Wg/Wnt signal can be transmitted through arrow/LRP5,6 and Axin independently of Zw3/Gsk3beta activity

Activation of the Wnt signaling cascade provides key signals during development and in disease. Here we provide evidence, by designing a Wnt receptor with ligand-independent signaling activity, that physical proximity of Arrow (LRP) to the Wnt receptor Frizzled-2 triggers the intracellular signaling cascade. We have uncovered a branch of the Wnt pathway in which Armadillo activity is regulated concomitantly with the levels of Axin protein. The intracellular pathway bypasses Gsk3beta/Zw3, the kinase normally required for controlling beta-catenin/Armadillo levels, suggesting that modulated degradation of Armadillo is not required for Wnt signaling. We propose that Arrow (LRP) recruits Axin to the membrane, and that this interaction leads to Axin degradation. As a consequence, Armadillo is no longer bound by Axin, resulting in nuclear signaling by Armadillo.     

The Adenomatous polyposis coli tumour suppressor is essential for Axin complex assembly and function and opposes Axin's interaction with Dishevelled

Most cases of colorectal cancer are linked to mutational inactivation of the Adenomatous polyposis coli (APC) tumour suppressor. APC downregulates Wnt signalling by enabling Axin to promote the degradation of the Wnt signalling effector β-catenin (Armadillo in flies). This depends on Axin's DIX domain whose polymerization allows it to form dynamic protein assemblies ('degradasomes'). Axin is inactivated upon Wnt signalling, by heteropolymerization with the DIX domain of Dishevelled, which recruits it into membrane-associated 'signalosomes'. How APC promotes Axin's function is unclear, especially as it has been reported that APC's function can be bypassed by overexpression of Axin. Examining apc null mutant Drosophila tissues, we discovered that APC is required for Axin degradasome assembly, itself essential for Armadillo downregulation. Degradasome assembly is also attenuated in APC mutant cancer cells. Notably, Axin becomes prone to Dishevelled-dependent plasma membrane recruitment in the absence of APC, indicating a crucial role of APC in opposing the interaction of Axin with Dishevelled. Indeed, co-expression experiments reveal that APC displaces Dishevelled from Axin assemblies, promoting degradasome over signalosome formation in the absence of Wnts. APC thus empowers Axin to function in two ways-by enabling its DIX-dependent self-assembly, and by opposing its DIX-dependent copolymerization with Dishevelled and consequent inactivation.     

Wingless modulates the ligand independent traffic of Notch through Dishevelled

Here we investigate the structural and functional basis of the interactions between Notch and Wingless signalling in Drosophila. Using yeast-two-hybrid and pull-down assays we show that Notch can bind directly a form of Dishevelled that is stabilized upon Wingless signalling. Moreover, we show that the mechanism by which Wingless signalling is able to downregulate Notch is by promoting its ligand-independent traffic to a compartment where it is degraded and that this activity depends on Dishevelled.     

GSKIP is homologous to the Axin GSK3beta interaction domain and functions as a negative regulator of GSK3beta

Although prominent FRAT/GBP exhibits a limited degree of homology to Axin, the binding sites on GSK3 for FRAT/GBP and Axin may overlap to prevent the effect of FRAT/GBP in stabilizing beta-catenin in the Wnt pathway. Using a yeast two-hybrid screen, we identified a novel protein, GSK3beta interaction protein (GSKIP), which binds to GSK3beta. We have defined a 25-amino acid region in the C-terminus of GSKIP that is highly similar to the GSK3beta interaction domain (GID) of Axin. Using an in vitro kinase assay, our results indicate that GSKIP is a good GSK3beta substrate, and both the full-length protein and a C-terminal fragment of GSKIP can block phosphorylation of primed and nonprimed substrates in different fashions. Similar to Axin GID(381-405) and FRATtide, synthesized GSKIPtide is also shown to compete with and/or block the phosphorylation of Axin and beta-catenin by GSK3beta. Furthermore, our data indicate that overexpression of GSKIP induces beta-catenin accumulation in the cytoplasm and nucleus as visualized by immunofluorescence. A functional assay also demonstrates that GSKIP-transfected cells have a significant effect on the transactivity of Tcf-4. Collectively, we define GSKIP as a naturally occurring protein that is homologous with the GSK3beta interaction domain of Axin and is able to negatively regulate GSK3beta of the Wnt signaling pathway.     

