Comparative phosphorylation map of Dishevelled 3 links phospho-signatures to biological outputs.

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 ß-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.

Binding of DEP domain to phospholipid membranes: More than just electrostatics.

Over the past decades an extensive effort has been made to provide a more comprehensive understanding of Wnt signaling, yet many regulatory and structural aspects remain elusive. Among these, the ability of Dishevelled (DVL) protein to relocalize at the plasma membrane is a crucial step in the activation of all Wnt pathways. The membrane binding of DVL was suggested to be mediated by the preferential interaction of its C-terminal DEP domain with phosphatidic acid (PA). However, due to the scarcity and fast turnover of PA, we investigated the role on the membrane association of other more abundant phospholipids. The combined results from computational simulations and experimental measurements with various model phospholipid membranes, demonstrate that the membrane binding of DEP/DVL constructs is governed by the concerted action of generic electrostatics and finely-tuned intermolecular interactions with individual lipid species. In particular, while we confirmed the strong preference for PA lipid, we also observed a weak but non-negligible affinity for phosphatidylserine, the most abundant anionic phospholipid in the plasma membrane, and phosphatidylinositol 4,5-bisphosphate. The obtained molecular insight into DEP-membrane interaction helps to elucidate the relation between changes in the local membrane composition and the spatiotemporal localization of DVL and, possibly, other DEP-containing proteins.

Primary Cilia Formation Does Not Rely on WNT/ß-Catenin Signaling.

Primary cilia act as crucial regulators of embryo development and tissue homeostasis. They are instrumental for modulation of several signaling pathways, including Hedgehog, WNT, and TGF-ß. However, gaps exist in our understanding of how cilia formation and function is regulated. Recent work has implicated WNT/ß-catenin signaling pathway in the regulation of ciliogenesis, yet the results are conflicting. One model suggests that WNT/ß-catenin signaling negatively regulates cilia formation, possibly via effects on cell cycle. In contrast, second model proposes a positive role of WNT/ß-catenin signaling on cilia formation, mediated by the re-arrangement of centriolar satellites in response to phosphorylation of the key component of WNT/ß-catenin pathway, ß-catenin. To clarify these discrepancies, we investigated possible regulation of primary cilia by the WNT/ß-catenin pathway in cell lines (RPE-1, NIH3T3, and HEK293) commonly used to study ciliogenesis. We used WNT3a to activate or LGK974 to block the pathway, and examined initiation of ciliogenesis, cilium length, and percentage of ciliated cells. We show that the treatment by WNT3a has no- or lesser inhibitory effect on cilia formation. Importantly, the inhibition of secretion of endogenous WNT ligands using LGK974 blocks WNT signaling but does not affect ciliogenesis. Finally, using knock-out cells for key WNT pathway components, namely DVL1/2/3, LRP5/6, or AXIN1/2 we show that neither activation nor deactivation of the WNT/ß-catenin pathway affects the process of ciliogenesis. These results suggest that WNT/ß-catenin-mediated signaling is not generally required for efficient cilia formation. In fact, activation of the WNT/ß-catenin pathway in some systems seems to moderately suppress ciliogenesis.

Phosphorylation-induced changes in the PDZ domain of Dishevelled 3.

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.

Roles of individual human Dishevelled paralogs in the Wnt signalling pathways.

Dishevelled (DVL) proteins are key mediators of most Wnt pathways. In all vertebrates, three DVL paralogs are present (DVL1, DVL2 and DVL3) but it is poorly defined to what extent they are functionally redundant. Here, we generated T-REx HEK 293 cells with only one DVL paralog (i.e., DVL1-only, DVL2-only, and DVL3-only) and compared their response to Wnt-3a and Wnt-5a ligands with wild type and DVL triple knockout cells. We show that DVL is essential, in addition to the previously shown Wnt-3a-induced phosphorylation of LRP6 and transcriptional activation of TCF/LEF-dependent reporter, also for Wnt-3a-induced degradation of AXIN1 and Wnt-5a-induced phosphorylation of ROR1. We have quantified the molar ratios of DVL1:DVL2:DVL3 in our model to be approximately 4:80:16. Interestingly, DVL-only cells do not compensate for the lack of other paralogs and are still fully functional in all analyzed readouts with the exception of Wnt-3a-induced transcription assessed by TopFlash assay. In this assay, the DVL1-only cell line was the most potent; on the contrary, the DVL3-only cell line exhibited only the negligible capacity to mediate Wnt signals. Using a novel model system - complementation assays in T-REx HEK 293 with amplified Wnt signal response (RNF43/ZNRF3/DVL1/DVL2/DVL3 penta KO cells) we demonstrate that it is not the total amount of DVL but ratio of individual paralogs what decides the signal strength. In sum, this study contributes to our better understanding of the role of individual human DVL paralogs in the Wnt pathway.

