells compared with proximal cells in the larval salivary gland. When ssp was ectopically expressed in the salivary gland, dE2F-1-lacZ expression was markedly elevated in distal cells, whereas it was only slightly elevated in proximal cells. However, PCNA-lacZ expression was markedly elevated throughout the salivary gland. By contrast, dE2F-1-lacZ and PCNA-lacZ expression were not elevated in distal cells of ssp mutant salivary glands compared with in wild-type salivary glands, and dfz3-lacZ expression in ssp mutant and Ssp-overexpressing salivary glands was not changed compared with in wild-type salivary glands, suggesting that Ssp is not involved in Arm-dTCF-mediated transactivation of Wg target genes. In addition, overexpression of Wg resulted in a decrease in the expression levels of dE2F-1-lacZ and PCNA-lacZ in distal cells. Thus, Ssp is active in the distal region where Wg signaling is not active, and Ssp is aggregated in the nucleus. Conversely, Ssp is not very active in the proximal region where Wg signaling is active, and Ssp is accumulated in the nuclear envelope (Taniue, 2010).

The expression of dE2F-1, PCNA and dfz3 was examined in the wing disc. Clones of cells lacking Ssp function were generated by FLP/FRT-mediated somatic recombination. Clones of ssp mutant cells underwent only a few divisions after they were generated in the presumptive wing blade: the mutant cells proliferated slowly and either died or were actively eliminated from the disc epithelium. Therefore, a Minute mutation, M(3)65F, was used to confer a growth advantage upon cells homozygous for ssp. When mitotic recombination was induced in a M(3)65F background using enhancer trap lines, ssp mutant cells exhibited reduced levels of dE2F-1-lacZ and PCNA-lacZ expression but did not show any change in the levels of dfz3-lacZ and Arm expression. These results suggest that ssp regulates the expression of dE2F-1 and PCNA, but is not involved in Arm-dTCF-mediated Wg signaling (Taniue, 2010).

To confirm these results, endogenous expression of dE2F-1 and PCNA was examined by quantitative real-time RT-PCR analysis using RNA from late third instar larvae. Flies carrying heat-shock-inducible Gal4 (hs-Gal4) were crossed with transgenic flies carrying UAS-GFP, UAS-ssp or UAS-wg. Consistent with the above results, overexpression of ssp resulted in elevated steady state levels of dE2F-1 and PCNA transcripts. Furthermore, overexpression of Wg induced decreases in the numbers of dE2F-1 and PCNA transcripts. These results suggest that dE2F-1 and PCNA expression is regulated positively by Ssp and negatively by Wg (Taniue, 2010).

Also whether Ssp regulates the expression of dE2F-1 by binding directly to its promoter region was examined. Electrophoretic mobility-shift assays (EMSA) showed that GST-Ssp, but not GST, bound to a 40-mer oligonucleotide corresponding to a region in the dE2F-1 promoter that contains three BBEs. By contrast, GST-Ssp barely bound to a mutated probe in which CG in each BBE had been replaced with AA. Binding of Ssp to the wild-type probe was inhibited in the presence of an excess amount of unlabeled wild-type probe, whereas the mutated probe did not inhibit the interaction significantly. When anti-Ssp antibody was included in the reaction mixture, the Ssp band was not detected. Furthermore, it was found that GST-SspδBFD, a mutant Ssp lacking the BED finger domain, did not bind to the wild-type probe. These results suggest that Ssp regulates dE2F-1 expression by binding directly to the BBEs in the dE2F-1 promoter region via its BED finger domain (Taniue, 2010).

To further elucidate the function of Ssp and Wg, the third instar salivary glands of ssp and wg mutants were examined. In the third instar salivary gland, the distal region undergoes greater endoreplication than does the proximal region. As a result, the nuclear size of distal gland cells is markedly larger than that of proximal gland cells. However, the nuclear size of ssp mutant distal cells was found to be smaller than that of wild-type distal cells. By contrast, the nuclear size of wg mutant proximal cells was larger than that of wild-type proximal cells. Thus, the difference in nuclear size between proximal and distal cells was also small in the salivary glands of wg mutants (Taniue, 2010).

To confirm these results, the effects were examined of Ssp and/or Wg overexpression on the nuclear size of salivary gland cells. When Ssp was overexpressed, the nuclear size of both proximal and distal cells was heterogenous. Overexpression of Wg decreased the nuclear size of distal cells: the difference in nuclear size between Wg-overexpressing proximal and distal cells was small. However, when Wg was overexpressed along with Ssp, the effect of Ssp was suppressed and the heterogeneity of nuclear size was not observed. Furthermore, to confirm that Ssp and Wg play important roles in the regulation of endoreplication, δArm-expressing clones were generated using the flip-out technique. It was found that the nuclear size of δArm-expressing cells is much smaller than that of surrounding cells. This result suggests that δArm mislocalizes Ssp to the plasma membrane, thereby negatively regulating Ssp activity for endoreplication (Taniue, 2010).

To directly show that ssp mutant cells undergo fewer endoreplications than do wild-type cells, BrdU-labeling experiments were performed. When wild-type salivary glands were labeled with BrdU, distal cells efficiently incorporated BrdU, indicating that they underwent at least one round of DNA replication during the labeling period. By contrast, very few nuclei of ssp mutant cells and Wg-overexpressing cells were labeled with BrdU (Taniue, 2010).

dMyc has also been reported to be required for the endoreplication of salivary gland cells. It is therefore interesting to examine the relationship between dMyc, Wg and Ssp in endoreplication. It was found that dMyc expression was unchanged in both ssp mutant and Ssp-overexpressing salivary glands. Thus, Ssp might not be involved in the regulation of dMyc (Taniue, 2010).

Taken together, these results suggest that Ssp and Wg play important roles in the regulation of endoreplication in the third instar salivary gland, and that Wg might exert its effect by negatively regulating the function of Ssp. It is interesting to speculate that Ssp plays a general role for endoreplication in all larval endocycling tissues (Taniue, 2010).