The segmentation clock in mice: interaction between the Wnt and Notch signalling pathways

In the last few years, the efforts to elucidate the mechanisms underlying the segmentation clock in various vertebrate species have multiplied. Early evidence suggested that oscillations are caused by one of the genes under the Notch signalling pathway (like those of the her or Hes families). Recently, Aulehla et al. [Wnt3a plays a major role in the segmentation clock controlling somitogenesis. Dev. Cell 4, 395-406] discovered that Axin2 (a gene under the Wnt3a signalling pathway) also oscillates in the presomitic mesoderm (PSM) of mice embryos and proposed some mechanisms through which the Notch and Wnt3a pathways may interact. They further suggested that a decreasing concentration of Wnt3a along the PSM may be the gradient the segmentation clock interacts with to form somites. These results were reviewed by Rida et al. [A notch feeling of somite segmentation and beyond. Dev. Biol. 265, 2-22], who introduced a complex clockwork comprising genes Hes1, Lfng (under the Notch pathway), and Axin2, as well as their multiple interactions. In the present work we develop a mathematical model based on the Rida et al. review and use it to tackle some of the questions raided by the Aulehla et al. paper: can the Axin2 feedback loop constitute a clock? Could a decreasing Wnt3a signaling constitute the wavefront, where phase is recorded and the spatial pattern laid down? What is the master oscillator?     

Wingless modulates the effects of dominant negative notch molecules in the developing wing of Drosophila

The development and patterning of the wing in Drosophila relies on a sequence of cell interactions molecularly driven by a number of ligands and receptors. Genetic analysis indicates that a receptor encoded by the Notch gene and a signal encoded by the wingless gene play a number of interdependent roles in this process and display very strong functional interactions. At certain times and places, during wing development, the expression of wingless requires Notch activity and that of its ligands Delta and Serrate. This has led to the proposal that all the interactions between Notch and wingless can be understood in terms of this regulatory relationship. Here we have tested this proposal by analysing interactions between Delta- and Serrate-activated Notch signalling and Wingless signalling during wing development and patterning. We find that the cell death caused by expressing dominant negative Notch molecules during wing development cannot be rescued by coexpressing Nintra. This suggests that the dominant negative Notch molecules cannot only disrupt Delta and Serrate signalling but can also disrupt signalling through another pathway. One possibility is the Wingless signalling pathway as the cell death caused by expressing dominant negative Notch molecules can be rescued by activating Wingless signalling. Furthermore, we observe that the outcome of the interactions between Notch and Wingless signalling differs when we activate Wingless signalling by expressing either Wingless itself or an activated form of the Armadillo. For example, the effect of expressing the activated form of Armadillo with a dominant negative Notch on the patterning of sense organ precursors in the wing resembles the effects of expressing Wingless alone. This result suggests that signalling activated by Wingless leads to two effects, a reduction of Notch signalling and an activation of Armadillo.     

Role of STRAP in regulating GSK3β function and Notch3 stabilization

Glycogen synthase kinase 3β (GSK3β) can regulate a broad range of cellular processes in a variety of cell types and tissues through its ability to phosphorylate its substrates in a cell- and time-specific manner. Although it is known that Axin and presenilin help to recruit β-catenin/Smad3 and tau protein to GSK3β, respectively, it is not clear how many of the other GSK3β substrates are recruited to it. Here, we have established the binding of GSK3β with a novel scaffold protein, STRAP, through its WD40 domains. In a new finding, we have observed that STRAP, GSK3β and Axin form a ternary complex together. We show for the first time that intracellular fragment of Notch3 (ICN3) binds with GSK3β through the ankyrin repeat domain. This binding between STRAP and GSK3β is reduced by small-molecule inhibitors of GSK3β. Further studies revealed that STRAP also binds ICN3 through the ankyrin repeat region, and this binding is enhanced in a proteasomal inhibition-dependent manner. In vivo ubiquitination studies indicate that STRAP reduces ubiquitination of ICN3, suggesting a role of STRAP in stabilizing ICN3. This is supported by the fact that STRAP and Notch3 are co-upregulated and co-localized in 59% of non-small cell lung cancers, as observed in an immunohistochemical staining of tissue microarrays. These results provide a potential mechanism by which STRAP regulates GSK3β function and Notch3 stabilization and further support the oncogenic functions of STRAP.     

Two functionally distinct Axin-like proteins regulate canonical Wnt signaling in C. elegans

Axin is a central component of the canonical Wnt signaling pathway that interacts with the adenomatous polyposis coli protein APC and the kinase GSK3beta to downregulate the effector beta-catenin. In the nematode Caenorhabditis elegans, canonical Wnt signaling is negatively regulated by the highly divergent Axin ortholog PRY-1. Mutation of pry-1 leads to constitutive activation of BAR-1/beta-catenin-dependent Wnt signaling and results in a range of developmental defects. The pry-1 null phenotype is however not fully penetrant, indicating that additional factors may partially compensate for PRY-1 function. Here, we report the cloning and functional analysis of a second Axin-like protein, which we named AXL-1. We show that despite considerable sequence divergence with PRY-1 and other Axin family members, AXL-1 is a functional Axin ortholog. AXL-1 functions redundantly with PRY-1 in negatively regulating BAR-1/beta-catenin signaling in the developing vulva and the Q neuroblast lineage. In addition, AXL-1 functions independently of PRY-1 in negatively regulating canonical Wnt signaling during excretory cell development. In contrast to vertebrate Axin and the related protein Conductin, AXL-1 and PRY-1 are not functionally equivalent. We conclude that Axin function in C. elegans is divided over two different Axin orthologs that have specific functions in negatively regulating canonical Wnt signaling.     

