Dishevelled Research Articles

Structural and Functional Insights into Dishevelled-Mediated Wnt Signaling

Dishevelled (DVL) proteins precisely control Wnt signaling pathways with many effectors. While substantial research has advanced our understanding of DVL’s role in Wnt pathways, key questions regarding its regulatory mechanisms and interactions remain unresolved. Herein, we present the recent advances and perspectives on how DVL regulates signaling. The experimentally determined conserved domain structures of DVL in conjunction with AlphaFold-predicted structures are used to understand the DVL’s role in Wnt signaling regulation. We also summarize the role of DVL in various diseases and provide insights into further directions for research on the DVL-mediated signaling mechanisms. These findings underscore the importance of DVL as a pharmaceutical target or biological marker in diseases, offering exciting potential for future biomedical applications.

Class-Wide Analysis of Frizzled-Dishevelled Interactions Using BRET Biosensors Reveals Functional Differences among Receptor Paralogs.

Wingless/Int-1 (WNT) signaling is mediated by WNT binding to 10 Frizzleds (FZD1-10), which propagate the signal inside the cell by interacting with different transducers, most prominently the phosphoprotein Dishevelled (DVL). Despite recent progress, questions about WNT/FZD selectivity and paralog-dependent differences in the FZD/DVL interaction remain unanswered. Here, we present a class-wide analysis of the FZD/DVL interaction using the DEP domain of DVL as a proxy in bioluminescence resonance energy transfer (BRET) techniques. Most FZDs engage in a constitutive high-affinity interaction with DEP. Stimulation of unimolecular FZD/DEP BRET sensors with different ligands revealed that most paralogs are dynamic in the FZD/DEP interface, showing distinct profiles in terms of ligand selectivity and signal kinetics. This study underlines mechanistic differences in terms of how allosteric communication between FZDs and their main signal transducer DVL occurs. Moreover, the unimolecular sensors represent the first receptor-focused biosensors to surpass the requirements for high-throughput screening, facilitating FZD-targeted drug discovery.

Structural insights into Frizzled3 through nanobody modulators

The Wnt receptor Frizzled3 (FZD3) is important for brain axonal development and cancer progression. We report structures of FZD3 in complex with extracellular and intracellular binding nanobodies (Nb). The crystal structure of Nb8 in complex with the FZD3 cysteine-rich domain (CRD) reveals that the nanobody binds at the base of the lipid-binding groove and can compete with Wnt5a. Nb8 fused with the Dickkopf-1 C-terminal domain behaves as a FZD3-specific Wnt surrogate, activating β-catenin signalling. The cryo-EM structure of FZD3 in complex with Nb9 reveals partially resolved density for the CRD, which exhibits positional flexibility, and a transmembrane conformation that resembles active GPCRs. Nb9 binds to the cytoplasmic region of FZD3 at the putative Dishevelled (DVL) or G protein-binding site, competes with DVL binding, and inhibits GαS coupling. In combination, our FZD3 structures with nanobody modulators map extracellular and intracellular interaction surfaces of functional, and potentially therapeutic, relevance.

Semaphorin Receptors Antagonize Wnt Signaling Through Beta-Catenin Degradation.

Precise control of morphogen signaling levels is essential for proper development. An outstanding question is: what mechanisms ensure proper morphogen activity and correct cellular responses? Previous work has identified Semaphorin (SEMA) receptors, Neuropilins (NRPs) and Plexins (PLXNs), as positive regulators of the Hedgehog (HH) signaling pathway. Here, we provide evidence that NRPs and PLXNs antagonize Wnt signaling in both fibroblasts and epithelial cells. Further, Nrp1/2 deletion in fibroblasts results in elevated baseline Wnt pathway activity and increased maximal responses to Wnt stimulation. Notably, and in contrast to HH signaling, SEMA receptor-mediated Wnt antagonism is independent of primary cilia. Mechanistically, PLXNs and NRPs act downstream of Dishevelled (DVL) to destabilize β-catenin (CTNNB1) in a proteosome-dependent manner. Further, NRPs, but not PLXNs, act in a GSK3β/CK1-dependent fashion to antagonize Wnt signaling, suggesting distinct repressive mechanisms for these SEMA receptors. Overall, this study identifies SEMA receptors as novel Wnt pathway antagonists that may also play larger roles integrating signals from multiple inputs.

