Wnk kinases are positive regulators of canonical Wnt/β-catenin signalling

Wnt/β-catenin signalling is central to development and its regulation is essential in preventing cancer. Using phosphorylation of Dishevelled as readout of pathway activation, we identified Drosophila Wnk kinase as a new regulator of canonical Wnt/β-catenin signalling. WNK kinases are known for regulating ion co-transporters associated with hypertension disorders. We demonstrate that wnk loss-of-function phenotypes resemble canonical Wnt pathway mutants, while Wnk overexpression causes gain-of-function canonical Wnt-signalling phenotypes. Importantly, knockdown of human WNK1 and WNK2 also results in decreased Wnt signalling in mammalian cell culture, suggesting that Wnk kinases have a conserved function in ensuring peak levels of canonical Wnt signalling.     

Phase separation of Axin organizes the β-catenin destruction complex

In Wnt/β-catenin signaling, the β-catenin protein level is deliberately controlled by the assembly of the multiprotein β-catenin destruction complex composed of Axin, adenomatous polyposis coli (APC), glycogen synthase kinase 3β (GSK3β), casein kinase 1α (CK1α), and others. Here we provide compelling evidence that formation of the destruction complex is driven by protein liquid–liquid phase separation (LLPS) of Axin. An intrinsically disordered region in Axin plays an important role in driving its LLPS. Phase-separated Axin provides a scaffold for recruiting GSK3β, CK1α, and β-catenin. APC also undergoes LLPS in vitro and enhances the size and dynamics of Axin phase droplets. The LLPS-driven assembly of the destruction complex facilitates β-catenin phosphorylation by GSK3β and is critical for the regulation of β-catenin protein stability and thus Wnt/β-catenin signaling.     

Testing Models of the APC Tumor Suppressor/β-Catenin Interaction Reshapes Our View of the Destruction Complex in Wnt Signaling

The Wnt pathway is a conserved signal transduction pathway that contributes to normal development and adult homeostasis, but is also misregulated in human diseases such as cancer. The tumor suppressor adenomatous polyposis coli (APC) is an essential negative regulator of Wnt signaling inactivated in >80% of colorectal cancers. APC participates in a multiprotein “destruction complex” that targets the proto-oncogene β-catenin for ubiquitin-mediated proteolysis; however, the mechanistic role of APC in the destruction complex remains unknown. Several models of APC function have recently been proposed, many of which have emphasized the importance of phosphorylation of high-affinity β-catenin-binding sites [20-amino-acid repeats (20Rs)] on APC. Here we test these models by generating a Drosophila APC2 mutant lacking all β-catenin-binding 20Rs and performing functional studies in human colon cancer cell lines and Drosophila embryos. Our results are inconsistent with current models, as we find that β-catenin binding to the 20Rs of APC is not required for destruction complex activity. In addition, we generate an APC2 mutant lacking all β-catenin-binding sites (including the 15Rs) and find that a direct β-catenin/APC interaction is also not essential for β-catenin destruction, although it increases destruction complex efficiency in certain developmental contexts. Overall, our findings support a model whereby β-catenin-binding sites on APC do not provide a critical mechanistic function per se, but rather dock β-catenin in the destruction complex to increase the efficiency of β-catenin destruction. Furthermore, in Drosophila embryos expressing some APC2 mutant transgenes we observe a separation of β-catenin destruction and Wg/Wnt signaling outputs and suggest that cytoplasmic retention of β-catenin likely accounts for this difference.     