It is believed that Wg/Wnt target genes are transactivated by Arm/β-catenin associated with TCF. However, expression of some human genes is transactivated by β-catenin that is associated with proteins other than TCF. For example, β-catenin interacts with the androgen receptor in an androgen-dependent manner and enhances androgen-mediated transactivation. In the present study, it was shown that Arm interacts with Ssp and negatively regulates its function. Ssp transactivates dE2F-1 and PCNA expression, and positively regulates the endoreplication of salivary gland cells. Furthermore, the Wg signal represses the function of Ssp by altering the subcellular localization of Ssp in the salivary gland: the Wg signal induces the accumulation of Ssp at the nuclear envelope. Interestingly, recent studies indicate that the nuclear membrane provides a platform for sequestering transcription factors away from their target genes. For example, it has been shown that the tethering of transcription factors such as c-Fos and R-Smads to the nuclear envelope prevents transcription of their target genes. The results appear to be consistent with these findings. Although the precise mechanism remains to be investigated, the interaction between Arm and Ssp appears to be required for the regulation of Ssp localization by Wg signaling. It remains to be investigated whether the mechanisms identified in the salivary gland are applicable to other tissues (Taniue, 2010).

A dual function of Drosophila capping protein on DE-cadherin maintains epithelial integrity and prevents JNK-mediated apoptosis

E-cadherin plays a pivotal role in epithelial cell polarity, cell signalling and tumour suppression. However, how E-cadherin dysfunction promotes tumour progression is poorly understood. This study shows that the actin-capping protein heterodimer, which regulates actin filament polymerization, has a dual function on DE-cadherin in restricted Drosophila epithelia. Knocking down Capping Protein in the distal wing disc epithelium disrupts DE-cadherin and Armadillo localization at adherens junctions and upregulates DE-cadherin transcription. In turn, DE-cadherin provides an active signal, which prevents Wingless signalling and promotes JNK-mediated apoptosis. However, when cells are kept alive with the Caspase inhibitor P35, the activity of the JNK pathway and of the Yorkie oncogene trigger massive proliferation of cells that fail to stably retain associations with their neighbours. Moreover, loss of capping protein cooperates with the Ras oncogene to induce massive tissue overgrowth. Taken together, these findings argue that in some epithelia, the dual effect of capping protein loss on DE-cadherin triggers the elimination of mutant cells, preventing them from proliferating. However, the appearance of a second mutation that blocks cell death may allow for the development of some epithelial tumours (Jezowska, 2011).

Actin filament (F-actin) turnover and organization is a critical regulator of AJs assembly, maintenance, and remodelling. F-actin growth, stability, disassembly and also their organization into functional higher-order networks are controlled by a plethora of actin-binding proteins (ABPs), strongly conserved between species. Capping protein (CP), composed of an α (Cpa) and β (Cpb) subunits, acts as a functional heterodimer by restricting accessibility of the filament barbed end, inhibiting addition or loss of actin monomers. In Drosophila, removing either cpa or cpb, promotes accumulation of F-actin within the cell and gives rise to identical developmental phenotypes. In the whole larval wing disc epithelium, loss of CP activity reduces Hpo pathway activity and leads to ectopic expression of several Yki target genes that promote cell survival and proliferation. However, inappropriate growth can only be observed in the proximal wing domain. In the distal wing primordium, cpa or cpb mutant cells mislocalize the AJs components DE-Cad and Arm, upregulate puc expression, extrude and die. This indicates that while loss of CP can under certain conditions result in tissue overgrowth due to inhibition of Hpo pathway activity, other factors such as the polarity status, also determine the survival and growth of the mutant tissue (Jezowska, 2011).

This study investigated the role of the actin-CP heterodimer in survival of cells in the distal wing disc epithelium. CP has a dual function in regulating DE-Cad: it stabilizes DE-Cad at cell-cell junctions, thereby preventing loss of epithelial integrity and inhibits upregulation of the DE-cad gene. DE-Cad would otherwise provide an active signal, which affects Wg signalling and promotes JNK-mediated apoptosis. However, when cells lacking CP are kept alive, JNK is converted into a potent inducer of proliferation (Jezowska, 2011).

This study demonstrates that in the distal wing disc epithelium, JNK signalling triggers apoptosis of cells with reduced CP expression but induces massive proliferation when apoptosis is blocked with P35. Yki activity is also required to allow overgrowth of 'undead' Cpa-depleted tissues. Induction of apoptosis has been shown to activate Yki through the JNK pathway and triggers compensatory cell proliferation. Thus, in CP-depleted cells kept alive with P35, Yki may act downstream of JNK signalling. Consistent with this, targeting Yki degradation in these tissues fully suppresses ectopic N-Cad expression but not MMP1 upregulation. Because CP also prevents Yki activity in the whole wing disc epithelium, independently of its effect on JNK signalling, (Fernandez, 2011; Sansores-Garcia, 2011), in the distal wing domain, excess Yki activity of 'undead' CP-depleted tissues may result from a dual effect, which involves a JNK-dependent and independent mechanisms (Jezowska, 2011).

JNK signalling has been reported to propagate from cell to cell in the wing disc, where it could trigger apoptosis or Yki-dependent compensatory proliferation. Neither non-autonomous apoptosis nor activation of JNK signalling was observed when patches of CP mutant cells were induced or dsRNA for CP was expressed with or without P35. Therefore, the propagation of JNK activation might be impaired in tissues knocked down for CP. However, increase proliferation was observed of wild-type cells apposed to 'undead' Cpa-depleted tissues. This suggests that JNK propagation is not required to trigger compensatory cell proliferation (Jezowska, 2011).

Several observations argue that in cells lacking CP, a DE-Cad-dependent signal promotes JNK-mediated apoptosis by inhibiting Wg signalling. First, knocking down Cpa affects Wg signalling, which has been shown to prevent JNK-dependent cell death in this region. Second, removing one copy of DE-cad in Cpa-depleted cells partially suppresses apoptosis and ectopic MMP1 expression and restores Wg target genes expression. Third, loss of CP is associated with upregulation of the DE-cad gene and increased levels of the DE-Cad protein. One way by which DE-Cad may block Wg signalling is by tethering Arm. In agreement with this possibility, in the distal wing disc epithelium, overexpression of DE-cad compromises Wg signalling, while co-expression of Arm rescues the DE-cad overexpression phenotype. Moreover, in mouse, overexpression of E-Cad induces apoptosis and sequesters the transcriptionally competent pool of β-cat, effectively shutting off expression of Lef/TCF/β-cat-responsive genes. Interestingly, in Cpa-depleted tissues, the faster mobility form of Arm is enriched. Because this form was proposed to correspond to the cytoplasmic pool of Arm, following CP loss, increase DE-Cad levels might tether and stabilize Arm in the cytoplasm, preventing it to transduce Wg signalling. How a defect in Wg signalling triggers JNK-mediated cell death is not known. In cells lacking CP, JNK activation may occur in response to loss of DIAP1 since overexpressing DIAP1 strongly reduces ectopic MMP1 expression. However, it cannot be excluded that JNK signalling reduces DIAP1 levels since JNK signalling can also function upstream of DIAP1 (Jezowska, 2011).

