Dishevelled2 activates WGEF via its interaction with a unique internal peptide motif of the GEF.

The Wnt-planar cell polarity (Wnt-PCP) pathway is crucial in establishing cell polarity during development and tissue homoeostasis. This pathway is found to be dysregulated in many pathological conditions, including cancer and autoimmune disorders. The central event in Wnt-PCP pathway is the activation of Weak-similarity guanine nucleotide exchange factor (WGEF) by the adapter protein Dishevelled (Dvl). The PDZ domain of Dishevelled2 (Dvl2PDZ) binds and activates WGEF by releasing it from its autoinhibitory state. However, the actual Dvl2PDZ binding site of WGEF and the consequent activation mechanism of the GEF have remained elusive. Using biochemical and molecular dynamics studies, we show that a unique "internal-PDZ binding motif" (IPM) of WGEF mediates the WGEF-Dvl2PDZ interaction to activate the GEF. The residues at P2, P0, P-2 and P-3 positions of IPM play an important role in stabilizing the WGEFpep-Dvl2PDZ interaction. Furthermore, MD simulations of modelled Dvl2PDZ-WGEFIPM peptide complexes suggest that WGEF-Dvl2PDZ interaction may differ from the reported Dvl2PDZ-IPM interactions. Additionally, the apo structure of human Dvl2PDZ shows conformational dynamics different from its IPM peptide bound state, suggesting an induced fit mechanism for the Dvl2PDZ-peptide interaction. The current study provides a model for Dvl2 induced activation of WGEF.

Dishevelled Segment Polarity Protein 3 (DVL3) Induced by Bacterial LPS Promotes the Proliferation and Migration of Prostate Cancer Cells through the TLR4 Pathway.

BACKGROUND: Chronic inflammation is associated with various malignant tumors. Bacterial lipopolysaccharides (LPSs) play a significant part in the event and development of prostate cancer. Dishevelled segment polarity protein 3 (DVL3) is a shared component of the Wnt/ß-catenin and Notch signaling pathways, which are involved in tumor progression, chemoresistance, and maintenance of stem cell-like properties. According to reports, prostatic cancer cell invasion and proliferation are mediated by toll-like receptor 4 (TLR4). However, the role and regulation of DVL3 in prostate cancer and its relationship with TLR4 remain unclear. METHODS: Survival curves were plotted to evaluate the relationship between DVL3 expression and prognosis in patients with prostate cancer. DVL3 was silenced in PC3 and DU145 cells using small interfering RNAs (siRNAs). Subsequently, cell counting kit-8 (CCK-8) assay, colony formation assay, transwell migration assay, and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) were performed to investigate the role of DVL3 in cell proliferation and migration in vitro. The protein markers of potential pathways were analyzed via western blotting. RESULTS: DVL3 expression was linked to prognosis in patients with prostate cancer; In particular, patients with high DVL3 expression had a poor prognosis. LPS stimulation increased (p < 0.01) the expression of DVL3 in PC3 cells. DVL3 regulated tumor cell proliferation and migration by mediating the increase (p < 0.01) in TLR4 expression. Knockout of TLR4 validated that TLR4 played a crucial role in LPS-induced DVL3 expression. Silencing of DVL3 decreased (p < 0.01) the LPS-induced proliferation and migration of PC3 cells. CONCLUSIONS: Bacterial LPS-induced DVL3 promoted the multiplication and migration of prostate cancer cells through the TLR4 pathway. This study offers a valuable reference for the development and clinical application of targeted drugs for prostate cancer.

A vertebrate Vangl2 translational variant required for planar cell polarity.