Hypoxia-inducible Factor-2α-dependent Hypoxic Induction of Wnt10b Expression in Adipogenic Cells

Adipocyte hyperplasia and hypertrophy in obesity can lead to many changes in adipose tissue, such as hypoxia, metabolic dysregulation, and enhanced secretion of cytokines. In this study, hypoxia increased the expression of Wnt10b in both human and mouse adipogenic cells, but not in hypoxia-inducible factor (HIF)-2α-deficient adipogenic cells. Chromatin immunoprecipitation analysis revealed that HIF-2α, but not HIF-1α, bound to the Wnt10b enhancer region as well as upstream of the Wnt1 gene, which is encoded by an antisense strand of the Wnt10b gene. Hypoxia-conditioned medium (H-CM) induced phosphorylation of lipoprotein-receptor-related protein 6 as well as β-catenin-dependent gene expression in normoxic cells, which suggests that H-CM contains canonical Wnt signals. Furthermore, adipogenesis of both human mesenchymal stem cells and mouse preadipocytes was inhibited by H-CM even under normoxic conditions. These results suggest that O₂ concentration gradients influence the formation of Wnt ligand gradients, which are involved in the regulation of pluripotency, cell proliferation, and cell differentiation.     

Stabilisation of β-Catenin Downstream of T Cell Receptor Signalling

Background

The role of TCF/β-catenin signalling in T cell development is well established, but important roles in mature T cells have only recently come to light.

Methodology/Principal Findings

Here we have investigated the signalling pathways that are involved in the regulation of β-catenin in primary human T cells. We demonstrate that β-catenin expression is upregulated rapidly after T cell receptor (TCR) stimulation and that this involves protein stabilisation rather than an increase in mRNA levels. Similar to events in Wnt signalling, the increase in β-catenin coincides with an inhibition of GSK3, the kinase that is required for β-catenin degradation. β-catenin stabilisation in T cells can also be induced by the activation of PKC with phorbol esters and is blocked by inhibitors of phosphatidylinositol 3-kinase (PI3K) and phospholipase C (PKC). Upon TCR signalling, β-catenin accumulates in the nucleus and, parallel to this, the ratio of TCF1 isoforms is shifted in favour of the longer β-catenin binding isoforms. However, phosphorylated β-catenin, which is believed to be inactive, can also be detected and the expression of Wnt target genes Axin2 and dickkopf is down regulated.

Conclusions/Significance

These data show that in mature human T cells, TCR signalling via PI3K and PKC can result in the stabilisation of β-catenin, allowing β-catenin to migrate to the nucleus. They further highlight important differences between β-catenin activities in TCR and Wnt signalling.     

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

The tumor suppressor adenomatous polyposis coli (APC) is an essential negative regulator of Wnt signaling through its activity in the destruction complex with Axin, GSK3β, and CK1 that targets β-catenin/Armadillo (β-cat/Arm) for proteosomal degradation. The destruction complex forms macromolecular particles we termed the destructosome. Whereas APC functions in the complex through its ability to bind both β-cat and Axin, we hypothesize that APC proteins play an additional role in destructosome assembly through self-association. Here we show that a novel N-terminal coil, the APC self-association domain (ASAD), found in vertebrate and invertebrate APCs, directly mediates self-association of Drosophila APC2 and plays an essential role in the assembly and stability of the destructosome that regulates β-cat degradation in Drosophila and human cells. Consistent with this, removal of the ASAD from the Drosophila embryo results in β-cat/Arm accumulation and aberrant Wnt pathway activation. These results suggest that APC proteins are required not only for the activity of the destructosome, but also for the assembly and stability of this macromolecular machine.     

Oncogenic mutations in adenomatous polyposis coli (Apc) activate mechanistic target of rapamycin complex 1 (mTORC1) in mice and zebrafish

Truncating mutations in adenomatous polyposis coli (APC) are strongly linked to colorectal cancers. APC is a negative regulator of the Wnt pathway and constitutive Wnt activation mediated by enhanced Wnt–β-catenin target gene activation is believed to be the predominant mechanism responsible for APC mutant phenotypes. However, recent evidence suggests that additional downstream effectors contribute to APC mutant phenotypes. We previously identified a mechanism in cultured human cells by which APC, acting through glycogen synthase kinase-3 (GSK-3), suppresses mTORC1, a nutrient sensor that regulates cell growth and proliferation. We hypothesized that truncating Apc mutations should activate mTORC1 in vivo and that mTORC1 plays an important role in Apc mutant phenotypes. We find that mTORC1 is strongly activated in apc mutant zebrafish and in intestinal polyps in Apc mutant mice. Furthermore, mTORC1 activation is essential downstream of APC as mTORC1 inhibition partially rescues Apc mutant phenotypes including early lethality, reduced circulation and liver hyperplasia. Importantly, combining mTORC1 and Wnt inhibition rescues defects in morphogenesis of the anterior-posterior axis that are not rescued by inhibition of either pathway alone. These data establish mTORC1 as a crucial, β-catenin independent effector of oncogenic Apc mutations and highlight the importance of mTORC1 regulation by APC during embryonic development. Our findings also suggest a new model of colorectal cancer pathogenesis in which mTORC1 is activated in parallel with Wnt/β-catenin signaling.KEY WORDS: APC, Wnt, mTOR, mTORC1, Zebrafish, Colon cancer, Polyposis, GSK-3     

