Wnt/Beta-Catenin Signaling Regulation and a Role for Biomolecular Condensates

Schaefer, Kristina N.; Peifer, Mark

doi:10.1016/j.devcel.2019.01.025

Cited by 155 publications

(169 citation statements)

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“…The destruction complex has many of the properties of a biomolecular condensate (Schaefer and Peifer, 2019). One of these is the ability of individual components to rapidly exchange with the cytoplasmic pool-Axin, APC2, and Dsh all can move into and out of puncta (Schwarz-Romond et al, 2005;Kunttas-Tatli et al, 2014;Pronobis et al, 2015).…”

Section: Slimb Is a Dynamic Component Of The Destruction Complexmentioning

confidence: 99%

“…Instead, Axin directs assembly of destruction complex proteins into what the field originally described as "puncta". We now recognize these as examples of supermolecular, non-membrane bound cellular compartments (reviewed in (Gammons and Bienz, 2017;Schaefer and Peifer, 2019), referred to as biomolecular condensates (Banani et al, 2017). Condensate formation is driven by Axin polymerization via its DIX domain, by APC function, and by other multivalent interactions e.g.…”

Section: Introductionmentioning

confidence: 99%

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Wnt Regulation: Exploring Axin-Disheveled interactions and defining mechanisms by which the SCF E3 ubiquitin ligase is recruited to the destruction complex

Schaefer

Pronobis

Williams

et al. 2019

Preprint

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Wnt signaling plays key roles in embryonic development and adult stem cell homeostasis, and is altered in human cancer. Signaling is turned on and off by regulating stability of the effector β-catenin. The multiprotein destruction complex binds and phosphorylates β-catenin, and transfers it to the SCF-TrCP E3-ubiquitin ligase, for ubiquitination and destruction. Wnt signals act though Dishevelled to turn down the destruction complex, stabilizing β-catenin. Recent work clarified underlying mechanisms, but important questions remain. We explore β-catenin transfer from the destruction complex to the E3 ligase, and test models suggesting Dishevelled and APC2 compete for association with Axin. We find that Slimb/TrCP is a dynamic component of the destruction complex biomolecular condensate, while other E3 proteins are not. Recruitment requires Axin and not APC, and Axin's RGS domain plays an important role. We find that elevating Dishevelled levels in Drosophila embryos has paradoxical effects, promoting the ability of limiting levels of Axin to turn off Wnt signaling. When we elevate Dishevelled levels, it forms its own cytoplasmic puncta, but these do not recruit Axin. SIM imaging in mammalian cells suggests that this may result by promoting Dishevelled: Dishevelled interactions at the expense of Dishevelled:Axin interactions when Dishevelled levels are high.part of the destruction complex, either by direct interaction with destruction complex proteins or using phosphorylated βcat as a bridge. In this model, once βcat is phosphorylated it could be directly transferred to the E3 ligase, thus preventing dephosphorylation of βcat by cellular phosphatases during transit. Immunoprecipitation (IP) experiments in animals and cell culture revealed that βTrCP can coIP with Axin, APC, βcat, and GSK3, and that Wnt signals reduce Axin: βTrCP coIP (Hart et al., 1999;Kitagawa et al., 1999;Liu et al., 1999;Li et al., 2012). However, these studies did not examine whether βTrCP or other components of the E3 are recruited to the destruction complex, leaving both models an option, especially if βTrCP acts as a shuttling protein between complexes. Here we address this issue.A second set of outstanding questions concern the mechanisms by which Wnt signaling down-regulates βcat destruction. Wnt signaling is initiated when Wnt ligands interact with complex multi-protein receptors, comprised of Frizzled family members plus LRP5/6 (reviewed in (DeBruine et al., 2017;Nusse and Clevers, 2017). This receptor complex recruits the destruction complex to the plasma membrane, via interaction of Axin with the phosphorylated LRP5/6 tail and with the Wnt effector Disheveled (Dvl in mammals/Dsh in Drosophila).This leads to downregulation of the destruction complex, reducing the rate of βcat destruction. Current data suggest destruction complex downregulation occurs via multiple mechanisms (reviewed in (MacDonald and He, 2012;Nusse and Clevers, 2017), some rapid and others initiated more slowly. These include direct inhibition of GSK3 by the phosphor...

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Section: Slimb Is a Dynamic Component Of The Destruction Complexmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Wnt Regulation: Exploring Axin-Disheveled interactions and defining mechanisms by which the SCF E3 ubiquitin ligase is recruited to the destruction complex

Schaefer

Pronobis

Williams

et al. 2019

Preprint

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“…In this complex, Axin-bound GSK-3 phosphorylates proline/serine-rich motifs in the LRP5/6 cytoplasmic tail, which then inhibit GSK-3 and hence b-catenin phosphorylation and destruction (MacDonald and He, 2012;Stamos et al, 2014;Stamos and Weis, 2013). Dvl self-association may contribute to formation of a multimeric and possibly phase-separated "signalosome" containing multiple copies of the receptor complex and its associated cytoplasmic components (Gammons and Bienz, 2018;Schaefer and Peifer, 2019).…”

Section: Introductionmentioning

confidence: 99%

Limited Dishevelled/Axin oligomerization determines efficiency of Wnt/β-catenin signal transduction