First described in the milkweed bug Oncopeltus fasciatus, planar cell polarity (PCP) is a developmental process essential for embryogenesis and development of polarized structures in Metazoans. This signaling pathway involves a set of evolutionarily conserved genes encoding transmembrane (Vangl, Frizzled, Celsr) and cytoplasmic (Prickle, Dishevelled) molecules. Vangl2 is of major importance in embryonic development as illustrated by its pivotal role during neural tube closure in human, mouse, Xenopus, and zebrafish embryos. Here, we report on the molecular and functional characterization of a Vangl2 isoform, Vangl2-Long, containing an N-terminal extension of about 50 aa, which arises from an alternative near-cognate AUA translation initiation site, lying upstream of the conventional start codon. While missing in Vangl1 paralogs and in all invertebrates, including Drosophila, this N-terminal extension is conserved in all vertebrate Vangl2 sequences. We show that Vangl2-Long belongs to a multimeric complex with Vangl1 and Vangl2. Using morpholino oligonucleotides to specifically knockdown Vangl2-Long in Xenopus, we found that this isoform is functional and required for embryo extension and neural tube closure. Furthermore, both Vangl2 and Vangl2-Long must be correctly expressed for the polarized distribution of the PCP molecules Pk2 and Dvl1 and for centriole rotational polarity in ciliated epidermal cells. Altogether, our study suggests that Vangl2-Long significantly contributes to the pool of Vangl2 molecules present at the plasma membrane to maintain PCP in vertebrate tissues.

Presenilin-2 knock-In mice show severe depressive behavior via DVL3 downregulation.

INTRODUCTION: Alzheimer's disease (AD) is the most common form of dementia. Depression is one of the most critical psychiatric complications of AD, and 20%-30% of patients with AD experience symptoms of depression. Phospho-glycogen synthase kinase-3 beta (GSK3ß) is known to be associated with AD and depression. Furthermore, the role of disheveled (DVL) is known to regulate GSK3ß. Moreover, presenilin-2 (PS2) and DVL have cross-talk with each other. Also, it is widely hypothesized that stress leads to hypersecretion of cortisol and is thus associated with depression. Dickkopf WNT signaling pathway inhibitor-1 (DKK-1) is a crucial factor regulating depression and both amyloid beta (Aß) and phosphorylation of tau are widely known as a biomarker of AD. METHODS: To investigate the relationship between AD and depression, and possible pathways connecting the two diseases, we examined memory function and depression-related behavior test results in PS2 knock-in AD mice (PS2 MT). Next, we confirmed that there are relationships between DVL, depression, and cognitive disease through the comparative toxicogenomics database (https://ctdbase.org) and STRING (https://string-db.org) database. RESULTS: PS2 knock-in mice showed much more severe memory impairment and depression than PS2 wild-type mice (PS2 WT). In AD-related behavioral experiments, PS2 MT mice showed more memory dysfunction compared with PS2 WT group mice. Moreover, Aß and phosphorylation of tau showed higher expression in PS2 MT mice than in PS2 WT mice. Depression-related behavioral tests showed that PS2 MT mice exhibited more depressive behaviors than PS2 WT mice. Furthermore, both higher cortisol levels and higher expression of DKK-1 were found in PS2 MT mice relative to PS2 WT mice. The results indicated that there is a relationship between DVL and the release of AD-related mediators and expression of the depression-related glucocorticoid receptor and DKK-1. In the PS2 knock-in group, DVL was significantly decreased compared with the PS2 WT group. CONCLUSION: Depression increases the risk of developing AD and other forms of dementia. Recent evidence indicates that depression symptoms could trigger changes in memory and thinking over time. However, it is recognized that there are no drugs to facilitate a full recovery for both AD and depression. However, our results suggest that AD and depression could be associated, and DVL could be a significant target for the association between AD and depression.

Delivery of microRNA-423-5p by exosome from adipose-derived stem/stromal cells inhibits DVL3 to potentiate autologous fat graft survival through adipogenesis and inflammatory response.

Autologous fat grafting represents a reconstructive technique but is limited by unstable graft retention. Based on existing reports and bioinformatics prediction, we hypothesized that delivering exosomes from human adipose-derived stem/stromal cells (hADSC-Exo) would increase fat graft survival and further explore the mechanism. hADSC-Exo were extracted and identified. An autologous fat grafting model was established using donor and recipient mice, followed by hADSC-Exo treatment. hADSC-Exo promoted the retention of autologous fat grafts in mice, along with increased adipocyte activity, angiogenesis, and decreased inflammation in grafts. Moreover, hADSC-Exo potentiated the adipose differentiation of 3T3-L1 cells, enhanced the angiogenic and migratory capacity of human umbilical vein endothelial cells, and inhibited the inflammation and viability of RAW 264.7 cells. The therapeutic effect of hADSC-Exo on fat grafting was associated with the delivery of microRNA (miR)-423-5p. Deletion of miR-423-5p in Exo impaired the function of hADSC-Exo on fat retention. miR-423-5p bound to DVL3 to suppress DVL3 expression, and DVL3 deletion promoted adipose differentiation of 3T3-L1 cells. In conclusion, our findings further widen the theoretical basis of the clinical application of hADSC-Exo in autologous fat grafts.

