Activation of the Wnt/β-catenin pathway crucially depends on the polymerization of dishevelled 2 (DVL2) into biomolecular condensates. However, given the low affinity of known DVL2 self-interaction sites and its low cellular concentration, it is unclear how polymers can form. Here, we detect oligomeric DVL2 complexes at endogenous protein levels in human cell lines, using a biochemical ultracentrifugation assay. We identify a low-complexity region (LCR4) in the C-terminus whose deletion and fusion decreased and increased the complexes, respectively. Notably, LCR4-induced complexes correlated with the formation of microscopically visible multimeric condensates. Adjacent to LCR4, we mapped a conserved domain (CD2) promoting condensates only. Molecularly, LCR4 and CD2 mediated DVL2 self-interaction via aggregating residues and phenylalanine stickers, respectively. Point mutations inactivating these interaction sites impaired Wnt pathway activation by DVL2. Our study discovers DVL2 complexes with functional importance for Wnt/β-catenin signaling. Moreover, we provide evidence that DVL2 condensates form in two steps by pre-oligomerization via high-affinity interaction sites, such as LCR4, and subsequent condensation via low-affinity interaction sites, such as CD2.

• Keywords
• DVL2, Wnt signaling, biochemistry, biomolecular condensates, cell biology, chemical biology, dishevelled, human, paralogs,
• MeSH
• beta Catenin metabolism   genetics   MeSH
• HEK293 Cells MeSH
• Humans MeSH
• Protein Multimerization MeSH
• Dishevelled Proteins * metabolism   genetics   MeSH
• Wnt Signaling Pathway * MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• beta Catenin MeSH
• DVL2 protein, human MeSH Browser
• Dishevelled Proteins * MeSH

Actomyosin contractility represents an ancient feature of eukaryotic cells participating in many developmental and homeostasis events, including tissue morphogenesis, muscle contraction and cell migration, with dysregulation implicated in various pathological conditions, such as cancer. At the molecular level, actomyosin comprises actin bundles and myosin motor proteins that are sensitive to posttranslational modifications like phosphorylation. While the molecular components of actomyosin are well understood, the coordination of contractility by extracellular and intracellular signals, particularly from cellular signalling pathways, remains incompletely elucidated. This study focuses on WNT/planar cell polarity (PCP) signalling, previously associated with actomyosin contractility during vertebrate neurulation. Our investigation reveals that the main cytoplasmic PCP proteins, Prickle and Dishevelled, interact with key actomyosin components such as myosin light chain 9 (MLC9), leading to its phosphorylation and localized activation. Using proteomics and microscopy approaches, we demonstrate that both PCP proteins actively control actomyosin contractility through Rap1 small GTPases in relevant in vitro and in vivo models. These findings unveil a novel mechanism of how PCP signalling regulates actomyosin contractility through MLC9 and Rap1 that is relevant to vertebrate neurulation.

• Keywords
• MDCK cells, Xenopus embryos, actomyosin contractility, neurulation, planar cell polarity, vertebrates,
• MeSH
• Actomyosin * metabolism   MeSH
• Phosphorylation MeSH
• Myosin Light Chains metabolism   MeSH
• Humans MeSH
• Mice MeSH
• Neurulation * MeSH
• Vertebrates metabolism   MeSH
• Cell Polarity * MeSH
• Dishevelled Proteins metabolism   genetics   MeSH
• Signal Transduction MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Actomyosin * MeSH
• Myosin Light Chains MeSH
• Dishevelled Proteins MeSH

Polyglutamylation is a reversible posttranslational modification that is catalyzed by enzymes of the tubulin tyrosine ligase-like (TTLL) family. Here, we found that TTLL11 generates a previously unknown type of polyglutamylation that is initiated by the addition of a glutamate residue to the free C-terminal carboxyl group of a substrate protein. TTLL11 efficiently polyglutamylates the Wnt signaling protein Dishevelled 3 (DVL3), thereby changing the interactome of DVL3. Polyglutamylation increases the capacity of DVL3 to get phosphorylated, to undergo phase separation, and to act in the noncanonical Wnt pathway. Both carboxy-terminal polyglutamylation and the resulting reduction in phase separation capacity of DVL3 can be reverted by the deglutamylating enzyme CCP6, demonstrating a causal relationship between TTLL11-mediated polyglutamylation and phase separation. Thus, C-terminal polyglutamylation represents a new type of posttranslational modification, broadening the range of proteins that can be modified by polyglutamylation and providing the first evidence that polyglutamylation can modulate protein phase separation.

