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Protein Interactions (part 1/2)

A series of Armadillo mutants were generated and examined and expressed in Drosophila embryos. Although DE-cadherin and alpha-catenin bind to Armadillo independent of one another, binding of both is required for the function of adherens junctions, that is, mutations that block alpha-Catenin and Cadherin-binding block junction formation. E-cadherin appears to bind in the Armadillo repeat region; this region is required for localization to the adherins junction. alpha-Catenin binding is eliminated by deletion of the region between the N-terminus and the repeats. There are two separate regions of Armadillo critical for Wingless signaling. Mutant Arm proteins deleted in the central repeats or lacking the N-terminal alpha-catenin-binding site all localize prominently at higher levels to cell nuclei in cells responding to Wingless signal. Some of the proteins deleted for parts of the central repeat region require Wingless signal to accumulate in the nucleus while others do not. Endogenous Armadillo normally accumulates in the nucleus and it may act there in transducing Wingless signal. Armadillo's roles in adherens junctions and Wingless signaling are independent. Mutant proteins lacking the domain between the C-terminal region and the Arm repeats retain function in adherens junctions but lack function in Wingless signal transduction. Phosphorylation changes in the Arm protein are detected only in mutant proteins with deletions in the Arm repeats region. It is concluded that the region essential for alpha-catenin binding is not essential for Wingless signaling. The Arm repeats region are essential both for Adherins junction function and for Wingless effector function (Orsulic, 1996).

DE-cadherin, the transmembrane cell adhesion protein and component of the adherins junction, associates with alpha-Catenin and beta-Catenin (Armadillo), and is protected from trypsin digestion only in the presence of Ca2+, as is the case for many of classic cadherins. Transfection of S2 cells with the DE-cadherin cDNA enhances their Ca(2+)-dependent cell aggregation. Antibodies to this molecule inhibited aggregation of not only the transfectants but also early embryonic cells (Oda, 1994).

Immunoprecipitation of Cadherin-N shows that it binds not only to alpha-Catenin but also to beta-Catenin/Armadillo (Arm). Alternative splicing generates two Arm isoforms: the 105 kDa ubiquitous form and the 82 kDa neural form (Loureiro and Peifer, personal communication to Iwai, 1997). Cadherin-N associates predominantly with the 82 kDa Arm, whereas Shotgun preferentially binds to the 105 kDa isoform. Transfection of Drosophila cultured cells with Cadherin-N cDNA induces cells to form aggregates. The activity of Cadherin-N is comparable to that of Shotgun (Iwai, 1997).

Cadherin-N seems to be the major cadherin that assembles catenins in axons. Mutants were isolated that produce only a small amount of Cadherin-N. Alpha-Catenin expression was compared between these mutant and wild-type embryos. Axonal expression of alpha-Catenin is greatly down-regulated in mutants although neuronal cell bodies retain a low level of alpha-catenin signals. In contrast, even in these mutants, alpha-Catenin is normally present in the midline glial cells and epithelia that synthesize Shotgun. The level of 82 kDa Arm, as opposed to that of the 105 kDa ubiquitous form, is preferentially reduced in the mutants (Iwai, 1997).

A Drosophila homolog of the human tumor suppressor gene adenomatous polyposis coli (APC) contains 2416-amino acid proteins, with seven complete armadillo repeats, one ß-catenin binding site and up to 7 copies of a 20-amino acid repeat (involved in ß catenin binding). Drosophila-APC, like its human counterpart, also contains a basic domain. Lacking in the Drosophila protein is a region homologous to a human protein region known to bind Drosophila Discs large. Expression of the Drosophila APC domain homologous to the region required for ß-catenin down-regulation results in a reduction in the concentration of cytoplasmic ß-catenin in a mammalian cell line. This same region binds to the Armadillo protein. Drosophila-APC expression is low, if detectable at all, during stages when ARM protein accumulates in a stripe pattern in the epidermis of fly embryos, suggesting that D-APC does not play a role in wingless signaling, which is involved in the establishment of segment polarity. Removing zygotic Drosophila-APC expression does not alter ARM protein distribution. High levels of Drosophila-APC expression in the central nervous system suggest that the protein plays a role in central nervous system formation. D-APC localizes in axon fiber tracts and motor neurons. A possible interaction between D-APC and ARM is supported by the late expression of both proteins in the CNS The high level of D-APC in the fly's CNS parallels similar observations in the rat. In both organisms, the protein appears to be restricted to postmitotic neurons. Mutants have thinner and less developed longitudinal fiber tracts. Lack of wingless expression in late CNS development suggests that WG and D-APC might have distinct functions in CNS development (Hayasi, 1997).

Wnt/Wingless directs many cell fates during development. Wnt/Wingless signaling increases the amount of beta-catenin/Armadillo, which in turn activates gene transcription. The Drosophila protein Axin is shown to interact with Armadillo and Drosophila APC. D-Axin was identified in a yeast two-hybrid screen for proteins that bind the Armadillo repeat domain of Arm. d-axin codes for a protein of 743 amino acids. A region near its N-terminus shows similarity to the regulator of G protein signaling (RGS domain), whereas its C-terminus contains a region homologous to a conserved sequence near the N-terminus of Dishevelled. Thus D-Axin has a domain structure very similar to that of proteins of the mammalian Axin family. Unlike mammalian Axin family members, which bind to GSK-3beta, D-Axin does not bind to the homologous protein Shaggy/Zeste white3. d-axin is expressed maternally and is ubiquitously expressed during development. Embryos devoid of maternal and zygotic d-axin have completely naked ventral cuticle, lacking all denticles (Hamada, 1999).

During wing disc development, Wg signaling is induced along the dorsoventral compartment boundary in the wing imaginal disc. Arm accumulates in the cytoplasm, associates with its partner Pangolin, and activates expression of target genes such as Distal-less. Mutation of d-axin results in the accumulation of cytoplasmic Armadillo and results in elevation of Distal-less. Ectopic expression of d-axin inhibits Wingless signaling. Hence, D-Axin negatively regulates Wingless signaling by down-regulating the level of Armadillo. It is speculated that the Axin family of proteins functions to establish a threshold to prevent premature signaling events caused by Wg/Wnt and to restrict areas that are capable of responding to Wg/Wnt. These results establish the importance of the Axin family of proteins in Wnt/Wingless signaling in Drosophila (Hamada, 1999).

Drosophila Armadillo and its vertebrate homolog beta-catenin are key effectors of Wingless/Wnt signaling. In the current model, Wingless/Wnt signal stabilizes Armadillo/beta-catenin, that then accumulates in nuclei and binds TCF/LEF family proteins, forming bipartite transcription factors which activate transcription of Wingless/Wnt responsive genes. This model was recently challenged. Overexpression in Xenopus of membrane-tethered beta-catenin or its paralog plakoglobin activates Wnt signaling, suggesting that nuclear localization of Armadillo/beta-catenin is not essential for signaling. Tethered plakoglobin or beta-catenin might signal on their own or might act indirectly by elevating levels of endogenous beta-catenin. These hypotheses were tested in Drosophila by removing endogenous Armadillo. A series of mutant Armadillo proteins with altered intracellular localizations were generated, and these were expressed in wild-type and armadillo mutant backgrounds. Membrane-tethered Armadillo cannot signal on its own; however it can function in adherens junctions. Mutant forms of Armadillo were generated carrying either heterologous nuclear localization or nuclear export signals. Although these signals alter the subcellular localization of Arm when overexpressed in Xenopus, in Drosophila they have little effect on localization and only subtle effects on signaling. This supports a model in which Armadillo’s nuclear localization is key for signaling, but in which Armadillo intracellular localization is controlled by the availability and affinity of its binding partners (Cox, 1999).

Data in vivo suggest that among Arm’s known partners, cadherins have the highest affinity, with APC and dTCF (Pangolin) having lower and lowest affinities, respectively. Thus, in embryos with reduced levels of Arm, the remaining Arm is exclusively associated with cadherins, as assayed by immunolocalization and by function. About 70% of cellular Arm is cadherin-associated. When cadherin binding sites are saturated, excess Arm binds to APC/Axin, leading to its destruction and thus preventing accumulation of free Arm. While APC levels, at least in mammalian cells, are low, relative to the total pool of beta catenin, Arm bound to APC is rapidly targeted for destruction, thus opening the way for the binding of additional Arm. Normally the destruction machinery can not only dispose of all non-junctional Arm, but its resources will not even be fully employed, since Arm synthesis can be increased several-fold without biological consequences. However, when the destruction machinery is inactivated either by Wg signal or mutation, Arm is synthesized but not destroyed, and thus levels of Arm rise. APC can bind Arm but in all probability, the APC is rapidly saturated, allowing accumulation of sufficient Arm to allow dTCF to effectively compete for binding. DE-cadherin, dAPC, dTCF and any other possible unknown partners together account for virtually all the Arm in a normal embryo; little if any free Arm is present. This model helps explain the differences in localization of the Armadillo attached to a nuclear localization sequence (Arm-NLS) and Armadillo attached to a nuclear export signal (Arm-NES) in flies and frogs. In Xenopus, added NLS or NES signals dramatically altered Arm’s intracellular distribution as expected, while in Drosophila the distribution of wild type Armadillo, Arm-NLS and Arm-NES are indistinguishable. It is proposed that this reflects differences in the level of expression. In flies, mutant Arm accumulates at near wild-type levels, so its binding partners can accommodate the additional protein. Arm bound to cadherin at the plasma membrane is unavailable for nuclear import; likewise Arm in a complex with dTCF is not available for export. Thus Arm-NLS and Arm-NES localization is primarily determined by their binding partners, resulting in a near normal localization. In contrast, Arm-NLS and Arm-NES expression levels in Xenopus likely exceed those of either endogenous beta-catenin or its binding partners. Free Arm is thus accessible to the nuclear import and export machinery, allowing alteration of its localization. Given this, is nuclear localization of Arm a regulated step in Wg signaling in normal cells? The fact that a subset of cells accumulate cytoplasmic but not nuclear Arm suggests that nuclear import may be regulated. In the simplest situation, addition of an NLS ought to promote Arm nuclear accumulation and trigger signaling, while addition of an NES should antagonize signaling. However, heterologous targeting signals have only subtle effects on signaling. Arm-NES signals in the same fashion as does Arm-WT, while only a subset of the Arm-NLS lines are activated for signaling. In the case of Arm-NLS: in cells in which the destruction machinery is on, no free Arm is available for nuclear import or export. In cells with intermediate levels of Wg signaling, the destruction machinery may be slowed, allowing accumulation of cytoplasmic Arm in complex with APC, but not to sufficient levels to saturate APC and allow nuclear import. Only when signaling is fully activated would sufficient free Arm accumulate for nuclear import. Addition of an NLS would thus only alter the balance in cells near the signaling threshold. Further, if nuclear Arm is bound to dTCF, it may be inaccessible to the nuclear export machinery. The mechanisms by which Arm/beta-catenin enters nuclei remain unclear; dTCF-dependent and independent pathways may exist. The recent observation that beta Catenin may mediate its own nuclear transport, independent of importins, further complicates the issue. Additional levels of regulation may occur, beyond the simple regulation of Arm/beta Catenin stability (Cox, 1999 and references).

Unlike Armadillo RNA, Armadillo protein accumulates non-uniformly in different cells of each embryonic segment. Cells alter their intracellular distribution of Armadillo in response to Wingless signal, accumulating increased levels of cytoplasmic Armadillo relative to those of membrane-associated protein. Levels of cytoplasmic Armadillo are also regulated by shaggy/ Zeste-White 3 kinase (Peifer, 1994a).

Armadillo’s level of phosphorylation varies both during embryonic development and from tissue to tissue. Phosphorylation occurs on both serine or threonine and tyrosine residues. Wingless signal negatively regulates Armadillo phosphorylation, while the segment polarity gene product Zeste-white 3, a serine/threonine protein kinase, promotes Armadillo phosphorylation (Peifer, 1994b).

The extracellular signals encoded by the Wnt family of genes regulate growth and differentiation in several developmental processes in both vertebrates and invertebrates. Genetic studies of the signaling pathway of the Drosophila Wnt homolog, Wingless, have identified a number of genes, including zeste white 3, that function to transduce the Wingless signal. zeste white 3 encodes a serine/threonine kinase. zw3 is expressed maternally and uniformally in the early embryo. It has been proposed that the Wingless signal is mediated by repression of this kinase activity. This hypothesis was tested by overexpressing zeste white 3 in a tissue-specific fashion using the UAS/GAL4 binary expression system. The wild-type zw3 cDNA was placed under transcriptional control of the yeast GAL4 upstream activating sequence (UAS). UAS-zw3 flies were mated to flies that express the yeast transcriptional activator GAL4 in either a cell- or tissue-specific fashion to drive chronic expression of zw3. Elevated levels of zeste white 3 in the ectoderm and mesoderm result in phenotypes that resemble a loss of wingless. Overexpression of zeste white 3 in the mesoderm disrupts several Wingless-dependent processes, including the specification of a unique cell type in the larval midgut (the copper cell), the formation of the second midgut constriction, and the expression of Wingless target genes Ultrabithorax and decapentaplegic in the mesoderm, and labial in the endoderm. Interstitial cells normally found interspersed with the copper cells are still present. This loss of copper cells is similar to the phenotypes observed due to a loss of labial expression or wg expression, both required for the specification of the copper cells. The second midgut constriction is dependent on Wg signaling; in wg, dishevelled, or armadillo mutant embryos, this constriction does not form. Interestingly, in zw3 mutant embryos the second midgut constriction does form, but it is abnomal, appearing to have multiple folds. Zeste white 3 regulates the stability of Armadillo, which is essential for transducing the Wingless signal to the nucleus. zeste white 3 overexpression blocks Wingless signaling through the modulation of Armadillo since expression of a constitutively active form of Armadillo, which is independent of Zeste white 3 regulation, is epistatic to overexpression of zeste white 3 (Seitz, 1998).

Wingless signaling generates a hyperphosphorylated form of Dishevelled, which is associated with a membrane fraction. Overexpressed DSH becomes hyperphosphorylated in the absence of extracellular WG and increases levels of the Armadillo protein, thereby mimicking the WG signal (Yanagawa, 1995).

Armadillo is required autonomously and continuously to mediate the response of wing cells to Wingless-Secreting cells located at a distance. Clones of arm mutant cells were generated in wing discs. These cells stop dividing and either die or are actively eliminated from the disc epithelium. When stained for either VG or DLL expression 36 hours after mitotic recombination is induced, none of the cells within such clones express either protein (Zecca, 1996).

Direct wg autoregulation differs from wg signaling to adjacent cells in the importance of fused, smoothened and cubitus interruptus relative to zw3 and armadillo. wg autoregulation during this early hh-dependent phase differs from later wg autoregulation by lack of gooseberry participation (Hooper, 1994).

The murine transcription factor lymphocyte enhancer binding factor 1 (LEF-1) recognizes a minimal wingless response sequence in the midgut enhancer of Ultrabithorax. This visceral mesoderm enhancer, located 2.9 kb from the Ubx start site contains adjacent elements that respond to wg and dpp signaling. The DPP response sequence within this enhancer is a cAMP-response element (CRE). Wingless and DPP act independently but synergistically through this enhancer to stimulate Ubx expression in the midgut. The LEF-1-binding site contains an excellent match to the LEF-1 binding site first identified in the T cell receptor alpha chain enhancer. LEF-1 binds the Ubx wingless response sequence (WRS) with high affinity and specificity (Riese, 1997).

Mouse LEF-1 was used in these experiments because the endogenous protein (now known to be Pangolin) had not yet been identified. The WRS is recognized by LEF-1 in a ternary complex with Armadillo protein. Expressing LEF-1 throughout the mesoderm results in an anterior expansion of Ubx expression in the visceral mesoderm. A similar anterior expansion is observed after the expression of arm throughout the visceral mesoderm. Under these circumstances the second midgut constriction appears precociously and tends to form as a double constriction. LEF-1 activity depends on arm, since LEF-1 fails to stimulate Ubx transcription in arm mutants. In contrast, LEF-1 expressing wg mutants show a moderate level of Ubx transcription in LEF-1 expressing embryos. This implies that LEF-1, perhaps by virtue of being overexpressed, bypasses the need for Wingless stimulation (Riese, 1997).

Pangolin is a HMG domain transcription factor involved in wingless signaling. Two pangolin mutants encode PAN proteins with amino-acid substitutions in domain of Pan corresponding to the domain of vertebrate Lef-1 that binds to ß-catenin. A test was made whether the wild-type or mutant N-terminal portions of PAN (amino acids 1-133) can bind to purified ß-catenin. The pan mutant proteins bind ß-catenin four-to-five fold less, as compared with wild type. These results indicate (1) that PAN can physically interact with the Armadillo homolog ß-catenin and (2) that mutations in this domain compromise wingless signaling in vivo and have a corresponding deleterious affect on the affinity of the binding interaction in vitro (Brunner, 1997).

T-cell factor (TCF), a high-mobility-group domain protein, is the transcription factor activated by Wnt/Wingless signaling. When signaling occurs, TCF binds to its coactivator, beta-catenin/Armadillo, and stimulates the transcription of the target genes of Wnt/Wingless by binding to TCF-responsive enhancers. Inappropriate activation of TCF in the colon epithelium and other cells leads to cancer. It is therefore desirable for unstimulated cells to have a negative control mechanism to keep TCF inactive. Drosophila CREB-binding protein (dCBP) binds to Drosophila TCF (Pangolin). dCBP mutants show mild Wingless overactivation phenotypes in various tissues. Consistent with this, dCBP loss-of-function suppresses the effects of armadillo mutation. Moreover, dCBP is shown to acetylate a conserved lysine in the Armadillo-binding domain of dTCF, and this acetylation lowers the affinity of Armadillo binding to dTCF. Although CBP is a coactivator of other transcription factors, these data show that CBP represses TCF (Waltzer, 1998).

The Armadillo protein of Drosophila and its vertebrate homologs, beta-catenin and plakoglobin, are implicated in cell adhesion and wnt signaling. The conservation of these two functions was examined by assaying the activities of mammalian beta-catenin and plakoglobin in Drosophila. In the female germ line, both mammalian beta-catenin and plakoglobin complement an armadillo mutation. shotgun mutant germ cells (which lack Drosophila E-cadherin) have a phenotype identical to that of armadillo mutant germ cells. Defects include random positioning of the oocyte within the egg chamber, irregular shape and size of nurse cells and nuclei, floating ring canals, and actin inclusions. It therefore appears that Armadillo's only role in the germ line is to be found in a complex with Drosophila E-cadherin (possibly an adhesion complex); both beta-catenin and plakoglobin can function in Drosophila cadherin complexes. Alternatively, Armadillo may have a role in organizing the cytoskeleton. Mammalian beta-Catenin and Plakoglobin can form complexes with Drosophila E-cadherin and alpha-Catenin. Both mammalian proteins, when provided zygotically, have enough activity to form adherens junctions in arm mutants and rescue a dorsal closure phenotype but are unable to restore cuticle patterning, resulting in a phenotype that resembles that of wingless. In embryonic signaling assays, plakoglobin has no detectable activity whereas beta-catenin's activity is weak. Surprisingly, when overexpressed, either in embryos or in wing imaginal disks, both beta-catenin and plakoglobin have a dominant negative activity on signaling, an effect also obtained with COOH-terminally truncated Armadillo. It is suggested that the signaling complex, which has been shown by others to comprise Armadillo and a member of the lymphocyte enhancer binding factor-1/T cell factor-family, may contain an additional factor that normally binds to the COOH-terminal region of Armadillo (White, 1998).

Pontin52 and reptin52 function as antagonistic regulators of beta-catenin signalling activity

In Wnt-stimulated cells, beta-catenin becomes stabilized in the cytoplasm, enters the nucleus and interacts with HMG box transcription factors of the lymphoid-enhancing factor-1 (LEF-1)/T-cell factor (TCF) family, thereby stimulating the transcription of specific target genes. Pontin52 has been identified as a nuclear protein interacting with beta-catenin and the TATA-box binding protein (TBP), suggesting its involvement in regulating beta-catenin-mediated transactivation. This study reports the identification of Reptin52 (see Drosophila Reptin) as an interacting partner of Pontin52. Highly homologous to Pontin52, Reptin52 likewise binds beta-catenin and TBP. Using reporter gene assays, it was shown that the two proteins antagonistically influence the transactivation potential of the beta-catenin-TCF complex. Furthermore, the evolutionary conservation of this mechanism is demonstrated in Drosophila: pontin and reptin are essential genes that act antagonistically in the control of Wingless signalling in vivo. These results indicate that the opposite action of Pontin52 and Reptin52 on beta-catenin-mediated transactivation constitutes an additional mechanism for the control of the canonical Wingless/Wnt pathway (Bauer, 2000; full text of article).

Drosophila APC2 and Armadillo participate in tethering mitotic spindles to cortical actin

Proper positioning of mitotic spindles ensures equal allocation of chromosomes to daughter cells. This often involves interactions between spindle and astral microtubules and cortical actin. In yeast and C. elegans, some of the protein machinery that connects spindles and cortex has been identified but, in most animal cells, this process remains mysterious. This study reports that the tumour suppressor homologue APC2 and its binding partner Armadillo both play roles in spindle anchoring during the syncytial mitoses of early Drosophila embryos. Armadillo, alpha-catenin and APC2 all localize to sites of cortical spindle attachment. APC2-Armadillo complexes often localize with interphase microtubules. Zeste-white 3 kinase, which can phosphorylate Armadillo and APC, is also crucial for spindle positioning and regulates the localization of APC2-Armadillo complexes. Together, these data suggest that APC2, Armadillo and alpha-catenin provide an important link between spindles and cortical actin, and that this link is regulated by Zeste-white 3 kinase (McCartney, 2001).

Wnt signals across the plasma membrane to activate the ß-catenin pathway by forming oligomers containing its receptors, Frizzled and LRP

Wnt-induced signaling via ß-catenin plays crucial roles in animal development and tumorigenesis. Both a seven-transmembrane protein in the Frizzled family and a single transmembrane protein in the LRP family (LDL-receptor-related protein 5/6 or Arrow) are essential for efficiently transducing a signal from Wnt, an extracellular ligand, to an intracellular pathway that stabilizes ß-catenin by interfering with its rate of destruction. However, the molecular mechanism by which these two types of membrane receptors synergize to transmit the Wnt signal is not known. Mutant and chimeric forms of Frizzled, LRP and Wnt proteins, small inhibitory RNAs, and assays for ß-catenin-mediated signaling and protein localization in Drosophila S2 cells and mammalian 293 cells were used to study transmission of a Wnt signal across the plasma membrane. The findings are consistent with a mechanism by which Wnt protein binds to the extracellular domains of both LRP and Frizzled receptors, forming membrane-associated hetero-oligomers that interact with both Disheveled (via the intracellular portions of Frizzled) and Axin (via the intracellular domain of LRP). This model takes into account several observations reported here: the identification of intracellular residues of Frizzled required for ß-catenin signaling and for recruitment of Dvl to the plasma membrane; evidence that Wnt3A binds to the ectodomains of LRP and Frizzled, and demonstrations that a requirement for Wnt ligand can be abrogated by chimeric receptors that allow formation of Frizzled-LRP hetero-oligomers. In addition, the ß-catenin signaling mediated by ectopic expression of LRP is not dependent on Disheveled or Wnt, but can also be augmented by oligomerization of LRP receptors (Cong, 2004).

What is the mechanism by which Frizzled transduces a Wnt signal? Mutations that disrupt the signaling activity of Frizzled also affect the ability of Frizzled to induce membrane translocation of Dvl and reduce physical interaction between Frizzled and Dvl, suggesting that a physical interaction between Frizzled and Dvl is required for the signaling activity of Frizzled. It is proposed that Frizzled might function as a docking site for Dvl in ß-catenin signaling. The results are consistent with the finding that the Lys-Thr-x-x-x-Trp motif at the C-terminal tail of Frizzled is not only required for activating ß-catenin signaling, but also for inducing Dvl membrane translocation. The PDZ domain of Dvl has been shown to directly bind to a peptide of C-terminal region of Frizzled containing the Lys-Thr-x-x-x-Trp motif, and this peptide can inhibit Wnt/ß-catenin signaling in Xenopus. However, the binding is relatively weak (Kd~10 microM). The current results suggest that multiple regions of Frizzled might be involved in the binding with Dvl and could increase the binding affinity (Cong, 2004).

The same structural elements may be required for Frizzled to function in both the planar polarity and the ß-catenin pathways, since membrane translocation of Dvl has been implicated in planar polarity signaling, and residues essential for the activity of Frizzled in ß-catenin signaling are also important for Frizzled-induced translocation of Dvl to the plasma membrane. It is possible that other proteins in the Frizzled-Dvl complex, such as LRP in ß-catenin signaling and Flamingo in planar polarity signaling, determine the signaling consequences of interaction between Frizzled and Dvl (Cong, 2004).

What is the role of LRP in transmitting the Wnt signal and what is the function of its extracellular domain of LRP for receiving the Wnt signal? An in vitro binding assay has suggested that Wnt1 is able to bind to the extracellular domain of LRP, but analogous binding was not observed in studies with Wg protein. Results from in vitro binding assays need to be treated cautiously, as the concentrations of ligands and receptors in these assays could be significantly higher than in physiological situations, and certain components normally involved in formation of the receptor complex could be missing in these assays. Therefore, functional data are necessary to address the significance of potential binding between Wnt and LRP. The extracellular domain of LRP can be functionally replaced by the extracellular domain of Frizzled, suggesting a physiological role for a direct, or indirect, interaction of Wnt with the extracellular domain of LRP (Cong, 2004).

LRP can also transmit a signal via ß-catenin without a requirement for Wnt. Advantage was taken of two commonly used inducible oligomerization strategies to demonstrate that oligomerization of LRP6 increases its signaling activity and its interaction with Axin. Interestingly, it has been shown that the second cysteine-rich domain of DKK2 stimulates ß-catenin signaling via LRP independently of Dvl. Further experiments are needed to determine whether this DKK2 fragment activates LRP by altering the oligomerization status of LRP (Cong, 2004).

The role of the cysteine-rich domain of Frizzled in Wingless-Armadillo signaling

The Frizzled (Fz) receptors contain seven transmembrane helices and an amino-terminal cysteine-rich domain (CRD) that is sufficient and necessary for binding of the Wnt ligands. Recent genetic experiments have suggested, however, that the CRD is dispensable for signaling. fz CRD mutant transgenes were generated and tested for Wg signaling activity. None of the mutants was functional in cell culture or could fully replace fz in vivo. Replacing the CRD with a structurally distinct Wnt-binding domain, the Wnt inhibitory factor, reconstitutes a functional Wg receptor. It is therefore hypothesized that the function of the CRD is to bring Wg in close proximity with the membrane portion of the receptor. This model was tested by substituting Wg itself for the CRD, a manipulation that results in a constitutively active receptor. It is proposed that Fz activates signaling in two steps: Fz uses its CRD to capture Wg, and once bound Wg interacts with the membrane portion of the receptor to initiate signaling (Povelones, 2005).

The principle finding of this study is that the Fz CRD is required for efficient Arm signaling. Fz transgenes carrying CRD mutations have compromised Arm signaling function in cell culture and cannot fully restore Arm signaling to fz,fz2 mutants in vivo. In addition, adding a heterologous Wnt-binding domain (WIF) to a CRD-deleted fz restores its ability to activate Arm signaling via Wg in cell culture. Based on the manipulations and results, it is hypothesized that the function of the CRD is to bring Wg in close proximity with the membrane portion of the receptor, a function that can be taken over by other Wnt-binding domains. This idea was tested by creating a transgene fusing Wg to Fz, eliminating the CRD in the process; this results in a constitutively active receptor (Povelones, 2005).