In vivo analysis in Drosophila reveals differential requirements of contact residues in Axin for interactions with GSK3β or β-catenin

Proper regulation of the Wingless/Wnt signaling pathway is essential for normal development. The scaffolding protein Axin plays a key role in this process through interactions with Drosophila Shaggy and Armadillo. In the current studies, we used a yeast two-hybrid assay to identify ten amino acids in Axin that are critical for in vitro interaction with Shaggy and two for interaction with Armadillo. We then generated five Axin variants in which individual putative contact amino acids were mutated and compared their activity, as assayed by rescue of axin null mutant flies, to that of Axin lacking the entire Shaggy (AxinΔSgg) or Armadillo (AxinΔArm) binding domain. Although we expected these mutants to function identically to Axin in which the entire binding domain was deleted, we instead observed a spectrum of phenotypic rescue. Specifically, two point mutants within the Shaggy binding domain showed loss of activity similar to that of AxinΔSgg and dominantly interfered with complex function, whereas a third mutant allele, AxinK446E, retained most function. Two Axin point mutants within the Armadillo binding domain were weak alleles and retained most function. These findings demonstrate the importance of in vivo verification of the role of specific amino acids within a protein.     

Amer1/WTX couples Wnt-induced formation of PtdIns(4,5)P2 to LRP6 phosphorylation

Phosphorylation of the Wnt receptor low-density lipoprotein receptor-related protein 6 (LRP6) by glycogen synthase kinase 3β (GSK3β) and casein kinase 1γ (CK1γ) is a key step in Wnt/β-catenin signalling, which requires Wnt-induced formation of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)). Here, we show that adenomatous polyposis coli membrane recruitment 1 (Amer1) (also called WTX), a membrane associated PtdIns(4,5)P(2)-binding protein, is essential for the activation of Wnt signalling at the LRP6 receptor level. Knockdown of Amer1 reduces Wnt-induced LRP6 phosphorylation, Axin translocation to the plasma membrane and formation of LRP6 signalosomes. Overexpression of Amer1 promotes LRP6 phosphorylation, which requires interaction of Amer1 with PtdIns(4,5)P(2). Amer1 translocates to the plasma membrane in a PtdIns(4,5)P(2)-dependent manner after Wnt treatment and is required for LRP6 phosphorylation stimulated by application of PtdIns(4,5)P(2). Amer1 binds CK1γ, recruits Axin and GSK3β to the plasma membrane and promotes complex formation between Axin and LRP6. Fusion of Amer1 to the cytoplasmic domain of LRP6 induces LRP6 phosphorylation and stimulates robust Wnt/β-catenin signalling. We propose a mechanism for Wnt receptor activation by which generation of PtdIns(4,5)P(2) leads to recruitment of Amer1 to the plasma membrane, which acts as a scaffold protein to stimulate phosphorylation of LRP6.     

Activated macrophages promote Wnt signalling through tumour necrosis factor-alpha in gastric tumour cells

The activation of Wnt/beta-catenin signalling has an important function in gastrointestinal tumorigenesis. It has been suggested that the promotion of Wnt/beta-catenin activity beyond the threshold is important for carcinogenesis. We herein investigated the role of macrophages in the promotion of Wnt/beta-catenin activity in gastric tumorigenesis. We found beta-catenin nuclear accumulation in macrophage-infiltrated dysplastic mucosa of the K19-Wnt1 mouse stomach. Moreover, macrophage depletion in Apc(Delta716) mice resulted in the suppression of intestinal tumorigenesis. These results suggested the role of macrophages in the activation of Wnt/beta-catenin signalling, which thus leads to tumour development. Importantly, the conditioned medium of activated macrophages promoted Wnt/beta-catenin signalling in gastric cancer cells, which was suppressed by the inhibition of tumour necrosis factor (TNF)-alpha. Furthermore, treatment with TNF-alpha induced glycogen synthase kinase 3beta (GSK3beta) phosphorylation, which resulted in the stabilization of beta-catenin. We also found that Helicobacter infection in the K19-Wnt1 mouse stomach caused mucosal macrophage infiltration and nuclear beta-catenin accumulation. These results suggest that macrophage-derived TNF-alpha promotes Wnt/beta-catenin signalling through inhibition of GSK3beta, which may contribute to tumour development in the gastric mucosa.