• Keywords
• Dishevelled 3, Noncanonical Wnt Signaling, Polyglutamylation, Protein Condensates, TTLL11,
• MeSH
• Phosphorylation MeSH
• HEK293 Cells MeSH
• Polyglutamic Acid metabolism   analogs & derivatives   MeSH
• Humans MeSH
• Peptide Synthases * metabolism   genetics   MeSH
• Protein Processing, Post-Translational * MeSH
• Dishevelled Proteins * metabolism   genetics   MeSH
• Phase Separation MeSH
• Wnt Signaling Pathway MeSH
• Signal Transduction MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• DVL3 protein, human MeSH Browser
• Polyglutamic Acid MeSH
• Peptide Synthases * MeSH
• Dishevelled Proteins * MeSH
• tubulin polyglutamylase MeSH Browser

RNF43 is an E3 ubiquitin ligase and known negative regulator of WNT/β-catenin signaling. We demonstrate that RNF43 is also a regulator of noncanonical WNT5A-induced signaling in human cells. Analysis of the RNF43 interactome using BioID and immunoprecipitation showed that RNF43 can interact with the core receptor complex components dedicated to the noncanonical Wnt pathway such as ROR1, ROR2, VANGL1, and VANGL2. RNF43 triggers VANGL2 ubiquitination and proteasomal degradation and clathrin-dependent internalization of ROR1 receptor and inhibits ROR2 activation. These activities of RNF43 are physiologically relevant and block pro-metastatic WNT5A signaling in melanoma. RNF43 inhibits responses to WNT5A, which results in the suppression of invasive properties of melanoma cells. Furthermore, RNF43 prevented WNT5A-assisted development of resistance to BRAF V600E and MEK inhibitors. Next, RNF43 acted as melanoma suppressor and improved response to targeted therapies in vivo. In line with these findings, RNF43 expression decreases during melanoma progression and RNF43-low patients have a worse prognosis. We conclude that RNF43 is a newly discovered negative regulator of WNT5A-mediated biological responses that desensitizes cells to WNT5A.

• Keywords
• BRAF V600E, RNF43, ROR1, VANGL1, WNT5A, cancer biology, cell biology, human, melanoma, mouse,
• MeSH
• Neoplasm Invasiveness genetics   MeSH
• Melanoma * genetics   pathology   prevention & control   MeSH
• Mice, Inbred NOD MeSH
• Mice MeSH
• Wnt-5a Protein genetics   metabolism   MeSH
• Signal Transduction * MeSH
• Ubiquitin-Protein Ligases genetics   metabolism   MeSH
• Animals MeSH
• Check Tag
• Male MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Wnt-5a Protein MeSH
• RNF43 protein, human MeSH Browser
• Ubiquitin-Protein Ligases MeSH
• WNT5A protein, human MeSH Browser

Dishevelled (DVL) is the central signal transducer in both Wnt/β-catenin-dependent and independent signalling pathways. DVL is required to connect receptor complexes and downstream effectors. Since proximal Wnt pathway components and DVL itself are upregulated in many types of cancer, DVL represents an attractive therapeutic target in the Wnt-addicted cancers and other disorders caused by aberrant Wnt signalling. Here, we discuss progress in several approaches for the modulation of DVL function and hence inhibition of the Wnt signalling. Namely, we sum up the potential of modulation of enzymes that control post-translational modification of DVL - such as inhibition of DVL kinases or promotion of DVL ubiquitination and degradation. In addition, we discuss research directions that can take advantage of direct interaction with the protein domains essential for DVL function: the inhibition of DIX- and DEP-domain mediated polymerization and interaction of DVL PDZ domain with its ligands.