While both in vivo and in vitro tests reveal that mutants with a defective Wnt interaction domain are compromised for Arm signaling, the requirement for the CRD is most evident in cell culture where all of the mutants show a reduced activity, particularly the one where the entire CRD is lacking. In the cell culture experiments, where the Wg signaling can be measured in a quantitative manner, a range of responses were found to the CRD mutants, corresponding to the differences in Wnt-binding strength. A range of phenotypes was noticed after examining cuticles in vivo and the abilities of the CRD mutants to restore signaling. While these rescue data are more difficult to measure, the phenotypes correspond in strength to the in vitro signaling levels. It is inferred from this relationship that signaling operates through the same mechanism in vivo as in cell culture. As an extension of this argument, it is suggested that the CRD plays a similar role in cell culture as in the embryo. However, signaling in vivo is less stringently dependent on the presence of the CRD, suggesting that its absence is being compensated for by other factors. If the function of the CRD (or other Wnt-binding domains such as the WIF) is, as proposed, to bring Wg in close proximity to the membrane domain of Fz, it is possible this function is taken over by other molecules acting in trans and that these factors are not present in vitro. Candidates for such molecules are members of the CRD containing ROR family and the RYK receptor tyrosine kinase, which has a WIF domain. It is also possible that extracellular matrix molecules provide such an accessory function, by presenting or concentrating Wg close to the Fz signaling domain (Povelones, 2005).

Is the only function of the CRD (or another Wg-binding domain, such as WIF) to capture Wg and to present it to the coreceptor Arrow? In that view, there would be no need for the seven-transmembrane domain of the Fz receptors; Fz would solely act to promote Wg interacting with Arrow. This was found to be unlikely; there are several studies that point to a requirement of specific residues in the Fz membrane domain in signaling. Mutations in those residues, either engineered or present in natural alleles, disrupt signaling. In addition, it has been recently proposed that in Drosophila, fz activates PCP and Arm signaling through heterotrimeric G proteins. Finally, expressing the CRD on the cell's surface as a GPI-linked membrane molecule does not promote signaling, but instead acts as a dominant negative. Taken together, these data suggest that the transmembrane portion of fz is a dynamic signal activating molecule and not merely a Wg presentation module (Povelones, 2005).

Overexpression of fzWIF in the Drosophila wing leads to both gain-of-function PCP and Arm signaling phenotypes. This is the composite of the consequences of fz and fz2 overexpression, which individually activate PCP and Arm signaling, respectively. There is much interest in determining how each receptor couples to a particular pathway. Although there is some disagreement in these studies, it is generally concluded that the transmembrane portion of fz, including the cytoplasmic tail, couples it to PCP signaling. Since fzWIF contains this portion of fz, it is not surprising that it too affects PCP signaling. What structural feature of fz2 is responsible for coupling it exclusively to Arm signaling? It was found that specifically replacing the fz CRD with the WIF domain results in a receptor that, like fz2, can activate Arm signaling. This finding is consistent with a study of fz/fz2 chimeras where the ability to activate Arm signaling was shown to be a property of the fz2 CRD. It was proposed that the feature conferring Arm coupling was the 10-fold higher affinity of the fz2 CRD for the Wg protein. By analogy, the WIF domain, like the fz2 CRD, may have a higher affinity for Wg than the fz CRD (Povelones, 2005).

Protein Interactions : Interaction of Armadillo with Legless

Wnt transduction is mediated by the association of ß-catenin with nuclear TCF DNA binding factors. The products of two newly identified Drosophila segment polarity genes, legless (lgs), and pygopus (pygo) are required for Wnt signal transduction at the level of nuclear ß-catenin. Lgs encodes the homolog of human BCL9; genetic and molecular evidence is provided that these proteins exert their function by physically linking Pygo to ß-catenin. These results suggest that the recruitment of Pygo permits ß-catenin to transcriptionally activate Wnt target genes and raise the possibility that a deregulation of these events may play a causal role in the development of B cell malignancies (Kramps, 2002).

The yeast two-hybrid system and GST pull-down assays were used to examine the possibility that Lgs physically interacts with Armadillo (Arm). Indeed, the N-terminal half of Lgs binds to the Arm protein. The Arm binding domain of Lgs was subsequently fine-mapped to the HD2 region and the Lgs binding domain of Arm to armadillo repeats 1-4. Consistent with these results, BCL9 also binds to Arm, as well as to ß-catenin, and the domain required for the interactions with ß-catenin again maps precisely to the conserved HD2 sequence (Kramps, 2002).

Since lgs17P and lgs17E encode amino acid substitutions in HD2, whether their protein products can bind to Arm protein in vitro was tested. The binding of Lgs17P and Lgs17E to Arm is reduced at least 10-fold compared to wild-type Lgs protein. This finding, which reinforces the observations of genetic interactions between arm and lgs alleles, is interpreted as evidence that Wnt/Wg signal transduction normally depends on molecular interactions between the Lgs/BCL9 and Arm/ß-catenin proteins (Kramps, 2002).

It can be concluded that Lgs and Pygo are required for the signaling activity of Arm, and that this function depends on the ability of Lgs to interact molecularly with Arm and on the ability of Pygo to molecularly interact with Lgs. Based on their subcellular localization and epistatic relationship with ArmS10, Lgs and Pygo are unlikely to exert their function by impeding proteasome-mediated degradation of Arm. They could play a role in permitting nuclear import or preventing nuclear export of Arm. However, this is thought to also be unlikely, because no difference is detected in subcellular localization of Arm in lgs mutant embryos. An alternative possibility is that Lgs and BCL9, respectively, function to tie Pygo to the ß-catenin-TCF complex, perhaps to allow Pygo to activate and sustain the expression of Wnt target genes. This hypothesis raises several predictions. (1) This model implies that the main task of Lgs/BCL9 is to serve as an adaptor to tether Pygo to Arm/ß-catenin. Thus, most of the Lgs/BCL9 protein should be dispensable, as long as Lgs HD1 is covalently linked to HD2, allowing the formation of a bridge between Arm/ß-catenin and Pygo. Lgs might even be entirely superfluous if Pygo is endowed with the ability to directly bind ß-catenin. (2) This model would require that Arm/ß-catenin is able to bind simultaneously, and stably, with both Pan/TCF and Lgs/BCL9. This in turn would necessitate separate binding sites on Arm/ß-catenin for Pan/TCF and Lgs/BCL9. (3) Pygo proteins would have to possess the ability to stimulate transcription when recruited to promoters of Wnt target genes (Kramps, 2002).

The primary structure of Arm and its mammalian homolog ß-catenin consists of an N-terminal and a C-terminal tail flanking a central domain of ~500 residues composed of 12 armadillo repeats. These repeats pack against one another to form a superhelix that features a positively charged groove. The armadillo repeat domain mediates the binding of ß-catenin to cadherins, APC, Axin, and TCF. Despite their lack of significant sequence homologies, these proteins bind competitively to ß-catenin, presumably because they contact the same surface area of ß-catenin. If Lgs/BCL9 also binds to this surface, it would be expected to compete with Pan/TCF for the interaction with ß-catenin and could not be recruited to Wnt target genes. This issue was addressed by using peptide competition and coimmunoprecipitation experiments (Kramps, 2002).

Biotinylated peptides representing the N-terminal domains of hTCF4 (or Pan) were used to pull down labeled ß-catenin (or Arm protein) with avidin beads. This peptide-protein interaction was effectively disrupted by an excess of nonbiotinylated TCF or Pan peptides, but not by an excess of HD2 peptides. GST-BCL9 protein was used to pull down labeled hTCF4 in the presence of ß-catenin protein. hTCF4 is efficiently retained on BCL9-charged glutathion beads in the presence, but not absence, of ß-catenin, which apparently can function as a bridge between the two proteins. These results indicate that Lgs/BCL9 and Pan/TCF do not compete for their interaction with Arm/ß-catenin, but rather bind it simultaneously. This in turn suggests that Lgs/BCL9 is recruited to TCF binding sites of Wnt target genes (Kramps, 2002).

To address the role of Pygo in ß-catenin-mediated transcription, a TCF reporter gene (TOP-Flash) was used in immortalized human embryo kidney cells (HEK 293 cells). Low levels of a stable mutant form of ß-catenin (DeltaN-ß-catenin) were introduced into these cells to partially stimulate the pathway. The additional expression of hPYGO1 or hPYGO2 leads to a large increase in luciferase activity (30-fold). These levels are significantly higher than the sum of those produced by either treatment alone. This potentiation of ß-catenin activity by hPYGO1 and 2 appears to be mediated by the interaction of endogenous TCF protein with its DNA target sites, as it is only observed with TOP-Flash, which contains five optimal TCF binding sites, but not with the control reporter FOP-Flash, which contains five mutated sites (Kramps, 2002).

Although less powerful per se than the genetic arguments, this experiment adds supportive evidence to the notion that Pygo proteins transduce Wnt signals by activating TCF target genes in a ß-catenin-dependent manner (Kramps, 2002).

The Wnt signalling system controls many fundamental processes during animal development and its deregulation has been causally linked to colorectal cancer. Transduction of Wnt signals entails the association of ß-catenin with nuclear TCF DNA-binding factors and the subsequent activation of target genes. Using genetic assays in Drosophila, a presumptive adaptor protein, Legless (Lgs), has been identifed that binds to ß-catenin and mediates signalling activity by recruiting the transcriptional activator Pygopus (Pygo). This study characterizes the ß-catenin/Lgs interaction and shows: (1) that it is critically dependent on two acidic amino acid residues in the first Armadillo repeat of ß-catenin; (2) that it is spatially and functionally separable from the binding sites for TCF factors, APC and E-cadherin; (3) that it is required in endogenous as well as constitutively active forms of ß-catenin for Wingless signalling output in Drosophila, and (4) that in its absence animals develop with the same phenotypic consequences as animals lacking Lgs altogether. Based on these findings, and because Lgs and Pygo have human homologues that can substitute for their Drosophila counterparts, it is inferred that the ß-catenin/Lgs binding site may thus serve as an attractive drug target for therapeutic intervention in ß-catenin-dependent cancer progression (Hoffmans, 2004).

This study is concerned with the question of how ß-catenin and Lgs interact molecularly with each other. The analysis addressed three issues: localization of the binding site on ß-catenin, specificity of this site vis-a-vis other partners of ß-catenin and in vivo significance of this interaction for Wg signal transduction. By means of site-directed mutagenesis the role of conspicuous ß-catenin residues in the binding to human LGS1 was examined. Two amino acids, D162 and D164, were identified that are both necessary for human LGS1 binding. Because substitutions of these residues with other amino acids did not affect the binding of several other proteins to ß-catenin, the role of these amino acids is interpreted as contact sites for human LGS1, rather than a structural function enhancing stability and/or three-dimensional conformation of ß-catenin. This conclusion, however, will need to be confirmed by determining the crystal structure of the ß-catenin/human LGS1 complex (Hoffmans, 2004).

Neither D162 nor D164 is required for binding to APC, E-cadherin or TCF4. Substitutions of these amino acids reduce binding to alpha-catenin twofold, but in vivo data suggest that this reduction does not prevent the assembly of adherens junctions. The specificity of the ß-catenin/human LGS1 interaction vis-a-vis that of ß-catenin and APC, E-cadherin or TCF4 is consistent with their respective locations on the surface of ß-catenin. While crystallographic studies show that APC, E-cadherin and TCF4 all bind to a common, extended surface within the groove of ß-catenin formed by Arm repeats 3-10, this analysis indicates that human LGS1 binds an acidic knob in Arm repeat 1. This knob is not only located more N terminally, it is also situated on the side of ß-catenin, which is opposite the groove. The spatial separation of these binding sites is in agreement with their separable functions observed in yeast binding assays, as well as with previous GST pull-down assays, in which simultaneous binding of TCF4 and human LGS1 to ß-catenin is observed (Kramps, 2002).

Thus, to assess the role of D162 and D164 in Wg transduction, mutant forms of Arm were subjected to various assays designed to reveal their in vivo function. Simple rescue and overexpression experiments have shown that transgenic Arm-D164A cannot substitute for endogenous Arm, and that the D164A mutation significantly reduces the constitutive signalling activity associated with N-terminal deletions of Arm. When tested in more advanced assays, it was found that D164 is required by wing disc cells to maintain Wg target gene expression and by developing embryos for segmentation. Together, these experiments support the conclusion that Arm signalling function relies on its capability to bind to Lgs throughout development (Hoffmans, 2004).

Although it is straightforward to interpret the results as a qualitative indication for the significance of the Arm/Lgs interaction, it is more difficult to assess their outcome in a quantitative manner. For example, the apparent residual expression of Dll in Arm-D164A cells may reflect perdurance of wild-type Arm or Dll proteins, but it could also indicate that a fraction of the Wg signal can be transmitted despite the D164A mutation. This latter scenario could in turn be attributed to some residual binding of Arm to Lgs, but it could also be explained by a partial redundancy of Lgs function. Lgs may be required for efficient Arm-mediated activation of Wg targets, but some activation may also occur in its absence. Consistent with this latter view, it was observed that animals lacking maternal and zygotic lgs product exhibit phenotypes equivalent to animals in which the sole source of Arm is the D164A transgene, yet neither of the two phenotypes are quite as severe as that of wg-null mutants (Hoffmans, 2004).

The Wnt pathway is highly conserved between Drosophila and vertebrates. The human homologues of Lgs (LGS1/BCL9) and Pygo (PYGO1 and PYGO2) can rescue lgs and pygo mutant flies, respectively. This suggests that these proteins have the same function in vertebrates and in Drosophila. It is possible therefore, that in vivo data can be extrapolated to Wnt signalling in mammals. Mutations in APC occur in more than 80% of inherited and sporadic colorectal cancers. These mutations lead to accumulation of free ß-catenin and as a result to overexpression of Wnt target genes. A chemical compound that interferes with the formation of the nuclear TCF/ß-catenin/Lgs/Pygo complex should in theory halt the progression of cancer. Such an anti-cancer drug must be highly specific though, since it should only disrupt the nuclear ß-catenin complex, but should not disrupt either the cytoplasmic ß-catenin/APC/Axin complex or the ß-catenin/E-cadherin complex at the cell membrane. APC, Axin and E-cadherin functions should not be compromised, since all three of them have tumour suppressor roles. This is not the case, however, for TCF and Lgs. Crystal structure data indicates that APC, Axin, E-cadherin and TCF4 partly use the same contact sites of ß-catenin for their binding. Therefore, designing an inhibitor that specifically disrupts the ß-catenin/TCF interaction is a difficult task. On the contrary, mapping and specificity results indicate that the ß-catenin/Lgs interaction site could be targeted without interfering with the binding of ß-catenin to APC and E-cadherin. Moreover, this analysis shows that genetic disruption of the Arm/Lgs interaction leads to severely reduced Wg signalling, suggesting that the protein-protein interaction between ß-catenin and Lgs may provide an attractive target for therapeutic intervention (Hoffmans, 2004).

A complex of Armadillo, Legless, and Pygopus coactivates dTCF to activate Wingless target genes

Upon receiving a Wnt signal, cells accumulate ß-catenin (Armadillo in Drosophila), which binds directly to TCF transcription factors, leading to the transcription of Wnt target genes. It is generally thought that ß-catenin/Armadillo is a transcriptional coactivator when bound to TCF in the nucleus and that this function is mediated by its C terminus. However, recent findings in Drosophila indicated that Armadillo may activate dTCF in the cytoplasm. This study reexamines the mechanism of Armadillo's signaling function in light of Legless and Pygopus, two nuclear factors recently discovered to be essential for this function. Armadillo, in order to activate dTCF, must enter the nucleus and form a complex with Legless and Pygopus. The ability of this complex to stimulate TCF-mediated transcription can be altered by linkage of a strong transcriptional activator or repressor to Armadillo. Furthermore, Armadillo is a strong transcriptional activator when fused to the yeast GAL4 DNA binding domain -- an activity that depends on regions of the Armadillo repeat domain that mediates binding to Legless and to chromatin modifying and remodeling factors. Finally, linkage of the N-terminal region of Pygopus, but not the C terminus of Armadillo, to dominant-negative dTCF can restore its signaling activity in transgenic flies. This evidence argues in favor of a revised coactivator factor model in which Armadillo's coactivator function depends on regions within its Armadillo repeat domain to which Legless/Pygopus and other transcriptional coactivators can bind. In contrast, the C terminus of Armadillo plays a less direct role in this function (Thompson, 2004).

The model that Arm functions in the nucleus as a transcriptional activator of dTCF clearly predicts that exclusion of Arm from the nucleus by tethering to membranes should render it unable to signal. Two such nuclear-excluded, membrane-tethered forms of Arm have been examined in Drosophila: Sev-Arm, a fusion of the extracellular and transmembrane domains of Sevenless to Arm's N terminus, and Arm-CAAX, which features a CAAX-type palmitoylation sequence at its C terminus. The signaling activity of Arm transgenes can be measured by examining their ability to rescue Drosophila embryos that are maternally and zygotically mutant (henceforth: mutant) for arm. A severe impediment to this analysis is that arm null mutants (eg: armXP33 and arm4 also called armYD35) have adhesion defects in addition to defective Wingless signaling and, consequently, do not develop beyond oogenesis. Thus, mutant conditions that affect signaling, but not adhesion, must be used. The most commonly used signaling-mutant (but adhesion-competent) allele is armXM19, a truncation of the Arm C terminus that generates embryos with defective Wingless signaling (Thompson, 2004).

Surprisingly, both Sev-Arm and Arm-CAAX were reported to substantially rescue Wingless signaling in armXM19 mutants. The two possible interpretations of these results are (1) that these proteins signal independently of endogenous Arm and (2) that the ArmXM19 mutant protein can be induced to signal in the presence of these transgenes. Discrimination between these two possibilities requires examination of these transgenes in alternative arm mutant backgrounds. In the case of Arm-CAAX it was possible to use an effectively null mutant, armXP33 (which does not express detectable Arm protein), because Arm-CAAX is able to function in adhesion. Arm-CAAX was found to rescue the adhesion, but not the signaling defect of armXP33. In the case of Sev-Arm, analysis in a null mutant background is not possible because this transgene is not competent to rescue the adhesion defect. Attempts were made with arm043A01, an allele that produces both signaling and mild adhesion defects, but the results are unclear, because mutant embryos do not secrete a cuticle. Therefore, alternative mutant conditions were generated by expressing signaling-mutant (but adhesion-competent) Arm transgenes, ArmS6 and ArmS12, in an arm4 null-mutant background. These conditions (henceforth: ArmS6 and ArmS12 mutants) generated embryos whose cuticle phenotype was a lawn of denticles, indicating that Wingless signaling was inactive. Ubiquitous expression of Sev-Arm with the Gal4-UAS system was unable to rescue the Wingless-signaling defect of these embryos, whereas similar ubiquitous expression of Sev-Arm was able to rescue the cuticular phenotype of armXM19 mutants considerably. Similarly, as a control, an activated form of Armadillo, ArmS10, was able to rescue all three signaling-mutant conditions. It is concluded that Sev-Arm, like Arm-CAAX, is unable to signal in the absence of functional endogenous Arm and that the C-terminally truncated ArmXM19 protein retains significant signaling activity that is revealed by the expression of membrane-tethered forms of Arm (Thompson, 2004).

In addition to dTCF, two other ubiquitous factors, Legless (Lgs) and Pygopus (Pygo), are essential for Arm's signaling activity in Drosophila. In lgs or pygo mutants, Arm is unable to signal, even when it accumulates at unusually high levels throughout the cell. The localization of these proteins (either the endogenous protein or epitope-tagged versions expressed from a transgene) were examined in the embryonic epidermis where high levels of Wingless induce accumulation of Arm in stripes of cells. dTCF, Lgs, and Pygo are predominantly nuclear in all cells regardless of their state of signaling. Notably, no evidence was found for nuclear export of tagged, expressed dTCF in response to Wingless in the embryonic epidermis (Thompson, 2004).

Although genetic analysis of Lgs and Pygo has demonstrated that they are essential for Arm's signaling activity, it remains possible that these proteins simply provide an essential function for dTCF. No evidence was found that dTCF stability or localization are affected in pygo mutants. Note that both Lgs and Pygo function are compromised in pygo mutants, because Lgs depends on Pygo for its nuclear localization (F.M. Townsley, A. Cliffe, and M. Bienz, unpublished data, cited in Thompson, 2004). If Lgs and Pygo provide an essential function for Arm rather than dTCF, then providing dTCF with a strong transcriptional activator should bypass the requirement for Lgs and Pygo. A fusion protein of dTCF with the VP16 transcriptional activation domain (dTCF-VP16) that had been shown to rescue armXM19 mutants was therefore expressed in wild-type and pygo mutant embryos with the GAL4-UAS system. Unfortunately, expression of dTCF-VP16 arrests embryogenesis prior to cuticular differentiation. Therefore the expression of the engrailed gene, a target of Wingless signaling in the embryo that is downregulated in pygo mutants was examined. Expression of dTCF-VP16 is able to restore engrailed expression in these embryos. It is concluded that Lgs and Pygo are not required for dTCF's stability, localization, or DNA binding activity but, rather, for activation of dTCF by Arm (Thompson, 2004).

Consistent with this view, in vitro binding experiments have shown that the Lgs HD2 domain binds directly to the first four Armadillo repeats of Arm, while the Lgs HD1 domain binds to the PHD domain of Pygo. On this basis, it was proposed that Arm, Lgs, and Pygo may form a complex in vivo. To test this proposal, an HA-tagged version of Pygo was expressed in Drosophila embryos that also expressed Wingless to activate signaling in all cells. The tagged Pygo was immunoprecipitated with αHA antibodies. Both Arm and Lgs were found to be readily coimmunoprecipitated from embryos expressing HAPygo, but not from control embryos. It is concluded that Arm, Lgs, and Pygo form a nuclear complex in Wingless-stimulated cells in vivo. These findings strongly support the view that Arm activates dTCF in the nucleus, since Lgs and Pygo, two binding partners for Arm that are essential for this process, are nuclear proteins (Thompson, 2004).

The the Arm/dTCF transcription factor model of Wingless signal transduction need to be reconsidered in light of the discovery of Legless and Pygopus. This model was originally prompted by the findings that (1) activation of dTCF depends on a direct binding interaction with Arm; (2) TCF transcription factors are constitutively localized to the nucleus, whereas Arm enters the nucleus only upon signaling; and (3) the C terminus of Arm, which is absent in armXM19 mutants, can function as a transcriptional activator when tethered to DNA (Thompson, 2004).

The model for Arm function predicts that Arm must enter the nucleus in order to form an active transcription factor with dTCF on DNA. The results of this study show that membrane-tethered forms of Arm cannot directly activate dTCF, supporting the notion that Arm must enter the nucleus to do so. The ability of membrane-tethered Arm to signal in an armXM19 mutant background must therefore reflect that the armXM19 mutation is not a null and must retain some signaling activity that is enhanced by the presence of membrane-tethered Arm. A plausible explanation for this phenomenon is that membrane-tethered Arm recruits negative regulators of Arm, thereby stabilizing and/or promoting nuclear translocation of endogenous Arm. In support of this explanation, effects of this kind have, in fact, been observed with several different types of membrane-targeted Arm and β-catenin. Consideration of these results reveals a point of conflict with the original form of the Arm/dTCF transcription factor model, which proposes that the Arm C terminus is necessary and sufficient for Arm's coactivator function. The armXM19 mutation encodes an Arm protein that lacks its C terminus. If this truncated protein retains some signaling activity, then the C terminus cannot be the sole mediator of Arm's coactivator function. In support of this view, several different C-terminally truncated Arm and β-catenins appear to retain significant signaling activity under conditions of overexpression. Furthermore, Arm's C terminus can be substituted without loss of function by the C terminus of a different Armadillo repeat domain protein, Pendulin. Unlike the Arm C terminus, the Pendulin C terminus lacks transactivating activity when fused to the GAL4 DNA binding domain. It is concluded that the C terminus is not sufficient to mediate Arm's coactivator function but instead, is likely to be required in some way for the stability or activity of the Armadillo repeat domain. These findings undermine one block of evidence upon which the Arm coactivator model was originally founded (Thompson, 2004).

Evidence was therefore sought that Arm functions as a transcriptional activator. Arm's ability to activate TCF-mediated transcription, as measured in the Topflash assay, is enhanced by addition of a strong transcriptional activator and reduced by addition of a strong transcriptional repressor. Tethering of Arm to DNA with the GAL4 DNA binding domain reveals that Arm functions as a strong transcriptional activator. Furthermore, this activity of Arm was suppressed by mutations in the Armadillo repeat domain (S6 and S12) that prevent Arm from transducing Wingless signals in vivo. The results indicate that Arm indeed functions as a coactivator and that this function depends on regions in the Armadillo repeat domain that may recruit additional coactivating factors (Thompson, 2004).

Two candidates that may mediate Arm's coactivator function are Lgs and Pygo. Lgs and Pygo are constitutively nuclear proteins that bind to the Armadillo repeat domain upon signaling and are essential for Arm to activate dTCF. Furthermore, Lgs and Pygo appear to be present in the coactivator complex. The N terminus of Pygo (PygoΔPHD) is sufficient to mediate the function of Lgs and Pygo in Wingless signaling when targeted to Arm by fusion to the Lgs HD2 domain. The same region of Pygo has the capacity to function as a transcriptional activator and, when fused to dTCF, can partially bypass the requirement for Armadillo in Wingless signal transduction (Thompson, 2004).

The results argue that Lgs and Pygo directly contribute to transcriptional activation of the Arm/dTCF transcription factor. Although the Pygo N terminus has been defined as a transactivator, it is possible that other regions of Lgs and Pygo may also possess this activity. It is further possible that Lgs and Pygo may contribute indirectly to Arm's coactivator activity: for example, by facilitating nuclear import or retention of Arm (F.M. Townsley, A. Cliffe, and M. Bienz, unpublished data cited in Thompson, 2004).

In any case, it is unlikely that Lgs and Pygo are the sole mediators of Arm's coactivator activity. For example, while the Arm S6 mutation (in repeat 1) might be predicted to affect Lgs binding, the Arm S12 mutation affects the C-terminal repeats (repeats 10 and 11). It is inferred that an additional, essential coactivating factor(s) is recruited to the C-terminal Arm repeats. Two obvious candidate factors are the histone acetyltransferase CBP/p300 and the chromatin remodeling enzyme Brahma, both of which have been found to bind to C-terminal regions of the Armadillo repeat domain (Thompson, 2004 and references therein). The evidence presented in this study argues in favor of an extended Arm/dTCF transcription factor model in which Arm coactivates dTCF by recruiting Lgs, Pygo, and other factors to its Armadillo repeat domain. (Thompson, 2004).

Pygopus and legless provide essential transcriptional coactivator functions to armadillo/β-catenin

Wnt signaling controls important aspects of animal development, and its deregulation has been causally linked to cancer. Transduction of Wnt signals entails the association of β-catenin with nuclear TCF DNA binding proteins and the subsequent activation of target genes. The transcriptional activity of Armadillo (Arm, the Drosophila β-catenin homolog) largely depends on two recently discovered components, Legless (Lgs) and Pygopus (Pygo). Lgs functions as an adaptor between Arm/β-catenin and Pygo, but different mechanisms have been proposed as to how Arm/β-catenin is controlled by Lgs and Pygo. Although Lgs and Pygo were originally thought to serve as nuclear cofactors for Arm/β-catenin to enhance its transactivation capacity, a recent analysis argued that they function instead to target Arm/β-catenin to the nucleus. This study used genetic assays in cultured cells and in vivo to discriminate between the two paradigms. Regardless of the measures taken to maintain the nuclear presence of Arm/β-catenin, a transcriptional-activation function of Pygo could not be bypassed. These findings therefore indicate that Arm/β-catenin depends on Lgs and Pygo primarily for its transcriptional output rather than for its nuclear import (Hoffmans, 2005).