In the distal wing domain, cells lacking CP mislocalize DE-Cad and Arm at AJs, upregulate expression of DE-cad and extrude from the epithelium (Janody, 2006). DE-cad appears to be a direct transcriptional target of the Hpo signalling pathway. CP inhibits Yki activity (Fernandez, 2011; Sansores-Garcia, 2011) and prevents shg-LacZ upregulation, even in mutant clones that maintain a polarized epithelial architecture in the proximal wing domain. Thus, increased DE-cad expression likely results from inhibition of Hpo pathway activity. However, while mutant clones for Hpo pathway components accumulate DE-Cad, mutant cells do not extrude from the wing disc epithelium. Therefore, the polarity defect of cells lacking CP is unlikely to result from increased DE-Cad levels. Different observations also argue that altered cell-cell adhesion does not result from a defect in Wg signalling or from ectopic activation of JNK signalling, as previously reported. First, reducing DE-cad levels do not restore Arm localization at AJs. Second, in Cpa-depleted tissues in which JNK signalling is blocked, dividing nuclei surrounded by dense F-actin patches are recovered on the basal surface of the distal wing disc epithelium. Third, unlike cells lacking CP, tissues expressing P35 and defective for Wg signalling or overexpressing DE-cad or in which high apoptotic levels were induced maintain a polarized epithelial architecture). Therefore, following loss of CP, the mislocalization of DE-Cad and Arm and the loss of cell-cell contacts are likely upstream or parallel events to DE-cad upregulation and JNK-mediated cell death. Because disruption of apical-basal polarity can trigger JNK activation, a model is favored by which CP prevents JNK-mediated cell death though a dual function on DE-Cad: it promotes DE-Cad-mediated cell adhesion and restricts DE-cad expression (Jezowska, 2011).

While the effect of CP loss on DE-cad transcription is not context dependent, the polarity defect is mainly observed in the distal wing domain. Different regions of the wing disc may have specific requirements in terms of AJs stability and remodelling. Because the distal wing disc is under higher mechanical stress, this epithelium may require higher dynamics of DE-Cad remobilization. CP might be critical to control this kinetic, making distal wing cells lacking CP more prone to lose cell-cell adhesion and extrude from the epithelium (Jezowska, 2011).

Interestingly, the proto-oncogene of the Src family kinases Src42A antagonizes DE-Cad-mediated cell adhesion and stimulates the transcription of DE-cad. Moreover, in the distal wing disc epithelium, the major inhibitor of Src family kinases C-terminal Src kinase (Csk), maintains AJs stability, prevents JNK-mediated apoptosis, whereas halving the genetic dose of DE-cad suppresses the apoptotic phenotype of dCsk-depleted cells. CP and mammalian c-Src both regulate F-actin. Conversely, the control of F-actin impacts on the kinase activity of c-Src. Thus, whether the main role of CP is to regulate Src activity in the distal wing disc is an exciting possibility to be tested in the future (Jezowska, 2011).

This study and others have previously shown that the CP heterodimer acts as tumour suppressor through its control of Hpo pathway activity. This study now shows that in specific epithelia, loss of CP also affects cell-cell adhesion, which is a fundamental step to an epithelial-to-mesenchymal transition (EMT), triggers MMP1 expression, which degrades the basal extracellular matrix, induces cell invasion and promotes massive proliferation of cells that fail to stably retain associations with their neighbours when cell death is blocked with P35. Moreover 'undead' CP-depleted cells show ectopic N-Cad expression, whose de novo expression promotes the transition from a benign to a malignant tumour phenotype. Finally, like other tumour suppressors, loss of CP cooperates with RasV12 in tissue overgrowth. These findings argue that in some epithelia in which CP activity is affected, the appearance of a second mutation that prevents apoptotic cell death may trigger the development of aggressive tumours in humans. However, in contrast to tumour progression, which correlates with loss of overall E-Cad expression and stimulation of canonical Wnt signalling, this study observed increase DE-Cad levels and inhibition of Wg signalling in tissues knocked down for CP. Interestingly, in flies, shg-LacZ expression is also enhanced in response to ectopic expression of the two oncogenes Src42A and Yki. This suggests the interesting hypothesis that transcriptional stimulation of DE-cad is an early mechanism of tumour suppression, which would promote the elimination of deleterious cells, possibly through inhibition of Wg signalling, rather than allowing them to proliferate and form tumours. Malignant cells that become resistant to cell death may compete successfully by losing the overall E-Cad expression and upregulating mesenchymal cadherins such as N-Cad to reinforce their fitness (Jezowska, 2011).

Erect Wing facilitates context-dependent Wnt/Wingless signaling by recruiting the cell-specific Armadillo-TCF adaptor Earthbound to chromatin

During metazoan development, the Wnt/Wingless signal transduction pathway is activated repetitively to direct cell proliferation, fate specification, differentiation and apoptosis. Distinct outcomes are elicited by Wnt stimulation in different cellular contexts; however, mechanisms that confer context specificity to Wnt signaling responses remain largely unknown. Starting with an unbiased forward genetic screen in Drosophila, a novel mechanism was recently uncovered by which the cell-specific co-factor Earthbound 1 (Ebd1), and its human homolog jerky, promote interaction between the Wnt pathway transcriptional co-activators β-catenin/Armadillo and TCF to facilitate context-dependent Wnt signaling responses (Benchabane, 2011). In the same genetic screen an unanticipated requirement was found for Erect Wing (Ewg), the fly homolog of the human sequence-specific DNA-binding transcriptional activator nuclear respiratory factor 1 (NRF1), in promoting contextual regulation of Wingless signaling. Ewg and Ebd1 functionally interact with the Armadillo-TCF complex and mediate the same context-dependent Wingless signaling responses. In addition, Ewg and Ebd1 have similar cell-specific expression profiles, bind to each other directly and also associate with chromatin at shared genomic sites. Furthermore, recruitment of Ebd1 to chromatin is abolished in the absence of Ewg. These findings provide in vivo evidence that recruitment of a cell-specific co-factor complex to specific chromatin sites, coupled with its ability to facilitate Armadillo-TCF interaction and transcriptional activity, promotes contextual regulation of Wnt/Wingless signaling responses (Xin, 2011).