Dishevelled Has Anti-Viral Activity in Rift Valley Fever Virus Infected Aedes aegypti.

Mosquitoes in the genera Aedes and Culex are vectors of Rift Valley fever virus (RVFV), which emerges in periodic epidemics in Africa and Saudi Arabia. Factors that influence the transmission dynamics of RVFV are not well characterized. To address this, we interrogated mosquito host-signaling responses through analysis of differentially expressed genes (DEGs) in two mosquito species with marked differences in RVFV vector competence: Aedes aegypti (Aae, low competence) and Culex tarsalis (Cxt, high competence). Mosquito-host transcripts related to three different signaling pathways were investigated. Selected genes from the Wingless (Wg, WNT-beta-catenin) pathway, which is a conserved regulator of cell proliferation and differentiation, were assessed. One of these, dishevelled (DSH), differentially regulates progression/inhibition of the WNT and JNK (c-Jun N-terminal Kinase) pathways. A negative regulator of the JNK-signaling pathway, puckered, was also assessed. Lastly, Janus kinase/signal transducers and activators of transcription (JAK-STAT) are important for innate immunity; in this context, we tested domeless levels. Here, individual Aae and Cxt were exposed to RVFV MP-12 via oral bloodmeals and held for 14 days. Robust decreases in DEGs in both Aae and Cxt were observed. In particular, Aae DSH expression, but not Cxt DSH, was correlated to the presence/absence of viral RNA at 14 days post-challenge (dpc). Moreover, there was an inverse relationship between the viral copy number and aaeDSH expression. DSH silencing resulted in increased viral copy numbers compared to controls at 3 dpc, consistent with a role for aaeDSH in antiviral immunity. Analysis of cis-regulatory regions for the genes of interest revealed clues to upstream regulation of these pathways.

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.

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.

DVL1 and DVL3 require nuclear localisation to regulate proliferation in human myoblasts

WNT signalling is essential for regulating a diverse range of cellular processes. In skeletal muscle, the WNT pathway plays crucial roles in maintenance of the stem cell pool and myogenic differentiation. Focus is usually directed at examining the function of central components of the WNT pathway, including β-CATENIN and the GSK3β complex and TCF/LEF transcription factors, in tissue homeostasis and cancer. Other core components of the WNT pathway though, are three dishevelled (DVL) proteins: membrane associated proteins that propagate WNT signalling from membrane to nucleus. Here we examined DVL function in human myogenesis and the muscle-related cancer alveolar rhabdomyosarcoma. We demonstrate that DVL1 and DVL3 are necessary for efficient proliferation in human myoblasts and are important for timely myogenic differentiation. DVL1 and DVL3 also contribute to regulation of proliferation in rhabdomyosarcoma. DVL1 or DVL3 must be present in the nucleus to regulate proliferation, but they operate through different protein domains: DVL3 requires the DIX and PDZ domains, while DVL1 does not. Importantly, DVL1 and DVL3 activity is independent of markedly increased translocation of β-CATENIN to the nucleus, normally a hallmark of active canonical WNT signalling.

Untangling Frizzled functions

Wnt signaling is a complex network of cellular signals with multiple downstream pathways regulated by signaling complexes comprised of combinations of the 19 Wnt ligands, 10 Frizzled (FZD) receptors, various co‐receptors and Dishevelled (DVL) second messengers. Up until now, Wnt receptors have either been functionally defined as whole families or individually by how they affect specific disease states when they are dysregulated, but the specific roles each receptor plays in Wnt signaling and activating the various pathways have yet to be fully understood.CRISPR is a powerful tool that has allowed whole families of protein isomers to be knocked out. By using cellular models that have been modified by CRISPR/Cas9 and are missing key proteins in the Wnt signaling complex, we can determine their individual functions and redundancies.We have determined that a FZD is necessary but any of the individual receptors is sufficient for DVL phosphorylation. Additionally, we have found that some Wnt signals can be rescued by single receptors, or just part of a receptor, while others seem to require multiple FZDs. The expression of these receptors is crucial for not only regulation within the Wnt family of pathways, but we also discovered a new arm of crosstalk with mTOR signaling.Wnt signaling is known to play key roles in many aspects of development, but erroneous signaling can cause diseases such as cancer and osteoporosis. Current therapeutics act as a sledgehammer and target all Wnt signaling, which leads to many off‐target effects and unintended side‐effects. Since cell surface receptors are typically the easiest to therapeutically target, FZD receptors have considerable therapeutic potential. Understanding the roles of each FZD receptor will allow us to better target individual disease mechanisms.