Wingless signals are secreted glycoproteins controlling many fundamental processes during animal development. Whereas several responses to Wnt ligands appear to entail direct cytoplasmic responses organizing planar cell polarity and organ morphogenesis, a significant fraction of Wnt responses concern transcriptional changes in the nucleus. This latter aspect of Wnt-signal transduction is mediated by β-catenin and is often referred to as “canonical” or β-catenin-dependent Wnt signaling. The canonical Wnt pathway plays important roles in embryonic-cell-fate determination, and its constitutive activation is oncogenic in several adult mammalian tissues, most notably in the intestinal epithelium. Hence, it is of prime interest to understand how β-catenin activity can upregulate transcription of Wnt target genes. Although cytoplasmic β-catenin was originally discovered through its role in cell adhesion, a large body of evidence indicates that it is degraded in the absence of a Wnt signal but stabilized in its presence. As a consequence, β-catenin can sufficiently accumulate, translocate to the nucleus, and be directed to Wnt target genes by associating with DNA-binding TCF/LEF proteins. However, it is less clear how a cell-adhesion component, relocated to the nucleus, can promote and sustain the transcriptional activity of these targets (Hoffmans, 2005).

Using genetic assays in Drosophila, a presumptive adaptor protein, Legless (Lgs) has been identified, that binds to β-catenin and its Drosophila homolog, Armadillo (Arm), as well as to the nuclear protein Pygopus (Pygo). On the basis of biochemical and phenotypic analysis, it is proposed that nuclear β-catenin/Arm assembles a quaternary complex, consisting of TCF, β-catenin, Lgs, and Pygo, in which Pygo serves as a transcriptional activator to induce and/or maintain the transcription of Wnt/Wg target genes. Alternatively, however, the requirement for Lgs and Pygo in Wnt/Wg signaling could be attributed to a role in targeting and retaining β-catenin in the nucleus, increasing its net nuclear concentration and, hence, its activity. This latter view has recently gained recognition and experimental support by a cell-biological analysis of these components. This study set out to address the mechanistic role of Pygo by subjecting the two models to three different tests; each case comes to the conclusion, that Pygo functions mainly in the transcriptional output of β-catenin (Hoffmans, 2005).

In the first approach, the consequences of disrupting the molecular interaction between β-catenin and Lgs was examined. β-catenin/Arm amino acid residues required for Lgs binding have been identified and it was observed that mutant β-catenin forms lacking these residues are severely compromised in their signaling activity. This reduction in activity could be caused either by a failure of β-catenin/Arm to recruit the “transcriptional mediator” Pygo or by a reduced (as a result of diminished nuclear anchoring) nuclear-cytoplasmic ratio of β-catenin/Arm. An experiment was repeated in which N-terminally truncated and therefore constitutively active forms of Arm, ArmS10-wt and ArmS10-D164A (differing solely in one critical amino acid residue necessary for Lgs binding), were expressed in the embryonic epidermis of Drosophila. Expression of ArmS10-wt suppresses denticle formation—a read-out for a gain of Wg signaling activity—whereas the D164A mutation, which impairs binding to Lgs, efficiently abolished this gain-of-function activity. When subjected to an immunohistochemical analysis, however, the two genotypes visually differed neither in amount nor subcellular localization of the ArmS10 proteins. The D164A mutation was further used in a cellular assay in which a constitutively active form of β-catenin (S33Y) was tethered to the enhancer of a reporter gene by the DNA binding domain of Gal4. Whereas β-catenin caused strong transcriptional activation, the D164A form lost this activity almost completely. Importantly, however, both forms were expressed at equivalent levels in human cells and did not differ in their ability to localize in nuclei. Because Lgs mediates the binding of β-catenin to Pygo, these results are interpreted as evidence that a failure of Arm/β-catenin to recruit Pygo impedes the transcriptional activity of the former despite the fact that it is nuclearly localized (Hoffmans, 2005).

A second test was devised on the assumption that Lgs appears to function merely as an adaptor between Arm/β-catenin and Pygo, thereby linking Arm/β-catenin either to a transcriptional activator or a nuclear anchor. Such a passive role for Lgs can be inferred from the observations that Lgs is dependent on Pygo both for its signaling activity and for its nuclear localization. If the main role of Lgs would be to link Arm/β-catenin to the constitutively nuclear anchor Pygo, it should gain functional independence of Pygo when bestowed with a nuclear-localization signal (NLS). Lgs was therefore modified by replacing a C-terminal portion with sequences of a green fluorescent protein (LgsN-eGFP) and adding the NLS of SV40 large T-antigen N-terminally (NLS-LgsN-eGFP). These altered forms of Lgs were examined for their subcellular distribution and signaling function. The addition of a single NLS effectively conferred nuclear localization, as assessed in transfected cells. When tested for their signaling capacity in Drosophila S2 cells, LgsN and NLS-LgsN were found to be equally active in rescuing the RNAi-mediated knockdown of endogenous Lgs. However, these two forms of Lgs were equally inactive in rescuing the knockdown of endogenous Pygo. Consistent with this result, it was also found that the Lgs-rescuing activity of NLS-LgsN still depends on the HD1 domain, through which it binds Pygo. Together, these results indicate that constitutive nuclear targeting of Lgs does not bypass the requirement for Pygo in Wg signaling, suggesting that Pygo must provide a function beyond ensuring availability of Lgs and β-catenin in the nucleus of Wg-transducing cells (Hoffmans, 2005).

The third test aimed at assessing the role of the N-terminal homology domain (NHD) of Pygo. Drosophila Pygo and its two mammalian homologs, Pygo1 and Pygo2, share—in addition to their C-terminal plant homology domain (PHD) finger domain, through which they bind Lgs—a short N-terminally located sequence of amino acids. On the basis of the conservation of Pygo function and absence of further common domains, the NHD was proposed to serve as transactivation domain. It was first confirmed that the NHD core domain (amino acids 91 to 101) is not required for nuclear localization of Pygo because neither the deletion of the core nor the change of a conserved and functionally required amino acid (F99A) affected the nuclear localization of Pygo in cultured cells. Importantly, these alterations also had no discernible effect on the capacity of Pygo to bind Lgs. If Pygo and Lgs primarily function to target Arm/β-catenin to the nucleus, then NHD mutations should not seriously affect Wnt/Wg signaling. It was found, however, that Pygo-F99A—in contrast to wild-type Pygo—failed to rescue Pygo function in cultured cells and in vivo. The endogenous pygo gene was replaced with a genomic pygo-F99A transgene in vivo, and it was observed that both mutant and wild-type Pygo proteins are expressed at comparable levels without detectable differences in nuclear-cytoplasmic distribution. The most explicit argument for a role of the NHD in transactivation was obtained by analyzing mutant clones of imaginal cells in which either the pygo-wt or the pygo-F99A transgenes were the only source of full-length Pygo protein. Both transgenes rescued Lgs nuclear localization in the mutant clones to a similar extent; however, pygo-F99A—but not pygo-wt—showed severely reduced transcription of the Wg target gene senseless. Because Pygo protein bearing a mutant NHD retains the capacity to localize Lgs (and, by inference, Arm), it is inferred that the key function of the Pygo NHD is to confer transcriptional activity to Arm (Hoffmans, 2005).

In summary, the functions of Lgs and Pygo were tested in β-catenin-dependent Wnt/Wg signaling by devising experiments that separate a role in transcriptional activation of targets from a role in nuclear targeting or retention of Arm/β-catenin. In all three situations examined, the transcriptional output of Arm/β-catenin depended on Pygo activity despite measures to grant Arm/β-catenin such alleged nuclear retention. When Arm/β-catenin was tethered directly to DNA via the Gal4 DNA binding domain, or when Lgs was endowed with an NLS of its own, Arm/β-catenin activity was still dependent on the recruitment of Pygo. Likewise, in vivo, when the nuclear retention activity of Pygo was left intact, Arm was not able to transduce Wg and activate target genes without the Pygo NHD. Although it cannot be rule out that Lgs and Pygo function as a nuclear anchor for β-catenin, the results collectively argue that the primary requirement for the two Arm/β-catenin partners must be attributed to a transcriptional role that allows Arm/β-catenin to activate and/or sustain the expression of Wnt/Wg target genes. Although information is lacking on the biochemical nature of this transactivation activity, it is tempting to assume that it involves the NHD-mediated recruitment of a chromatin-modification complex or of factors mediating transcription initiation or elongation (Hoffmans, 2005).

Wingless-independent association of Pygopus with dTCF target genes

The Wnt signaling pathway controls numerous cell fates during animal development. Its inappropriate activity can lead to cancer in many human tissues. A key effector of the canonical Wnt pathway is β-catenin (or Drosophila Armadillo), a highly unstable phosphorylated protein that shuttles rapidly between nucleus and cytoplasm. Wnt signaling inhibits its phosphorylation and degradation; this allows it to associate with TCF/LEF factors bound to Wnt target genes and to stimulate their transcription by recruiting chromatin modifying and remodeling factors. The transcriptional activity of Armadillo/β-catenin also depends on Pygopus (Pygo), a nuclear protein with which it associates through the Legless/BCL9 adaptor. It has been proposed that Pygo associates with TCF target genes during Wnt signaling through Armadillo and Legless to recruit a transcriptional coactivator through its Nbox motif. This study reports that Pygo is associated constitutively with dTCF target genes in Drosophila salivary glands and tissue-culture cells. The evidence indicates that this association depends on dTCF and on the Nbox motif of Pygo, but not on Legless. An alternative model is proposed according to which Pygo functions at the onset of Wnt signaling, or at low signaling levels, to capture Armadillo at dTCF target genes, thus enabling the interaction between Armadillo and dTCF and, consequently, the Armadillo-mediated recruitment of transcriptional coactivators (de la Roche, 2007).

Pygo could act as an Armadillo-loading factor whose function might be essential at limiting levels of activated Armadillo, either at low Wingless signaling levels or during the early phase of a Wingless response. Thus, Pygo could target even low levels of nuclear Armadillo to dTCF loci, thereby facilitating the efficient interaction between DNA-bound dTCF and Armadillo and enabling the subsequent recruitment of transcriptional cofactors. It is conceivable that the adaptor chain would rearrange after the capture of Armadillo, which might enable a putative transactivation function of the Nbox binding factor, consistent with a dual role of Pygo. In essence, this model envisages that Pygo predisposes dTCF target genes for efficient activation in response to Wingless. It explains why Pygo is required for efficient nuclear accumulation of Legless and Armadillo and why this requirement is bypassed by high levels of nuclear Armadillo. Note that some dTCF target genes in Drosophila or mammals may not rely on this predisposing function of Pygo, and some modes of Wnt-induced transcription may proceed without it. The ultimate test of this model will depend on the identification of the Nbox binding factor and its proposed role in predisposing TCF target genes to Wnt-induced transcription (de la Roche, 2007).

The PHD domain is required to link Drosophila Pygopus to Legless/β-catenin and not to histone H3

In Drosophila Pygopus (Pygo) and Legless (Lgs)/BCL9 are integral components of the nuclear Wnt/Wg signaling machine. Despite intense research, ideas that account for their mode of action remain speculative. One proposition, based on a recently discovered function of PHD fingers, is that Pygo, through its PHD, may decipher the histone code. This study found that human, but not Drosophila, Pygo robustly interacts with a histone-H3 peptide methylated at lysine-4. The different binding behavior is due to a single amino acid change that appears unique to Drosophilidae Pygo proteins. Rescue experiments with predicted histone binding mutants showed that in Drosophila the ability to bind histones is not essential. Further experiments with Pygo-Lgs fusions instead demonstrated that the crucial role of the PHD is to provide an interaction motif to bind Lgs. The results reveal an interesting evolutionary dichotomy in Pygo structure-function, as well as evidence underpinning the chain of adaptors model (Kessler, 2009).

This study has carefully tested the idea that histone binding contributes to the function of Pygo. The results demonstrate that in Drosophila the ability to bind H3K4me is not required for Pygo function. Instead the results suggest that the sole crucial function of the dPygoPHD finger is as a link in the chain of adaptors, which recruits the transcriptional activator potential of the NHD to Wg target genes (Kessler, 2009).

The findings do not exclude that in other species histone binding by the PygoPHD has a functional importance. Indeed it is tempting to speculate that the different behavior of mammalian and Drosophila Pygo with respect to histone binding accounts for their different requirement in vivo. In mice, where histone binding appears convincing, Pygo loss of function has a 'mild' phenotype. In Drosophila, where histone binding is apparently absent, Pygo has a positive role that is reflected in a strong loss of function phenotype. Since the effect of Pygo loss of function in other organisms has not been documented, it remains unclear if mice represent the rule and Drosophila the exception, or vice versa. If the conservation of the W in the K4 cage is an indication then Drosophila is the exception. One paradigm that would account for the observations is that Pygo has both positive and negative modes of action; histone binding providing the negative role. A possible mechanism for this model was suggested by Mosimann (2009): Pygo binds H3K4me via its PHD finger. By using BCL9 as adaptor it can retain β-catenin in the proximity of a Wingless response independently of TCF. Pygo-dependent tethering of β-catenin to methylated histones frees TCF to recruit co-repressors (e.g. Groucho and CtBP), which then counteract the β-catenin-nucleated activating chromatin remodeling processes. However, this mechanism relies on the existence of a Histone/Pygo/BCL9 complex, which the results suggest may not form (Kessler, 2009).

Whatever the case, the demonstration that dPygo’s role in Wg signaling can be reduced to the N-terminal Homology Domain (NHD) of Pygopus suggests that its primary positive function lies in this domain. The list of transcriptional complexes which interact with the NHD is small but growing, a trend which should continue. Via the interaction of the PHD with HD1 of Lgs this polygamy of interactions is targeted to Wnt target genes. In this way Pygo may act co-operatively with the C-terminus of Arm/β-catenin -- recruiting complexes required for Wg/Wnt target gene activation (Kessler, 2009).

Drosophila APC2 is a cytoskeletally-associated protein that regulates wingless signaling in the embryonic epidermis

Human APC (hAPC) and Drosophila Apc-like bind to ßcatenin (ßcat) and Armadillo (Arm), respectively. A test was performed to see whether Adenomatous polypopsis coli tumor suppressor homolog 2 (Apc2) also interacts with Arm in vivo. Arm was immunoprecipitated (IPed) from embryonic extracts, and, in parallel, proteins were IPed with anti-myc, a control mAb. Apc2 specifically co-IPs with Arm from both early and older embryos, but does not co-IP with the control anti-myc antibody. Arm could not be detected in anti-Apc2 IPs. Because the antigen for the Apc2 antisera includes the Arm binding region, these sera might not recognize an Apc2-Arm complex. An Apc2 fragment containing the putative ßcat binding sites co-IPs with ßcat when expressed in the human colorectal cancer cell line SW480 (McCartney, 1999).

The hAPC-ßcat interaction is direct, and is mediated by the 15 and 20 amino acid repeats of hAPC and the Arm repeats of ßcat; the analogous region of Drosophila Apc-like binds Arm (Hayashi, 1997). To test whether Apc2 directly interacts with Arm, the yeast two-hybrid system was used to examin whether the 15 and 20 amino acid repeats of Apc2 interact with the full set of Arm repeats of Arm (R1-13), or with the centralmost Arm repeats (R3-8; the binding site for Drosophila E-cadherin and dTCF). For comparison, the 15 and 20 amino acid repeats of Drosophila Apc-like were tested. The full 15 and 20 amino acid repeat regions of both Drosophila Apc-like and Apc2 strongly interact with the entire Arm repeat region and with R3-8. Thirty one and thirty four amino acid fragments carrying individual 15 or 20 amino acid repeats of dAPC and Apc2 (selected as good matches to the consensus) were also tested. Individual 15 amino acid repeats of either Drosophila Apc-like or Apc2 interact with both the entire Arm repeat region of Arm and with R3-8. An individual 20 amino acid repeat of Drosophila Apc-like also interacts with both Arm fragments. A single 20 amino acid repeat of Apc2 interacts strongly with Arm repeats 1-13; its interaction with R3-8 is much weaker (McCartney, 1999).

Since hAPC is phosphorylated, it was thought that various Drosophila Apc2 isoforms might be phosphorylation variants. To test this, Apc2 was immunoprecipitated (IPed) from embryos and the IPs were treated with protein phosphatase 2A (PP2A), a serine/threonine-specific phosphatase. PP2A treatment reduces the apparent molecular mass of Apc2; this effect is abolished if the PP2A inhibitor okadaic acid is included during incubation. Further, if embryonic cells are dissociated and incubated in tissue culture medium, the apparent molecular mass of Apc2 decreases; this effect is also abolished by okadaic acid, suggesting that it is mediated by endogenous phosphatases. Parallel alterations in Arm phosphorylation support this hypothesis. Taken together, these data suggest that the Apc2 isoforms reflect, at least in part, differential phosphorylation (McCartney, 1999).

Biochemical analyses suggest that Apc2 associates with the cell cortex. When 0 to 6 hour old embryos are fractionated into soluble (S100) and membrane-associated (P100) fractions, Apc2 partitions almost equally into these two fractions. In contrast, Arm is almost exclusively in the membrane fraction at this stage. The isoforms of Apc2 in the membrane fraction migrate more rapidly on SDS-PAGE than those in either the soluble fraction or the total cell lysate; because these isoforms are not detectable in total lysate, it is suspected that they may arise during fractionation by dephosphorylation. To examine whether Apc2 might associate with the membrane via a glycoprotein, Con A-Sepharose. Con A-Sepharose can be used to isolate membrane glycoproteins as well as proteins associated with them (e.g., Arm). A subset of Apc2 specifically binds to Con A in extracts from 0-6-h embryos. Thus, Apc2 may be anchored to the cortex via a transmembrane glycoprotein (McCartney. 1999).

Using a yeast two-hybrid screen for proteins that bind to Armadillo, the Drosophila beta-catenin homolog, a new Drosophila APC homolog, Apc2, has been identified. Apc2 also binds to Shaggy, the Drosophila GSK-3 homolog. Interference with Apc2 function produces embryonic phenotypes like those of shaggymutants. Interestingly, Apc2 is concentrated in apicolateral adhesive zones of epithelial cells, along with Armadillo and E-cadherin, which are both integral components of the adherens junctions in these zones. Various mutant conditions that cause dissociation of Apc2 from these zones also obliterate the segmental modulation of free Armadillo levels that is normally induced by Wingless signaling. It is proposed that the Armadillo-destabilizing protein complex, consisting of Apc2, Shaggy, and a third protein, Axin, is anchored in adhesive zones, and that Wingless signaling may inhibit the activity of this complex by causing dissociation of Apc2 from these zones (Yu, 1999).

Actin-dependent membrane association of a Drosophila epithelial APC protein and its effect on junctional Armadillo

The adenomatous polyposis coli (APC) protein is an important tumor suppressor in the colon. It promotes the destabilization of free cytoplasmic ß-catenin (the vertebrate homolog of the Drosophila protein Armadillo), a critical effector of the Wnt signaling pathway. The ß-catenin protein is also a component of adherens junctions, linking these to the actin cytoskeleton. The fruit fly has two APC genes: one encodes the ubiquitous E-APC (also known as dAPC2) and the other is mainly expressed in neuronal cells. In Drosophila epithelial cells, the ubiquitous form of APC, E-APC, is associated with adherens junctions. This association appears to be necessary for E-APC to function in destabilizing Armadillo. Using actin-depolymerizing drugs, it has been established that an intact actin cytoskeleton is required for the association of E-APC with adherens junctions in the Drosophila embryo. From an analysis of profilin mutants in which the actin cytoskeleton is disrupted, it was found that E-APC also requires actin filaments to associate with adhesive cell membranes in the ovary. Notably, conditions that delocalize E-APC from membranes, including a mutation in E-APC itself, cause partial detachment of Armadillo from adhesive membranes. It is concluded that actin filaments are continuously required for E-APC to be associated with junctional membranes. These filaments may serve as tracks for E-APC to reach the adherens junctions. The failure of E-APC to do so appears to affect the integrity of junctional complexes (Townsley, 2000).

The discovery of a link between Drosophila E-APC and the actin cytoskeleton contrasts with the work in vertebrate cells that uncovered a link between APC and microtubules. This may be explained as follows: (1) there may be genuine differences between APC proteins in their ability to utilize cytoskeletal elements. Notably, the carboxy-terminal third of human APC, which spans the microtubule-binding domain (but which, however, does not mediate tracking), is conserved in other vertebrate APCs, and is also found in the neuronal Drosophila APC, but is absent in E-APC. It is not known whether the neuronal Drosophila APC binds to or colocalizes with microtubules. (2) Evidence for the ability of vertebrate APC to utilize the actin cytoskeleton for its subcellular localization may have been missed so far. This could be because, in the vertebrate studies, cytochalasin D was used and its actin-depolymerizing effect is much weaker than that of latrunculin A. Indeed, there is a significant effect of latrunculin A on the subcellular distribution of human APC in transfected mammalian cells. Also, there may be a subtle effect of cytochalasin D on the subcellular distribution of APC in mammalian cells. (3) Perhaps most important, the cells in which the various APC proteins have been studied are substantially different from one another. The vertebrate work was carried out in migrating tissue culture cells whereas the Drosophila work has focused on stationary cells that adhere tightly to one another within tissues; these are cells that do not exhibit any obvious migratory behavior. Although human and mouse APC are associated with cell membranes in the intestinal epithelium, the requirement for this association is not known. Using a polarized tissue-culture cell model, it has been discovered that human APC associates in an actin-dependent way with the apical cell membrane compartment. Perhaps the mechanism mediating the fast transport of APC to, and the transient association with, distal sites in migrating cells is fundamentally different from the mechanism mediating its stable association with junctional membrane compartments in tissue. Microtubules may be more suitable for the former; actin filaments for the latter (Townsley, 2000).

In the embryo, the ability of E-APC to associate with junctional compartments appears to be critical for the destabilization of Armadillo, perhaps because the Armadillo-destabilizing Axin complex is localized in these apical compartments. The failure of E-APC to reach the Axin complex would explain the observed embryonic phenotypes that mimic stabilization of Armadillo; according to the shuttling model, this would result in a failure of E-APC to deliver Armadillo to this complex, and consequently in a failure of Armadillo to be earmarked by this complex for degradation. Ultimately, stabilized Armadillo would translocate into the nucleus and alter the transcription of TCF target genes (Townsley, 2000).

This work provides evidence that the failure of E-APC to associate with membranes may not only elicit an indirect nuclear response, but may also directly affect the junctional integrity of these membranes. The delocalization of junctional E-APC correlates with detachment of junctional Armadillo in three different situations: in chic mutant ovaries, in LMB-treated embryos and, most importantly, in E-APC mutant ovaries and embryos. Furthermore, a mild effect on junctional Armadillo has been observed in embryos in which E-APC is depleted by RNA interference. These observations indicate that the failure of E-APC to associate with junctional compartments may affect the junctional integrity. Ultimately, this would also affect the associated actin filaments, an expectation that is borne out by the observations in the E-APC mutants. In any case, the loss of Armadillo and actin filaments from cellular junctions appears to be a consequence of the failure of E-APC to associate with, or to reach, these junctions. This is consistent with the shuttling model, which ascribes a function to APC in shuttling Armadillo from the cytoplasmic to the junctional compartment, for incorporation into cadherin junctions. Note that this putative effect of the delocalized mutant E-APC on the junctional integrity might weaken the junctional anchorage of the Axin complex. This would thus aggravate further its own junctional delocalization, and the cytoplasmic Armadillo would accumulate to yet higher levels (Townsley, 2000).

The mild mutant phenotypes in E-APC mutant ovaries could indeed be due to failure of adhesion between germ cells. Adhesion mediated by E-cadherin and Armadillo is critical for normal shaping and positioning of the nurse cells and of the oocyte during oogenesis. Furthermore, oogenesis involves massive growth of the germ cells, and it is thus reasonable to assume that the adhesive junctional zones in the germ-cell membranes undergo considerable remodelling during oogenesis. The association of E-APC with these junctional membranes may therefore reflect a function of E-APC in the process of junctional growth and/or remodelling. Strong loss-of-function mutations of E-APC are required to establish whether this is the case (Townsley, 2000).

Generation of cell-fate diversity in Metazoan depends in part on asymmetric cell divisions in which cell-fate determinants are asymmetrically distributed in the mother cell and unequally partitioned between daughter cells. The polarization of the mother cell is a prerequisite to the unequal segregation of cell-fate determinants. In the Drosophila bristle lineage, two distinct mechanisms are known to define the axis of polarity of the pI and pIIb cells. Frizzled (Fz) signaling regulates the planar orientation of the pI division, while Inscuteable (Insc) directs the apical-basal polarity of the pIIb cell. The orientation of the asymmetric division of the pIIa cell is identical to the orientation of its mother cell, the pI cell, but, in contrast, is regulated by an unknown Insc- and Fz-independent mechanism. Drosophila E-Cadherin-Catenin (Shotgun-Armadillo) complexes are shown to localize at the cell contact between the two cells born from the asymmetric division of the pI cell. The mitotic spindle of the dividing pIIa cell rotates to line up with asymmetrically localized Shotgun-Armadillo complexes. While a complete loss of Shotgun function disrupts the apical-basal polarity of the epithelium, both a partial loss of Shotgun function and expression of a dominant-negative form of Shotgun affect the orientation of the pIIa division. Furthermore, expression of dominant-negative Shotgun also affects the position of Partner of Inscuteable (Pins) and Bazooka, two asymmetrically localized proteins known to regulate cell polarity. These results show that asymmetrically distributed Shotgun regulates the orientation of asymmetric cell division (Le Borgne, 2002).

The polar formation of junctional complexes close to the cytokinesis site could constitute a general mechanism to regulate the orientation of an asymmetric cell division relative to the axis of the previous division. For instance, in the Drosophila larval brain, each neuroblast divides asymmetrically in a stem-cell mode with a fixed orientation to generate a series of ganglion mother cells (GMCs), leading to the accumulation of GMCs on one side of the neuroblast. Arm and dAPC2, a Drosophila homolog of the Adenomatous Poliposis Coli protein, colocalize at the cell contact region between the neuroblast and its progeny GMCs. This study raises the possibility that, following the first round of neuroblast division, junctional complexes localizing specifically at the cell-cell contact between the neuroblast and its sister cell may orient the next neuroblast division (Le Borgne, 2002).

Echinoid is a component of adherens junctions that cooperates with DE-Cadherin to mediate cell adhesion

Echinoid is an immunoglobulin domain-containing transmembrane protein that modulates cell-cell signaling by Notch and the EGF receptors. In the Drosophila wing disc epithelium, Echinoid is a component of adherens junctions that cooperates with DE-Cadherin in cell adhesion. Echinoid and β-catenin (a DE-Cadherin interacting protein) each possess a C-terminal PDZ domain binding motif that binds to Bazooka/PAR-3; these motifs redundantly position Bazooka to adherens junctions. Echinoid also links to actin filaments by binding to Canoe/AF-6/afadin. Moreover, interfaces between Echinoid- and Echinoid+ cells, like those between DE-Cadherin- and DE-Cadherin+ cells, are deficient in adherens junctions and form actin cables. These characteristics probably facilitate the strong sorting behavior of cells that lack either of these cell-adhesion molecules. Finally, cells lacking either Echinoid or DE-Cadherin accumulate a high density of the reciprocal protein, further suggesting that Echinoid and DE-Cadherin play similar and complementary roles in cell adhesion (Wei, 2005).

Several observations prompted the study of Ed as a canonical CAM in the monolayered wing imaginal disc. Thus, mitotic recombination clones of cells mutant for the null allele ed1x5 exhibit rounded and smooth contours, in contrast to clones of wild-type cells that show wiggly shapes. This indicated that ed- /- cells have distinct adhesive properties and assort with themselves rather than with the surrounding ed+/- M+/- cells. (ed1x5 clones were M+, since without a growth advantage they hardly survive). It was also observed that Ed was absent from the membrane of the heterozygous cells that contacted the mutant cells, a finding consistent with the observation that Ed forms homophilic interactions and that these are required to incorporate/stabilize Ed at the cell membrane. Finally, Ed was found to localize basally to the apical marker Crb and apically to the basolateral marker Dlg. In fact, Ed colocalizes with both DE-Cad and Arm, and, therefore, it might be part of AJs. AJs are structures important for cell-cell contact and recognition. So, these results suggested that Ed plays a role in cell-cell adhesion (Wei, 2005).