Genome-wide expression profiling has revealed that the vast majority of Wnt target genes are context specific, and, by contrast, globally activated target genes are quite rare. Factors that confer context-dependent target gene activation in response to activation of this widely used signal transduction pathway are of fundamental clinical importance, as they provide putative targets for therapeutic intervention; however, the identity of these factors, and their mechanism of action remain poorly understood, and have posed a daunting challenge for the Wnt signaling field (Xin, 2011).

An unbiased forward genetic approach was undertaken to identify genes that promote Wingless signal transduction. The design of the genetic screen allowed identification of any gene that impacts Wingless signaling downstream, or at the level of the Apc/Axin destruction complex, which in principle could encompass a wide range of functions. Surprisingly, however, among the relatively small number of genetic complementation groups revealed in the screen, two cell-specific chromatin-associated co-factors were identified that had not been implicated in Wingless signaling previously, but directly associate with each other and probably mediate the same context-specific Wingless-dependent developmental process. The combined activities of the two factors in this complex not only result in its recruitment to discrete chromatin sites, but also promote activity of the Armadillo-TCF transcription complex. Together, these unanticipated findings uncover a novel mechanism that facilitates context-specific regulation of Wingless signaling (Xin, 2011).

It was recently discovered that the novel Drosophila CENPB domain protein Ebd1 and its human homolog Jerky contribute to Wnt/Wingless target gene activation by enhancing β-catenin-TCF complex formation and β-catenin recruitment to chromatin (Benchabane, 2011). Since Ebd1 is expressed in a cell-restricted pattern, these findings provided evidence that context-dependent responses to Wnt/Wingless stimulation are facilitated by cell-specific co-factors that interact with both β-catenin/Armadillo and TCF to enhance complex formation and activity. However, whether Ebd1 conferred context specificity solely by functioning as a cell-specific Armadillo-TCF adaptor or also by associating with distinct chromatin sites was not known (Xin, 2011).

This study provides multiple lines of evidence consistent with the model that the putative sequence-specific DNA-binding transcriptional activator Ewg is a physical and functional partner of Ebd1 in regulating context-specific Wingless signaling, and is required for chromatin recruitment of the Ewg-Ebd1 complex. Together, these studies suggest that Ebd1 and Ewg act together in the same context-specific Wingless-dependent developmental process, interact functionally with Armadillo-TCF, are expressed in similar cell-restricted patterns, associate physically with each other and are recruited to similar chromatin sites throughout the genome. Furthermore, all recruitment of Ebd1 to chromatin is abolished in the absence of Ewg. Based on these findings, a model is proposed in which context specificity in Wingless signaling is achieved in part through association of Ewg with distinct enhancer sites, and recruitment of Ebd1 to these enhancers. In addition, previous work suggested that by binding both Armadillo and TCF, Ebd1 stabilizes the Armadillo-TCF complex (Benchabane, 2011). Together, these findings support the model that Ebd1 promotes recruitment of the Armadillo-TCF complex to enhancers containing Ewg-binding sites, thereby facilitating context specificity in Wingless signal transduction. Alternatively, Ebd1 might also function in an Ewg-independent manner by solely providing an Armadillo-TCF adaptor function that stabilizes the Armadillo-TCF complex and thereby facilitates transcriptional activation of a subset of Wingless target genes requiring relatively high Armadillo-TCF levels (Xin, 2011).

The ability to visualize endogenous Ebd1 and Ewg on larval salivary gland polytene chromosomes reveals that Ewg is essential for association of Ebd1 with chromatin, and that nearly all, if not all, chromatin sites with which Ebd1 associates are also bound by Ewg. By contrast, the association of Ewg with chromatin is not dependent on Ebd1; furthermore, not all Ewg-associated chromatin sites are bound by Ebd1, which probably reflects the broader role for Ewg in muscle and neuronal development. Together, these findings suggest that Ewg is necessary but not sufficient for recruitment of Ebd1 to chromatin, and either additional factors or specific DNA sequences are important for association of Ebd1 with chromatin. In particular, the presence of CENPB-type DNA-binding domains in Ebd1 indicates that Ebd1 may bind DNA directly, and therefore the association of Ebd1 with chromatin may require not only Ewg, but also sequence-specific Ebd1 DNA-binding sites. If so, Ewg might bind and remodel specific chromatin sites, thereby allowing Ebd1 to access nearby cognate binding sites or, alternatively, physical association with Ewg may result in a conformational change in Ebd1 that enables DNA binding. Similarly, association of Ebd1 with the Armadillo-TCF complex may facilitate association of Ebd1 and/or Armadillo-TCF with distinct chromatin sites; currently, this hypothesis could not be tested in vivo, since fixation and immunostaining conditions do not detect the indirect association of endogenous Armadillo with polytene chromosomes. Nonetheless, the extensive physical and genetic interactions between Ewg, Ebd1 and Wingless pathway transcriptional components, coupled with association of Ewg-Ebd1 with distinct chromatin sites support the model that context-specific activation of Wingless target genes is facilitated by interaction of Armadillo-TCF with cell-specific co-activator complexes at specific sites in chromatin (Xin, 2011).