Kan

Enos

Korkmazhan

et al. 2020

Preprint

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SUMMARYIn Wnt/β-catenin signaling, the transcriptional coactivator β-catenin is regulated by its phosphorylation in a complex that includes the scaffold protein Axin and associated kinases. Wnt binding to its coreceptors activates the cytosolic effector Dishevelled (Dvl), leading to the recruitment of Axin and the inhibition of β-catenin phosphorylation. This process requires interaction of homologous DIX domains present in Dvl and Axin, but is mechanistically undefined. We show that Dvl DIX forms antiparallel, double-stranded oligomers in vitro, and that Dvl in cells forms oligomers typically <10 molecules at endogenous expression levels. Axin DIX (DAX) forms small single-stranded oligomers, but its self-association is stronger than that of DIX. DAX caps the ends of DIX oligomers, such that a DIX oligomer has at most four DAX binding sites. The relative affinities and stoichiometry of the DIX-DAX interaction provide a mechanism for efficient inhibition of β-catenin phosphorylation upon Axin recruitment to the Wnt receptor complex.

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“…Overexpression, which is widely used, comes with caveats of potential artifacts. The Wnt pathway scaffold proteins have dimerization (12,13) or oligomerization (14,15) domains, which carry with them the potential of forming large nonphysiological structures when expressed at high concentration (16)(17)(18). Whether these structures exist in the physiological context remains a matter of debate.…”

Section: Introductionmentioning

confidence: 99%

“…The Dvl oligomers in our study is far smaller than the "puncta" in some other studies that could reach the diameter of 1~2 um, potentially containing millions of proteins. We must point out that we didn't rule out the possibility that other Wnt pathway proteins like Axin and APC to form large protein condensations(18) or Dvl to form super complexes in other biological systems. It's possible that the different oligomerization forms of Dvl are used to perform different functions.We do not yet understand why Dvl dynamicity is so carefully programmed into the system.…”

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confidence: 99%

Single molecule dynamics of Dishevelled at the plasma membrane and Wnt pathway activation

Chen

Kang

et al. 2019

Preprint

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Dvl (Dishevelled) is one of several essential non-enzymatic components of the Wnt signaling pathway. In most current models, Dvl forms complexes with Wnt ligand receptors, Fzd and LRP5/6 at the plasma membrane, which then recruits other components of the destruction complex leading to inactivation of β -catenin degradation. Although this model is widespread, direct evidence for this process is lacking. In this study, we tagged mEGFP to C-terminus of dishevlled2 gene using CRISPR/Cas9 induced homologous recombination and observed its dynamics directly at the single molecule level with Total Internal Reflection Fluorescence (TIRF) microscopy. We focused on two questions: 1) What is the native size and the dynamic features of membrane-associated Dvl complexes during Wnt pathway activation? 2) What controls the behavior of these complexes? We found that membrane bound Dvl2 is predominantly monomer in the absent of Wnt (mean size 1.10). Wnt3a stimulation leads to an increase in the total concentration of membrane-bound Dvl2 from 0.08/μm 2 to 0.34/μm 2 . Wnt3a also leads to increased oligomerization which raises the weighted averaged mean size of Dvl2 complexes to 1.4; with 65% of Dvl still as monomers. The driving force for Dvl2 oligomerization is the increased concentration of Dvl2 at the membrane caused by increased affinity of Dvl2 for Fzd, the Dvl2 and Fzd binding is independent of LRP5/6. The oligomerized Dvl2 complexes have greatly increased dwell time, 2~3 minutes compared to less than 1 second for monomeric Dvl2.These properties make Dvl a unique scaffold dynamically changing its state of assembly and stability at the membrane in response to Wnt ligands. Significance Statement:Canonical Wnt signaling is one of the most widely distributed pathways in metazoan development. Despite intense genetic and biochemical study for over 35 years, the major features of signaling across the plasma membrane are still poorly understood. Dishevelled serves as an essential bridge between the membrane receptors and downstream signaling components.Attempts to reconstruct the pathway and analyze its biochemical features in vitro have been hampered by Dishevelled's tendency to aggregate in vitro and to form large aggregates of dubious significance in vivo. To obtain a molecular understanding of the role of Dvl in Wnt signaling, while circumventing these aggregation problems we have expressed a fluorescent tagged Dishevelled in cells at their physiological concentration and quantified the size distribution of Dishevelled before and after Wnt treatment. We found that limited oligomerization in response to the Wnt ligand is very dynamic and provides a key step of signal transduction.

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Wnt/Beta-Catenin Signaling Regulation and a Role for Biomolecular Condensates

Cited by 155 publications

References 184 publications

Wnt Regulation: Exploring Axin-Disheveled interactions and defining mechanisms by which the SCF E3 ubiquitin ligase is recruited to the destruction complex

Wnt Regulation: Exploring Axin-Disheveled interactions and defining mechanisms by which the SCF E3 ubiquitin ligase is recruited to the destruction complex

Limited Dishevelled/Axin oligomerization determines efficiency of Wnt/β-catenin signal transduction

Single molecule dynamics of Dishevelled at the plasma membrane and Wnt pathway activation

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