Whether Ed affects components of AJs was examined by analyzing the localization of Arm within ed mutant clones. Arm strongly accumulates at the apical membranes of ed- /- cells, and these cells have a reduced apical surface. Both effects are clear in small clones, but cells within larger clones (over hundreds of cells) had both the density of Arm and the apical surface more similar to those of the wild-type cells. Similar observations were made with DE-Cad and Actin. It is suggested that the increased concentration of these molecules in small clones most probably results from the apical constriction as supported by the accumulation of nonmuscle myosin II, without a net per cell increment of these proteins. Alternatively, it could result from increased stability of these proteins. The apical constriction continued through the SJs and ended at the planes just below the GJs as revealed by an Innexin antibody. Hence, these ed- /- cells adopt a bottle shape. In contrast, the apposed ed- /- and ed+/- cells that form the border of the clone enlarge and adopte a rectangular shape. At this interface, the ed- /- cells often contacted the heterozygous cells by their long sides, as if in an attempt to minimize the number of cells that formed the interface (Wei, 2005).

Interestingly, Arm and DE-Cad, but not Actin, are depleted at the interface membrane of both small and large clones. This suggests that ed- /- and ed heterozygous cells discriminate one another and that AJs do not form properly in between them (Wei, 2005).

ed clones are surrounded by an Actin 'cable'. High-magnification images suggest that the cable is contained within the ed heterozygous cells surrounding the clone and that it is therefore generated by these cells. Several observations suggest that this Actin cable exerts a force. The cells surrounding an ed clone elongate toward the clone and accumulate nonmuscle myosin II at the interface membrane, as if attempting to cover the space exposed by the apically constricted ed- /- cells. This effect is reminiscent of the stretching of the leading-edge cells that will cover the underlying amnioserosa during dorsal closure of the embryos. In the wing disc, the boundary that separates the dorsal (D) and ventral (V) regions of the wing pouch has the shape of a smooth arc and contains an actin 'fence'. After the second instar, this boundary corresponds to a compartment border that imposes absolute restrictions to cell lineages. Large ed- /- clones close to or touching this boundary displace it toward the clones. In contrast, ed clones that straddle the boundary do not overtly distorted it, although the boundary could be less smooth within the clone. (Straddling clones might be originated before the compartment border was established or might be formed of D and V clones that fuse together). Moreover, the Actin cable surrounding the clones fuse with the Actin fence at the D/V boundary, suggesting that the distortion of this boundary is effected through this Actin linkage. Control ed+ M+ clones do not induce such distortions. These observations suggest that the Actin cable may contribute to the roundish shape of the ed clones and help confine their cells (Wei, 2005).

DE-Cad is a classical homophilic cell adhesion molecule of AJs. It interacts with β-catenin/Arm, which in turn binds α-catenin. Through the association between α-catenin and F-Actin, DE-Cad establishes links between cells that connect to the Actin cytoskeleton. This study shows that Ed is another CAM that, at the resolution of confocal microscopy, is also located at the AJs of imaginal disc cells. While cells in clones mutant for ed still seem to form normal AJs, the cells at the border of the clone seem impaired in forming them. It is hypothesized that this may help them segregate from surrounding ed+/- cells. Ed was identified as a binding partner for PDZ proteins that, similarly to Arm, helps localize Baz to AJs. Moreover, it was found that through the binding of Cno, Ed, like DE-Cad/β-catenin, may link to F-Actin. Hence, Ed has functions in cell-cell adhesion similar to those of DE-Cad (Wei, 2005).

The differential adhesion hypothesis proposes that cell sorting may be driven by differences in the quantity and/or quality of adhesive molecules displayed on the surface of cells. In keeping with this hypothesis, it was found that ed- /- cells sort out from ed+/- cells, as shown by the remarkably round shapes and smooth contours of the ed clones. Moreover, their differential adhesiveness is also manifest by the fusion of different ed clones to yield composite but still roundish clones. It is suggested that contraction of the apically enriched Actin network and of the actin cable surrounding the clone, possibly by interaction with nonmuscle myosin II also present there, may contribute to the the apical constriction of the ed- /- cells. It was also observed that the interface between ed+/- and ed- /- cells is depleted of DE-Cad, Arm and Baz, besides completely lacking Ed. This strongly suggests that this interface is deficient in AJs and probably helps to insulate ed- /- cells from the surrounding ed heterozygous cells. It is hypothesized that this deficiency of AJs, which may reduce adhesion between ed+/- and ed- /- cells, and the inward-pulling force generated by apical constriction and the actin cable may help create the smooth and rounded contour of the clones at the level of AJs. At the plane of SJs, the clonal boundary is not as smooth. This may be due to the presence of normal levels of SJs, since seemingly wild-type amounts of Dlg were detected at the interface membrane. Normal levels of SJs may allow the clones to remain integrated in the epithelium. It is stressed that when ed clones grow large, the apical constriction disappears, suggesting that the forces responsible for this constriction become insufficient or no longer operate. If the force is exerted, at least in part, by the Actin cable surrounding the clone, as in a purse-string mechanism, it would make sense that this force becomes ineffectual as the number of cells within the clone increases. Remarkably, these differences of apical cell constriction observed in small and large ed clones have a correlate on the adult wing blade: small clones display an increased density of trichomes, implying that their cells are small or more tightly packed, whereas large clones have cells of normal size. This indicates that the apical constriction is retained through imaginal disc eversion, when the disc epithelium changes from columnar to planar (Wei, 2005).

In the embryonic epithelium, Baz, localized to both AJs and the marginal zone, is the initial apical regulator. How is Baz recruited to the apical domain? In the follicular epithelium, Baz is localized to this domain through lateral exclusion mediated by PAR-1/14-3-3 and apical anchoring by Crb/Sdt/Patj. The data support an additional mechanism to localize Baz to the apical domain. Both Ed and Arm can bind Baz through their C-terminal PDZ binding motif and therefore they may redundantly localize Baz to AJs. Indeed, the localization of Baz to AJs is relatively normal in the absence of either one. Most Baz is lost only when both Arm and Ed are depleted, as occurs at the interface membrane of ed clones or in large shg clones where Ed gradually breaks down. In the latter case, there is good colocalization between Baz and the sites maintaining residual Ed. It is suggested that in the epithelium of the wing disc, Baz localizes to AJs by the combined effects of its binding to Ed/Arm and the lateral exclusion of PAR-1/14-3-3. Additionally, apical anchoring of Baz may be mediated by direct association between the Baz and Crb apical complexes. During early embyogenesis, Ed is also present at pseudocleavage furrows. This observation, together with the ability of Ed to localize Baz to AJs, may explain the finding that during cellularization, Baz can accumulate apically in the absence of Arm. Ed also binds to the PDZ domain of Cno and mediates its localization to AJs, where Cno interacts with F-Actin either directly or indirectly through the association with Polychaetoid/ZO-1. Interestingly, the evolutionally conserved EIIV domain of Ed binds Baz and Cno in a mutually exclusive manner. Thus, the concentrations of and differential affinities between Ed, Baz, and Cno should determine their dynamic equilibrium at AJs (Wei, 2005).

Although Baz is critical to form AJs in the blastoderm and in the follicular epithelium, removal of Baz (or Par-6) from cells of the wing disc does not affect the localization of DE-Cad or Ed to AJs. This is consistent with the report that in imaginal discs, Baz does not affect the localization of DE-Cad and Dlg but is required for the asymmetric localization of cell fate determinants. Together, these results suggest that in wing discs, the Baz complex is not critical for the formation of AJs, and that the effect of the loss of Ed on AJs formation/maintenance is not due to Baz depletion (Wei, 2005).

Several similarities between the roles of DE-Cad and Ed in the wing disc epithelium are worth noting. Both Ed and DE-Cad are CAMs that establish homophilic interactions and localize to AJs. The absence of either Ed or of DE-Cad in cells of small clones causes their apical constriction and strong segregation from wild-type cells, giving rise to smooth round borders. In both cases, the mutant cells are impaired in forming AJs with neighboring wild-type or heterozygous cells and are surrounded by an Actin cable. Ed interacts with Cno, and DE-Cad with Arm, and both Cno and Arm directly or indirectly associate with F-Actin. Thus, Ed and DE-Cad represent two distinct classes of CAMs, with widely different chemical compositions, that connect to F-Actin, contribute to cell adhesion in the wing disc, and seem to have partially overlapping functions (Wei, 2005).

In contrast, DE-Cad and Ed differ in their ability to regulate the apical/basal cell polarity. Ed affects components of AJs, but not those of the apical Crb and the basolateral Dlg complexes. In contrast, DE-Cadherin is necessary for Crb localization, but similarly to Ed, it is not required for Dlg localization. Furthermore, the maintenance of Ed at AJs requires DE-Cad. In contrast, localization of DE-Cad to AJs is independent of Ed. Interestingly, the DE-Cad/Arm complex is not essential for the formation of the follicular epithelium, but upon removal of this complex, the integrity of the epithelium is lost slowly over the period of several days. This suggests that other molecules may be maintaining the epithelial structure. During stages 1 to 10 of oogenesis Ed is mainly expressed in the follicle cells, and these cells, if mutant for ed, show at low frequency a multilayered structure with disrupted expression of some polarity markers. Thus, it will be of interest to elucidate whether, in this epithelium, Ed and DE-Cad/Arm also play partially redundant roles in cell adhesion and apical/basal polarity. While both Ed and DE-Cad contribute to cell adhesion and recognition, it is unclear whether each molecule imparts specific recognition properties to cells, so that the final cell-cell affinity results from the sum of distinct affinities mediated by these different CAMs. More specifically, can an increased level (density) of DE-Cad replace the absence of Ed? The results showing that ed- /- cells, with either normal levels (in large clones) or high density (in small clones) of DE-Cad, do not intermix with wild-type cells suggests that the binding specificity provided by a given CAM is not overruled by a higher level (density) of a different CAM. Moreover, the cell sorting properties conferred by Ed cannot account for the separation of cells at both sides of the A/P compartment boundary of the wing disc because A and P cells do not intermingle within composite ed, smo double mutant clones. (Similarly, DE-Cad is not responsible for the sorting out of A and P cells. Hence, cell-cell adhesion in the wing disc appears to depend on multiple CAMs (Ed, DE-Cad, etc.), each imparting specific cell recognition properties. Although Ed and its C-terminal EIIV motif are conserved in invertebrates, no clear vertebrate homolog with 7 Ig domains and a PDZ domain binding motif has been found. Nectin1-4 comprises a family of 3 Ig domain-containing CAM that have several differentially spliced forms and localize to AJs. Most spliced forms share a conserved C-terminal E/A-X-Y-V that binds the PDZ domain of Afadin. Moreover, this motif also interacts with Par-3, the vertebrate homolog of Baz. In spite of these similarities, overexpression of either nectin 1-α or 3-α does not rescue the remarkable clonal phenotype of ed (Wei, 2005).

Regulation of Armadillo nuclear import

Drosophila Armadillo plays two distinct roles during development. It is a component of adherens junctions, and functions as a transcriptional activator in response to Wingless signaling. In the current model, Wingless signal causes stabilization of cytoplasmic Armadillo allowing it to enter the nucleus where it can activate transcription. However, the mechanism of nuclear import and export remains to be elucidated. Two gain-of-function alleles of Armadillo are shown to activate Wingless signaling by different mechanisms. The S10 allele localizes to the nucleus, where it activates transcription. In contrast, the DeltaArm allele localizes to the plasma membrane, and forces endogenous Arm into the nucleus. Therefore, DeltaArm is dependent on the presence of a functional endogenous allele of arm to activate transcription. DeltaArm may function by titrating Axin protein to the membrane, suggesting that Axin acts as a cytoplasmic anchor keeping Arm out of the nucleus. In axin mutants, Arm is localized to the nuclei. Nuclear retention is dependent on dTCF/Pangolin. This suggests that cellular distribution of Arm is controlled by an anchoring system, where various nuclear and cytoplasmic binding partners determine its localization (Tolwinski, 2001).

Evidence is provided for the titration model, but focus is on potential cytoplasmic anchors that retain ß-catenin/Arm in the cytoplasm. Endogenous Arm accumulates in the nucleus in response to expression of DeltaArm, and the underlying mechanism appears to be independent of protein levels. DeltaArm functions downstream of zw3, and does not increase endogenous protein levels appreciably. These results point to a mechanism by which DeltaArm affects some component of the cytoplasmic retention machinery. axin may be this component, since its mutation leads to nuclear Arm accumulation, and its overexpression prevents it. Axin appears to be amenable to a titration model, because its function is highly dose dependent. Only maternal mutation of axin leads to a naked cuticle with a partial rescue by a paternal copy. Zygotic mutation doesn’t produce an embryonic phenotype. Overexpression leads to a wg phenotype only if expressed very early. Observations in tissue culture show that Axin is localized to the cytoplasmic membrane and the cytoplasm, but is excluded from the nucleus. Also, mutant forms of Arm lacking repeats that are required for Axin binding localize to the nucleus. Therefore, a model is favored in which DeltaArm directly titrates out Axin, leading to nuclear localization of endogenous Arm. DeltaArm retains arm repeats 3 through 8, shown to be required for Axin binding, and may sequester Axin away from endogenous Arm. This suggests a dual role for Axin, both as a scaffold for degradation and as a component of the cytoplasmic retention machinery (Tolwinski, 2001).

Nuclear import of Armadillo/ß-catenin is crucial for activation of the transcriptional response to Wg signaling. Wg stabilizes cytoplasmic pools of Arm/ß-catenin that must subsequently be imported into the nucleus to activate Wg targets. The mechanism of Arm/ß-catenin stabilization has been studied extensively, but the understanding of nuclear import of Arm/ß-catenin remains vague. Studies have shown that ß-catenin nuclear import is independent of importinß/ß-karyopherin; instead, it depends on the direct interaction of the central Armadillo (Arm) repeats to the nuclear pore complex. ß-catenin contains 12 tandem Arm repeats that are necessary and sufficient for nuclear accumulation. Arm repeats are fundamentally similar to the HEAT repeats of importinß/ß-karyopherin, suggesting that ß-catenin may interact directly with the pore complex as does importinß/ß-karyopherin. Indeed, ß-catenin binds directly to a yeast nucleoporin, Nup1. These studies suggest that ß-catenin does not use the standard NLS/importin dependent import pathway, but instead supplies an importin-like activity itself (Tolwinski, 2001).

Studies have found that ß-catenin import is constitutive. They suggest a system of cytoplasmic and nuclear anchors that control the flow of ß-catenin into and out of the nucleus. However, prevention of import by cytoplasmic anchoring may be the regulated step, since export is probably controlled by APC. In resting cells, ß-catenin is observed mostly at the cell membrane, therefore it seems likely that localization of ß-catenin to this compartment prevents it from entering the nucleus. Axin has been observed to localize to the plasma membrane, as well as the cytoplasm, and is thus well positioned to function as an anchor. A strong nuclear localization of Arm is observed in experiments where no Axin protein is present. In contrast, overexpressed Axin prevents the nuclear accumulation of Arm normally associated with DeltaArm expression (Tolwinski, 2001).

Since Arm import and export have been reported to be highly dynamic, a second mechanism must be in place to retain the imported Arm within the nucleus. One possibility is that dTCF/Pan anchors nuclear Arm to the DNA. By expressing a dominant negative form of TCF that interacts with DNA but no longer binds Arm, the nuclear accumulation observed following DeltaArm expression alone is blocked. Overexpressed dTCFDeltaN may occupy many of the DNA binding sites that Arm normally uses to stay in the nucleus, making it susceptible to export. Expression of dTCFDeltaN does not lead to complete exclusion of endogenous Arm from the nucleus, suggesting that there may be more relevant nuclear factors, possibly groucho. Overexpression of full-length dTCF does not lead to nuclear accumulation of endogenous Arm, suggesting that dTCF levels are not limiting. This is consistent with overexpression of dTCF having only a very subtle cuticle phenotype. However, overexpression of LEF-1 (a mammalian homolog of dTCF) in tissue culture cells, does lead to nuclear accumulation of ß-catenin. This was not observed in Drosophila embryos, suggesting that limiting levels of nuclear anchor may be a feature of specific cell types that have yet to be observed in Drosophila (Tolwinski, 2001).

A model is favored where the dynamic import and export of Arm is controlled by binding partners in the cytoplasm and the nucleus. Axin is involved in cytoplasmic anchoring, and dTCF/Pan is involved in nuclear retention. Arm retained in the cytoplasm is degraded unless it enters adherens junctions. In response to Wg, degradation stops, and Arm accumulates in the cytoplasm bound to Axin. Some Arm enters the nucleus where it binds dTCF/Pan. An equilibrium is reached as a result of active import and export, and inactive degradation. This is the situation in Arm stripes where diffuse staining throughout the cell is observed. However, the existence of anchoring offers a second level of signaling control that could induce a rapid and concentrated nuclear accumulation of Arm with no change in levels. Specific nuclear accumulation has been observed in Xenopus and sea urchin. Though levels were not measured, the striking lack of cytoplasmic ß-catenin is suggestive of a lack of cytoplasmic anchoring. Another response of this type may be what is observed in the epithelial to mesenchyme transition. Here, ILK is overexpressed in epithelial cells resulting in very high nuclear accumulation of ß-catenin without an increase in levels, suggesting the possibility of inhibition of cytoplasmic anchoring (Tolwinski, 2001).

Recently, two studies have suggested that APC is involved in the nuclear export of Arm/ß-catenin. APC contains a nuclear export signal (NES) which is required for efficient export of ß-catenin from the nucleus. Combining this result with the current data, it is proposed that there are at least two levels of control of Arm/ß-catenin localization involving cytoplasmic anchoring and active export. APC may play a role in preventing Arm/ß-catenin from accumulating in the nucleus due to dTCF binding. Both controls must be overcome to accumulate enough Arm/ß-catenin to activate transcription (Tolwinski, 2001).

ß-Catenin is the nuclear effector of the Wnt signaling cascade. The mechanism by which nuclear activity of ß-catenin is regulated is not well defined. Therefore, the nuclear marker RanGTP was used to screen for novel nuclear ß-catenin binding proteins. A cofactor of chromosome region maintenance 1 (CRM1)-mediated nuclear export, Ran binding protein 3 (RanBP3), was identified as a novel ß-catenin-interacting protein that binds directly to ß-catenin in a RanGTP-stimulated manner. RanBP3 inhibits ß-catenin-mediated transcriptional activation in both Wnt1- and ß-catenin-stimulated human cells. In Xenopus laevis embryos, RanBP3 interferes with ß-catenin-induced dorsoventral axis formation. Furthermore, RanBP3 depletion stimulates the Wnt pathway in both human cells and Drosophila melanogaster embryos. In human cells, this is accompanied by an increase of dephosphorylated ß-catenin in the nucleus. Conversely, overexpression of RanBP3 leads to a shift of active ß-catenin toward the cytoplasm. Modulation of ß-catenin activity and localization by RanBP3 is independent of adenomatous polyposis coli protein and CRM1. It is concluded that RanBP3 is a direct export enhancer for ß-catenin, independent of its role as a CRM1-associated nuclear export cofactor (Hendriksen, 2005).

The Drosophila RanBP3 homologue was identifed and RNAi was used to study its role in Drosophila development. At the end of embryogenesis, the ventral epidermis is covered by a cuticle that is built up by a repeating pattern of naked cuticle and denticles. Wingless signaling increases levels of Arm (ß-catenin) that specifies the fate of epidermal cells responsible for secreting naked cuticle. Therefore, loss of wg expression results in an embryo that is covered with denticles lacking naked cuticle and overexpression of wg results in a naked cuticle embryo. Likewise, loss of an inhibitor of Wnt signaling also results in naked cuticle embryos as shown by RNAi against Drosophila Axin. As a control, embryos were injected with ß-galactosidase double-stranded RNA (dsRNA), and the majority (97%) developed into larvae that were indistinguishable from noninjected wt larvae. 3% of these control embryos showed some very weak effects on denticle belt formation. RNAi against Axin resulted in a significant increase in naked cuticle phenotype in 24% of the Axin dsRNA-injected embryos, with phenotypes varying from partial loss of denticles to completely naked embryos. Injection of dsRNA against the Drosophila RanBP3 caused a partial or complete transformation of denticles into naked cuticle in 14% of the embryos. The most severe phenotypes of the RanBP3 RNAi embryos showed deformation of both the head and spiracles, resembling Axin RNAi. In addition, almost all RanBP3 RNAi embryos showing a strong naked cuticle phenotype were shorter than the embryos injected with Daxin dsRNA. To confirm that the RanBP3 dsRNA injections resulted in decreased RanBP3 levels, RT-PCR was performed on buffer and RanBP3 dsRNA-injected embryos. RanBP3 mRNA levels were indeed decreased in RanBP3 dsRNA-injected embryos, whereas RP49 control mRNA levels remained unaffected. The effects of RanBP3 dsRNA injection were assayed on wg target gene induction. For this, stage 10 RanBP3 or Daxin dsRNA-injected embryos were stained with anti-Engrailed antibody. Normal engrailed expression is present in segmental stripes that are two cells wide. Removal of the Wnt signaling inhibitor Axin by dsRNA injection resulted in a broader Engrailed expression pattern that extended from two to four rows of cells. In RanBP3 dsRNA-injected embryos, Engrailed expression expanded by one row of cells. These in vivo data show that removal of RanBP3 leads to a phenotype that is associated with Wnt signaling activation, suggesting that RanBP3 also acts as negative regulator of Wnt signaling in Drosophila. In conclusion, this study identified an unexpected role for RanBP3 as a novel inhibitor of Wnt signaling that enhances nuclear export of active ß-catenin. This function is separate from its role in CRM1-mediated nuclear export. The structural similarities between CRM1 and ß-catenin suggest that RanBP3 may be a more general cofactor for nuclear export of ARM repeat proteins (Hendriksen, 2005).

Casein kinase I phosphorylates the Armadillo protein and induces its degradation in Drosophila

Casein kinase I (CKI) is a positive regulator of Wnt signaling in vertebrates and Caenorhabditis elegans. To elucidate the function of Drosophila CKI in the wingless pathway, CKI was disrupted by double-stranded RNA-mediated interference (RNAi). While previous findings were mainly based on CKI overexpression, this is the first convincing loss-of-function analysis of CKI. Surprisingly, CKIalpha- or CKIepsilon-RNAi markedly elevates Armadillo (Arm) protein levels in Drosophila Schneider S2R+ cells, without affecting Arm mRNA levels. Pulse-chase analysis showed that CKI-RNAi stabilizes Arm protein. Moreover, Drosophila embryos injected with CKIalpha double-stranded RNA showed a naked cuticle phenotype, which is associated with activation of Wg signaling. These results indicate that CKI functions as a negative regulator of Wg/Arm signaling. Overexpression of CKIalpha induces hyper-phosphorylation of both Arm and Dishevelled in S2R+ cells and, conversely, CKIalpha-RNAi reduces the amount of hyper-modified forms. His-tagged Arm is phosphorylated by CKIalpha in vitro on a set of serine and threonine residues that are also phosphorylated by Zeste-white 3. Thus, it is proposed that CKI phosphorylates Arm and stimulates its degradation (Yanagawa, 2002).

Since loss-of-function studies are the key to revealing the actual function of Drosophila CKI in the Wg pathway, RNAi was used to disrupt the CKI gene expression in Drosophila Schneider S2R+ cells. S2R+ cells were cultured in the presence of double-stranded (ds)RNA for CKIalpha, CKIepsilon, alpha-Catenin, casein kinase II catalytic (alpha) subunit (CKII-alpha) or LacZ for 3 days and then the protein levels in the cell lysates were analyzed by Western blotting. Addition of dsRNA for CKIepsilon, alpha-Catenin and CKII-alpha causes a selective decrease in the corresponding proteins. While previous studies with Xenopus, Caenorhabditis elegans and mammalian systems have reported that CKI is a positive regulator of Wnt signaling, both CKIalpha- and CKIepsilon-RNAi markedly elevate Arm protein levels, suggesting that CKI functions as a negative regulator of Arm protein in Drosophila. Since CKIalpha-RNAi induces higher levels of Arm protein accumulation than CKIepsilon-RNAi, CKIalpha was mainly used for subsequent analyses (Yanagawa, 2002).

To search for the sequence in Arm that responds to CKIalpha-RNAi, stable S2R+ cell lines expressing wild-type and various mutant forms of myc-tagged Arm were established and the effects of CKIalpha-RNAi on accumulation of these Arm mutant proteins were examined by Western blotting. Similar to endogenous Arm, wild-type Arm with the myc-tag is markedly stabilized by CKIalpha-RNAi. Since phosphorylation of Arm at the N-terminus is known to determine its stability, Arm mutants lacking the N-terminal 58 or 138 amino acids were analyzed. These two mutants, which are more stable than the wild-type, no longer respond to CKIalpha-RNAi, indicating that the target sequence for CKIalpha-RNAi resides in the N-terminal 58 amino acids. Therefore, a series of N-terminal mutants was made. In the serine/threonine to alanine mutant, the Ser and Thr residues originally identified as phosphorylation target sites for ZW3 (S at codon 44, 48, 56 and T at 52) were changed to Ala. In S56A and S58A, the Ser at 56 and 58, respectively, was changed to Ala. In the ED to QN mutant, a stretch of acidic amino acids (E and D) was replaced with Q and N (E at 61, 63, 64, 66 to Q and D at 62 to N). This mutant was produced because CKI is known to phosphorylate a Ser or Thr residue close to the acidic residues and this stretch of acidic amino acids is also conserved in ß-catenin and plakoglobin (Yanagawa, 2002).

Analyses with this series of Arm mutants has revealed that protein levels of the S58A mutant are somewhat elevated even without CKI-RNAi, but this mutant responds to CKI-RNAi similarly to the wild-type Arm, while, the S56A mutant responds slightly less than the wild-type Arm. The S/T to A mutant no longer responds to CKIalpha-RNAi, while the ED to QN mutant response is much weaker than that of the wild-type Arm. These results suggest that CKIalpha directly or indirectly stimulates phosphorylation of Ser44, 48 and 56, as well as Thr52, thereby destabilizing Arm and that the stretch of acidic amino acids may facilitate this process. If so, the ED to QN mutant would be expected to be more stable than the wild-type Arm. Hence, the stabilities of the wild-type, S/T to A, S56A and ED to QN forms of Arm were compared. The S/T to A mutant is the most stable, with the S56A mutant second. The ED to QN mutant is more stable than the wild-type Arm, but less stable than the S/T to A mutant (Yanagawa, 2002).

Next to be examined was whether CKIalpha directly phosphorylates a set of Ser and Thr residues in the N-terminal region of Arm phosphorylated by ZW3. The results indicate that phosphorylation sites for CKIalpha are Ser44, 48 and 56, as well as Thr52 residues (among these, S56 seems to be the major phosphorylation site, whose phosphorylation affects those of the other three sites). A cluster of acidic amino acids is also required for this phosphorylation. The cluster of acidic amino acids described above is conserved in ß-catenin (amino acid sequence from 53 to 58: EEEDVD). Notably, mutations in this region have been reported in tumors. Of 37 independent anaplastic thyroid carcinoma samples, four had mutations. One hepatoblastoma has been reported that had a 42 base pair deletion in ß-catenin exon 3, which leads to deletion of amino acids from S45 to D58. Clearly, CKI mutations in certain tumors remain to be explored (Yanagawa, 2002).