HCMV UL8 interaction with ß-catenin and DVL2 regulates viral reactivation in CD34+ hematopoietic progenitor cells.

IMPORTANCE: CD34+ hematopoietic progenitor cells (HPCs) are an important cellular reservoir for latent human cytomegalovirus (HCMV). Several HCMV genes are expressed during latency that are involved with the maintenance of the viral genome in CD34+ HPC. However, little is known about the process of viral reactivation in these cells. Here, we describe a viral protein, pUL8, and its interaction and stabilization with members of the Wnt/ß-catenin pathway as an important component of viral reactivation. We further define that pUL8 and ß-catenin interact with DVL2 via a PDZ-binding domain, and loss of UL8 interaction with ß-catenin-DVL2 restricts viral reactivation. Our findings will be instrumental in understanding the molecular processes involved in HCMV reactivation in order to design new antiviral therapeutics.

A Van Gogh/Vangl tyrosine phosphorylation switch regulates its interaction with core Planar Cell Polarity factors Prickle and Dishevelled.

Epithelial tissues can be polarized along two axes: in addition to apical-basal polarity they are often also polarized within the plane of the epithelium, known as planar cell polarity (PCP). PCP depends upon the conserved Wnt/Frizzled (Fz) signaling factors, including Fz itself and Van Gogh (Vang/Vangl in mammals). Here, taking advantage of the complementary features of Drosophila wing and mouse skin PCP establishment, we dissect how Vang/Vangl phosphorylation on a specific conserved tyrosine residue affects its interaction with two cytoplasmic core PCP factors, Dishevelled (Dsh/Dvl1-3 in mammals) and Prickle (Pk/Pk1-3). We demonstrate that Pk and Dsh/Dvl bind to Vang/Vangl in an overlapping region centered around this tyrosine. Strikingly, Vang/Vangl phosphorylation promotes its binding to Prickle, a key effector of the Vang/Vangl complex, and inhibits its interaction with Dishevelled. Thus phosphorylation of this tyrosine appears to promote the formation of the mature Vang/Vangl-Pk complex during PCP establishment and conversely it inhibits the Vang interaction with the antagonistic effector Dishevelled. Intriguingly, the phosphorylation state of this tyrosine might thus serve as a switch between transient interactions with Dishevelled and stable formation of Vang-Pk complexes during PCP establishment.

Dishevelled segment polarity protein 2 promotes gastric cancer progression through Wnt/ß-catenin pathway.

Dishevelled family proteins (DVL1-3), key scaffold proteins, act on canonical and non-canonical Wnt/ß-catenin signaling pathway. DVL has been implicated in various tumor progression. However, its role and underlying mechanisms in gastric cancer (GC) remain unclear. The aim of this study was to investigate the role of DVL in GC development using cell lines and 209 GC specimens. We analyzed three orthologs of DVL in GC tissues and paired adjacent non-tumor tissues, and only DVL2 is highly expressed in GC tissues. We also analyzed clinicopathological data on DVL2 expression in gastric cancer specimens. In immunohistochemistry, DVL2 expression was up-regulated in GC tissues compared with paired adjacent non-tumor tissues (153/209, 73.2%). DVL2 expression level was significantly correlated with many clinicopathological parameters such as T stage (P < 0.001) and N stage (P < 0.001). Survival analysis showed that the overall survival (OS) of patients with high expression of DVL2 was significantly shorter than those with low expression. Multivariate Cox regression analysis revealed that DVL2 expression was an important and independent prognostic factor for gastric cancer patients (P = 0.011, HR=1.78, 95%CI (1.14-2.79). Depletion of endogenous DVL2 using short hairpin RNA (shRNA) inhibited GC cell proliferation, migration, and invasion. The abnormal activation of Wnt/ß-catenin signaling pathway is mainly achieved through the abnormal expression of DVL2. DVL2 is highly expressed in gastric cancer tissues, which may be a new independent risk factor for the prognosis of gastric cancer patients. In gastric cancer, DVL2 overexpression plays a crucial role in the occurrence and development of gastric cancer, so it may become a new, effective and complementary therapeutic target for gastric cancer.