Anticancer Activity of (S)-5-Chloro-3-((3,5-dimethylphenyl)sulfonyl)-N-(1-oxo-1-((pyridin-4-ylmethyl)amino)propan-2-yl)-1H-indole-2-carboxamide (RS4690), a New Dishevelled 1 Inhibitor.

Simple SummaryThe WNT/β-catenin pathway regulates a huge number of cellular functions, and its dysregulation is correlated to the development of cancer. In this work, we focused on the interaction between Dishevelled 1 (DVL1) protein, an important player in this pathway, and its cognate receptor Frizzled via a shared PDZ domain. Computational studies led to the discovery of racemate RS4690 (1) showing selective inhibition of DVL1 binding. After separation of the racemic mixture, enantiomer (S)-1 inhibited DVL1 with an EC50 of 0.49 ± 0.11 μM and the growth of HCT116 cells that did not present the APC mutation with an EC50 value 7.1 ± 0.6 μM, and caused a high level of ROS production. Compound (S)-1 shows potential as a new therapeutic agent against WNT-dependent colon cancer.Wingless/integrase-11 (WNT)/β-catenin pathway is a crucial upstream regulator of a huge array of cellular functions. Its dysregulation is correlated to neoplastic cellular transition and cancer proliferation. Members of the Dishevelled (DVL) family of proteins play an important role in the transduction of WNT signaling by contacting its cognate receptor, Frizzled, via a shared PDZ domain. Thus, negative modulators of DVL1 are able to impair the binding to Frizzled receptors, turning off the aberrant activation of the WNT pathway and leading to anti-cancer activity. Through structure-based virtual screening studies, we identified racemic compound RS4690 (1), which showed a promising selective DVL1 binding inhibition with an EC50 of 0.74 ± 0.08 μM. Molecular dynamic simulations suggested a different binding mode for the enantiomers. In the in vitro assays, enantiomer (S)-1 showed better inhibition of DVL1 with an EC50 of 0.49 ± 0.11 μM compared to the (R)-enantiomer. Compound (S)-1 inhibited the growth of HCT116 cells expressing wild-type APC with an EC50 of 7.1 ± 0.6 μM and caused a high level of ROS production. These results highlight (S)-1 as a lead compound for the development of new therapeutic agents against WNT-dependent colon cancer.

Abstract P5-10-04: Exploring novel connections between dishevelled (DVL) proteins and HER2-positive breast cancer-road to translation