• Keywords
• Casein kinase 1, DIX oligomerization, Dishevelled, PDZ inhibitors, Wnt signalling-related diseases,
• MeSH
• Adaptor Proteins, Signal Transducing MeSH
• Phosphoproteins * metabolism   MeSH
• Humans MeSH
• Dishevelled Proteins * metabolism   MeSH
• Wnt Signaling Pathway * MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Adaptor Proteins, Signal Transducing MeSH
• Phosphoproteins * MeSH
• Dishevelled Proteins * MeSH

The PDZ domain of Dishevelled 3 protein belongs to a highly abundant protein recognition motif which typically binds short C-terminal peptides. The affinity of the PDZ towards the peptides could be fine-tuned by a variety of post-translation modifications including phosphorylation. However, how phosphorylations affect the PDZ structure and its interactions with ligands remains elusive. Combining molecular dynamics simulations, NMR titration, and biological experiments, we explored the role of previously reported phosphorylation sites and their mimetics in the Dishevelled PDZ domain. Our observations suggest three major roles for phosphorylations: (1) acting as an on/off PDZ binding switch, (2) allosterically affecting the binding groove, and (3) influencing the secondary binding site. Our simulations indicated that mimetics had similar but weaker effects, and the effects of distinct sites were non-additive. This study provides insight into the Dishevelled regulation by PDZ phosphorylation. Furthermore, the observed effects could be used to elucidate the regulation mechanisms in other PDZ domains.

• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

BACKGROUND: Dishevelled (DVL) is an essential component of the Wnt signaling cascades. Function of DVL is controlled by phosphorylation but the molecular details are missing. DVL3 contains 131 serines and threonines whose phosphorylation generates complex barcodes underlying diverse DVL3 functions. In order to dissect the role of DVL phosphorylation we analyzed the phosphorylation of human DVL3 induced by previously reported (CK1ε, NEK2, PLK1, CK2α, RIPK4, PKCδ) and newly identified (TTBK2, Aurora A) DVL kinases. METHODS: Shotgun proteomics including TiO2 enrichment of phosphorylated peptides followed by liquid chromatography tandem mass spectrometry on immunoprecipitates from HEK293T cells was used to identify and quantify phosphorylation of DVL3 protein induced by 8 kinases. Functional characterization was performed by in-cell analysis of phospho-mimicking/non-phosphorylatable DVL3 mutants and supported by FRET assays and NMR spectroscopy. RESULTS: We used quantitative mass spectrometry and calculated site occupancies and quantified phosphorylation of > 80 residues. Functional validation demonstrated the importance of CK1ε-induced phosphorylation of S268 and S311 for Wnt-3a-induced β-catenin activation. S630-643 cluster phosphorylation by CK1, NEK2 or TTBK2 is essential for even subcellular distribution of DVL3 when induced by CK1 and TTBK2 but not by NEK2. Further investigation showed that NEK2 utilizes a different mechanism to promote even localization of DVL3. NEK2 triggered phosphorylation of PDZ domain at S263 and S280 prevents binding of DVL C-terminus to PDZ and promotes an open conformation of DVL3 that is more prone to even subcellular localization. CONCLUSIONS: We identify unique phosphorylation barcodes associated with DVL function. Our data provide an example of functional synergy between phosphorylation in structured domains and unstructured IDRs that together dictate the biological outcome. Video Abtract.

• Keywords
• CK1, DVL3, Dishevelled, Kinase, Mass spectrometry, NEK2, Phosphorylation, TTBK2, Wnt,
• MeSH
• Phosphorylation MeSH
• HEK293 Cells MeSH
• Mass Spectrometry MeSH
• NIMA-Related Kinases metabolism   MeSH
• Protein Conformation MeSH
• Cells, Cultured MeSH
• Humans MeSH
• Dishevelled Proteins chemistry   metabolism   MeSH
• Signal Transduction MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Comparative Study MeSH
• Names of Substances
• DVL3 protein, human MeSH Browser
• NIMA-Related Kinases MeSH
• NEK2 protein, human MeSH Browser
• Dishevelled Proteins MeSH