The findings also suggest that Ebd1-Ewg complex function is required for only a context-specific subset of the Wingless-dependent processes that direct indirect flight muscles (IFM) development. For example, Wingless secreted from larval wing imaginal disc epithelia specifies the fate of myoblasts associated with the wing disc that are important for IFM development. In addition, Wingless signaling regulates the specification of tendon cells and the position of flight muscle attachment sites. Further evidence for Wingless signaling in IFM development was revealed by hypomorphic wingless mutants, some of which display a morphologically normal external appearance, but are nonetheless flightless. Indeed, analysis of these hypomorphic wingless mutants reveals defects in late steps of muscle differentiation that result in partial loss of IFMs (Benchabane, 2011). In addition, strong genetic interactions was found between Ewg and the Armadillo-TCF complex. Together with the genetic and physical interactions between Ewg and Ebd1, as well as previous findings that Ebd1 facilitates Wingless signaling in adult muscle development (Benchabane, 2011), the genetic interaction between Ewg and TCF provides further evidence supporting the model that Ewg and Ebd1 facilitate Wingless-dependent adult flight muscle development. However, no Ewg expression is detected in IFM precursors until pupation, suggesting that Ewg does not participate in early steps of Wingless-dependent specification of IFM myoblasts or tendon cells, but instead promotes later steps in IFM growth and differentiation. Previous studies revealed that context specificity in Wnt signaling responses is facilitated by either crosstalk between signal transduction pathways or replacement of TCF with tissue-specific DNA-binding transcription co-factors that associate with β-catenin. Thus, these findings suggest a novel mechanism in which a sequence-specific DNA-binding transcription factor recruits a cell-specific Armadillo-TCF adaptor to modulate context-specific Wingless signaling responses (Xin, 2011).

Ewg activity in neuronal differentiation can be functionally replaced by its human homolog nuclear respiratory factor 1 (NRF1) (Haussmann, 2008), a sequence-specific DNA-binding transcription factor important for activation of genes required in mitochondrial biogenesis and respiration, and for regulation of histone gene expression. Functional analysis of vertebrate NRF1 during development has been hampered by early lethality, as mouse Nrf1-null embryos die near the time of uterine implantation. Fortuitously, however, inactivation of the zebrafish NRF1 homolog, not really finished (nrf), has improved understanding of NRF1 function during vertebrate development; fish nrf-null mutants survive up to 14 days of development with the primary developmental defect restricted to markedly smaller brains and retinas. The tissue-restricted nature of the fish nrf-null mutant phenotype was unexpected, given the broader expression of the fish nrf gene and the global roles for NRF1 in mitochondrial and histone function revealed by in vitro studies. However, these in vivo analyses revealed that fish Nrf, like fly Ewg, acts in specific tissue-restricted developmental processes. Of note, Wnt signaling is required for patterning of the mouse central nervous system. Similarly, Wnt signaling is also crucial for brain and eye development in zebrafish, thus raising the possibility that, like Ewg, vertebrate Nrf may also facilitate a context-specific subset of Wnt-dependent developmental processes (Xin, 2011).

Jerky/Earthbound facilitates cell-specific Wnt/Wingless signalling by modulating beta-catenin-TCF activity

Wnt/Wingless signal transduction directs fundamental developmental processes, and upon hyperactivation triggers colorectal adenoma/carcinoma formation. Responses to Wnt stimulation are cell specific and diverse; yet, how cell context modulates Wnt signalling outcome remains obscure. In a Drosophila genetic screen for components that promote Wingless signalling, Earthbound 1 (Ebd1), a novel member in a protein family containing Centromere Binding Protein B (CENPB)-type DNA binding domains, was identified. Ebd1 is expressed in only a subset of Wingless responsive cell types, and is required for only a limited number of Wingless-dependent processes. In addition, Ebd1 shares sequence similarity and can be functionally replaced with the human CENPB domain protein Jerky, previously implicated in juvenile myoclonic epilepsy development. Both Jerky and Ebd1 interact directly with the Wnt/Wingless pathway transcriptional co-activators β-catenin/Armadillo and T-cell factor (TCF). In colon carcinoma cells, Jerky facilitates Wnt signalling by promoting association of β-catenin with TCF and recruitment of β-catenin to chromatin. These findings indicate that tissue-restricted transcriptional co-activators facilitate cell-specific Wnt/Wingless signalling responses by modulating β-catenin-TCF activity (Benchabane, 2011).

The absence of Ebd1 expression in many tissues is likely critical for proper development, as attempts to ectopically express Ebd1 using several tissue-specific promoters resulted in cell and/or organismal lethality. Further, within Ebd1-expressing cells, some Wingless-dependent developmental processes proceed normally upon Ebd loss, suggesting that only a subset of Wingless target genes is dependent on Ebd. Specifically, flight muscle loss resulting from Wingless pathway inactivation is more severe than that resulting from Ebd loss, even upon simultaneous elimination of all five Drosophila CENPB proteins. Together, these data provide genetic evidence that cell-specific responses to Wingless signalling are mediated in part by tissue-specific co-factors that modulate activity of the Armadillo/TCF transcription complex (Benchabane, 2011).

In vivo epistasis experiments support the model that Ebd1 functions at the level of the Armadillo-TCF transcriptional complex, and in vitro data indicate that Jerky and Ebd1 contribute to Wnt/Wingless target genes activation by enhancing β-catenin-TCF complex formation and β-catenin recruitment to chromatin. Thus, Jerky and Ebd likely serve an adaptor function similar to that previously documented for human transducin β-like protein 1 (TBL-1). However, by contrast with TBL-1, which promotes activation of all Wnt target genes analysed, only certain Wingless-dependent developmental events (and by extension, associated target genes) require Ebd. How does an Arm-TCF adaptor protein such as Ebd1 promote the transcription of only a subset of Wingless target genes? The data are consistent with two distinct models. Ebd may act solely as an Arm-TCF adaptor, such that Wingless target genes requiring relatively high Arm-TCF complex levels may be dependent on Ebd for activation, whereas activation of target genes requiring lower Arm-TCF levels may be Ebd independent. Alternatively, given the presence of its CENPB DNA binding domains, Ebd1 may provide two functions -- to act as an Armadillo-TCF adaptor and to also associate with cognate sites in enhancer elements. Thus, Ebd1 DNA-binding specificity may also contribute directly to Wingless target gene selection. The existence of cell-specific factors such as Ebd that modulate Arm-TCF activity was presaged by previous studies, which indicated that binding of DNA by TCF is a regulated process; not all TCF-binding site clusters bind TCF in vivo, and TCF binding at a particular locus varies between cell types, suggesting that optimal TCF binding to cognate sites likely requires recruitment by cell-specific transcription co-factors (Benchabane, 2011).