WNT stimulation induces dynamic conformational changes in the Frizzled-Dishevelled interaction.

Frizzleds (FZDs) are G protein-coupled receptors (GPCRs) that bind to WNT family ligands. FZDs signal through multiple effector proteins, including Dishevelled (DVL), which acts as a hub for several downstream signaling pathways. To understand how WNT binding to FZD stimulates intracellular signaling and influences downstream pathway selectivity, we investigated the dynamic changes in the FZD5-DVL2 interaction elicited by WNT-3A and WNT-5A. Ligand-induced changes in bioluminescence resonance energy transfer (BRET) between FZD5 and DVL2 or the isolated FZD-binding DEP domain of DVL2 revealed a composite response consisting of both DVL2 recruitment and conformational dynamics in the FZD5-DVL2 complex. The combination of different BRET paradigms enabled us to identify ligand-dependent conformational dynamics in the FZD5-DVL2 complex and distinguish them from ligand-induced recruitment of DVL2 or DEP to FZD5. The observed agonist-induced conformational changes at the receptor-transducer interface suggest that extracellular agonist and intracellular transducers cooperate through transmembrane allosteric interaction with FZDs in a ternary complex reminiscent of that of classical GPCRs.

Mechanistic studies in Drosophila and chicken give new insights into functions of DVL1 in dominant Robinow syndrome.

The study of rare genetic diseases provides valuable insights into human gene function. The autosomal dominant or autosomal recessive forms of Robinow syndrome are genetically heterogeneous, and the common theme is that all the mutations lie in genes in Wnt signaling pathways. Cases diagnosed with Robinow syndrome do survive to adulthood with distinct skeletal phenotypes, including limb shortening and craniofacial abnormalities. Here, we focus on mutations in dishevelled 1 (DVL1), an intracellular adaptor protein that is required for both canonical (ß-catenin-dependent) or non-canonical (requiring small GTPases and JNK) Wnt signaling. We expressed human wild-type DVL1 or DVL1 variants alongside the endogenous genome of chicken and Drosophila. This design is strategically suited to test for functional differences between mutant and wild-type human proteins in relevant developmental contexts. The expression of variant forms of DVL1 produced a major disorganization of cartilage and Drosophila wing morphology compared to expression of wild-type DVL1. Moreover, the variants caused a loss of canonical and gain of non-canonical Wnt signaling in several assays. Our data point to future therapies that might correct the levels of Wnt signaling, thus improving skeletal growth.

The Wnt pathway protein Dvl1 targets somatostatin receptor 2 for lysosome-dependent degradation.

The Somatostatin receptor 2 (Sstr2) is a heterotrimeric G protein-coupled receptor that is highly expressed in neuroendocrine tumors and is a common pharmacological target for intervention. Unfortunately, not all neuroendocrine tumors express Sstr2, and Sstr2 expression can be downregulated with prolonged agonist use. Sstr2 is rapidly internalized following agonist stimulation and, in the short term, is quantitatively recycled back to the plasma membrane. However, mechanisms controlling steady state expression of Sstr2 in the absence of agonist are less well described. Here, we show that Sstr2 interacts with the Wnt pathway protein Dvl1 in a ligand-independent manner to target Sstr2 for lysosomal degradation. Interaction of Sstr2 with Dvl1 does not affect receptor internalization, recycling, or signaling to adenylyl cyclase but does suppress agonist-stimulated ERK1/2 activation. Importantly, Dvl1-dependent degradation of Sstr2 can be stimulated by overexpression of Wnts and treatment of cells with Wnt pathway inhibitors can boost Sstr2 expression in neuroendocrine tumor cells. Taken together, this study identifies for the first time a mechanism that targets Sstr2 for lysosomal degradation that is independent of Sstr2 agonist and can be potentiated by Wnt ligand. Intervention in this signaling mechanism has the potential to elevate Sstr2 expression in neuroendocrine tumors and enhance Sstr2-directed therapies.

Hyperoxia exposure upregulates Dvl-1 and activates Wnt/ß-catenin signaling pathway in newborn rat lung.

