armadillo
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 Armadilloís
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 Armís 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 Armís
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).
Armadilloís 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).
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).
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 þ-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 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).
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 þ-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 et al., 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).
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).
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).
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).
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).
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 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).
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 (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 þ-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).
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,