Drosophila Twins regulates Armadillo levels in response to Wg/Wnt signal

Protein Phosphatase 2A (PP2A) has a heterotrimeric-subunit structure, consisting of a core dimer of ~36 kDa catalytic and ~65 kDa scaffold subunits complexed to a third variable regulatory subunit. Several studies have implicated PP2A in Wg/Wnt signaling. However, reports on the precise nature of the PP2A role in Wg/Wnt pathway in different organisms are conflicting. twins (tws), which codes for the B/PR55 regulatory subunit of PP2A in Drosophila, is shown to be a positive regulator of Wg/Wnt signaling. In tws- wing discs both short- and long-range targets of Wingless morphogen are downregulated. Analyses of tws- mitotic clones suggest that requirement of Tws in Wingless pathway is cell-autonomous. Epistatic genetic studies indicate that Tws functions downstream of Dishevelled and upstream of Sgg and Armadillo. These results suggest that Tws is required for the stabilization of Armadillo/ß-catenin in response to Wg/Wnt signaling. Interestingly, overexpression of, otherwise normal, Tws protein induces dominant-negative phenotypes. The conflicting reports on the role of PP2A in Wg/Wnt signaling could be due to the dominant-negative effect caused by the overexpression of one of the subunits (Bajpai, 2004).

Results of these studies show that Twins is involved in modifying Wg signaling. Partial to complete downregulation of short- (Ct and Sca) and long-range (Dll and Vg) targets of Wg pathway is observed in tws- background. The downregulation of Wg signaling in wing discs is reflected in adult phenotypes, such as serrated wing margin in mitotic clones of tws. Loss-of-Wg phenotypes (induced by the overexpression of DN-TCF/pan or Sgg or Cadintra) are enhanced in tws heterozygous mutant background. In addition, mutation in tws suppresses the phenotypes induced by Dsh, a positive component of Wg signaling. Finally, some of the phenotypes induced by the overexpression of Tws are characteristic of gain-of-Wg phenotypes. These results suggest that Tws functions as a positive regulator of Wg signaling (Bajpai, 2004).

Overexpression of otherwise normal Tws protein induces dominant-negative phenotypes. The dominant-negative phenotype is unlikely to be neomorphic or antimorphic, since UAS-Tws rescues tws alleles (at the levels of both Wingless-dependent and independent developmental events) and also induces gain-of-Wg phenotypes. The dominant-negative phenotype is probably due to imbalance in the relative amounts of the three subunits in the heterotrimeric complex, proper formation of which is obligatory for PP2A function. Thus, the conflicting reports on the role of PP2A in Wnt signaling could be due to the dominant negative effect caused by the overexpression of one of the subunits (Bajpai, 2004).

In tws mutant background, cytoplasmic Arm levels are downregulated. Even overexpressed Arm is degraded in tws- background. Furthermore, loss of tws had no effect on the degradation-resistant form of Arm, which suggests that Tws functions upstream of Arm to mediate Wg signaling. These results could not be confirmed directly by Western blotting, since only a very small fraction (such as DV cells) of wing disc shows changes in Arm levels in response to Wg signaling. Nevertheless, results presented in this report suggest that stabilization of the cytoplasmic form of Arm by Wg signaling is dependent on Tws function (Bajpai, 2004).

A dominant-negative form of Sgg/GSK-3ß is able to rescue tws- phenotype at the level of Dll expression. However, overexpression of Dsh failed to rescue Dll expression in tws- discs, suggesting that Tws functions downstream of Dsh and upstream of Sgg to stabilize cytoplasmic Arm in response to Wg signaling. Preliminary results presented here suggest that function of Tws in Wg pathway is inactivation of Sgg. Normally, overexpressed APC sequesters Arm only in those cells in which Sgg activity is downregulated. In other cells, APC participates in Arm-degradation machinery. In tws- wing discs, overexpressed APC fails to sequester Arm in DV cells, suggesting that loss of tws results in upregulation of Sgg activity. However, it has been reported that PR/B56epsilon functions upstream of Dsh to regulate Wnt signaling in Xenopus embryos. The PR/B56epsilon homolog in Drosophila is widerborst (with 80% identity at the protein level), which is involved in the determination of planar cell polarity. widerborst is also known to be functional upstream of Dsh, but not in the canonical Wg/Wnt pathway. Although Tws homologs in other organisms have not been well characterized, the current studies are consistent with a role for PP2A in dephosphorylation of Axin (Bajpai, 2004).

The next question regards the substrate of PP2A function in the Wg pathway. In mammalian cells, Axin is dephosphorylated in response to Wnt signaling. Furthermore, dephosphorylated Axin binds ß-catenin less efficiently than the phosphorylated form. Thus, dephosphorylation of Axin would free ß-catenin from the degradation machinery. Thus, Tws may function by inhibiting the activity of Axin, which acts a scaffold protein to bring Sgg and Arm to close proximity. Further biochemical work is in progress to determine phosphorylated status of Arm in tws- background and to determine if Tws directly binds to Sgg or Axin or both (Bajpai, 2004).

The APC tumor suppressor binds to C-terminal binding protein to divert nuclear ß-Catenin from TCF

Adenomatous polyposis coli (APC) is an important tumor suppressor in the colon. APC antagonizes the transcriptional activity of the Wnt effector ß-catenin by promoting its nuclear export and its proteasomal destruction in the cytoplasm. This study reports a third function of APC in antagonizing ß-catenin involving C-terminal binding protein (CtBP). APC is associated with CtBP in vivo and binds to CtBP in vitro through its conserved 15 amino acid repeats. Failure of this association results in elevated levels of ß-catenin/TCF complexes and of TCF-mediated transcription. Notably, CtBP is neither associated with TCF in vivo nor does mutation of the CtBP binding motifs in TCF-4 alter its transcriptional activity. This questions the idea that CtBP is a direct corepressor of TCF. The evidence indicates that APC is an adaptor between ß-catenin and CtBP and that CtBP lowers the availability of free nuclear ß-catenin for binding to TCF by sequestering APC/ß-catenin complexes (Hamada, 2004).

To identify proteins that bind to APC in Drosophila embryos, crude embryonic extracts were incubated with bacterially expressed Drosophila E-APC fused to glutathione-S-transferase (GST). Analysis of associated proteins by MALDI mass spectrometry reveals dCtBP as an unexpected binding partner of E-APC. CtBP was initially discovered as a cellular protein binding to the C terminus of the adenovirus E1A protein, which suppresses its transformation potential. CtBP is a transcriptional corepressor in mammals and binds to various DNA binding proteins via a short conserved motif P-h-D-L-S-x-R/K. Mammals have a second CtBP relative, CtBP2, which also recognizes this motif and whose function overlaps that of CtBP (Hamada, 2004).

Intriguingly, a motif similar to P-h-D-L-S-x-R/K is found in each of the 15 amino acid repeats (15R) of APC and of Drosophila E-APC. These repeats can bind to ß-catenin but cannot promote its proteasomal destruction; the latter requires the Axin binding motifs of APC. Therefore, there is no known function of the 15Rs in the downregulation of ß-catenin. The interaction between an individual 15R and ß-catenin has been characterized at the structural level. The presumed CtBP binding motif shares some but not all of the residues in the C-terminal half of the 15R that are engaged in the interaction with ß-catenin (Hamada, 2004).

Binding between E-APC and dCtBP was confirmed in vitro by pull-down assays between bacterially expressed GST-dCtBP and in vitro translated E-APC. This binding is comparable to that between E-APC and Armadillo (Drosophila ß-catenin); however, Armadillo does not bind directly to GST-dCtBP. A small region spanning the two 15Rs of E-APC fused to GST is sufficient for binding to in vitro translated dCtBP, while a triple alanine substitution ('AxAxA') in the P-h-D-L-S motif of each 15R (in the context of the C-terminal half of E-APC) almost completely abolishes binding to dCtBP. The same is true for the binding between human CtBP and a central fragment of APC (residues 918-1698) that binds efficiently to GST-CtBP, while its mutant version AxAxA binds poorly. APC(918-1698) contains two further putative CtBP binding motifs that were substituted in addition ('AxAxAplus'). This further reduced the binding to GST-CtBP (by >16%); no binding whatsoever was detectable with a GST-LEF-1 control. Importantly, both APC mutants bind to ß-catenin equally well as the wild-type. Likewise, both mutants retain the ability to reduce the overall levels of coexpressed HA-tagged ß-catenin in transfected APC mutant cancer cells, though a low level of endogenous ß-catenin can still be detected by immunofluoresence in these transfected cells. Thus, the binding between APC and CtBP is specific and conserved and neither appears to affect APC's binding to ß-catenin nor its ability to promote the destruction of cytoplasmic ß-catenin (Hamada, 2004).

APC is also associated with CtBP in mammalian cells: endogenous CtBP can be coimmunoprecipitated with endogenous APC, and vice versa, in 293T cells and in HCT116 colorectal cancer cells that express wild-type APC. Furthermore, in APC mutant cancer cells, the resident APC truncations can be coimmunoprecipitated in SW480 cells, but not in COLO320 cells. Notably, the 15Rs are retained only in the APC truncation of the former, but not of the latter. Thus, the association of APC with CtBP in mammalian cells depends on its 15Rs (Hamada, 2004).

Few colorectal carcinomas express APC truncations that lack the 15Rs. COLO320 is one of the rare colorectal cancer cell line of this type. Interestingly, this line exhibits exceptionally high TCF-mediated transcription. This suggests that the 15Rs may harbor an activity that is critical for the downregulation of the transcriptional activity of TCF (Hamada, 2004).

To test whether the binding of CtBP to the 15Rs is functionally relevant, a complementation assay was used of APC mutant cancer cells based on a luciferase reporter linked to TCF binding sites (pTOPFLASH). This quantitative assay is highly specific for TCF-mediated transcription and serves as a fairly direct readout of exogenous APC function in restoring low levels of TCF transcription. COLO320 cells show very high TOPFLASH values, >2× higher than those of SW480 cells and up to 5× higher than those of other APC mutant colorectal cancer cells. These values are reduced substantially after cotransfection with APC(918-1698), which spans the 15Rs and the 5'-most nuclear export signal (NES1506) and Axin binding site. Similar APC fragments have previously been found to efficiently reduce the ß-catenin levels in SW480 cells. In contrast, the AxAxA mutant is less active in reducing TOPFLASH values, and AxAxAplus is even less active. The control values of pFOPFLASH (containing mutant TCF sites) are low and unchanged by the mutants. It is concluded that the binding between APC and CtBP is critical for the APC-mediated downregulation of the transcriptional activity of ß-catenin. The residual activities of AxAxA and AxAxAplus in this assay are likely to reflect their ability to promote Axin-mediated destruction and nuclear export of ß-catenin; note that APC(918-1698) and its mutant versions shuttle in and out of the nucleus, as judged by their nuclear accumulation after exposure to leptomycin B (Hamada, 2004).

Evidence has indicated that APC can sequester nuclear ß-catenin and keep it from binding to TCF and activating transcription. This sequestration can be demonstrated experimentally if an APC fragment is targeted to the nucleus by linkage to a nuclear localization signal (NLS): this causes a dramatic nuclear accumulation of endogenous ß-catenin, but these high levels of nuclear ß-catenin are ineffective in stimulating TCF-mediated transcription. This therefore provides an assay for measuring the sequestration of nuclear ß-catenin by APC (Hamada, 2004).

NLS-fusions of the AxAxA and AxAxAplus mutants were tested in this sequestration assay. Interestingly, the mutant NLS-fusions are less active in reducing TOPFLASH values than their wild-type controls. These differences are significant since the expression levels of wild-type and mutant NLS-fusions are essentially the same. Notably, the loss of function of the AxAxA and AxAxAplus mutants in reducing ß-catenin activity is exacerbated in this sequestration assay where the levels of nuclear ß-catenin are high. This suggests a role of the APC-CtBP interaction in sequestering nuclear ß-catenin (Hamada, 2004).

A possible model is that APC binds to free nuclear ß-catenin in competition with TCF and targets ß-catenin to CtBP (by being an adaptor between these two proteins), thus diverting ß-catenin away from TCF. CtBP, being anchored at specific sites within the nucleus, could act as a "sink" for APC/ß-catenin complexes, thus shifting the binding equilibrium of ß-catenin yet further away from TCF (Hamada, 2004).

Three lines of evidence support this model: (1) ß-catenin can be detected in a complex with CtBP in SW480, but not in COLO320 cells, whose APC truncation can bind neither CtBP nor ß-catenin; (2) in COLO320 cells transfected with NLS-fusions of APC, it is estimated that the levels of endogenous TCF-4/ß-catenin complexes are 1.5×-2× higher in the case of AxAxAplus compared to the wild-type control. These increased levels of TCF-4/ß-catenin complexes are likely to be the basis for the high TCF-mediated transcription in the complementation assays. (3) In CtBP mutant mouse cells expressing tagged LEF-1, 2×-3× more endogenous ß-catenin can be coimmunoprecipitated with LEF-1 than in the corresponding parental control cells (heterozygous for both alleles). The total levels of ß-catenin are the same in the two cell lines, as are the amounts of APC bound ß-catenin. The latter two lines of evidence indicate that CtBP reduces the availability of ß-catenin for binding to TCF (Hamada, 2004).

If so, absence of CtBP should result in elevated levels of TCF-mediated transcription. Indeed, the basal TOPFLASH activity (due to endogenous TCF/ß-catenin) in CtBP mutant cells is increased ~3.7× compared to their control cells. Furthermore, cotransfection of activates ß-catenin (S33A mutant) and Lef-1 stimulate TOPFLASH activity to higher levels in CtBP mutant cells compared to the control. By comparison, <2× differences are detected in transcriptional activity between mutant and wild-type cells if FOPFLASH or an SV40-based control reporter (pRL-SV) are tested. Indeed, the activity levels of the internal control renilla reporter (pRL-CMV) are the same in both cell lines. Therefore, Lef-1-mediated transcription is more sensitive to CtBP loss than the transcription mediated by other transcription factors. Thus, CtBP appears to antagonize TCF-mediated transcription in a relatively specific way (Hamada, 2004).

It has been reported that Xenopus CtBP can bind to XTcf-3 and antagonize the transcription of TCF target genes in the early Xenopus embryo. It was noted that TCF-3 and TCF-4 factors possess CtBP binding motifs and suggested that CtBP may be a corepressor of these TCFs. Potentially, this could explain the increased basal levels of TCF-mediated transcription in CtBP mutant cells compared to their parental controls. However, it is unlikely to explain the increased levels of Lef-1-stimulated transcription, given that Lef-1 is a TCF factor that lacks CtBP binding motifs (Hamada, 2004).

In vivo association between CtBP and TCF had never been demonstrated, so this was examined in comparison to the in vivo association between CtBP and APC. First, it was asked whether endogenous CtBP and TCF-4 coimmunoprecipitate in colorectal cancer cells, given that TCF-4 is expressed in these cells. ß-catenin coimmunoprecipitates with TCF-4, as expected; however, CtBP is not detectable in the same TCF-4 immunoprecipitate. Conversely, while APC coimmunoprecipitates with CtBP, TCF-4 does not. Thus, endogenous CtBP is associated with APC, but not with TCF, in colorectal cancer cells. Notably, the same is true in 293T cells in which TCF is transcriptionally inactive: endogenous CtBP is associated with APC and ß-catenin, but not with endogenous TCF-4. It is concluded that TCF is not detectable in a complex with CtBP, regardless of cell type and transcriptional activity (Hamada, 2004).

It has been reported that exogenous TCF-4 can repress TOPFLASH transcription in transfected simian COS cells (that lack E1A expression) in a CtBP-dependent manner, while a C-terminal truncation of TCF-4 without the CtBP binding motifs (such as those arising from frameshift mutations in TCF-4 in some microsatellite-unstable colorectal carcinomas) does not respond to overexpressed CtBP in this assay. These experiments were repeated by comparing the activities of mutant TCF-4, whose two CtBP binding motifs were mutated in the same way as those of APC (TCF-4 AxAxA with triple alanine substitutions in residues 1, 3, and 5 of the P-h-D-L-S-x-R/K motif) and its wild-type control in TOPFLASH assays, and in their response to overexpressed CtBP. Overexpressed TCF-4 can repress TOPFLASH transcription in a dose-dependent manner in transfected SW480 and COS cells. However, the AxAxA TCF-4 mutant was similarly inhibitory, despite being expressed at slightly higher levels than wild-type TCF (especially at low doses of transfected plasmid). Furthermore, the mutant was equally responsive to coexpressed CtBP as the wild-type TCF-4. Therefore, although the AxAxA mutation affects the activity of APC(918-1698) in TCF-specific transcription assays, the same mutation in TCF-4 does not affect its activity in these assays. In agreement with this, a comparable double mutation of the CtBP binding motifs in XTcf-3 does not reduce its repressive potential in Xenopus embryos. Note that this double mutation does reduce the in vitro binding of XTcf-3 to CtBP, and so does the AxAxA double mutant of TCF-4. However, the in vitro binding between CtBP and TCF-4 is ~10× less strong than that between TCF-4 and ß-catenin. Thus, the in vitro binding between CtBP and TCF, although apparently specific, is very weak indeed. It may be spurious, given the lack of a detectable association between these proteins in vivo (Hamada, 2004).

In summary, no evidence was obtained for a significant physical or functional interaction between CtBP and TCF. These results thus question the idea that CtBP functions generally as a corepressor of TCF factors. It is agreed that the TCF-4 frameshift mutations observed in microsatellite-unstable colorectal carcinomas are passenger mutations without any functional relevance for TCF-mediated transcription or tumorigenesis (Hamada, 2004).

It was asked whether dCtBP might antagonize Armadillo-mediated transcription during Drosophila development. However, this is not straightforward to test, since dCtBP mutants show highly pleiotropic mutant phenotypes: null mutant embryos are grossly abnormal and do not develop beyond early stages, due to failing interactions between dCtBP and segmentation gene products. This precludes a meaningful analysis of dTCF target gene expression in these mutants. And although dCtBP has been implicated in antagonizing dTCF transcription in the developing midgut, this is an indirect effect mediated by the DNA binding protein Brinker to which CtBP can bind. Likewise, CtBP loss in the mouse causes pleiotropic mutant phenotypes, one of which, unexpectedly, mimics loss of Wnt signaling, but this could also be an indirect effect of CtBP binding to another target protein outside the Wnt pathway (Hamada, 2004).

Thus, to explore the regulatory relationship between dCtBP and Armadillo during development, it was asked whether dCtBP loss would affect the phenotypic consequences of overactive or depleted Armadillo. This is indeed the case: lowering the dose of dCtBP enhances the rough eye phenotype caused by activated Armadillo, but the same condition suppresses the wing nick phenotype due to Armadillo depletion in cells whose stimulation by Wingless is required for normal wing margin formation. These genetic interactions are similar to those of negative components of the Wnt pathway that downregulate Armadillo, such as Drosophila Axin and APC, consistent with dCtBP antagonizing Armadillo. Again, it is emphasized that this antagonism is unlikely to be due to dCtBP being a direct corepressor of dTCF, given that the latter does not contain any CtBP binding motifs. The results suggest that the antagonism between CtBP and Armadillo/ß-catenin is conserved and operates in multiple tissues and cell types (Hamada, 2004).

This study has presented evidence that CtBP binds to APC directly and specifically via the conserved 15Rs of APC and that the association of the two proteins in vivo is functionally relevant since it is required for the full activity of APC in reducing TCF-mediated transcription in colorectal cancer cells. In contrast, no evidence was found for a direct physical or functional interaction between CtBP and TCF in mammalian cells, calling into question whether CtBP acts generally as a transcriptional corepressor of TCF factors (Hamada, 2004).

Instead, the evidence suggests that CtBP antagonizes TCF-mediated transcription by cooperating with APC to sequester nuclear ß-catenin. This sequestration could be a safeguard function of APC, operating in parallel to (and to some extent redundantly with) its other functions in promoting nuclear export and degradation of ß-catenin. It is proposed that APC sequesters ß-catenin by targeting it to CtBP, thus lowering the pool of free nuclear ß-catenin that is available for binding to TCF. The sequestration of the APC/ß-catenin complex by CtBP may be based on spatial segregation within the nucleus (e.g., anchoring of the complex at specific subnuclear bodies). Whatever the precise mechanism, the observed functional cooperation between CtBP and APC in colorectal cancer cells suggests a role of CtBP as a tumor suppressor in the colon (Hamada, 2004).

Drosophila exocyst components Sec5, Sec6, and Sec15 regulate E-Cadherin trafficking from recycling endosomes to the plasma membrane; Armadillo interacts with Sec15

Loss of function of the Drosophila exocyst components in epithelial cells results in E-Cadherin (Shotgun) accumulation in an enlarged Rab11 recycling endosomal compartment and inhibits Shotgun delivery to the membrane. Rab11 and Armadillo interact with Sec15 and Sec10, respectively. These results support a model whereby the exocyst regulates E-Cadherin trafficking, from recycling endosomes to sites on the epithelial cell membrane where Armadillo is located (Langevin, 2005).

In budding yeast, the exocyst has been proposed to tether post-Golgi vesicles to the membrane of the growing bud prior to fusion. This model is supported by several observations. (1) Exocyst components localize both on post-Golgi vesicles and on the bud membrane (Boyd, 2004). Analogously in Drosophila, Sec5 and Sec15 localize along the lateral membrane and on the REs. (2) Mutations in genes encoding components of the exocyst complex lead to the accumulation of post-Golgi vesicles (Novick, 1980). Analogously, Sec5, Sec6, and Sec15 loss of function leads to an enlargement of the recycling endosome (RE) compartment; this enlargement interpreted as an accumulation of RE vesicles. (3) The localization of Sec8p and Exo70p at the growing bud, i.e., the site of polarized exocytosis, depends on the function of the other exocyst components. Analogously, Sec5 is localized along the lateral membrane, where E-Cadherin delivery is affected, and its localization along the cortex depends on Sec6. It is therefore proposed that in Drosophila epithelial cells, Sec5, Sec6, and Sec15 act by tethering vesicles originating from the recycling endosomal compartment to the lateral membrane of epithelial cells, as a prerequisite for their exocytosis (Langevin, 2005).

In epithelial cells, Arm and E-Cadherin colocalize to the AJs of the ZA as well as along the lateral membrane. In the absence of Sec5, Sec6, and Sec15 function, E-Cadherin trafficking is affected and E-Cadherin accumulates in the RE. Similarly, in the absence of arm, E-Cadherin fails to localize at the membrane and localizes in the RE. The identification of an interaction between Arm and Sec10 is therefore consistent with a model whereby this interaction provides a landmark at the site where Arm is enriched in order to deliver E-Cadherin from the recycling endosomes. Nevertheless, Arm may play an additional role in stabilizing E-Cadherin at the AJs. A direct demonstration of the function of Arm in regulating the delivery of E-Cadherin will therefore require the identification of arm mutant alleles that do not perturb its function as a regulator of E-Cadherin stabilization and only affects its interaction with Sec10 (Langevin, 2005).

In the absence of Sec5, Sec6, or Sec15 function, E-Cadherin delivery to the lateral membrane is inhibited and E-Cadherin accumulates in the REs. Furthermore, E-Cadherin was found to transcytose in a Sec5-dependent manner from the lateral membrane of epithelial cells to the apical AJs. Therefore, this study reveals at least a role of the exocyst in the recycling of E-Cadherin from the lateral membrane to the apical AJs. Furthermore, the strong reduction of E-Cadherin present on the lateral membrane is interpreted as a failure to recycle E-Cadherin from the lateral membrane back to the lateral membrane, which cannot be compensated for by the delivery of newly synthesized E-Cadherin to the lateral membrane. The loss of E-Cadherin on the lateral membrane may also lead to a reduction of E-Cadherin delivery at the AJs. This may have also contributed to the loss of epithelial cell polarity observed in some of the sec5 mutant epithelial cells (Langevin, 2005).

In polarized MDCK cells, the apical REs are well known as a site of sorting during endocytic and transcytotic transport. The REs have also been shown to serve as an intermediate during the transport of newly synthesized proteins from the Golgi to the plasma membrane in nonpolarized MDCK cells. Similarly, upon overexpression of GFP-E-Cad in HeLa cells, E-Cad transits from the Golgi to the Rab11 endosomes. Nevertheless, the existence of such a pathway remains to be established in polarized MDCK cells. In fact, the overexpression of a dominant-negative form of Rab11 leads to sequestration of E-Cadherin in the REs, but whether sequestered E-Cadherin represented newly synthesized or recycled E-Cadherin was not determined. The existence of such a Golgi-to-RE pathway also remains to be established in Drosophila epithelial cells. If so, a role of the exocyst in regulating the delivery of newly synthesized E-Cadherin from the Golgi to the lateral membrane via the REs remains plausible (Langevin, 2005).

Whether the exocyst regulates E-Cadherin localization in mammalian cells has not been directly analyzed. However, E-Cadherin is proposed to act as a regulator of the localization of the exocyst complex in polarizing mammalian cells since E-Cad- and Nectin-2α-dependent cell-cell contacts were proposed to recruit the exocyst complex in order to promote the growth of the lateral epithelial cell domain. The current study suggests that upon the recruitment of the exocyst complex by E-Cadherin, the exocyst promotes the delivery of more E-Cadherin to the lateral membrane during the establishment of apico-basal polarity. In fact, several reports can be reconciled with a function of the exocyst in regulating the transport of E-Cadherin in mammalian cells. Thus, polarized exocytosis of E-Cad to the lateral membrane is dependent upon its interaction with Arm. And, as stated above, REs have shown to serve as an intermediate during the transport of E-Cad from the Golgi to the lateral membrane where E-Cadherin, β-Catenin, and α-Catenin form the AJs. Furthermore, the overexpression of a dominant-negative form of Rab11 impairs the delivery of E-Cadherin to the lateral membrane. Consistent with the exocyst regulating trafficking from the REs, exocyst components also localize on the REs, and Sec15 is an effector of Rab11. Finally, E-Cadherin and catenins are associated with exocyst components (Langevin, 2005 and references therein).

In conclusion, this work provides evidence for a conserved role of the exocyst in regulating the delivery of E-Cadherin from REs to sites on the plasma membrane and in thereby contributing to the maintenance of epithelial cell polarity (Langevin, 2005).

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. This study genetically identified a novel Wnt/Wg pathway component 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).

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).

The HRPT2 gene, encoding Parafibromin, the mammalian homolog of Hyrax, is ubiquitously expressed and encodes a predicted protein of 531 amino acids. The primary sequence of parafibromin neither closely resembles other known proteins nor reveals obvious structural motifs that might provide a direct clue as to its function. The exception is an ~200-amino-acid C-terminal segment of parafibromin, which displays modest homology (27%) to budding yeast Cdc73, a component of the Paf1 complex that functions at various stages during the yeast transcription cycle (Yart, 2005).

The Paf1 complex has been originally identified as an RNA polymerase II (RNAP II)-associated complex and minimally contains Paf1 (see Drosophila Paf1), Cdc73, Rtf1 (see Drosophila Rtf1), Leo1, and Ctr9. It has been implicated in the regulation of genes whose products function in metabolism and cell cycle control. Genetic and biochemical evidence in yeast suggest key roles for Paf1 complex components at various stages of the gene expression pathway, including transcript site selection, transcriptional elongation, histone H2B monoubiquitination and subsequent histone H3 methylation, and more recently poly(A) length control and the coupling of transcriptional and posttranscriptional events (Yart, 2005 and references therein).

The function of the Paf1 complex has also been intimately linked to site-specific phosphorylation events of RNAP II within its carboxy-terminal domain (CTD). Site-specific phosphorylation of RNAP II CTD is an important mechanism that contributes, at least in part, to the normal temporal coordination of the activities of the various protein assemblages involved in mRNA synthesis. For example, during the transition from transcription initiation to elongation, serine 5 of CTD is phosphorylated. As RNAP II elongates, serine 5 phosphorylation diminishes while serine 2 phosphorylation increases. The latter initiates the recruitment of factors involved in subsequent steps of RNA processing. For example, the protein URI is an unconventional member of the prefoldin (PFD) family of ATP-independent molecular chaperones. URI-associated proteins including the tumor suppressor parafibromin and human orthologs of the yeast Paf1 complex. Parafibromin is associated with the serine 5- and serine 2-phosphorylated forms of RNAP II CTD, and a naturally occurring tumor-derived mutant of parafibromin lacks PAF1 and RNAP II binding function. These data infer a potential role of the tumor suppressor parafibromin in transcriptional/posttranscriptional control pathways (Yart, 2005 and references therein).