Taken together with previous studies, these findings reveal that modulation of Wnt/Wingless responses is facilitated by several distinct modes of transcriptional regulation. For example, recruitment of β-catenin to chromatin can occur in a TCF-independent manner, through association of β-catenin with sequence-specific DNA-binding co-activators that extend the range of Wnt/β-catenin target genes. In addition, cooperative signalling between the Wnt/Wingless pathway and other signal transduction pathways also modulates target gene specificity. For instance, molecular cross-talk at the transcriptional level between the mammalian Wnt pathway and the TGFβ or JNK pathway is mediated by interaction of Lef-1 with Smads and TCF-4 with c-Jun, respectively. Signal transduction pathway cross-talk promotes Wingless target gene activation in Drosophila also. Further, some modulators of Wingless pathway activity, such as Lines and Split ends/Spenito, do not associate with Armadillo or TCF directly, but instead act downstream or in parallel with Armadillo-TCF to specify cell fate. The current findings reveal that context-dependent responses to Wnt/Wingless stimulation are also facilitated by cell-specific co-factors that interact with both β-catenin and TCF to enhance their activity (Benchabane, 2011).

The human CENPB domain protein Jerky shares functional homology with Ebd1. Mouse Jerky was originally identified as a neuronally enriched protein in a murine epilepsy model; heterozygous inactivation of jerky results in recurrent generalized seizures, whereas homozygous jerky inactivation also results in growth and fertility defects. The subsequent mapping of human Jerky (JH8/JRK) to chromosome 8p24 revealed linkage to a susceptibility locus for juvenile myoclonic epilepsy and childhood absence epilepsy. Recently, a genome-wide RNA interference screen identified human Jerky as an activator of a TCF-dependent reporter as well as endogenous Wnt/β-catenin target genes in colon carcinoma cells. Jerky was found to facilitate the activation of a subset, but not all endogenous Wnt/β-catenin target genes examined, paralleling the current in vivo analysis of Ebd function. These findings indicate that Jerky facilitates Wnt pathway-dependent transcription by stabilizing the β-catenin-TCF complex and promoting recruitment of β-catenin to chromatin. The findings also raise the possibility that attenuation of Wnt transduction underlies generalized seizures, as well as growth and fertility defects in mice with reduced Jerky activity, and may contribute to development of certain types of childhood epilepsy (Benchabane, 2011).

Hipk proteins dually regulate Wnt/Wingless signal transduction

The Wnt/Wingless (Wg) pathway is an evolutionarily conserved signaling system that is used reiteratively, both spatially and temporally, to control the development of multicellular animals. The stability of cytoplasmic β-catenin/Armadillo, the transcriptional effector of the pathway, is controlled by sequential N-terminal phosphorylation and ubiquitination that targets it for proteasome-mediated degradation. Orthologous members of the Homeodomain-interacting protein kinase family from Drosophila to vertebrates have been implicated in the regulation of Wnt/Wingless signaling. In Drosophila, as a consequence of Hipk activity, cells accumulate stabilized Armadillo that directs the expression of Wg-specific target genes. Hipk promotes the stabilization of Armadillo by inhibiting its ubiquitination (and hence subsequent degradation) by the SCF(Slimb) E3 ubiquitin ligase complex. Vertebrate Hipk2 impedes β-catenin ubiquitination to promote its stability and the Wnt signal in a mechanism that is functionally conserved. Moreover, this study describes that Hipk proteins have a role independent of their effect on β-catenin/Armadillo stability to enhance Wnt/Wingless signaling (Verheyen, 2012).

In summary, Hipk proteins play multiple important roles during developmental signaling events, and have the capacity to simultaneously impact the outcomes of both Wg and Hh signal transduction. The finding that Hipk proteins act by blocking Slimb/β-TrCP-mediated ubiquitination of substrates suggests that additional targets may also be affected through the action of these kinases. Additionally a nuclear role for Hipk was observed that is independent of its role in stabilizing the Wg pathway effector Arm. The data suggest that Hipk can enhance the transcriptional activation by the Arm/TCF complex. This role thought to be independent of its role in blocking substrate ubiquitination (Verheyen, 2012).

Multiple roles have been described for Hipk family members in regulation of Wnt/Wg pathway activity. Such findings are reminiscent of the work performed to decipher the roles of several other kinases in the Wnt pathway. Both GSK3 and CK1 have distinct roles at different steps in the transduction of Wnt signaling and act to either promote or inhibit pathway activity and as a result gene expression. The finding that Hipk-stabilized Arm is phosphorylated by GSK3 and CK1 reveals a specific point of action in the targeting and destruction of Arm/β- catenin. Future work should reveal whether the stabilized N-terminally phosphorylated Arm is associated with the destruction complex, in effect blocked from being transferred to the proteasome as a result of Hipk acting on Slimb. The regulation of β-catenin stability and activity has important implications for normal growth and patterning, tissue homeostasis and the development of cancer (Verheyen, 2012).

Kinesin-II recruits Armadillo and Dishevelled for Wingless signaling in Drosophila

Wingless (Wg)/Wnt signaling is fundamental in metazoan development. Armadillo (Arm)/beta-catenin and Dishevelled (Dsh) are key components of Wnt signal transduction. Recent studies suggest that intracellular trafficking of Wnt signaling components is important, but underlying mechanisms are not well known. This study shows that Klp64D, the Drosophila homolog of Kif3A kinesin II subunit, is required for Wg signaling by regulating Arm during wing development. Mutations in klp64D or RNAi cause wing notching and loss of Wg target gene expression. The wing notching phenotype by Klp64D knockdown is suppressed by activated Arm but not by Dsh, suggesting that Klp64D is required for Arm function. Furthermore, klp64D and arm mutants show synergistic genetic interaction. Consistent with this genetic interaction, Klp64D directly binds to the Arm repeat domain of Arm and can recruit Dsh in the presence of Arm. Overexpression of Klp64D mutated in the motor domain causes dominant wing notching, indicating the importance of the motor activity. Klp64D shows subcellular localization to intracellular vesicles overlapping with Arm and Dsh. In klp64D mutants, Arm is abnormally accumulated in vesicular structures including Golgi, suggesting that intracellular trafficking of Arm is affected. Human KIF3A can also bind β-catenin and rescue klp64D RNAi phenotypes. Taken together, it is proposed that Klp64D is essential for Wg signaling by trafficking of Arm via the formation of a conserved complex with Arm (Vuong, 2914).

