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

Abstract

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...

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

“…S7). Severing might also explain the observation that mild overexpression of Dvl did not significantly affect wingless signaling (Schaefer et al, 2020). Crucially, capping and/or severing are distinct from models in which the Axin interaction with Dvl occurs by co-polymerization of DAX and DIX (Bienz, 2014), which would not limit the number of Axin molecules associated with Dvl.…”

Quantitative live-cell imaging and computational modeling shed new light on endogenous WNT/CTNNB1 signaling dynamics

“…It is, however, in line with the notion that CTNNB1 still accumulates in a destruction complex after WNT signaling. One line of evidence proposes that WNT traps CTNNB1 in a complex by inhibiting its transfer to the E3 ligase, rather than by inhibiting its phosphorylation and release ( Li et al, 2012 ; Pronobis et al, 2015 ; Schaefer et al, 2020 ). However, other reports strongly suggest that CTNNB1 phosphorylation is at least partially inhibited during the WNT response ( Hernández et al, 2012 ; Mukherjee et al, 2018 ).…”

“…The fact that cells in which GSK3 phosphorylation is inhibited through S45F mutation or CHIR99021 treatment show similar behavior, suggests that the size of the cytoplasmic complex is directly linked to the phosphorylation status of CTNNB1. The destruction complex has been shown to associate with (parts of) the ubiquitin and proteasome machinery ( Li et al, 2012 ; Lui et al, 2011 ; Schaefer et al, 2020 ; Thorvaldsen et al, 2015 ). One interesting possibility, therefore, is that phosphorylated CTNNB1 is required for coupling the destruction complex to the ubiquitination and proteasome machinery.…”

Multivalent tumor suppressor adenomatous polyposis coli promotes Axin biomolecular condensate formation and efficient β-catenin degradation

“…The biocondensate model of β-catenin destruction complex has been proposed recently 12 , 33 . The biocondensates create physically separated compartments in the cytosol that is specifically enriched of the components of the complex.…”