Activity of Smurf2 Ubiquitin Ligase Is Regulated by the Wnt Pathway Protein Dishevelled.

Wnt and BMP signaling pathways are two key molecular machineries regulating development and homeostasis. The efficient coordination of Wnt and BMP is essential in many developmental processes such as establishment of antero-posterior and dorso-ventral body axis, regulation of convergent extension, or development of various organ systems. SMAD ubiquitination regulatory factor (Smurf) family of E3 ubiquitin ligases are important and evolutionary conserved regulators of TGF-ß/BMP signaling pathways. Smurf2 has been previously shown to regulate Wnt/planar cell polarity (PCP) signaling pathway by ubiquitinating Prickle1, one of the key components of PCP. We explored the role of Smurf2 in Wnt pathways in further detail and identified that Smurf2 is also a ubiquitin ligase of Dishevelled (DVL), the key cytoplasmic signal transducer in the Wnt pathway. Interestingly, the Smurf2 and DVL relationship expands beyond substrate-E3 ligase. We can show that DVL activates Smurf2, which allows Smurf2 to ubiquitinate its substrates from Wnt/PCP (Prickle1) as well as TGF-ß/BMP (Smad2) pathways more efficiently. Using SMAD7 as an example of Smurf2 activator we show that DVL and SMAD7 both activates Smurf2 activity. In HEK293 cells the deficiency of DVL phenocopies absence of Smurf2 and leads to the increased phosphorylation of R-Smads. Smurf2-DVL connection provides a novel and intriguing point of crosstalk for Wnt and BMP pathways.

Dishevelled has a YAP nuclear export function in a tumor suppressor context-dependent manner.

Phosphorylation-dependent YAP translocation is a well-known intracellular mechanism of the Hippo pathway; however, the molecular effectors governing YAP cytoplasmic translocation remains undefined. Recent findings indicate that oncogenic YAP paradoxically suppresses Wnt activity. Here, we show that Wnt scaffolding protein Dishevelled (DVL) is responsible for cytosolic translocation of phosphorylated YAP. Mutational inactivation of the nuclear export signal embedded in DVL leads to nuclear YAP retention, with an increase in TEAD transcriptional activity. DVL is also required for YAP subcellular localization induced by E-cadherin, α-catenin, or AMPK activation. Importantly, the nuclear-cytoplasmic trafficking is dependent on the p53-Lats2 or LKB1-AMPK tumor suppressor axes, which determine YAP phosphorylation status. In vivo and clinical data support that the loss of p53 or LKB1 relieves DVL-linked reciprocal inhibition between the Wnt and nuclear YAP activity. Our observations provide mechanistic insights into controlled proliferation coupled with epithelial polarity during development and human cancer.

The N-Terminal Part of the Dishevelled DEP Domain Is Required for Wnt/ß-Catenin Signaling in Mammalian Cells.

Dishevelled (DVL) proteins are key mediators of the Wnt/ß-catenin signaling pathway. All DVL proteins contain three conserved domains: DIX, PDZ, and DEP. There is a consensus in the field that the DIX domain is critical for Wnt/ß-catenin signaling, but contradictory evidence regarding the function of the DEP domain exists. It has been difficult, until recently, to test the importance of the DEP domain rigorously because of the interference with endogenous DVL, expressed in all Wnt-responsive cell lines. In this study, we took advantage of DVL knockout (DVL1/DVL2/DVL3 triple knockout) cells fully deficient in Wnt3a-induced signaling events and performed a series of rescue experiments. Using these complementation assays, we analyzed the role of individual DVL isoforms. Further domain mapping of DVL1 showed that both the DVL1 DEP domain and especially its N-terminal region are required and sufficient for Wnt3a-induced phosphorylation of LRP6 and TopFlash reporter activation. On the contrary, multiple DEP domain mutants deficient in the planar cell polarity (PCP) pathway could fully rescue the Wnt3a response. This study provides conclusive evidence that the DVL DEP domain is essential for Wnt/ß-catenin signaling in mammalian cells and establishes an experimental system suitable for further functional testing of DVL.