BACKGROUND: Bronchopulmonary dysplasia is a serious and lifelong pulmonary disease in premature neonates that influences around one-quarter of premature newborns. The wingless-related integration site /ß-catenin signaling pathway, which is abnormally activated in the lungs with pulmonary fibrosis, affects cell differentiation and lung development. METHODS: Newborn rats were subjected to hyperoxia exposure. Histopathological changes to the lungs were evaluated through immunohistochemistry, and the activation of disheveled and Wnt /ß-catenin signaling pathway components was assessed by Western blotting and real-time PCR. The abilities of proliferation, apoptosis and migration were detected by Cell Counting Kit-8, flow cytometry and scratch wound assay, respectively. RESULTS: Contrasting with normoxic lungs, hyperoxia-exposed lungs demonstrated larger alveoli, fewer alveoli and thicker alveolar septa. Superoxide dismutase activity was significantly decreased (7th day: P < 0.05; 14th day: P < 0.01) and malondialdehyde significantly increased (7th day: P < 0.05; 14th day: P < 0.01) after hyperoxia exposure. Protein and mRNA expression levels of ß-catenin, Dvl-1, CTNNBL1 and cyclin D1 were significantly upregulated by hyperoxia exposure on 7th day (P < 0.01) and 14th day (P < 0.01). In hyperoxic conditions, Dvl-l downregulation and Dvl-l downregulation + MSAB treatment significantly increased the proliferation rates, decreased the apoptosis rates and improved the ability of cell migration. In hyperoxic conditions, Dvl-l downregulation could decrease the mRNA expression levels of GSK3ß, ß-catenin, CTNNBL1 and cyclin D1 and decrease the protein relative expression levels of GSK3ß, p-GSK3ß, ß-catenin, CTNNBL1 and cyclin D1. CONCLUSIONS: We confirmed the positive role of Dvl-1 and the Wnt/ß-catenin signaling pathway in promoting BPD in hyperoxia conditions and provided a promising therapeutic target.

Overexpressions of RHOA, CSNK1A1, DVL2, FZD8, and LRP5 genes enhance gastric cancer development in the presence of Helicobacter pylori.

BACKGROUND AND STUDY AIMS: Intestinal metaplasia (IM), and Helicobacter pylori (HP) infection can be shown as risk factors in the development of gastric cancer (GC). WNT signaling pathway plays a critical role in carcinogenesis. However, the literature studies are limited on the significance of this pathway for the transition from IM to GC. PATIENTS AND METHODS: We aimed to investigate the importance of the genes of WNT signaling pathways diagnostic and prognostic markers in the presence and absence of HP in conversion from IM to GC. 104 patients, (GC group n = 35, IM group n = 45, control group n = 25) were included in this case-control study. Expression of genes in WNT signalling were searched in study groups with qRT-PCR array and qRT-PCR method. Data were analysed using PCR array data analysis software. RESULTS: Statistically significant overexpression of RHOA, CSNK1A1, DVL2, FZD8 and LRP5 genes was detected in the GC and IM groups compared to the control group (p < 0.05). Statistically significant overexpression of RHOA, CSNK1A1, DVL2, FZD8 and LRP5 genes was observed in patients with metastatic GC compared to patients with GC without metastasis (p < 0.05). It was found that the RHOA, CSNK1A1, DVL2, FZD8 and LRP5 genes were statistically significantly over-expressed in diffuse GC patients compared to non-diffuse GC patients (p < 0.05). Statistically significant overexpression of RHOA, CSNK1A1, DVL2, FZD8 and LRP5 genes was detected in HP positive IM patients compared to HP negative IM patients (p < 0.05). CONCLUSION: Overexpression of RHOA, CSNK1A1, DVL2, FZD8 and LRP5 genes in IM may suggest that these genes are important markers in the development of IM and inflammation with HP. In addition, these genes are linked to tumor burden in the GC group. Consequently, we can conclude that these genes are poor prognosis biomarkers for GC and have the potential to be used as markers for future treatment monitoring.

Dvl proteins regulate SMAD1, AHR, mTOR, BRD7 protein expression while differentially regulating canonical and non-canonical Wnt signaling pathways in CML cell lines.