Abstract Aberrant Wnt signaling contributes to every stage of tumorigenesis from immunosuppression in the tumor microenvironment (TME) and reduced tumor infiltrating lymphocytes (TIL) activity in the tumor niche to modulating several cancer hallmarks such as unrestrained growth, increased neoplastic cell survival, invasion, and metastasis. The overactive Wnt pathway is critical for tumor progression and hence a therapeutic target in multiple clinical trials. Dishevelled (DVL)s are the major scaffold proteins which relay signals in both canonical and non-canonical Wnt pathways and help maintain constitutive oncogenic signaling. Though DVL proteins are significant drivers of oncogenic Wnt signaling, little is known about their role in modulating tumor immunity. Previously we demonstrated that nuclear localization of DVL proteins is associated with transcription of several cancer-associated genes as well promotion of in vivo tumor growth. Moreover, our preliminary studies revealed significant positive association between high DVL expression and poor patient survival in different aggressive subtypes of breast cancer including HER2-positive breast cancer. Therefore, we hypothesize that (i) DVL depletion will reduce expression of classic Wnt markers involved in cancer cell proliferation and invasion in HER2-overexpressed in vitro models of breast cancer and (ii) clinically, high DVL protein expression will modulate markers of tumor immunity such as TIL score, levels of cytotoxic (CD8α) T-cells, neutrophil/platelet to lymphocyte ratio (NLR, PLR), eventually resulting in poor survival outcomes in HER2-positive breast cancer.To test our hypothesis, we performed DVL2 loss of function studies in two different HER2-overexpressed breast cancer cell lines such as SKBR3 (ER/PR-, HER2+) and BT474 (ER/PR+, HER2+) via lentiviral shRNA transduction and siRNA transfection. We analyzed RNA (RTqPCR) and protein (western blot) expression of classic Wnt markers and performed cell proliferation and cell cycle analyses by live cell imaging and flow cytometry, respectively. Additionally, we performed a pilot study including 10 HER2-positive breast cancer patients to dissect the role of DVL2 in tumor immunity. Retrospective chart review on patient records and banked tissue histology were performed to investigate the markers of tumor immunity and DVL proteins. Data were analyzed in SPSS (version 25) and GraphPad Prism (version 7) at a significance p<0.05.Initially, we identify higher DVL2 protein levels in tissues from HER2-positive breast cancer compared to non-cancer and other subtypes of breast cancer. Next, DVL2 depletion (NTC vs. sh- or siDVL2) results in reduced mRNA expression of Wnt target genes such as CYCLIN D1, OCT4, MMP7, VEGFA classically involved in cell proliferation, stem cell renewal, migration, and invasion respectively in both SKBR3 and BT474 cells. Similarly, live cell proliferation and cell cycle analyses reveal that DVL2 knockdown resulted in reduced proliferation, higher growth arrest (G1), limited mitosis (G2/M) compared to non-targeted control. Analyses on patient tissues further demonstrate that higher DVL2 expression pose a weak negative correlation with % TIL score (r=-0.17, NS) and % CD8α levels (r=-0.32, NS) while have a positive correlation with NLR (r=0.65, p=0.05), where high NLR denotes worse cancer prognosis. Though not statistically significant, these results from our pilot study reveal crucial roles of DVL2 proteins in regulating immunity and survival of HER2-positive breast cancer patients. Our study is noteworthy since it demonstrates possible immune regulatory role of DVL proteins in HER2-positive breast cancer which in turn, will aid in designing future clinical trials and in-depth mechanistic studies benefitting patients of different subtypes of breast cancer. Citation Format: Fahmida Rasha, Mingxiao V Yang, Isabel Castro-Piedras, Kathryn L. Furr, Annie Snitman, Sonia Y. Khan, Luis Brandi, Hafiz Khan, Nusrat Jahan, Michael W. Melkus, Kevin Pruitt, Rakhshanda Layeequr Rahman. Exploring novel connections between dishevelled (DVL) proteins and HER2-positive breast cancer-road to translation [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-10-04.

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.

Nuclear Dishevelled targets gene regulatory regions and promotes tumor growth.

Dishevelled (DVL) critically regulates Wnt signaling and contributes to a wide spectrum of diseases and is important in normal and pathophysiological settings. However, how it mediates diverse cellular functions remains poorly understood. Recent discoveries have revealed that constitutive Wnt pathway activation contributes to breast cancer malignancy, but the mechanisms by which this occurs are unknown and very few studies have examined the nuclear role of DVL. Here, we have performed DVL3 ChIP-seq analyses and identify novel target genes bound by DVL3. We show that DVL3 depletion alters KMT2D binding to novel targets and changes their epigenetic marks and mRNA levels. We further demonstrate that DVL3 inhibition leads to decreased tumor growth in two different breast cancer models in vivo. Our data uncover new DVL3 functions through its regulation of multiple genes involved in developmental biology, antigen presentation, metabolism, chromatin remodeling, and tumorigenesis. Overall, our study provides unique insight into the function of nuclear DVL, which helps to define its role in mediating aberrant Wnt signaling.

USP42 protects ZNRF3/RNF43 from R-spondin-dependent clearance and inhibits Wnt signalling.

The tumour suppressors RNF43 and ZNRF3 play a central role in development and tissue homeostasis by promoting the turnover of the Wnt receptors LRP6 and Frizzled (FZD). The stem cell growth factor R‐spondin induces auto‐ubiquitination and membrane clearance of ZNRF3/RNF43 to promote Wnt signalling. However, the deubiquitinase stabilising ZNRF3/RNF43 at the plasma membrane remains unknown. Here, we show that the USP42 antagonises R‐spondin by protecting ZNRF3/RNF43 from ubiquitin‐dependent clearance. USP42 binds to the Dishevelled interacting region (DIR) of ZNRF3 and stalls the R‐spondin‐LGR4‐ZNRF3 ternary complex by deubiquitinating ZNRF3. Accordingly, USP42 increases the turnover of LRP6 and Frizzled (FZD) receptors and inhibits Wnt signalling. Furthermore, we show that USP42 functions as a roadblock for paracrine Wnt signalling in colon cancer cells and mouse small intestinal organoids. We provide new mechanistic insights into the regulation R‐spondin and conclude that USP42 is crucial for ZNRF3/RNF43 stabilisation at the cell surface.