Three lines of evidence argue for the notion that Hyx represents a component of the Drosophila 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. 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 and references therein).

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 have been 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).

Adenomatous polyposis coli is present near the minimal level required for accurate graded responses to the Wingless morphogen

The mechanisms by which the Wingless (Wg) morphogen modulates the activity of the transcriptional activator Armadillo (Arm) to elicit precise, concentration-dependent cellular responses remain uncertain. Arm is targeted for proteolysis by the Axin/Adenomatous polyposis coli (Apc1 and Apc2)/Zeste-white 3 destruction complex, and Wg-dependent inactivation of destruction complex activity is crucial to trigger Arm signaling. In the prevailing model for Wg transduction, only Axin levels limit destruction complex activity, whereas Apc is present in vast excess. To test this model, Apc activity was reduced to different degrees, and the effects were analyzed on three concentration-dependent responses to Arm signaling that specify distinct retinal photoreceptor fates. It was found that both Apc1 and Apc2 negatively regulate Arm activity in photoreceptors, but that the relative contribution of Apc1 is much greater than that of Apc2. Unexpectedly, a less than twofold reduction in total Apc activity, achieved by loss of Apc2, decreases the effective threshold at which Wg elicits a cellular response, thereby resulting in ectopic responses that are spatially restricted to regions with low Wg concentration. It is concluded that Apc activity is not present in vast excess, but instead is near the minimal level required for accurate graded responses to the Wg morphogen (Benchabane, 2008).

Previous genetic studies have provided conclusive evidence that the two Drosophila Apc proteins are crucial negative regulators of Arm signaling. Simultaneous inactivation of both Apc proteins results in ectopic Arm signaling in nearly all, if not all, cells, indicating that Apc is required to prevent Arm signaling in the absence of Wg stimulation. In contrast with the prevailing model for Wg transduction, which proposes that Apc is present in vast excess, the work presented in this study reveals that a less than twofold reduction in Apc activity can shift the threshold for the response to Wg. It is concluded that by negatively regulating Arm, Apc prevents ectopic Arm activity not only where Wg is absent, but also within the range of the Wg gradient (Benchabane, 2008).

Translation of a gradient of Wg morphogen activity to quantitatively distinct levels of Arm signaling is required to induce concentration-dependent cellular responses, although the mechanisms by which this occurs remain uncertain. The current results reveal that in regions of low Wg concentration, reducing total Apc activity by less than twofold results in aberrant cell fate specification. A morphogen model predicts that the low Wg concentration present in this region of the gradient is below the threshold necessary to trigger a detectable cellular response. This is the only region within the Wg gradient where a relatively small reduction in total Apc activity elicits an ectopic cellular response, and this response is characteristic of intermediate-level Arm signaling. Thus, these results reveal that Apc activity is in excess in regions where Wg is absent, but is not in vast excess within the range of the Wg gradient. Together, these data indicate that Apc activity is present near the minimal level required to prevent ectopic Arm signaling and thereby ensure accurate graded responses (Benchabane, 2008).

In Xenopus egg extracts, the levels of Axin are several magnitudes lower than the levels of other proteins in the destruction complex, suggesting that only Axin is a limiting component in Arm proteolysis, whereas Apc is present in vast excess. How can these biochemical data be reconciled with the current in vivo data, which indicate that Apc is not present in excess within the range of the Wg gradient? One possibility is that the levels of Apc in Xenopus eggs are much greater than those present in Drosophila photoreceptors. Alternatively, total Apc levels could be present in excess regardless of cell type or organism, but the relevant pool contributing to destruction complex activity, distinguished by either post-translational modification and/or intracellular localization, might be present near threshold levels. A correlation between the degree of reduction in the activity of the fly and mammalian Apc proteins with the level of β-catenin/Arm signaling has been demonstrated in several other developmental contexts and in tumorigenesis. Thus data from diverse experimental models indicate that the level of Apc contributes to the level of β-catenin/Arm signaling (Benchabane, 2008).

How is a gradient of Wg concentration translated into quantitatively distinct levels of Arm activity? Upon Wg stimulation, inactivation of the Axin/Zw3/Apc destruction complex is the primary event that triggers Arm signaling. Inactivation of Axin is important for downstream signal transduction in response to Wg stimulation, and is likely to be mediated by the translocation of Axin to the plasma membrane, and/or the degradation of Axin. Thus the local Axin concentration is likely to have a significant role in determining whether the destruction complex is assembled, and consequently is important in regulating Arm stability. The current findings provide in vivo evidence that the level of destruction complex activity is crucial for accurate patterning in response to Wg, and is dependent not only on Axin, but also on the maintenance of Apc activity above a minimal level. It is concluded that within the range of the Wg gradient, both Axin and Apc are present near threshold levels, and that, together, they achieve the precise levels of destruction complex activity required for accurate graded responses (Benchabane, 2008).

Modulation of the ligand-independent traffic of Notch by Axin and Apc contributes to the activation of Armadillo in Drosophila

There is increasing evidence for close functional interactions between Wnt and Notch signalling. In many instances, these are mediated by convergence of the signalling events on common transcriptional targets, but there are other instances that cannot be accounted for in this manner. Studies in Drosophila have revealed that an activated form of Armadillo, the effector of Wnt signalling, interacts with, and is modulated by, the Notch receptor. Specifically, the ligand-independent traffic of Notch serves to set up a threshold for the amount of this form of Armadillo and therefore for Wnt signalling. In the current model of Wnt signalling, a complex assembled around Axin and Apc allows GSK3 (Shaggy) to phosphorylate Armadillo and target it for degradation. However, genetic experiments suggest that the loss of function of any of these three elements does not have the same effect as elevating the activity of β-catenin. This study shows that Axin and Apc, but not GSK3, modulate the ligand-independent traffic of Notch. This finding helps to explain unexpected differences in the phenotypes obtained by different ways of activating Armadillo function and provides further support for the notion that Wnt and Notch signalling form a single functional module (Muñ-Descalzo, 2011).

Cells expressing ArmS10, a form of Arm that is insensitive to phosphorylation by GSK3, do not overgrow and remain integrated in the epithelium. Clones of cells mutant for Axin, a central element of the Arm destruction complex, exhibit very high levels of Arm, some of which can be found in the nucleus, and exhibit overgrowths and round edges suggestive of defects in cellular recognition. These phenotypes are related to, but distinct from, those caused by expression of ArmS10 and support the contention that Axin exerts controls on the activity of Arm that are additional to those mediated through its role as a scaffold for GSK3. The effects of Axin loss of function are reminiscent of those caused by expression of ArmS10 in cells with compromised Notch function. Since these effects are caused by the loss of the ligand-independent traffic of Notch, this study tested whether Axin exerts some effect on the traffic of Notch (Muñ-Descalzo, 2011).

Clones of cells mutant for Axin did not show alterations in ligand-dependent Notch signalling, although they exhibited a mild but reproducible increase in Notch protein on the apical side, and overexpression of Axin reduced the amount of Notch present at the cell surface. These observations suggest that Axin regulates the amount of Notch at the cell surface. To test whether this control is exerted by targeting the endocytosis and traffic of Notch, label and chase experiments were performed with Notch. Under the experimental conditions and focusing the analysis in the pouch of the wing imaginal disc, labelled Notch disappeared from the cell surface within 10 minutes of the chase and could be found in punctate intracellular structures, presumably vesicles associated with endocytic traffic. Performing the same assay in the absence of Axin revealed that the endocytosis and traffic of Notch is impaired in Axin mutant cells, and after 30 minutes a substantial amount of Notch could still be detected on the cell surface. This suggests that Axin is involved in, or can influence, the traffic of Notch. Performing the same experiment in discs overexpressing Axin, a decrease was observed in the amount of Notch over time. Altogether, these results suggest that Axin contributes to the removal of Notch from the cell surface and to targeting it for degradation (Muñ-Descalzo, 2011).

Regulation of the activity of Arm by Notch is mediated by its ligand-independent traffic as shown by the activity of chimeric receptors in which the extracellular domain of Notch has been substituted by the extracellular domain of CD8 (CeN) or Torso (TN; Tor - FlyBase). Since Wingless signalling promotes the traffic and degradation of these receptors and cells lacking Axin have elevated levels of Wnt signalling, this study examined what would happen to the stability of CeN in this situation. Surprisingly, the levels of CeN remained largely unchanged in clones of cells mutant for Axin, suggesting that in the absence of Axin, despite high levels of Wnt signalling, CeN cannot be degraded . This could be because Axin is required for the degradation of CeN or because this degradation is dependent on Wnt and Dsh but not on Axin. A contribution of Axin is favoured by the observations that overexpression of Axin reduces, and Axin loss of function increases, Notch levels (Muñ-Descalzo, 2011).

A functional relationship between Axin and Notch is also highlighted by the observation that, in tissue culture, simultaneous reductions of Notch and Axin induce very high levels of Arm activity. However, in vivo, simultaneous loss of both Notch and Axin leads to a suppression of the growth induced by the loss of Axin alone, a phenotype that is associated with extensive cell death and perhaps reflects a synergy of the roles of each protein in apoptosis. For this reason, to test the synergy between the two proteins in determining Arm activity in vivo, a NotchRNAi construct was expressed that reduces, but does not abolish, Notch function in clones of cells mutant for Axin. Under these conditions, there is no apoptosis and larger outgrowths than those promoted by the loss of Axin alone were observed. These phenotypes indicate a synergistic effect of the mutations and suggest that Axin is involved in the modulation of Notch while it traffics through the cell (Muñ-Descalzo, 2011).

Apc, a second element of the Arm destruction complex, is encoded in Drosophila by Apc1 (Apc - FlyBase) and Apc2, which play redundant roles in the regulation of Wnt signalling. In order to test whether Apc is also involved in the traffic of Notch, clones of cells mutant for Apc1 and Apc2 were generated in wing imaginal discs and the traffic of Notch was assessed. In these clones, cells exhibited very similar phenotypes to those of Axin mutants in terms of growth, overall shape and levels of Arm. In addition, they exhibited altered traffic of Notch. However, instead of being clearly localised in vesicles or in the cell membranes, as in the case of Axin mutant cells, Notch protein appeared as a 'fuzzy' stain throughout the cytoplasm of the Apc1/2 mutant cells that was not associated with any subcellular structure. Axin and Apc have been shown to play functionally related, but distinct, roles in the regulation of Arm/δ-catenin and these differences might extend to their effects on Notch (Muñ-Descalzo, 2011).

The function of Axin and Apc is to provide a scaffold for the phosphorylation of Arm/β-catenin by Sgg/GSK3. Since, in mammalian systems, GSK3 has been shown to phosphorylate Notch and there are reports of interactions between Notch and Sgg in Drosophila, tests were performed to see whether Sgg has an effect on the traffic of Notch. Clones of cells mutant for sgg displayed elevated levels of Arm but no discernible effects on the endocytosis and traffic of Notch. This is consistent with the observation that Sgg is not required for the effects of Notch on Wnt signalling (Muñ-Descalzo, 2011).

In addition to their interactions with Wnt signalling, Axin and Apc display interactions with other signalling pathways and, in the case of Apc, with the cytoskeleton. These additional interactions might contribute to the differences between the effects of activated Arm and the loss of function of Axin and Apc. Notwithstanding this, the results reveal a function of Axin and Apc in the traffic of Notch. Previous studies have shown that compromising the traffic of Notch elevates the activity of an activated form of Arm. In Axin or Apc1,Apc2 mutant clones, in addition to the elevation of active Arm, the traffic of Notch is compromised and probably contributes to the increase in Arm activity. In this situation, the levels and activity of Arm would be higher than those resulting from the expression of an activated form of Arm alone. There is evidence that Axin functions in the regulation of Arm activity in a manner that is independent of its role as a scaffold for GSK3. Some of these effects could be mediated through its role in the endocytosis and traffic of Notch, which also could traffic with a GSK3-independent form of Arm (Muñ-Descalzo, 2011).

These results underscore the inadequacy of the notion that Wnt signalling flows through a linear pathway to target the destruction complex and promote β-catenin transcriptional activity. Although this framework helps to explain some of the effects associated with Wnt signalling, it is inconsistent with the observation that, in many instances, changes in the concentration of Arm/β-catenin are insufficient to promote transcriptional activity. While the axis Wnt-Dsh-Axin/Apc-β-catenin is the backbone of Wnt signalling, it is clear that there are additional elements that are not simply modulatory add-ons. In this regard, the interactions between Wnt and Notch signalling are a recurrent theme in developmental biology and disease and might not reflect a simple functional convergence in specific processes at the transcriptional level. The results presented in this study reinforce the notion that Wnt and Notch configure a molecular device (Wntch), in which the mutual control of their activities serves to regulate the assignation of cell fates with the effect of Notch providing a buffer to fluctuations in the resting levels of Arm (Muñ-Descalzo, 2011).

Nemo kinase phosphorylates β-catenin to promote ommatidial rotation and connects core PCP factors to E-cadherin-β-catenin

Frizzled planar cell polarity (PCP) signaling regulates cell motility in several tissues, including ommatidial rotation in Drosophila melanogaster. The Nemo kinase (Nlk in vertebrates) has also been linked to cell-motility regulation and ommatidial rotation but its mechanistic role(s) during rotation remain obscure. This study shows that nemo functions throughout the entire rotation movement, increasing the rotation rate. Genetic and molecular studies indicate that Nemo binds both the core PCP factor complex of Strabismus-Prickle, as well as the E-cadherin-β-catenin (E-cadherin-Armadillo in Drosophila) complex. These two complexes colocalize and, like Nemo, also promote rotation. Strabismus (also called Vang) binds and stabilizes Nemo asymmetrically within the ommatidial precluster; Nemo and β-catenin then act synergistically to promote rotation, which is mediated in vivo by Nemo's phosphorylation of β-catenin. These data suggest that Nemo serves as a conserved molecular link between core PCP factors and E-cadherin-β-catenin complexes, promoting cell motility (Mirkovic, 2011).

The data suggest that Nmo connects the core PCP Stbm-Pk complex to the activity of E-cad-β-cat. Consistent with this, mutations in stbm and pk enhance not only the nmoP rotation defects but also rotation defects of hypomorphic shg (E-cad) backgrounds. As the presence of the Stbm-Pk complex seems to increase the amount of Nmo at R4 membranes and junctional complexes, it is hypothesized that a rise in Stbm levels would increase the ability of sev>Nmo to cause an over-rotation phenotype. This is indeed the case. These data indicate that Nmo serves as a link from PCP factors to the E-cad-catenin complexes. The data are consistent with a model in which the Stbm-Pk complex helps to recruit and/or stabilize Nmo at membrane regions (where the PCP factors partially overlap with E-cad-β-cat complexes (Mirkovic, 2011).

The effect of Nmo on E-cad-β-cat complexes could be mediated either through the dynamics of lateral clustering (for example, formation or disassembly of higher-order E-cad-β-cat complexes) or through changes in the interaction of β-cat with other associated proteins. An E-cad::β-cat fusion protein (which bypasses a β-cat requirement and provides stable adhesion is not influenced by Nmo, suggesting that once β-cat is part of the E-cad-catenin complex Nmo cannot affect their activity. It is thus possible that phosphorylation of β-cat by Nmo affects the E-cad-β-cat complex activity (as an ArmS10AAA isoform with the Nmo target sites mutated no longer cooperates with Nmo) and this phosphorylation may also modulate interactions of the complex with other binding partners, such as β-cat. The interactions of adhesion and planar polarity during the early 'convergence-extension' rearrangements in the fly embryo suggest a mechanism in which a polarized pattern of junction remodeling drives cell intercalation. Polarized activity of RhoA and Myosin II (encoded by zipper) regulates adherens junction disassembly along the anterior-posterior axis, primarily by regulating lateral cadherin clustering without affecting surface levels of cadherins. The specific effect of RhoA on rotation, along with the interaction of nmo with zipper, supports the idea that actin-myosin contractility is downstream of Nmo. Loss of maternal contribution or Nmo overexpression in the embryonic epidermis phenocopies shg alleles or ArmS10 cuticle defects, respectively. Thus, Nmo may be generally required in epithelia undergoing morphogenetic movements, where it modulates polarized remodeling of adherens junctions in response to local asymmetries created by, for example, the activity of PCP signaling complexes (Mirkovic, 2011).

In conclusion, this study defines a framework in which Nmo serves as a link between PCP (Stbm) and the regulation of adhesive cell behavior at the level of adherens junction complexes. Although Nmo is recruited and/or maintained apically by the Stbm-Pk complex, other factors must affect Nmo activity or localization as well, because the set of cells requiring nmo (all outer R-cells) is broader than the set of cells requiring stbm (R4). First, the Nmo could regulate rate of rotation independently of the PCP complexes through Notch (N) signaling and/or Egfr signaling, as suggested by genetic data: N- alleles strongly suppress sev>Nmo, and N functions in all R-cells. Second, an asymmetric input or localization of Nmo by Stbm would provide a direction to rotation. Thus, a Notch-Nmo interaction in all cells and an asymmetric Stbm effect in R4 could combine to regulate both rate and direction of rotation. The observation that zebrafish Nlk enhances the PCP-specific Wnt11 cell migration defects in prechordal plates supports a general Nmo-mediated mechanism in PCP-associated cell movements (Mirkovic, 2011).

Yan, an ETS-domain transcription factor, negatively modulates the Wingless pathway in the Drosophila eye by interaction with Armadillo

yan, an ETS-domain transcription factor belonging to the Drosophila epidermal growth factor receptor (DER) pathway, is an antagonist of the Wingless signalling pathway. Cells lacking yan function in the Drosophila eye show increased Wingless pathway activity, and inhibition of Wingless signalling in yan-/- cells rescues the yan mutant phenotype. Biochemical analysis shows that Yan physically associates with Armadillo, a crucial effector of the Wingless pathway, thereby suggesting a direct regulatory mechanism. It is concluded that yan represents a new and unsuspected molecular link between the Wingless and DER pathways (Olson, 2012).

On the basis of in vivo and in vitro characterization of the antagonistic interaction between Yan and the Wingless pathway, it is proposed that yan has a dual role at the moving anterior/posterior boundary defined by the morphogenetic furrow. In addition to the previously defined function of Yan in inhibiting premature photoreceptor recruitment, it is suggested that Yan blocks Wingless signalling at the morphogenetic furrow, probably by regulating the activity of Armadillo protein. By maintaining low Wingless signalling activity, it is proposed that Yan maintains the competence of these cells to adopt a retinal fate. In the absence of Yan, Armadillo is free to either participate in adherens junctions, thereby causing the apical constriction phenotype, or interact with TCF in the nucleus to activate target genes. It is speculated that in yan−/− clones generated far from a source of Wingless, most of the liberated Armadillo that does not localize to adherens junctions is degraded and is unable to robustly signal in the nucleus. This could explain why apical constriction is observed in all yan−/− clones, but ectopic Fz3–RFP activation is seen only in yan−/− clones near the lateral margins of the eye disc. Similarly, expression of Axin—which can strongly downregulate both the membrane-directed and signalling-competent pool of liberated Armadillo—within yan−/− clones results in a stronger rescue of apical constriction than the expression of dnTCF, which only interferes with the signalling-competent pool of Armadillo. These data indicate that both the junctional and nuclear signalling functions of Armadillo probably contribute to the yan−/− phenotype (Olson, 2012).

In conclusion, this study shows a new and unsuspected function for yan in the negative regulation of Wingless signalling. These results might be relevant to the understanding of the molecular regulation of Wnt–RTK signalling crosstalk in human disease. The closest human homologue of yan, encoded by TEL/ETV6 is known to be associated with leukaemia, which can also result from uncontrolled activation of Wnt signalling (Olson, 2012).

Monomeric α-catenin links cadherin to the actin cytoskeleton

The linkage of adherens junctions to the actin cytoskeleton is essential for cell adhesion. The contribution of the cadherin-catenin complex to the interaction between actin and the adherens junction remains an intensely investigated subject that centres on the function of α-catenin, which binds to cadherin through β-catenin and can bind F-actin directly or indirectly. This study delineates regions within Drosophila α-Catenin (α-Cat) that are important for adherens junction performance in static epithelia and dynamic morphogenetic processes. Moreover, whether persistent α-catenin-mediated physical linkage between cadherin and F-actin is crucial for cell adhesion is addressed, and the functions of α-catenin monomers and dimers at adherens junctions is characterized. The data support the view that monomeric α-catenin acts as an essential physical linker between the cadherin-β-catenin complex and the actin cytoskeleton, whereas α-catenin dimers are cytoplasmic and form an equilibrium with monomeric junctional α-catenin (Desai, 2013).

α-catenin is conserved across the eukaryotic kingdom, where it functions broadly in intercellular adhesion during development and differentiation. In Drosophila melanogaster, cell adhesion is disrupted when α-catenin contains a mutation in the binding site for Armadillo, which is the D. melanogastor homologue of β-catenin. Adherens junctions are also present in the nematode Caenorhabditis elegans, which expresses the homologues HMR-1 (cadherin), HMP-1 (α-catenin) and HMP-2 (β-catenin). In mice, there are three α-catenins and one close relative, which all share substantial amino-acid sequence identity: αE-catenin is most prevalent in epithelial tissues; αN-catenin is restricted to neural tissues; αT-catenin is expressed primarily in heart tissue; and α-catulin, which is an α-catenin-like protein, is ubiquitously expressed. A more distant relative is vinculin, which is ubiquitously expressed and localizes to both focal adhesions and adherens junctions (Desai, 2013 and references therein).

Adherens junctions and their core constituents, the classic cadherin adhesion molecules, contribute significantly to animal development and tissue homeostasis. Adherens junction defects can lead to various human pathologies, including cancer. Adherens junction function relies on the association of cadherins with the microtubule and actin cytoskeleton through their cytoplasmic binding partners, the catenins. Elucidating the function of α-catenin, which operates at the interface of the cadherin- β-catenin complex and F-actin, is a major goal in the field (Desai, 2013).

Studies on mammalian αE-catenin have given rise to two models for α-catenin function: the physical linkage and the allosteric regulation model. αE-catenin can bind both β-catenin and F-actin suggesting that it can physically link the cadherin- β-catenin complex directly to F-actin. This simple model lacks direct experimental support because a quaternary complex between cadherin, β-catenin, αE-catenin and F-actin could not be documented. Complex formation with F-actin could be demonstrated in vitro only in the presence of EPLIN, one of several F-actin-associated proteins that bind to αE-catenin, such as vinculin, α-actinin, afadin, ZO-1 and formin. Thus, a more complex physical linkage model poses that αE-catenin links the cadherin/β-catenin complex to F-actin indirectly by interacting with actin-binding proteins. A role for αE-catenin as a physical linker between cadherin and actin is consistent with the discovery that αE-catenin acts as a tension sensor that is responsive to actomyosin contraction at adherens junctions (Desai, 2013 and references therein).

Alternatively, α-catenin was proposed to regulate actin organization to support adherens junction formation, rather than act as a physical linker. αE-catenin binds β-catenin as a monomer but shows high affinity for F-actin only as a homodimer. The β-catenin binding site and homodimerization domain of αE-catenin overlap, suggesting that it cannot interact with β-catenin and F-actin simultaneously. These findings precipitated the view that αE-catenin may act allosterically by binding β-catenin to increase its own local concentration at adherens junctions, which is required to promote αE-catenin dimerization after dissociation from β-catenin. This model does not adequately address how adherens junctions are physically linked to actin and resist tensile forces. One question that results from these contradictory models is whether α-catenin dimerization is critical for adherens junction function (Desai, 2013).

This study reports an in vivo structure-function analysis of Drosophila α-Catenin (α-Cat) to assess the roles of its domains in several developmental processes and to distinguish between the physical linkage and allosteric regulation models for α-catenin function (Desai, 2013).

The model that α-catenin acts as a physical linker between cadherin and the actin cytoskeleton seems strongly supported by the ability of cadherin- α-catenin chimaeras to substitute for the cadherin-catenin complex, both in tissue culture assays and during morphogenesis (see Sarpal, 2012). The analysis of these fusion proteins emphasizes that: α-catenin acts in the immediate sub-membranous space, excluding an essential cytosolic function suggested by other studies in the tissues this laboratory has examined (Sarpal, 2012). Further, a dynamic interaction between α-catenin and the cadherin-β-catenin complex is not required to support normal adherens junctions. Although recent examples suggest that β-catenin modulation can contribute to the dynamic regulation of the cadherin-catenin complex in certain situations, this is not a basic requirement of adherens junction assembly and function. That cadherin- α-catenin chimaeras can replace the endogenous complex suggests instead that much of the regulation of cadherin-catenin complex function takes place at the interface between α-catenin and the actin cytoskeleton. Physical linkage of α-catenin to cadherin does not interfere with its function. This does not imply, however, that α-catenin normally needs to be physically linked to the cadherin-β-catenin complex to function. Indeed, the estimated dissociation constant (Kd) of the α-catenin β-catenin interaction (~1 µM) is much weaker than the cadherin-β-catenin interaction. The allosteric regulation model for α-catenin suggests that an α-catenin homodimer modulates actin organization through interference with the Arp2/3 complex at adherens junctions. Homodimerization and β-catenin binding require the same binding interface and are mutually exclusive. Cadherins can dimerize or cluster and could therefore promote α-catenin dimerization even in chimaeric proteins that lack the α-catenin dimerization domain (for example, DEcad::αCatΔVH1). Several membrane-bound regulators of actin organization exist, including WASp, indicating that α-catenin could retain its Arp2/3 regulating activity despite its covalent linkage to cadherin. To gain further evidence into the molecular mechanism of α-catenin function this study investigated whether α-catenin function at adherens junctions can be decoupled from β-catenin binding, whether monomeric α-catenin can support adherens junctions and whether α-catenin dimerization promotes or inhibits adherens junction function (Desai, 2013).

To address the first point αCatΔVH1, lacking the first vinculin-homology region (VH1), was fused to either BazOD (BazOD::αCatΔVH1) or Baz (Baz::αCatΔVH1), which recruited αCatΔVH1 to adherens junctions. αCatΔVH1 does not support adherens junctions alone, but did so when fused to DEcad. BazOD::αCatΔVH1 and Baz::αCatΔVH1 showed weak biochemical interactions with Arm and DEcad and little rescue of adherens junctions. These findings suggest that the physical link between α-catenin and β-catenin not only recruits α-catenin to adherens junctions, but needs to persist for normal adherens junction function. Both the physical linkage and allosteric regulation models propose that α-catenin interacts with the actin cytoskeleton at adherens junctions; however, they differ on whether binding of α-catenin to β-catenin is required to physically link cadherin and the actin cytoskeleton. The data argue for persistent physical linkage as a core requirement for α-catenin function. It was also found that localization of αCatΔVH1 to adherens junctions through fusion to Ed did not support adherens junction integrity, in contrast to DEcad::αCatΔVH1, suggesting that cadherins have distinct properties that are important for α-catenin function (Desai, 2013).

Similar to C. elegans and D. discoideum α-catenin proteins, αN-catenin was found to be monomeric in solution. αN-catenin can functionally replace the Drosophila protein, which formed a large dimer fraction in solution similar to αE-catenin. These results indicate that monomeric α-catenin can support adherens junction function, and that the in vitro monomer/dimer ratio may not correlate with the in vivo function of α-catenins (Desai, 2013).