Dishevelled (Dvl) is a scaffold protein that transmits Wnt signals to downstream effector molecules via both canonical and non-canonical Wnt signaling pathways. Deregulated activation of Dvl proteins has been reported in various solid tumors. However, it is not clear which pathway and proteins are responsible for observed aberrant activities and their relevance in disease prognosis. In addition, there is relatively limited knowledge on the role Dvl proteins may have in hematologic malignancy etiopathogenesis. In this study, we demonstrated that Dvl genes are not expressed in normal bone marrow but are expressed at different levels in the bone marrow of patients with chronic myeloid leukemia. We showed SMAD1, AHR, mTOR, BRD7 protein expressions are significantly affected by Dvl silencing and overexpression in CML cell lines. Wnt/ß-catenin and Wnt/PCP signaling pathway components are effectively repressed after Dvl silencing in K562 cells, while regulator of Wnt/Ca2+ signaling showed increase in both CML cell lines. Targeting Dvl proteins increases imatinib susceptibility of the K562 and MEG-01 cell lines. In light of our data, Dvl could be a potential therapeutic target in the treatment of CML.

Exosomal circ_0091741 promotes gastric cancer cell autophagy and chemoresistance via the miR-330-3p/TRIM14/Dvl2/Wnt/ß-catenin axis.

The importance of cancer cell-released exosomes in the treatment of various cancers has been well-characterized. The current study aims to examine the potential biological functions of gastric cancer (GC) cell-released exosomes delivering a novel circRNA circ_0091741 in GC and the underlying molecular mechanism. Expression of circ_0091741 was examined in the GC cells, (OXA)-resistant HGC-27 (HGC-27/OXA) cells, and isolated exosomes, after which its downstream miRNA was analyzed. The role and mechanism of the circ_0091741 transmitted by GC cells-derived exosomes in GC cell autophagy and chemoresistance were assessed using various molecular biological methods. A mouse tumor xenograft model was prepared to discern the effect of circ_0091741 on tumorigenesis in vivo. GC cells and their exosomes were characterized by upregulated circ_0091741 expression. circ_0091741 transferred by GC cell-derived exosomes induced the autophagy and OXA resistance of GC cells. circ_0091741 obstructed the binding of miR-330-3p to TRIM14 and increased the expression of TRIM14. TRIM14 could cause activation of the Wnt/ß-catenin signaling pathway by stabilizing Dvl2. By this mechanism, the autophagy and OXA resistance of GC cells were augmented. In vivo assay unfolded that orthotopic implantation of exosomal circ_0091741 overexpressed GC cells into nude mice enhanced tumorigenesis. In conclusion, our study emphasized the promotive role of exosomal circ_0091741 in autophagy and chemoresistance of GC cells, thus laying the basis for the development of novel therapeutic targets for GC treatment.

Inhibiting von Hippel‒Lindau protein-mediated Dishevelled ubiquitination protects against experimental parkinsonism.

Dopaminergic neuron degeneration is a hallmark of Parkinson's disease (PD). We previously reported that the inactivation of von Hippel‒Lindau (VHL) alleviated dopaminergic neuron degeneration in a C. elegans model. In this study, we investigated the specific effects of VHL loss and the underlying mechanisms in mammalian PD models. For in vivo genetic inhibition of VHL, AAV-Vhl-shRNA was injected into mouse lateral ventricles. Thirty days later, the mice received MPTP for 5 days to induce PD. Behavioral experiments were conducted on D1, D3, D7, D14 and D21 after the last injection, and the mice were sacrificed on D22. We showed that knockdown of VHL in mice significantly alleviated PD-like syndromes detected in behavioral and biochemical assays. Inhibiting VHL exerted similar protective effects in MPP+-treated differentiated SH-SY5Y cells and the MPP+-induced C. elegans PD model. We further demonstrated that VHL loss-induced protection against experimental parkinsonism was independent of hypoxia-inducible factor and identified the Dishevelled-2 (DVL-2)/ß-catenin axis as the target of VHL, which was evolutionarily conserved in both C. elegans and mammals. Inhibiting the function of VHL promoted the stability of ß-catenin by reducing the ubiquitination and degradation of DVL-2. Thus, in vivo overexpression of DVL-2, mimicking VHL inactivation, protected against PD. We designed a competing peptide, Tat-DDF-2, to inhibit the interaction between VHL and DVL-2, which exhibited pharmacological potential for protection against PD in vitro and in vivo. We propose the therapeutic potential of targeting the interaction between VHL and DVL-2, which may represent a strategy to alleviate neurodegeneration associated with PD.

Letting go: Dishevelled phase separation recruits Axin to stabilize ß-catenin.