Chondrocyte Polarity During Endochondral Ossification Requires Protein-Protein Interactions Between Prickle1 and Dishevelled2/3.

The expansion and growth of the endochondral skeleton requires organized cell behaviors that control chondrocyte maturation and oriented division. In other organs, these processes are accomplished through Wnt/planar cell polarity (Wnt/PCP) signaling pathway and require the protein-protein interactions of core components including Prickle1 (PK1) and Dishevelled (DVL). To determine the function of Wnt/PCP signaling in endochondral ossification of the cranial base and limb, we utilized the Prickle1Beetlejuice (Pk1Bj ) mouse line. The Pk1Bj allele has a missense mutation in the PK1 LIM1 domain that results in a hypomorphic protein. Similar to human patients with Robinow syndrome, the Prickle1Bj/Bj mouse mutants lack growth plate expansion resulting in shorter limbs and midfacial hypoplasia. Within the Prickle1Bj/Bj limb and cranial base growth plates we observe precocious maturation of chondrocytes and stalling of terminal differentiation. Intriguingly, we observed that the growth plate chondrocytes have randomized polarity based on the location of the primary cilia and the location of PRICKLE1, DVL2, and DVL3 localization. Importantly, mutant PK1Bj protein has decreased protein-protein interactions with both DVL2 and DVL3 in chondrocytes as revealed by in vivo co-immunoprecipitation and proximity ligation assays. Finally, we propose a model where the interaction between the Prickle1 LIM1 domain and DVL2 and DVL3 contributes to chondrocyte polarity and contributes to proximal-distal outgrowth of endochondral elements. © 2021 American Society for Bone and Mineral Research (ASBMR).

Intersection of two key signal integrators in the cell: activator of G-protein signaling 3 and dishevelled-2.

Activator of G-protein signaling 3 (AGS3, encoded by GPSM1) was discovered as a one of several receptor-independent activators of G-protein signaling, which are postulated to provide a platform for divergence between canonical and noncanonical G-protein signaling pathways. Similarly, Dishevelled (DVL) proteins serve as a point of divergence for β-catenin-dependent and -independent signaling pathways involving the family of Frizzled (FZD) ligands and cell-surface WNT receptors. We recently discovered the apparent regulated localization of dishevelled-2 (DVL2) and AGS3 to distinct cellular puncta, suggesting that the two proteins interact as part of various cell signaling systems. To address this hypothesis, we asked the following questions: (1) do AGS3 signaling pathways influence the activation of β-catenin (CTNNB1)-regulated transcription through the WNT-Frizzled-Dishevelled axis, and (2) is the AGS3 and DVL2 interaction regulated? The interaction of AGS3 and DVL2 was regulated by protein phosphorylation, subcellular distribution, and a cell-surface G-protein-coupled receptor. These data, and the commonality of functional system impacts observed for AGS3 and DVL2, suggest that the AGS3-DVL2 complex presents an unexpected path for functional integration within the cell.This article has an associated First Person interview with the first author of the paper.

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-β/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-β/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.