To address the relationship between α-catenin dimerization and adherens junction function, the effects were tested of enhanced α-catenin dimerization. α-Cat or αCatΔVH1 fusion to BazOD or Baz probably causes enhanced multimerization, including dimerization of α-Cat. Although these chimaeras localize to adherens junctions they show reduced interactions with Arm and perform poorly. Dimerization was also enhanced by removing the N-terminal 56 or 64 amino acids from αN-catenin and α-Cat, respectively. Similar to αEcatΔ57, αCatΔ64 interacted with β-catenin/Arm. However, these constructs performed poorly when compared with their respective full-length proteins, suggesting that α-catenin dimers are inactive in adhesion and may represent a cytoplasmic pool that forms a dynamic equilibrium with α-catenin monomers. Monomers are recruited to adherens junctions through their interaction with β-catenin/ Arm, which probably stabilizes monomeric α-catenin that links cadherin to the actin cytoskeleton (Desai, 2013).

αE-catenin operates as a tension sensor and mechanotransducer at adherens junctions, changing conformation in response to pulling forces exerted by actomyosin. To achieve this, α-catenin needs to be suspended between two anchor points, which could be cadherin- β-catenin and F-actin, consistent with the physical linkage model. However, αE-catenin homodimers contain two actin-binding domains and can bundle actin filaments, raising the possibility that actin filament sliding as a result of myosin activity could apply tension to α-catenin. As the allosteric regulation model poses that α-catenin homodimers form preferentially at adherens junctions, tension sensing could be restricted to adherens junctions even without α-catenin linking cadherin to actin. However, actomyosin-generated tension also applies to E-cadherin and depends on the presence of αE-catenin, suggesting that at least part of the tension exerted on αE-catenin occurs when it physically links cadherin to the actin cytoskeleton. These data are complementary to the analysis of Drosophila α-Cat. Although it has not been possible to document a quartenary complex of the cadherin-catenin complex with F-actin, the data presented in this study are consistent with α-catenin physically linking cadherin to the actin cytoskeleton as a core requirement of α-catenin and cadherin-catenin complex function (Desai, 2013).

The results on the function of different regions within Drosophila α-Cat are in line with data from tissue culture studies on αE-catenin. The Arm and actin-binding regions at the N and C termini of α-Cat, respectively, are essential for function. The central region of α-Cat enhances adherens junction stability but does not contribute to α-Cat recruitment to adherens junctions, which relies only on the VH1-dependent binding to Arm. The central region between the VH1 and VH3 domains includes multiple parts that make partly independent contributions to α-Cat function, most likely through interactions with other binding partners. If the central region is activated by actomyosin pulling forces, then the tension-mediated conformational change in α-Cat would be expected to facilitate multiple interactions (Desai, 2013).

Although α-catenins can bind directly to F-actin in vitro, whether this occurs in vivo remains unresolved. It is possible that interactions with F-actin are indirect and mediated through F-actin-binding proteins. α-catenin organizes a complex interface between cadherin and the actin cytoskeleton. Uncovering how the multiple interactions between α-catenin and actin-binding proteins such as vinculin, formin, afadin or EPLIN contribute to adherens junction regulation during morphogenesis remains a major challenge for future investigation (Desai, 2013).

Brain tumor specifies intermediate progenitor cell identity by attenuating beta-catenin/Armadillo activity

During asymmetric stem cell division, both the daughter stem cell and the presumptive intermediate progenitor cell inherit cytoplasm from their parental stem cell. Thus, proper specification of intermediate progenitor cell identity requires an efficient mechanism to rapidly extinguish the activity of self-renewal factors, but the mechanisms remain unknown in most stem cell lineages. During asymmetric division of a type II neural stem cell (neuroblast) in the Drosophila larval brain, the Brain tumor (Brat) protein segregates unequally into the immature intermediate neural progenitor (INP), where it specifies INP identity by attenuating the function of the self-renewal factor Klumpfuss (Klu), but the mechanisms are not understood. This study reports that Brat specifies INP identity through its N-terminal B-boxes via a novel mechanism that is independent of asymmetric protein segregation. Brat-mediated specification of INP identity is critically dependent on the function of the Wnt destruction complex, which attenuates the activity of β-catenin/Armadillo (Arm) in immature INPs. Aberrantly increasing Arm activity in immature INPs further exacerbates the defects in the specification of INP identity and enhances the supernumerary neuroblast mutant phenotype in brat mutant brains. By contrast, reducing Arm activity in immature INPs suppresses supernumerary neuroblast formation in brat mutant brains. Finally, reducing Arm activity also strongly suppresses supernumerary neuroblasts induced by overexpression of klu. Thus, the Brat-dependent mechanism extinguishes the function of the self-renewal factor Klu in the presumptive intermediate progenitor cell by attenuating Arm activity, balancing stem cell maintenance and progenitor cell specification (Komori, 2013).

Asymmetric stem cell division provides an efficient mechanism to simultaneously self-renew a stem cell and to generate a progenitor cell that produces differentiated progeny. Because self-renewal proteins segregate into both daughter progeny of the dividing parental stem cell through the inheritance of its cytoplasmic content, rapidly downregulating the activity of these proteins is essential for the specification of progenitor cell identity. Brat plays a central role in specifying INP identity in the Ase- immature INP by antagonizing the function of the self-renewal transcription factor Klu (Xiao, 2012). These previous findings have been extended to show that Brat specifies INP identity in the Ase- immature INP through two separable, but convergent, mechanisms. A novel Brat-dependent mode of Wnt pathway regulation was identified that prevents Ase- immature INPs from reverting into supernumerary neuroblasts. Brat specifies INP identity by attenuating the transcriptional activity of Arm through its N-terminal B-boxes. This negative regulation of Arm is achieved through the activity of Apc2 and the destruction complex. Because increased arm function alone is insufficient to induce supernumerary neuroblasts, the ability of Wnt signaling to promote neuroblast identity is dependent on other signaling mechanisms that act downstream of Brat. Indeed, Arm function is essential for Klu to induce supernumerary neuroblasts. These two Brat-regulated mechanisms function to safeguard against the accidental reversion of an uncommitted progenitor cell into a supernumerary stem cell and to ensure that an uncommitted progenitor cell can only adopt progenitor cell identity (Komori, 2013).

Physical interaction with the cargo-binding domain of Mira is essential for the unequal segregation of Brat into the immature INP following the asymmetric division of neuroblasts. Previous studies concluded that the NHL domain of Brat directly interacts with the cargo-binding domain of Mira, but the roles of the B-boxes and the coiled-coil domain in the asymmetric segregation of Brat were unknown due to a lack of specific mutant alleles. By combining a yeast two-hybrid interaction assay and in vivo functional validation, it is concluded that both the coiled-coil domain and the NHL domain are indeed required for the asymmetric segregation of Brat into the Ase- immature INP following the asymmetric division of neuroblasts. It is speculated that the coiled-coil domain and the NHL domain of Brat function cooperatively to provide a more stable binding platform for Mira to ensure efficient protein segregation (Komori, 2013).

The severity of the supernumerary neuroblast phenotype in various brat mutant allelic combinations correlates with the level of endogenous brat inherited by the Ase- immature INP. The brat DG19310 mutation carries a transposable P-element inserted in the 5′ regulatory region of the brat gene. The brat11 mutation, however, results in a premature stop codon at amino acid 779, leading to a truncated form of the protein that lacks most of the NHL domain and is predicted to be unable to interact with Mira. The brat DG19310 or brat DG19310/11allelic combination most likely reduces Brat expression without affecting its binding to Mira. Thus, the minimal threshold of Brat necessary for the proper specification of INP identity in Ase- immature INPs is met most of the time, leading to a mild supernumerary neuroblast phenotype in brat DG19310 or brat DG19310/11 brains. By contrast, the brat11 homozygous or brat11/Df mutant allelic combination impairs the binding of Brat to Mira, rendering the Mira-based asymmetric protein-sorting mechanism unable to segregate Brat into the Ase- immature INP. As such, the threshold of Brat necessary for proper specification of INP identity in Ase- immature INPs is rarely met, leading to a severe supernumerary neuroblast phenotype in brat11 or brat11/Df brains. Overexpression of the bratΔC-coil or bratΔNHL transgene using the UAS/Gal4 system almost certainly results in an abnormally high level of the transgenic protein in the cytoplasm of neuroblasts. Thus, inheriting a portion of the neuroblast cytoplasm containing an overwhelming abundance of the mutant transgenic protein is likely to be sufficient to reach the threshold of Brat necessary for proper specification of INP identity in Ase- immature INPs. It is concluded that the mechanism that causes Brat to asymmetrically segregate into the Ase- immature INP is functionally separable from the mechanism that specifies INP identity (Komori, 2013).

Could the asymmetric protein segregation mechanism promote the specification of INP identity by depleting Brat from the neuroblast? Type II neuroblasts overexpressing brat, bratΔC-coil or brat<ΔNHL maintained their identity and generated similar numbers of progeny as wild-type control neuroblast. Thus, it is unlikely that Brat-dependent specification of INP identity occurs through asymmetric depletion of Brat from the neuroblast. Whether Brat acts redundantly with other asymmetrically segregating determinants to specify INP identity in Ase- immature INPs was also tested. Numb also exclusively segregates into the immature INP during asymmetric divisions of type II neuroblasts. However, asymmetric segregation of Numb is not dependent on Brat, and Numb-dependent specification of INP identity also occurs independently of Brat. Thus, it is unlikely that Brat acts redundantly with other asymmetric segregating determinants to specify INP identity in Ase- immature INPs (Komori, 2013).

A surprising finding revealed by the current study is that the B-boxes are uniquely required for the specification of INP identity. This raises a series of interesting questions. What are the roles of B-boxes in the function of Brat in embryonic neuroblasts? Embryos lacking both maternal and zygotic function of brat often lack RP2 neurons but never possess supernumerary neuroblasts. Since brat mutant alleles that specifically affect the function of B-boxes are unavailable, the roles of B-boxes in the function of Brat during the asymmetric division of embryonic neuroblasts remain unknown. Brat regulates embryonic pattern formation by repressing mRNA translation through the ternary complex that also contains Nanos and Pumilio. However, it is unlikely that Brat specifies INP identity through the Nanos-Pumilio-Brat translational repression complex for the following reasons. First, the NHL domain of Brat is required for binding to Pumilio and Nanos and for the assembly of the translational repressor complex. However, the NHL domain is dispensable for Brat-dependent specification of INP identity. Second, Nanos expression is undetectable in larval brains, and pumilio mutant larval brains do not possess supernumerary type II neuroblasts. Together, these results are consistent with the conclusion that Brat specifies INP identity via a novel Arm-mediated mechanism (Komori, 2013).

The amino acid sequence of the B-boxes is highly conserved among all TRIM family proteins, including Brat, and is predicted to adopt a 'RING-like' fold tertiary structure. The RING-like fold might facilitate protein-protein interactions. This is a particularly intriguing hypothesis in light of the fact that Apc2 and Brat both localize to the basal cortex in type II neuroblasts, and overexpression of brat, but not bratδB-boxes, can restore Apc2 protein localization in neuroblasts. However, epitope-tagged Brat and endogenous could not be coprecipitated Apc2 from the brain lysate extracted from brat null mutant larvae overexpressing a Myc-tagged Brat transgenic protein. Thus, Brat might maintain Apc2 protein localization indirectly through other factors. Future biochemical analyses of the Brat protein and identification of the proteins that directly interact with the B-boxes will provide insight into how Brat controls Apc2 localization (Komori, 2013).

The destruction complex targets β-catenin/Arm for degradation during canonical Wnt signaling, so reduced function of the destruction complex will lead to an increase in β-catenin/Arm, which forms a complex with Tcf/LEF family transcription factors to activate Wnt target gene expression. This study has concluded that the Brat-Apc2 mechanism specifies INP identity by preventing aberrant activation of Wnt target gene expression in Ase- immature INPs. The role of the Wnt ligand was tested in the Brat-dependent specification of INP identity by removing the function of the Wnt ligand using a temperature-sensitive mutant allele or by overexpressing a dominant-negative form of Frizzled (FzDN or GPIdFz2) in brat DG19310/11 mutant brains. Interestingly, neither of these manipulations modified the supernumerary neuroblast phenotype in the sensitized brat genetic background (data not shown). These results suggest that the Wnt ligand and its receptor Fz are irrelevant in the Brat-dependent specification of INP identity and that the Brat- Apc2 mechanism prevents Wnt target gene expression in Ase- immature INPs by negatively regulating the activity of Arm. However, these data do not exclude the possibility that a novel activating mechanism of Wnt signaling might be present in type II neuroblasts in Drosophila larval brains (Komori, 2013).

Attempts were made to directly demonstrate that loss of brat function indeed leads to derepression of Wnt target gene expression in supernumerary neuroblasts.The expression was examined of two distinct Wnt reporter transgenes, WRE-lacZ and Notum-lacZ in brat mutant brains. However, it was not possible to detect the expression of these transgenes in supernumerary neuroblasts in brat null mutant brains. Because genetic manipulations altering the activity of Arm efficiently modify the supernumerary neuroblast phenotype in brat mutant brains, these two transgenes are unlikely to have the necessary regulatory elements to reflect Wnt target gene activity in this tissue. Thus, it is proposed that the Brat-Apc2 mechanism specifies INP identity by antagonizing the transcriptional activity of Arm in Ase- immature INPs via a receptor-independent mechanism (Komori, 2013).

Wnt signaling regulation plays key roles in both stem cell renewal and the differentiation of progenitor cell types (Merrill, 2012; Habib et al., 2013). In the mammalian intestinal epithelium, for example, loss of Apc and activation of Wnt signaling results in the maintenance of stem cell properties in the progenitor cells, a failure to differentiate, and the production of intestinal polyps that progress to malignant tumors. In the intestine, the inappropriate activation of Wnt signaling is sufficient to elicit stem cell properties. In the progenitor cells of larval type II neuroblasts, the activation of Wnt signaling alone, through either the expression of stabilized Arm or the loss of Apc2, does not drive stem cell renewal in otherwise wild-type immature INPs. In this system, Brat is the key regulator attenuating self-renewal through two independent, but convergent, mechanisms in its regulation of both Klu and Wnt signaling. Although Arm activity is required for Klu-dependent self-renewal in immature INPs, its inability to promote self-renewal alone suggests that Wnt signaling is likely to be playing a permissive role rather than an instructive role in eliciting the neuroblast identity. It is proposed that Brat downregulates the function of Klu through both Arm-dependent and -independent mechanisms. Previous studies have demonstrated that TRIM32 and TRIM3, vertebrate orthologs of Brat, are essential regulators of neural stem cells during brain development and brain tumor formation (Boulay, 2009; Schwamborn, 2009). It will be interesting to test whether TRIM32 and TRIM3 regulate neural stem cells via a β-catenin-dependent mechanism (Komori, 2013).

Btk29A promotes Wnt4 signaling in the niche to terminate germ cell proliferation in Drosophila

Btk29A is the Drosophila ortholog of the mammalian Bruton's tyrosine kinase (Btk), mutations of which in humans cause a heritable immunodeficiency disease. Btk29A mutations stabilized the proliferating cystoblast fate, leading to an ovarian tumor. This phenotype was rescued by overexpression of wild-type Btk29A and phenocopied by the interference of Wnt4-β-catenin signaling or its putative downstream nuclear protein Piwi in somatic escort cells. Btk29A and mammalian Btk directly phosphorylate tyrosine residues of β-catenin, leading to the up-regulation of its transcriptional activity. Thus, this study identified a transcriptional switch involving the kinase Btk29A/Btk and its phosphorylation target, β-catenin, which functions downstream of Wnt4 in escort cells to terminate Drosophila germ cell proliferation through up-regulation of piwi expression. This signaling mechanism likely represents a versatile developmental switch (Hamada-Kawaguchi, 2014).

Stem cell maintenance and differentiation are not entirely autonomic, but instead are under strict control by supporting cells that form the 'niche'. Recent studies in Drosophila have shown that the dynamics of Piwi and its associated piRNAs, a protein-RNA complex for gene silencing, are required in not only germ cells but also distinct niche-forming somatic cells (escort cells for germ cell development); however, their regulatory mechanisms remain largely unknown. This study identified a transcriptional switch involving the factor Bruton's tyrosine kinase (Btk) and its phosphorylation target, β-catenin, operating downstream of Wnt4 in escort cells to terminate Drosophila germ cell proliferation through modulation of piwi expression (Hamada-Kawaguchi, 2014).

Drosophila Btk29A type 2 is the ortholog of human BTK. The type 1 isoform is present and the type 2 is absent in Btk29AficP mutants. Germ stem cells (GSCs) and transit amplifying cystoblasts (CBs) are localized in the germarium situated at the anterior tip of an ovariole, posteriorly flanked by region 2, in which each CB divides twice and differentiates into cystocytes. The 16 cystocytes originating from a single CB remain interconnected by the fibrous structure fusome, a derivative of the spectrosome. GSCs and CBs both carry the spectrosome, a round, tubulin-enriched structure. The Btk29A mutant germarium contains significantly more germ cells than does the wild-type germanium. Although supernumerary cells were observed with spectrosomes in the Btk29AficP germarium, many of the excess cells appear to be cystocytes, as they were accompanied by a branched fusome structure. A large excess of cystocytes in grossly deformed ovarioles has been observed in female Drosophila that are mutant for mei-P26, a gene encoding a TRIM-NHL (tripartite motif and Ncl-1, HT2A, and Lin-41 domain) protein that binds to the argonaute protein Ago-1 for microRNA regulation. In mei-P26 mutants, an ovarian tumor 'cystocytoma' is formed because cystocytes regain the ability to self-renew after they enter the differentiation path. This suggests that mei-P26 normally terminates CB proliferation. Intriguingly, the following phenotypes of mei-P26 were recapitulated in Btk29AficP. First, phospho-histone H3-positive mitotic germline cells, which were restricted to the anterior tip of the wild-type germarium, were detected throughout the ovarioles. Second, the expression of Bam, a protein that induces differentiation of GSCs into CBs in the wild type, was markedly increased in CB-like GSC daughters. Third, oo18 RNA-binding protein (Orb) remained expressed in multiple cells in a cyst, contrasting to a wild-type cyst, where Orb expression becomes restricted to an oocyte (Hamada-Kawaguchi, 2014).

The reduction in mei-P26 transcription in Btk29AficP places mei-P26 downstream of Btk29A. Notably, mei-P26 functions cell-autonomously in germ cells. However, the almost complete rescue of germ cell defects in Btk29AficP was attained by overexpression of Btk29A+ type 2 via bab1-Gal4, which showed high levels of expression in terminal filament cells and cap cells (TF and CPC, respectively) and lower levels of expression in escort cells (EC). bab1-Gal4 was effective in inducing germ cell overproduction when used to knockdown Btk29A. hh-Gal4 with expression in the terminal filament cells and cap cells and c587-Gal4 with expression in escort cells were also used to target UAS-Btk29ARNAi expression; c587-Gal4, but not hh-Gal4, led to the overproduction of spectrosome-bearing cells, and therefore, the escort cells were considered as likely sites of Btk29A action. These observations imply that Btk29A is required in the escort cells for soma-to-germ signaling to control the switch from proliferation to differentiation in germ cells, where mei-P26 functions as a core component of the switch (Hamada-Kawaguchi, 2014).

Bone morphogenetic protein (BMP) signaling and piwi-dependent signaling compose two different pathways in the niche to control proliferation and differentiation of GSCs and their daughters. BMPs are secreted morphogens, and Piwi is an argonaute protein regulating gene expression. The Btk29AficP mutation abrogated piwi expression with little effect on decapentaplegic (dpp) or glass bottom boat (gbb) expression, two BMPs operating in the germarium, and the BMP downstream component Mothers against Dpp (Mad) was normally phosphorylated in Btk29AficP GSCs. Furthermore, somatic piwi knockdown mimicked the Btk29AficP ovarian phenotypes (Hamada-Kawaguchi, 2014).

Immunohistochemistry revealed that the Btk29AficP mutation or somatic Btk29A knockdown abrogated Piwi expression in the niche, but not in germ cells. This reduction in Piwi expression was reversed by the somatic Btk29A+ overexpression. Furthermore, the loss-of-function piwi allele dominantly enhanced the Btk29A mutant phenotype. Moreover, somatic overexpression of piwi+ in Btk29AficP alleviated the germ cell hypertrophy and reduced Bam expression to the normal level. It is therefore considered that Btk29A regulates the Piwi-dependent pathway in the niche to control germ cell proliferation (Hamada-Kawaguchi, 2014).

Piwi and piRNAs constitute a major transposon-silencing pathway. Somatic knockdown of Btk29A resulted in an increase in the expression of gypsy-lacZ that monitored the activity of the gypsy transposon. Also, transcript levels of the ZAM, DM412, and mdg1 transposons were significantly increased in Btk29AficP. It is therefore concluded that the Piwi deficiency due to the impairment of Btk29A results in derepression of transposon activities (Hamada-Kawaguchi, 2014).

Genome instability associated with transposon mobilization may lead to the activation of a DNA double-strand break (DSB) checkpoint. A mutation in DSB signaling, mnk, did not ameliorate the germ cell phenotype induced by somatic Btk29A knockdown, indicating that the germ cell hypertrophy by the Btk29A deficiency is not a consequence of the DSB checkpoint activation (Hamada-Kawaguchi, 2014).

Next, potential substrates of Btk29A in the niche were sought. Btk29A type 2 was enriched in the interface between cells where Drosophila melanogaster epithelial (DE)-cadherin and associated Arm, the β-catenin ortholog, are the major structural components. No sign were found of tyrosine phosphorylation of DE-cadherin, whereas Arm contained a high level of phosphotyrosine, which was almost entirely absent from Btk29AficP ovaries. However, Arm immunoprecipitated from Btk29AficP was strongly phosphorylated in vitro by the exposure of Arm to active Btk29A protein that had been immunoprecipitated from wild-type ovaries. These results demonstrate that Btk29A mediates the tyrosine phosphorylation of Arm in vivo (Hamada-Kawaguchi, 2014).

The anti-Arm labeling intensity of cell adhesion sites was stronger in Btk29AficP than in the wild type. Immunoprecipitation assays revealed that the relative amount of Arm associated with DE-cadherin was greater in Btk29AficP than in the wild type , suggesting that the tyrosine phosphorylation of Arm facilitates its release from the membrane to the cytoplasm, as in mammalian cells (Hamada-Kawaguchi, 2014).

Mammalian β-catenin is tyrosine-phosphorylated at residues Y86, Y142, and Y654. When transfected into mammalian Cos7 cells, Drosophila Btk29A type 2 phosphorylated all these tyrosine residues of β-catenin. Moreover, the antibodies against phosphorylated Y142 (anti-pY142) and anti-pY654 recognized Arm phosphorylated at the conserved site Y150 and Y667, respectively, in the immunoprecipitates from ovarian lysates (Hamada-Kawaguchi, 2014).

Expression of unphosphorylatable Arm-Y150F in the escort cells via c587-GAL4 or bab1-GAL4, but not hh-Gal4, induced germ cell hypertrophy, whereas another unphosphorylatable mutant, Y667F, or wild-type Arm exerted little effect. In addition, somatic arm knockdown resulted in an increase in spectrosome-containing cells, reduced piwi expression in escort cells, and increased Bam expression in germ cells. Considering these results together, it is proposed that Btk29A acts on Arm, which in turn regulates piwi in the niche (Hamada-Kawaguchi, 2014).

Arm functions in the canonical Wnt pathway. Therefore the ovaries of wg, Wnt2, Wnt4, and Wnt5 mutants were examined; the germ cell overproduction was detected only in Wnt4. Somatic knockdown of Wnt4 aided by bab1-GAL4 resulted in a reduction in the expression of Piwi, accompanied by an accumulation of germ cells carrying spectrosomes with an increase in germline Bam expression. These findings support the hypothesis that Arm in the escort cells regulates germ cell proliferation under the control of Wnt4, which was likely derived from somatic cells other than cap cells and terminal filament cells, as hh-GAL4 selective for these cells was least effective to induce germ cell overproduction when used to drive Wnt4RNAi expression (Hamada-Kawaguchi, 2014).

To evaluate the ability of Arm to activate transcription, T cell factor (TCF) reporter assays were used with Cos7 cells transiently transfected with human Btk (hBtk). The wild-type hBtk alone was sufficient to induce phosphorylation at Y142 and Y654 of β-catenin, whereas the kinase-dead hBtk (Btk-K430E) was not. Tyrosine phosphorylation of β-catenin was completely blocked by two antagonists of hBtk. Similarly, Btk29A type 2 phosphorylated Y142 and Y654 of mammalian β-catenin. Notably, the TCF reporter activity was six times as high when hBtk was transfected into Cos7 cells compared with the mock-transfected control, indicating that hBtk modulates the TCF-dependent transcriptional activation mechanism, in which Arm-β-catenin is involved as a coactivator (Hamada-Kawaguchi, 2014).

The expression of an arm-dependent Ubx-lacZ reporter was examined in the embryonic midgut. Btk29A knockdown abrogated the expression of this reporter, demonstrating that Btk29A supports Arm-dependent transcription in vivo (Hamada-Kawaguchi, 2014).

Btk29A was shown to phosphorylate Arm-β-catenin on conserved tyrosine residues, one of which (Arm-Y150) is pivotal for the niche function to prevent GSC daughters from overproliferating. Notably, most GSCs in Btk29A mutants do not express Bam (fig. S1R). This suggests that the presumptive Btk29A-Arm-Piwi pathway selectively regulates the proliferation of differentiating GSC daughters without interfering with GSC maintenance. Without Btk29A type 2, cystoblasts fail to exit the cell cycle, leading to the overproduction of germ cells, many of which are unable to complete differentiation and contribute to the genesis of an ovarian tumor (Hamada-Kawaguchi, 2014).

β-Catenin exerts multiple functions through its promiscuous binding abilities in cell-to-cell interactions and transcription. This protein plays critical roles in stem cell biology, and β-catenin malfunction results in a variety of cancers. These findings add a new dimension to the study of β-catenin by highlighting the pivotal role of the tyrosine phosphorylation of β-catenin in the control of transcription in the nucleus, in addition to the regulated control of the stability and motility of cell adhesion (Hamada-Kawaguchi, 2014).

Self-association of the APC tumor suppressor is required for the assembly, stability, and activity of the Wnt signaling destruction complex

The tumor suppressor Adenomatous polyposis coli (APC) is an essential negative regulator of Wnt signaling through its activity in the destruction complex with Axin, GSK3beta and CK1 that targets β-catenin/Armadillo (β-cat/Arm) for proteosomal degradation. The destruction complex forms macromolecular particles termed the destructosome. While APC functions in the complex through its ability to bind both β-cat and Axin, it is hypothesized that APC proteins play an additional role in destructosome assembly through self-association. This study shows that a novel N-terminal coil, the APC Self-Association Domain (ASAD), found in vertebrate and invertebrate APCs, directly mediates self-association of Drosophila APC2 and plays an essential role in the assembly and stability of the destructosome that regulates β-cat degradation in Drosophila and human cells. Consistent with this, removal of the ASAD from the Drosophila embryo results in β-cat/Arm accumulation and aberrant Wnt pathway activation. These results suggest that APC proteins are required not only for the activity of the destructosome, but also for the assembly and stability of this macromolecular machine (Kunttas-Tatli, 2014).

Ras-activated Dsor1 promotes Wnt signaling in Drosophila development

Wnt/Wingless (Wg) and Ras/MAPK signaling both play fundamental roles in growth, cell-fate determination, and when dysregulated, can lead to tumorigenesis. Several conflicting modes of interaction between Ras/MAPK and Wnt signaling have been identified in specific cellular contexts, causing synergistic or antagonistic effects on target genes. Evidence was found that the dual specificity kinase MEK, Downstream of Raf1 (Dsor1), is required for Wnt signaling. Knockdown of Dsor1 results in loss of Wg target gene expression, as well as reductions in stabilized Armadillo (Arm; Drosophila β-catenin). A close physical interaction between Dsor1 and Arm was identified, and catalytically inactive Dsor1 was found to cause a reduction inactive Arm. These results suggest that Dsor1 normally counteracts the Axin-mediated destruction of Arm. Ras-Dsor1 activity was found to be independent of upstream activation by EGFR, rather it appears to be activated by the insulin-like growth factor receptor to promote Wg signaling. Together these results suggest novel crosstalk between Insulin and Wg signaling via Dsor1 (Hall, 2015).