Protection of Armadillo/beta-Catenin by Armless, a novel positive regulator of Wingless signaling

The Wingless (Wg/Wnt) signaling pathway is essential for metazoan development, where it is central to tissue growth and cellular differentiation. Deregulated Wg pathway activation underlies severe developmental abnormalities, as well as carcinogenesis. Armadillo/β-Catenin plays a key role in the Wg transduction cascade; its cytoplasmic and nuclear levels directly determine the output activity of Wg signaling and are thus tightly controlled. In all current models, once Arm is targeted for degradation by the Arm/β-Catenin destruction complex, its fate is viewed as set. This study identified a novel Wg/Wnt pathway component, Armless (Als; CG5469) that is required for Wg target gene expression in a cell-autonomous manner. Genetic and biochemical analyses showed that Als functions downstream of the destruction complex, at the level of the SCF/Slimb/βTRCP E3 Ub ligase. In the absence of Als, Arm levels are severely reduced. Biochemical and in vivo studies showed that Als interacts directly with Ter94, an AAA ATPase known to associate with E3 ligases and to drive protein turnover. It is suggested that Als antagonizes Ter94's positive effect on E3 ligase function, and it is proposed that Als promotes Wg signaling by rescuing Arm from proteolytic degradation, spotlighting an unexpected step where the Wg pathway signal is modulated (Reim, 2014).

The wingless (wg) gene was found nearly forty years ago with the characterization of a Drosophila mutant without wings. The gene encodes a secreted glycoprotein, the founding member of the Wnt family of signaling proteins. In the decades following its discovery, Wg/Wnt signaling has been shown to be essential during embryogenesis. Indeed, it is important throughout an organism's life, controlling also the homeostasis of different organs, for example, regeneration of epithelial cells in the intestine - the aberrant behavior of these cells in cancer is caused by constitutive Wg/Wnt signaling, which is consequently a key focus of medical and translational research (Reim, 2014).

The relay of the Wg signal is controlled at different levels. However, the pivotal step is the regulation of the levels of Armadillo (Arm)/β-Catenin, the key transducer of the Wg/Wnt pathway. A multiprotein complex consisting of the scaffold proteins Axin and APC and the kinases Shaggy/GSK3β and Casein kinase I (CKI) recruits and phosphorylates Arm/β-Catenin. This marks Arm/β-Catenin for ubiquitination by the SCF/Slimb/βTRCP E3 ubiquitin ligase and subsequent degradation by the ubiquitin-proteasome system (UPS). When Wg/Wnt binds its receptors at the cell membrane, degradation of Arm/β-Catenin is prevented, presumably by protein interactions that lead to the dissociation of the E3 ubiquitin ligase from the Arm/β-Catenin destruction complex. As a consequence, Arm/β-Catenin translocates into the nucleus, where it adopts its role as a transcriptional effector of Wg/Wnt signaling. Although this step is crucial, and is a potential point of regulation, little is known about the players involved in the processing of Arm/β-Catenin and its ultimate degradation (Reim, 2014).

In a genome-wide RNA interference (RNAi) screen Armless (Als) was isolated as a regulator of proximodistal growth of Drosophila limbs, and has been shown in subsequent analyses to exert its function in the Wg pathway. Detailed genetic studies demonstrate that Als acts downstream of the destruction complex, at the level of the SCF/Slimb/βTRCP E3 Ub ligase. Cells depleted for Als exhibit strongly reduced Arm protein levels. Importantly, the activity of a constitutively active form of Arm, which cannot be phosphorylated and hence escapes ubiquitination and proteasomal degradation, is insensitive to depletion of Als. Using immunopurification and mass spectrometry analysis this study found that Ter94 interacts with Als. Ter94 is an AAA ATPase associated with protein turnover and proteasomal degradation. In sum, these data suggest that Als acts downstream of the Arm/β-Catenin destruction complex to positively regulate Arm protein levels, possibly by rescuing Arm from ubiquitination via Slimb. The human ortholog of Als, UBXN6, can substitute for Als in Drosophila, and Wnt target gene expression was impaired upon knock-down of UBXN6 in HEK-293 cells. It is thus infered that Als and UBXN6 represent regulators of a conserved mechanism that ensures appropriate levels of Armadillo/β-Catenin by antagonizing its entry into the UPS (Reim, 2014).

A prevalent mechanism for controlling information flow in signaling pathways is the alteration of the protein levels of key components. In the Wg/Wnt pathway, the Arm/β-Catenin destruction complex targets Arm/β-Catenin for ubiquitination by the SCF/Slimb/βTRCP E3 Ub ligase, resulting in proteasomal degradation and low cytoplasmic levels of Arm/β-Catenin in the Wnt pathway off state. If the pathway is turned on, Slimb-mediated ubiquitination is prevented, thus rescuing Arm from its proteasomal fate and causing a concomitant increase in Arm protein levels. This study describes Als as a new component of this control system; Als was found to be required to prevent the degradation of Arm/β-Catenin (Reim, 2014).

This study has identified als in a genome-wide in vivo RNAi screen in Drosophila. Because no EMS- or P-element-induced null allele was isolated, and because another gene overlaps with als, particularly thorough evidence validating als gene function was obtained. (1) The als phenotypes could be reproduced by nine different UAS-RNAi transgenes encoding independent RNA target sites. Together with an extended off target analysis, unintentional RNAi was ruled out as a cause for the als phenotypes. (2) RNAi-mediated inhibition of als expression was ascertained by monitoring als mRNA expression via real-time PCR and antisense mRNA in situ hybridization. (3) Expression of Als with different RNAi-insensitive rescue transgenes, as well as with its human ortholog UBXN6, rescued als phenotypes (Reim, 2014).