WNT Receptor Requirements for Dishevelled Phosphorylation

The Dishevelled (DVL) protein is a key component of WNT signaling that relays signals from receptors to downstream effectors. It has been shown that following WNT ligand binding to Frizzled (FZD) receptors, DVL is recruited to the membrane and is phosphorylated. Downstream signals of WNT require the presence of DVL. Since FZD receptors are the known receptor for WNT ligands, it has been assumed, but not shown that Wnt‐stimulated DVL phosphorylation is dependent on a FZD receptor. Since there are 19 WNTs, 10 FZDs, 3 DVLs and co‐receptors, such as LRP5/6 and ROR1/2, that play a role in Wnt signaling, it is suspected that the specific combination that forms the signaling complex determines the downstream signaling output. Most WNTs have been shown to activate canonical, β‐catenin dependent signaling, whereas WNT5a can stimulate multiple non‐canonical effector pathways such as planar cell polarity and calcium signaling. We hypothesize that the specific receptors, whether it be ROR1/2 or a particular FZD, that bind to WNT5a determine the non‐canonical pathway that is activated. In order to look further downstream, our primary objective is to determine which receptors are required in response to WNT5a to activate DVL phosphorylation.By using cellular models missing key proteins in the WNT signaling complex, we can determine their individual functions and redundancies. To study WNT‐FZD‐DVL interactions, we are utilizing cell lines which lack all ten FZDs (FZDless), ROR1/2 (Rorless), or DVL1/2/3 (DVLless) and assessing relative expression and phosphorylation of DVL. DVL expression is decreased in RORless cells and ROR1 and ROR2 expression are decreased in DVLless cells. LGK974 Porcupine inhibitor treatment to inhibit endogenous WNT secretion also reduces DVL expression in wildtype cells. In comparison, overexpression of the FZD2 receptor, results in increased ROR2 expression. FZDless cells show no phosphorylation of DVL under endogenous WNT stimulation or in the presence of WNT conditioned media (CM). In contrast, RORless cells show DVL phosphorylation under endogenous WNT expression, but not with LGK974 and subsequent WNT5a CM treatment.In conclusion, expression and phosphorylation of DVL are dependent on the expression of specific WNT receptors. WNT‐induced DVL phosphorylation is dependent on the presence of FZD receptors but expression of ROR receptors is not necessary. However, these requirements are dependent on the particular WNT ligand in question. WNT5a not only requires FZD receptors to stimulate DVL phosphorylation, but also requires ROR receptors. Overall, these findings demonstrate the intricacies of WNT signaling and lends itself to further investigation of these receptor requirements.Support or Funding InformationVan Andel Institute Graduate School Fellowship

The Rho guanine nucleotide exchange factor Trio is required for neural crest cell migration and interacts with Dishevelled.

Directional migration during embryogenesis and tumor progression faces the challenge that numerous external signals need to converge to precisely control cell movement. The Rho guanine exchange factor (GEF) Trio is especially well suited to relay signals, as it features distinct catalytic domains to activate Rho GTPases. Here, we show that Trio is required for Xenopus cranial neural crest (NC) cell migration and cartilage formation. Trio cell-autonomously controls protrusion formation of NC cells and Trio morphant NC cells show a blebbing phenotype. Interestingly, the Trio GEF2 domain is sufficient to rescue protrusion formation and migration of Trio morphant NC cells. We show that this domain interacts with the DEP/C-terminus of Dishevelled (DVL). DVL - but not a deletion construct lacking the DEP domain - is able to rescue protrusion formation and migration of Trio morphant NC cells. This is likely mediated by activation of Rac1, as we find that DVL rescues Rac1 activity in Trio morphant embryos. Thus, our data provide evidence for a novel signaling pathway, whereby Trio controls protrusion formation of cranial NC cells by interacting with DVL to activate Rac1.

Acetylation of conserved DVL-1 lysines regulates its nuclear translocation and binding to gene promoters in triple-negative breast cancer

Dishevelled (DVL) proteins are central mediators of the Wnt signalling pathway and are versatile regulators of several cellular processes, yet little is known about their post-translational regulation. Acetylation is a reversible post-translational modification (PTM) which regulates the function of several non-histone proteins involved in tumorigenesis. Since we previously demonstrated that lysine deacetylase, SIRT-1, regulates DVL protein levels and its function, we reasoned that DVL could potentially be a substrate for SIRT-1 mediated deacetylation. To further examine the potential role of multiple families of lysine deacetylases in the post-translational regulation of DVL, we screened for novel acetylation sites using liquid chromatography mass-spectrometry (LC-MS/MS) analysis. Herein, we report 12 DVL-1 lysine residues that show differential acetylation in response to changes in oxygen tension and deacetylase inhibition in triple-negative breast cancer (TNBC). PTMs are well documented to influence protein activity, and cellular localization. We also identify that acetylation of two key lysine residues, K69 and K285, present on the DIX and PDZ domains respectively, promote nuclear over cytoplasmic localization of DVL-1, and influences its promoter binding and regulation of genes implicated in cancer. Collectively, these findings for the first time, uncover acetylation as a novel layer of regulation of DVL-1 proteins.