This study has demonstrated that Dsor1/MEK is a novel regulator of Wg/Wnt signaling. Dsor1 and the mammalian ortholog MEK are critical for full activation of Wg/Wnt signal transduction. Their role in Wg/Wnt signaling is likely independent of MAPK phosphorylation, challenging the current dogma of the MAPK signaling cascade. This study also identified that the currently known activator of Ras- Dsor1 in imaginal discs, EGFR, is non-essential for a role in Wg signal propagation. Activation of this novel cross talk mechanism may be initiated through insulin-like growth factors (IGFs)/Drosophila insulin-like peptides (DILPs) and the InR (Hall, 2015).

These findings have highlighted a new function for components of the MAPK signaling cascade in Wnt activation. These two critical signaling pathways have been found to influence each other in many carcinomas, yet have not been well established in normal developmental contexts. The current results uncover a role of Dsor1 in Wg signaling in the developing larval imaginal discs and salivary glands. Epistasis experiments identified that Dsor1 acts upon the Wg pathway in the receiving cells after Dsh recruitment to the co-receptors Fz and Arrow, and its presence is needed for the recruitment of the destruction complex components to the membrane surface. As Dsor1 showed a close physical interaction with Arm itself at both the apical and basal cell surface, it suggests that Dsor1 acts to promote and retain the destruction complex near the receptor complex. Future experiments will need to clarify if Dsor1 is utilized as a linker to promote destruction complex retention, or if its ability to phosphorylate specific target proteins within the complex is key. However, the utilization of catalytically inactive MEK1 resulted in reduced TCF reporter activity and significantly reduced active β-catenin, suggesting the phosphorylation activity of MEK is critical for Wnt signaling in mammalian cells (Hall, 2015).

Quite surprisingly the results identified that Dsor1/MEK activity was independent of its well-known function in Rl/ERK activation. The MAPK signaling cascade is one of the best characterized and understood phosphorylation pathways to date. Its central dogma has revolved around its simple and exclusively linear signaling series for the activation of Rl/ERK. A small number of previous studies have questioned this model, demonstrating that MEK is capable of phosphorylating other targets, even GSK3. The results are consistent with this alternative (Hall, 2015).

This study demonstrated through RNAi knockdown, ectopic expression, and genetic interaction studies that Rl does not influence Wg activity, and that the effect that was observe is specific to Dsor1. A mammalian cell culture experiment does support previous findings that vertebrate ERK can promote Wnt activity, suggesting the mechanism may diverge slightly between flies and vertebrates. Moreover, a novel MEK function was demonstrated for direct promotion of Wnt signaling. Dsor1 activation may require InR signaling The findings reveal that the predominant larval MAPK signaling cascade initiated by EGF is not required for the activation of this novel Ras-Dsor1-Wg interaction. Endogenous EGFR signaling is the only identified activator of di-phospho-Rolled in the developing wing. The results do not contradict the current understanding of MAPK activity in the developing wing, but demonstrate a novel pathway that utilizes several of the pathway proteins. It was surprising to identify that inhibition of the InR resulted in a striking phenocopy of Dsor1 disruption, suggesting InR may be the upstream activator of Ras-Dsor1. InR activation of Ras-MAPK signaling for a proliferation response has been previously identified. The results suggest that DILPs may play a role in patterning as well, via Wg signaling. It has been identified that insulin and IGF1 can promote Wnt activity by increasing β-catenin stability and nuclear accumulation, as well as up regulation of other pathway components through multiple distinct mechanisms. In future studies it will be interesting to identify if InR-Ras-MEK-Wnt crosstalk can also be elucidated in mammalian cells, as well as trying to distinguish if IGF/DILPs promote Dsor1 activity for Wg signaling (Hall, 2015).

Constitutive scaffolding of multiple Wnt enhanceosome components by Legless/BCL9

Wnt/β-catenin signaling elicits context-dependent transcription switches that determine normal development and oncogenesis. These are mediated by the Wnt enhanceosome, a multiprotein complex binding to the Pygo chromatin reader and acting through TCF/LEF-responsive enhancers. Pygo renders this complex Wnt-responsive, by capturing β-catenin via the Legless/BCL9 adaptor. This study used CRISPR/Cas9 genome engineering of Drosophila legless (lgs) and human BCL9 and B9L to show that the C-terminus downstream of their adaptor elements is crucial for Wnt responses. BioID proximity labeling revealed that BCL9 and B9L, like PYGO2, are constitutive components of the Wnt enhanceosome. Wnt-dependent docking of β-catenin to the enhanceosome apparently causes a rearrangement that apposes the BCL9/B9L C-terminus to TCF. This C-terminus binds to the Groucho/TLE co-repressor, and also to the Chip/LDB1-SSDP enhanceosome core complex via an evolutionary conserved element. An unexpected link between BCL9/B9L, PYGO2 and nuclear co-receptor complexes suggests that these β-catenin co-factors may coordinate Wnt and nuclear hormone responses (van Tienen, 2017).

The Wnt/β-catenin signaling cascade is an ancient cell communication pathway that operates context-dependent transcriptional switches to control animal development and tissue homeostasis. Deregulation of the pathway in adult tissues can lead to many different cancers, most notably colorectal cancer. Wnt-induced transcription is mediated by T cell factors (TCF1/3/4, LEF1) bound to Wnt-responsive enhancers, but their activity depends on the co-activator β-catenin (Armadillo in Drosophila), which is rapidly degraded in unstimulated cells. Absence of β-catenin thus defines the OFF state of these enhancers, which are silenced by Groucho/TLE co-repressors bound to TCF via their Q domain. This domain tetramerizes to promote transcriptional repression (Chodaparambil, 2014), which leads to chromatin compaction apparently assisted by the interaction between Groucho/TLE and histone deacetylases (HDACs) (van Tienen, 2017).

Wnt signaling relieves this repression by blocking the degradation of β-catenin, which thus accumulates and binds to TCF, converting the Wnt-responsive enhancers into the ON state. This involves the binding of β-catenin to various transcriptional co-activators via its C-terminus, most notably to the CREB-binding protein (CBP) histone acetyltransferase or its p300 paralog, resulting in the transcription of the linked Wnt target genes. Subsequent reversion to the OFF state (for example, by negative feedback from high Wnt signaling levels near Wnt-producing cells, or upon cessation of signaling) involves Groucho/TLE-dependent silencing, but also requires the Osa/ARID1 subunit of the BAF (also known as SWI/SNF) chromatin remodeling complex which binds to β-catenin through its BRG/BRM subunit. Cancer genome sequencing has uncovered a widespread tumor suppressor role of the BAF complex, which guards against numerous cancers including colorectal cancer, with >20% of all cancers exhibiting at least one inactivating mutation in one of its subunits, most notably in ARID1A. Thus, it appears that failure of Wnt-inducible enhancers to respond to negative feedback imposed by the BAF complex strongly predisposes to cancer (van Tienen, 2017).

How β-catenin overcomes Groucho/TLE-dependent repression remains unclear, especially since β-catenin and TLE bind to TCF simultaneously (Chodaparambil, 2014). Therefore, the simplest model envisaging competition between β-catenin and TLE cannot explain this switch, which implies that co-factors are required. One of these is Pygo, a chromatin reader binding to histone H3 tail methylated at lysine 4 (H3K4m) via its C-terminal PHD finger (Fiedler, 2008). In Drosophila where Pygo was discovered as an essential co-factor for activated Armadillo, its main function appears to be to assist Armadillo in overcoming Groucho-dependent repression. It has been discovered recently that Pygo associates with TCF enhancers via its highly conserved N-terminal NPF motif that binds directly to the ChiLS complex, composed of a dimer of Chip/LDB (LIM domain-binding protein) and a tetramer of SSDP (single-stranded DNA-binding protein, also known as SSBP). Notably, ChiLS also binds to other enhancer-bound NPF factors such as Osa/ARID1 and RUNX, and to the C-terminal WD40 domain of Groucho/TLE, and thus forms the core module of a multiprotein complex termed 'Wnt enhanceosome' (Fiedler, 2015). This study proposed that Pygo renders this complex Wnt-responsive by capturing Armadillo/β-catenin through the Legless adaptor (whose orthologs in humans are BCL9 and B9L, also known as BCL9-2). The salient feature of this model is that the Wnt enhanceosome keeps TCF target genes repressed prior to Wnt signaling while at the same time priming them for subsequent Wnt induction, and for timely shut-down via negative feedback depending on Osa/ARID1 (Fiedler, 2015; van Tienen, 2017 and references therein).

This study assessed the function of Legless and BCL9/B9L within the Wnt enhanceosome. Using a proximity-labeling proteomics approach (called BioID) in human embryonic kidney (HEK293) cells, a compelling association was uncovered between BCL9/B9L and the core Wnt enhanceosome components, regardless of Wnt signaling. Co-immunoprecipitation (coIP) and in vitro binding assays based on Nuclear Magnetic Resonance (NMR) revealed that BCL9 and B9L associate with TLE3 through their C-termini, and that they bind directly to Chip/LDB-SSDP via their evolutionary conserved homology domain 3 (HD3). These elements are outside the sequences mediating the adaptor function between Pygo and Armadillo/β-catenin, but they are similarly important for Wnt responses during Drosophila development and in human cells, as is shown by CRISPR/Cas9-based genome editing. The results consolidate and refine the Wnt enhanceosome model, indicating a constitutive scaffolding function of BCL9/B9L within this complex. The evidence further suggests that BCL9/B9L but not Pygo undergoes a β-catenin-dependent rearrangement within the enhanceosome upon Wnt signaling (see Model of the Wnt enhanceosome), gaining proximity to TCF, which might trigger enhanceosome switching (van Tienen, 2017).

This study has uncovered genetic and physical interactions between two constitutive core components of the Wnt enhanceosome and the C-terminus of Legless/BCL9. The first of these is ChiLS, the core module of the Wnt enhanceosome (Fiedler, 2015): ChiLS is a direct and specific ligand of the α-helical HD3 element of B9L and, likely, of other Legless/BCL9 orthologs, given the strong sequence conservation of this α-helix. The physiological relevance of this interaction with ChiLS is underscored by genetic analysis in flies. The evidence thus implicates HD3 as an evolutionary conserved contact point between Legless/BCL9 and ChiLS, although the primary link between these two proteins appears to be provided by Pygo (van Tienen, 2017).

A second link between the Legless/BCL9 C-terminus and the Wnt enhanceosome is mediated by the WD40 domain of TLE/Groucho. Given evidence from RIME, this link is also likely to be direct although, for technical reasons, it has not been possible to prove this. The function of the C-terminus of Legless/BCL9 for transducing Wnt signals was revealed by the wg-like phenotypes in Drosophila larvae and flies and by their defective transcriptional Wg responses, and by the loss of transcriptional Wnt responses in BCL9/B9L-deleted human cells. The evidence indicates that Legless/BCL9 undergoes three separate functionally relevant interactions with distinct components of the Wnt enhanceosomewith Pygo, ChiLS and Groucho/TLE. Importantly, BioID revealed that these interactions are constitutive, preceding Wnt signaling, and that they hardly change upon Wnt stimulation. Taken together with its multivalent interactions with the Wnt enhanceosome, this is consistent with Legless/BCL9 being a core component of this complex, providing a scaffolding function that facilitates its assembly and/or maintains its cohesion (van Tienen, 2017).

Following Wnt stimulation, Legless/BCL9 undergoes an additional physiologically relevant interaction, by binding to (stabilized) Armadillo/β-catenin via HD2. Legless/BCL9 thus confers Wnt-responsiveness on the Wnt enhanceosome through its ability to capture Armadillo/β-catenin. In other words, in addition to scaffolding the enhanceosome, Legless/BCL9 also earmarks this complex for Wnt responses. Intriguingly, the BioID data indicated that the capture of β-catenin by Legless/BCL9 triggers its rearrangement within the complex, apposing its C-terminus to TCF. This apparent β-catenin-dependent apposition is consistent with structural data showing that BCL9/B9L HD2 is closely apposed to TCF when in a ternary complex with β-catenin. The evidence supports the notion of Legless/BCL9 acting as an Armadillo loading factor, facilitating access of Armadillo/β-catenin to TCF, but argues against the original co-activator hypothesis which posited that Legless/BCL9 is recruited to TCF by Armadillo/β-catenin exclusively in Wnt-stimulated cells. Whatever the case, the β-catenin-dependent apposition of the Legless/BCL9 C-terminus to TCF is likely to trigger Wnt enhanceosome switching from OFF to ON, resulting in the relief of Groucho/TLE-dependent repression and culminating in the Wnt-dependent transcriptional activation of linked target genes (van Tienen, 2017).

This transition of the Wnt enhanceosome from OFF to ON is accompanied by a proximity gain between Legless/BCL9 and CBP/p300, likely to reflect at least in part its de novo binding to Armadillo/β-catenin. However, the evidence indicates that CBP/p300 is associated with the Wnt enhanceosome prior to Wnt signaling, possibly via direct binding to B9L as suggested by RIME, and that the docking of Armadillo/β-catenin to the Wnt enhanceosome strengthens its association with CBP/p300, and/or directs the histone acetyltransferase activity of CBP/p300 towards its substrates, primarily the histone tails. By acetylating these tails, CBP/p300 appears to promote Wnt-dependent transcription in flies and human cells. Indeed, CBP-dependent histone acetylation has been observed at Wg target enhancers in Drosophila although, interestingly, this preceded transcriptional activation. This is consistent with BioID data, indicating constitutive association of CBP/p300 with the Wnt enhanceosome (van Tienen, 2017).

It seems plausible that histone acetylation at Wnt target enhancers is instrumental in antagonizing the compaction of their chromatin imposed by Groucho/TLE, which depends on its tetramerization via its Q domain as well as its binding to HDACs. Indeed, HDACs were found near the bottom of the BioID lists, and one of the top hits identified by B9L was GSE1, a subunit of the BRAF-HDAC complex. However, CBP/p300 also has non-histone substrates within the Wnt enhanceosome, including dTCF in Drosophila whose Armadillo-binding site can be acetylated by dCBP, which thus blocks the binding between the two proteins and antagonizes Wg responses. It thus regulates Wnt-dependent transcription positively as well as negatively, similarly to Groucho/TLE which not only silences Wnt target genes but also earmarks them for Wnt inducibility, as a core component of the Wnt enhanceosome. It is intriguing that both bimodal regulators are associated constitutively with this complex. A corollary is that the docking of Armadillo/β-catenin to the Wnt enhanceosome changes their substrate specificities and/or activities (van Tienen, 2017).

An important refinement of the initial enhanceosome model is with regard to the BAF complex, which appears to be a constitutive component of the Wnt enhanceosome, as indicated by BioID data. This complex is highly conserved from yeast to humans, and it contains 15 subunits in human cells (Kadoch, 2015), including the DNA-binding Osa/ARID1 subunit. A wealth of evidence from studies in flies and mammals indicates that this complex primarily antagonizes Polycomb-mediated silencing of genes, most notably of the INK4A locus which encodes an anti-proliferative factor, which could explain why the BAF complex functions as a tumor suppressor in many tissues. However, recall that this complex also specifically antagonizes Armadillo/β-catenin-mediated transcription, likely via its BRG/BRM subunit which directly binds to β-catenin. Evidence from studies in Drosophila of Wg-responsive enhancers suggests that this complex mediates a negative feedback from high Wg signaling levels near Wg-producing cells which results in re-repression, imposed by the Brinker homeodomain repressor and its Armadillo-binding Teashirt co-repressor. The same factors may also install silencing on Wnt-responsive enhancers upon cessation of Wnt signaling. Notably, mammals do not have a Brinker ortholog, which could explain some of the apparent functional differences between flies and mammals with regard to the BAF complex (Kadoch, 2015). However, the closest mammalian relatives of Teashirt are the Homothorax/MEIS proteins, a family of homeodomain proteins whose expression can be Wnt-inducible. They are thus candidates for Wnt-induced repressors that confer BAF-dependent feedback inhibition (van Tienen, 2017).

Notably, none of BioID lists contained RUNX proteins. Based on functional evidence from Drosophila midgut enhancers, it is proposed that these proteins (which bind to both enhancers and Groucho/TLE) are pivotal for initial assembly of the Wnt enhanceosome at Wnt-responsive enhancers during early embryonic development, or in uncommitted progenitor cells of specific cell lineages (Fiedler, 2015). However, HEK293 cells are epithelial cells and may thus not express any RUNX factors. In any case, the negative BioID results suggest that RUNX factors function in a hit-and-run fashion. Evidently, the Wnt enhanceosome complex, once assembled at Wnt-responsive enhancers, can switch between ON and OFF states without RUNX (van Tienen, 2017).

In summary, this study has uncovered a fundamental role to Legless/BCL9 as a scaffold of the Wnt enhanceosome, far beyond its role in linking Armadillo/β-catenin to Pygo. Indeed, the function of Legless/BCL9 may extend beyond transcriptional Wnt responses, as indicated by the unexpected discovery of its strong association with nuclear co-receptor complexes. Potentially, these associations underlie the observed cross-talk between Wnt/β-catenin and nuclear hormone receptor signaling, documented extensively in the literature, including evidence for direct activation of the androgen receptor by β-catenin. Furthermore, a strong association between TLE1 and the estrogen receptor has been discovered in breast cancer cells, where TLE1 assists the estrogen receptor in its interaction with chromatin and its proliferation-promoting function. This is reminiscent of the role of Groucho/TLE as a cornerstone of the Wnt enhanceosome, proposed to earmark TCF enhancers for subsequent β-catenin docking and transcriptional Wnt responses (Fiedler, 2015). It will be interesting to test experimentally the putative roles of BCL9/B9L and Pygo in enabling cross-talk between β-catenin and nuclear hormone receptor signaling, both during normal development and in cancer (van Tienen, 2017).

Wnt-Dependent inactivation of the Groucho/TLE co-repressor by the HECT E3 ubiquitin ligase Hyd/UBR5

Extracellular signals are transduced to the cell nucleus by effectors that bind to enhancer complexes to operate transcriptional switches. For example, the Wnt enhanceosome is a multiprotein complex associated with Wnt-responsive enhancers through T cell factors (TCF; see Pangolin) and kept silent by Groucho/TLE co-repressors. Wnt-activated β-catenin (see Armadillo) binds to TCF to overcome this repression, but how it achieves this is unknown. This study discovered that this process depends on the HECT E3 ubiquitin ligase Hyd/UBR5, which is required for Wnt signal responses in Drosophila and human cell lines downstream of activated Armadillo/β-catenin. Groucho/TLE was identified as a functionally relevant substrate, whose ubiquitylation by UBR5 is induced by Wnt signaling and conferred by β-catenin. Inactivation of TLE by UBR5-dependent ubiquitylation also involves VCP/p97, an AAA ATPase regulating the folding of various cellular substrates including ubiquitylated chromatin proteins. Thus, Groucho/TLE ubiquitylation by Hyd/UBR5 is a key prerequisite that enables Armadillo/β-catenin to activate transcription (Flack, 2017).

An essential step enabling Wnt-dependent transcription is the conversion of the Wnt enhanceosome from silent to active. This involves the binding of the Wnt effector β-catenin to TCF, which releases the transcriptional silence imposed on the linked genes by TCF-bound Groucho/TLE. This study has discovered a crucial role of Hyd/UBR5 in this process, and the evidence suggests that β-catenin directs the activity of this HECT ubiquitin ligase toward Groucho/TLE, to block its repressive activity. The evidence also implicates VCP/p97 in this UBR5-dependent inactivation of Groucho/TLE during Wnt signaling (Flack, 2017).

By generating UBR5 null mutant cell lines, it was possible to resolve previous inconsistencies regarding the effects of UBR5 depletion on Wnt/β-catenin responses in human cell lines. UBR5 KO cell lines consistently showed reduced Wnt responses, but no changes in β-catenin levels. This parallels the results from hyd mutant clones in flies, providing unequivocal evidence for Hyd/UBR5 as a positive regulator of Wnt signaling in fly and human cells (Flack, 2017).

Three strands of evidence implicate Groucho/TLE as a physiologically relevant substrate of Hyd/UBR5 during Wnt signaling. First, epistasis analysis revealed that Hyd/UBR5 acts below Armadillo/β-catenin, and thus likely targets a substrate in the nucleus, consistent with its nuclear localization. Second, the activity of UBR5 in ubiquitylating Groucho/TLE is triggered by Wnt/β-catenin signaling. Third, in Drosophila wing discs, hyd is largely dispensable in the absence of Groucho (as revealed by hyd gro double mutant clones), which provides powerful evidence that Hyd acts by antagonizing Groucho (Flack, 2017).

Two possible mechanisms by which β-catenin might activate UBR5 toward TLE3 during Wnt signaling are considered. Either, β-catenin might disinhibit UBR5 if this enzyme were normally autoinhibited, like the NEDD4 family HECT ligases. Indeed, one of these ligases (WWP2) is disinhibited by Dishevelled, which, upon polymerization, engages in multivalent interactions with WWP2 to release its cognate binding sites from autoinhibitory contacts. However, the strong activity of UBR5 toward PAIP2 in the absence of Wnt signaling argues against this mechanism. An alternative mechanism is favored, namely that β-catenin apposes enzyme and substrate, e.g., via triggering a conformational change of the Wnt enhanceosome that results in proximity between UBR5 and Groucho/TLE. Support for this mechanism comes from previous proximity labeling experiments that revealed a β-catenin-dependent rearrangement of some of the components within the Wnt enhanceosome (van Tienen, 2017), and from coIP assays showing that β-catenin promotes the association between UBR5 and TLE3 (Flack, 2017).

How does UBR5-dependent ubiquitylation of Groucho/TLE inactivate its co-repressor function? The most obvious mechanism involves proteasomal turnover of Ub-TLE, given the specificity of UBR5 in generating K48-linked Ub chains, which are efficient proteasomal targeting signals. In support of this, the levels of UBR5-dependent Ub-TLE3 are elevated after proteasome inhibition. However, negative results from the cycloheximide chase experiments argue against rapid proteosomal degradation being the primary mechanism underlying the UBR5-dependent inactivation of Groucho/TLE (Flack, 2017).

It was also considered that the ubiquitylation of the WD40 domain might interfere with its binding to cognate ligands, and thus weaken the association of Groucho/TLE with the Wnt enhanceosome. However, this does not seem to be the case since Ub-TLE3 appears to bind to its ligands as efficiently as unmodified TLE, including a K-only mutant which can only be ubiquitylated at K720, a WD40 pore residue that is crucial for ligand binding and co-repression. Evidently, the extended C terminus through which ubiquitin is attached to K720 is flexible enough to allow simultaneous ligand binding. However, for technical reasons, it was not possible to test the binding of Ub-TLE to the key ligand through which Groucho/TLE exerts its repressive function -- namely the nucleosomes to which Groucho/TLE binds via both its structured domains, to promote chromatin compaction. Nevertheless, it is plausible that the attachment of multiple ubiquitin chains to the WD40 domain would loosen up the binding of Groucho/TLE to nucleosomes, and thus attenuate its ability to compact chromatin (Flack, 2017).

Evidence based on dominant-negative VCP/p97 and two distinct VCP/p97 inhibitors implicates this ATPase in the Wnt-dependent inactivation of Ub-TLE. Intriguingly, a recent proteomic screen for NMS-873-induced VCP/p97-associated proteins identified TLE1 and TLE3 as the only Wnt signaling components, along with VCP/p97 adaptors and other putative substrates, consistent with the notion of Groucho/TLE is a substrate of this ATPase. VCP/p97 regulates the folding of ubiquitylated proteins, to promote their segregation from large structures, such as endomembranes, and also from large protein complexes, including DNA repair and chromatin complexes. It is therefore conceivable that VCP/p97 unfolds Groucho/TLE upon its ubiquitylation, especially if this modification loosened the interaction of Groucho/TLE with nucleosomes. Whatever the case, unfolding of the Groucho/TLE tetramer by VCP/p97 is likely to destabilize it, which would disable its repressive function. This is consistent with a recent proposal that the relief of Groucho-dependent repression is based on kinetic destabilization of the Groucho complex (Chambers, 2017), which may be facilitated by its ubiquitylation and unfolding by VCP/p97 (Flack, 2017).

One other E3 ligase has been shown to ubiquitylate TLE3, namely the RING ligase XIAP, which constitutively monoubiquitylates the Q domain of TLE3, apparently stimulating Wnt-dependent transcription by blocking its binding to TCF4. This contrasts with the Wnt-induced activity of UBR5 toward TLE3 revealed by this study. Evidently, the two ligases act distinctly, and also independently, given that the UBR5-dependent polyubiquitylation of TLE3 is normal in XIAP KO cells. However, it is also noted that the reduction of Wnt-dependent transcription in the XIAP KO cells was modest at best, compared to the substantial reduction in UBR5 KO cells. Either XIAP plays a lesser role in promoting transcriptional Wnt responses or a compensating E3 ligase was upregulated during the process of establishing XIAP KO cells. It is noted that the XIAP KO mice are viable, and without any overt mutant phenotypes, and that the Drosophila XIAP mutants do not show wg-like phenotypes, in contrast to the hyd mutant clones that phenocopy strong wg-like mutant phenotypes. All in all, it appears that UBR5 has a more profound role than XIAP in enabling transcriptional Wnt responses (Flack, 2017).

Inactivation of Groucho/TLE by UBR5 and VCP/p97 could also underlie other signaling-dependent gene switches that involve Groucho/TLE-dependent repression, e.g., Notch signaling, which depends on binding of Groucho/TLE to HES repressors. Indeed, recent genetic screens in C. elegans have identified the UBR5 ortholog sog-1 as a negative regulator of Notch signaling during nematode development. Although it is conceivable that hyd also affects Notch responses in flies, this study found that the derepression of the Notch target gene wg in hyd mutant wing disc clones is not sensitive to blockade by dominant-negative Mastermind, which argues against a role of Hyd in Notch-dependent transcription in this tissue. It is also noted that Ubr5 has been linked to defective Hedgehog signaling in mice, following an earlier lead of Groucho as a putative Hyd target in the context of Hedgehog signaling, although these links between Hyd/Ubr5 and Hedgehog signaling appear to be indirect (Flack, 2017).

However, UBR5 clearly also modifies substrates other than Groucho/TLE, including proteins with PAM2 motifs that are recognized by its MLLE domain, e.g., PAIP2 involved in translational control. Furthermore, via its UBR domain, UBR5 may recognize substrates of the N-end rule pathway, though few of these have been identified to date. Given the nuclear location of UBR5, it seems highly likely that most of its physiologically relevant substrates are nuclear proteins, e.g., the RING E3 ligase RNF168, which is ubiquitylated and destabilized by UBR5 during the DNA damage response (Flack, 2017).

UBR5 has been heavily implicated in cancer, although it is somewhat unclear whether it promotes or antagonizes tumor progression, which may depend on context. However, UBR5 amplification is the predominant genetic alteration in many types of cancers (far more prevalent than loss-of-function UBR5 mutations), and amplified UBR5 correlates with poor outcomes in breast cancer. This implies a tumor-promoting role of UBR5, consistent with its role in relieving Groucho/TLE-dependent repression of Wnt responses. It will be interesting to test whether UBR5 loss-of-function inhibits β-catenin-dependent tumorigenesis, e.g., in the intestine. This might be expected, given the results from the colorectal cancer cell line HCT116 whose β-catenin-dependent transcription is attenuated by UBR5 KO and whose proliferation is slowed down by VCP/p97 inhibition. If this were to apply generally to other colorectal cancer lines, this would indicate the potential of UBR5 and VCP/p97 as new enzymatic targets for therapeutic intervention in colorectal and other β-catenin-dependent cancers. It could widen the application of CB-5083, an orally bioavailable VCP/p97 inhibitor currently in phase 1 clinical trials (Flack, 2017).

Protein Interactions

Continued: part 2/2

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

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