Deubiquitinase USP47/UBP64E Regulates β-Catenin Ubiquitination and Degradation and Plays a Positive Role in Wnt Signaling

Wnt signaling plays important roles in development and tumorigenesis. A central question about the Wnt pathway is the regulation of β-catenin. Phosphorylation of β-catenin by CK1α and GSK3 promotes β-catenin binding to β-TrCP, leading to β-catenin degradation through the proteasome. The phosphorylation and ubiquitination of β-catenin have been well characterized; however, it is unknown whether and how a deubiquitinase is involved. In this study, by screening RNA interference (RNAi) libraries, we identified USP47 as a deubiquitinase that prevents β-catenin ubiquitination. Inactivation of USP47 by RNAi increased β-catenin ubiquitination, attenuated Wnt signaling, and repressed cancer cell growth. Furthermore, USP47 deubiquitinates itself, whereas β-TrCP promotes USP47 ubiquitination through interaction with an atypical motif in USP47. Finally, in vivo studies in the Drosophila wing suggest that UBP64E, the USP47 counterpart in Drosophila, is required for Armadillo stabilization and plays a positive role in regulating Wnt target gene expression.     

Casein Kinase 1 α Phosphorylates the Wnt Regulator Jade-1 and Modulates Its Activity

Tight regulation of Wnt/β-catenin signaling is critical for vertebrate development and tissue maintenance, and deregulation can lead to a host of disease phenotypes, including developmental disorders and cancer. Proteins associated with primary cilia and centrosomes have been demonstrated to negatively regulate canonical Wnt signaling in interphase cells. The plant homeodomain zinc finger protein Jade-1 can act as an E3 ubiquitin ligase-targeting β-catenin for proteasomal degradation and concentrates at the centrosome and ciliary basal body in addition to the nucleus in interphase cells. We demonstrate that the destruction complex component casein kinase 1α (CK1α) phosphorylates Jade-1 at a conserved SLS motif and reduces the ability of Jade-1 to inhibit β-catenin signaling. Consistently, Jade-1 lacking the SLS motif is more effective than wild-type Jade-1 in reducing β-catenin-induced secondary axis formation in Xenopus laevis embryos in vivo. Interestingly, CK1α also phosphorylates β-catenin and the destruction complex component adenomatous polyposis coli at a similar SLS motif to the effect that β-catenin is targeted for degradation. The opposing effect of Jade-1 phosphorylation by CK1α suggests a novel example of the dual functions of CK1α activity to either oppose or promote canonical Wnt signaling in a context-dependent manner.     

β-Arrestin Promotes Wnt-induced Low Density Lipoprotein Receptor-related Protein 6 (Lrp6) Phosphorylation via Increased Membrane Recruitment of Amer1 Protein