Dishevelled exerts a molecular force that guides cell fate, but how it does so remains enigmatic. In this issue, Kang et al. (2022. J. Cell Biol.https://doi.org/10.1083/jcb.202205069) show Dvl2 undergoes liquid-liquid phase separation to stabilize ß-catenin by pulling Axin into its biomolecular condensate at the plasma membrane.

Ubiquitin-assisted phase separation of dishevelled-2 promotes Wnt signalling.

Dishvelled-2 (Dvl2) is an essential component of Wnt pathway, which controls several cell fate decisions during development, such as proliferation, survival and differentiation. Dvl2 forms higher-order protein assemblies in the cell that are critical for relaying the signal from upstream Wnt ligand-frizzled receptor binding to downstream effector ß-catenin activation. However, the precise molecular nature and contribution of Dvl2 protein assemblies during Wnt signalling is unknown. Here, we show that Dvl2 forms protein condensates driven by liquid-liquid phase separation. An intrinsically disordered region (IDR) at the N-terminus is essential for Dvl2 phase separation. Importantly, we identified the HECT-E3 ligase WWP2 as an essential driver of Dvl2 phase separation in vitro and in cells. We demonstrated that ubiquitylation of Dvl2 through K63 linkage by WWP2 is required for formation of Dvl2 condensates. Phase-separated Dvl2 activates Wnt signaling by sequestering the components of destruction complex and thus relieving ß-catenin. Together, our results reveal a ubiquitylation-dependent liquid-liquid phase separation as a new process through which Dvl2 forms condensates, which is necessary for transduction of Wnt signalling. This article has an associated First Person interview with the first author of the paper.

Dishevelled phase separation promotes Wnt signalosome assembly and destruction complex disassembly.

The amplitude of Wnt/ß-catenin signaling is precisely controlled by the assembly of the cell surface-localized Wnt receptor signalosome and the cytosolic ß-catenin destruction complex. How these two distinct complexes are coordinately controlled remains largely unknown. Here, we demonstrated that the signalosome scaffold protein Dishevelled 2 (Dvl2) undergoes liquid-liquid phase separation (LLPS). Dvl2 LLPS is mediated by an intrinsically disordered region and facilitated by components of the signalosome, such as the receptor Fzd5. Assembly of the signalosome is initiated by rapid recruitment of Dvl2 to the membrane, followed by slow and dynamic recruitment of Axin1. Axin LLPS mediates assembly of the ß-catenin destruction complex, and Dvl2 attenuates LLPS of Axin. Compared with the destruction complex, Axin partitions into the signalosome at a lower concentration and exhibits a higher mobility. Together, our results revealed that Dvl2 LLPS is crucial for controlling the assembly of the Wnt receptor signalosome and disruption of the phase-separated ß-catenin destruction complex.

PI(4,5)P2-stimulated positive feedback drives the recruitment of Dishevelled to Frizzled in Wnt-ß-catenin signaling.

In the Wnt-ß-catenin pathway, Wnt binding to Frizzled (Fzd) and LRP5 or LRP6 (LRP5/6) co-receptors inhibits the degradation of the transcriptional coactivator ß-catenin by recruiting the cytosolic effector Dishevelled (Dvl). Polymerization of Dvl at the plasma membrane recruits the ß-catenin destruction complex, enabling the phosphorylation of LRP5/6, a key step in inhibiting ß-catenin degradation. Using purified Fzd proteins reconstituted in lipid nanodiscs, we investigated the factors that promote the recruitment of Dvl to the plasma membrane. We found that the affinity of Fzd for Dvl was not affected by Wnt ligands, in contrast to other members of the GPCR superfamily for which the binding of extracellular ligands affects the affinity for downstream transducers. Instead, Fzd-Dvl binding was enhanced by increased concentration of the lipid PI(4,5)P2, which is generated by Dvl-associated lipid kinases in response to Wnt and which is required for LRP5/6 phosphorylation. Moreover, binding to Fzd did not promote Dvl DEP domain dimerization, which has been proposed to be required for signaling downstream of Fzd. Our findings suggest a positive feedback loop in which Wnt-stimulated local PI(4,5)P2 production enhances Dvl recruitment and further PI(4,5)P2 production to support Dvl polymerization, LRP5/6 phosphorylation, and ß-catenin stabilization.