These analyses show that als encodes an essential positive Wg signaling component. This conclusion is based on the following evidence. als depletion caused wings with notched wing margins and loss of sensory bristles, which is characteristic of impaired Wg signaling. The distal wing region is most sensitive to als levels, as is the case for other positive components of Wg signaling. In agreement with this, increased als expression was found in the central wing pouch, at least in earlier L3 larval stages. Stimulation of the Wg pathway in wing imaginal discs or Kc-167 cells caused higher als expression, suggesting that als can be positively controlled by Wg signaling. However, Als levels must be precisely controlled since already mild overexpression of UAS-als elicits a dominant-negative effect on Wg signaling. The function of als for Wg signaling is not restricted to the wing: also in other tissues, such as the thorax, eyes, legs, and the embryo, als phenotypes are identical to those seen when Wg signaling is disturbed. Also in human HEK-293 cells UBXN6/UBXD1, the ortholog of Als, was found to be required for Wnt signaling, and human UBXN6 largely rescues the als phenotypes in Drosophila, which suggests their functional conservation. Depletion of als also enhanced Wg-sensitized phenotypes, further supporting the notion that its product is a Wg pathway component. Moreover, the expression of positively regulated Wg target genes is reduced or abolished upon loss of als function, while Wg-repressed target gene expression is ectopically activated. Importantly, while interfering with als function suppressed Wg signaling, it did not affect other pathways, such as Notch and Hh, Jak/Stat, or EGFR signaling. However, it cannot be ruled out that als is not required in another pathway in a different biological context. In humans, UBXN6 is reported to play a role in diverse scenarios: for example, it was shown to play a role in Caveolin turnover in human osteosarcoma U2OS cells. This might indicate a broader role of UBXN6 in mammalians (Reim, 2014).

The data show that Als regulates Armadillo protein levels. Based on epistasis experiments, Als acts downstream of Shaggy/GSK3β and upstream of the SCF/Slimb/βTRCP E3 Ub ligase, which is known to ubiquitinate Arm, a prerequisite for proteasomal degradation. Consistent with this, the degradation-resistant form of Arm could completely bypass the requirement for als, in contrast to the wild-type form of Arm. This suggests that proteasomal degradation acts downstream of als; however, this cannot be taken as an unambiguous proof. Importantly, depletion of ubiquitin and overexpression of CSN6, a negative regulator of SCF/Slimb/βTRCP E3 Ub ligase, could ameliorate the als phenotype (as well as phenotypes based on the overexpression of Axin or Shaggy, which overactivate the destruction complex, thus resulting in enhanced Arm degradation). In contrast, altering these factors did not ameliorate the Lgs phenotype, which is caused by interfering more downstream in the Wg pathway. These findings suggest that als works upstream of proteasomal degradation. A further informative experiment was monitoring Wg pathway components with respect to protein levels: Arr, Fz, Axin, APC, Sgg, and Arm. The only change in the absence of Als function was Arm: its levels were strongly reduced upon als depletion. The effects on Arm levels could be due either to a direct effect on Arm or to an indirect effect on a negative component. Importantly, the rate-limiting factor Axin as well as other key negative components of the Arm/β-Catenin destruction complex were unaltered uponals depletion (Reim, 2014).

Some further mechanistic insight was obtained with the finding that Ter94 interacts in vitro and in vivo with Als. Interestingly, Als-Ter94 was found to localize at the cell cortex, as was similarly observed for the Arm/β-Catenin destruction complex. The studies are consistent with earlier work that showed that the human ortholog of Ter94, p97, interacts with UBXN6. Ter94/p97/Cdc48 is a conserved and highly abundant AAA ATPase that was found to associate with SCF/Slimb/βTRCP E3 Ub ligases or proteasomal shuttle factors to mediate UPS-mediated protein degradation. Specifications of the diverse activities of Ter94/p97 and the fate of its substrates are mainly exerted by UBX domain protein co-factors, which eventually either promote or hinder p97's function in protein turnover; an example of the latter involves the dissociation of the SCF/Slimb/βTRCP E3 Ub ligase complex, eventually leading to its inactivation. Interestingly, it was recently reported that inactivation of the E3 ligase complex upon Wnt signaling is achieved by its dissociation from the destruction complex. Based on the current experiments and what is known about Ter94/p97, a possible mechanism is suggested that Als antagonizes Ter94's positive effect on E3 ligase function, thereby rescuing Arm levels. No increased protein levels were observed of Slimb, Axin, Shaggy, or APC in this analyses; thus, the results favor a model in which Als antagonizes Ter94 to hinder the transfer of Arm to the proteasome by interfering with the SCF/Slimb/βTRCP E3 Ub ligase function or its assembly. Importantly, no interaction was found between Arm and Als. This is consistent with the finding that UBX domain family members lacking an UBA domain, such as UBXN6/Als, do not directly interact with substrate proteins, but are necessary for the activity or fate of the Ter94/p97 (Reim, 2014).

Interestingly, another study found that ter94 depletion affected the partial proteolysis of Ci. However, that study observed neither any typical consequence of disturbed Hh signaling per se (i.e., no alteration of Hh target gene expression in genes such as ptc) nor any phenotypical consequence upon overexpression of a dominant negative form of Ter94 (i.e., aberrant wing patterning and growth typical for Hh signaling). This is consistent with the current data that neither Ci target expression nor Hh signaling was affected upon als or ter94 depletion (Reim, 2014).

p97/Ter94 is known as a highly pleiotropic AAA ATPase associated with many cellular functions. Further, p97/Ter94 acts in multifaceted and large protein–protein complexes, and it is its regulatory co-factors, including UBX domain proteins, that render p97/Ter94 specific for a certain task in a particular cellular context. For example, p47/Shp1 is a co-factor of p97/Ter94 that blocks other co-factors from Ter94 binding. Interestingly, in Kc-167 cell mass spectroscopy experiments, this study found p47 in Ter94/Als protein complexes, but only in the absence of Wg stimulation. On the other hand, als transcript and Als protein levels were elevated upon Wg signaling. These findings suggest a dynamic regulation of the Ter94 complex upon signaling inputs. The identification and functional analysis of all key components of the Als-Ter94 complex will be needed to obtain a refined insight into Als-Ter94's molecular mechanism (Reim, 2014).

Critically, this work spotlights an underappreciated facet in the control of the output of the entire canonical Wg/Wnt pathway - how Arm/β-Catenin is handed over to the proteasome— and the potential for regulating this step; this works also indicates that this step, in contrast to the conventional wisdom, is tunable. The identification and characterization of the UBX protein Als as a positive regulator of Wg/Wnt signaling contributes to this layer of pathway control (Reim, 2014).

back to armadillo Protein Interactions part 1/2

armadillo continued: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

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