β-Arrestin is a scaffold protein that regulates signal transduction by seven transmembrane-spanning receptors. Among other functions it is also critically required for Wnt/β-catenin signal transduction. In the present study we provide for the first time a mechanistic basis for the β-arrestin function in Wnt/β-catenin signaling. We demonstrate that β-arrestin is required for efficient Wnt3a-induced Lrp6 phosphorylation, a key event in downstream signaling. β-Arrestin regulates Lrp6 phosphorylation via a novel interaction with phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P₂)-binding protein Amer1/WTX/Fam123b. Amer1 has been shown very recently to bridge Wnt-induced and Dishevelled-associated PtdIns(4,5)P₂ production to the phosphorylation of Lrp6. Using fluorescence recovery after photobleaching we show here that β-arrestin is required for the Wnt3a-induced Amer1 membrane dynamics and downstream signaling. Finally, we show that β-arrestin interacts with PtdIns kinases PI4KIIα and PIP5KIβ. Importantly, cells lacking β-arrestin showed higher steady-state levels of the relevant PtdInsP and were unable to increase levels of these PtdInsP in response to Wnt3a. In summary, our data show that β-arrestins regulate Wnt3a-induced Lrp6 phosphorylation by the regulation of the membrane dynamics of Amer1. We propose that β-arrestins via their scaffolding function facilitate Amer1 interaction with PtdIns(4,5)P₂, which is produced locally upon Wnt3a stimulation by β-arrestin- and Dishevelled-associated kinases.     

Independent interactions of phosphorylated β-catenin with E-cadherin at cell-cell contacts and APC at cell protrusions

Background

The APC tumour suppressor functions in several cellular processes including the regulation of β-catenin in Wnt signalling and in cell adhesion and migration.

Findings

In this study, we establish that in epithelial cells N-terminally phosphorylated β-catenin specifically localises to several subcellular sites including cell-cell contacts and the ends of cell protrusions. N-terminally phosphorylated β-catenin associates with E-cadherin at adherens junctions and with APC in cell protrusions. We isolated APC-rich protrusions from stimulated cells and detected β-catenin, GSK3β and CK1α, but not axin. The APC/phospho-β-catenin complex in cell protrusions appears to be distinct from the APC/axin/β-catenin destruction complex. GSK3β phosphorylates the APC-associated population of β-catenin, but not the cell junction population. β-catenin associated with APC is rapidly phosphorylated and dephosphorylated. HGF and wound-induced cell migration promote the localised accumulation of APC and phosphorylated β-catenin at the leading edge of migrating cells. APC siRNA and analysis of colon cancer cell lines show that functional APC is required for localised phospho-β-catenin accumulation in cell protrusions.

Conclusions

We conclude that N-terminal phosphorylation of β-catenin does not necessarily lead to its degradation but instead marks distinct functions, such as cell migration and/or adhesion processes. Localised regulation of APC-phospho-β-catenin complexes may contribute to the tumour suppressor activity of APC.     

NFAT5 represses canonical Wnt signaling via inhibition of β-catenin acetylation and participates in regulating intestinal cell differentiation

The intestinal mucosa undergoes a continual process of proliferation, differentiation, and apoptosis, which is regulated by multiple signaling pathways. The Wnt/β-catenin pathway has a critical role in this process. Previously, we have shown that the calcineurin-dependent nuclear factor of activated T cell (NFAT) is involved in the regulation of intestinal cell differentiation, as noted by the alteration of brush-border enzyme intestinal alkaline phosphatase (IAP) activity. Here, we show that calcineurin-independent NFAT5 interacts with β-catenin to repress Wnt signaling. We found that overexpression of NFAT5 inhibits, whereas knockdown of NFAT5 increases, TOPflash reporter activity and the expression of Wnt/β-catenin target genes, suggesting that NFAT5 inhibits Wnt signaling. In addition, we demonstrated that NFAT5 directly interacts with the C-terminal transactivation domain (TAD) of β-catenin, inhibits CBP interaction with β-catenin, and inhibits CBP-mediated β-catenin acetylation. Moreover, NFAT5 is expressed in the mucosa of human intestine, with the most pronounced staining in the most differentiated region near the epithelial surface. Knockdown of NFAT5 attenuated sodium butyrate (NaBT)-mediated induction of IAP and sucrase activities; overexpression of NFAT5 induced IAP promoter activity. In summary, we provide evidence showing that NFAT5 is a regulator of Wnt signaling. Importantly, our results suggest that NFAT5 regulation of intestinal cell differentiation may be through inhibition of Wnt/β-catenin signaling.