Regulation of Wnt signalling pathways by the Dishevelled DEP domain
Wnt signals bind to Frizzled receptors to trigger canonical and noncanonical signalling responses that control cell fates during animal development and tissue homeostasis. All Wnt signals are relayed by the hub protein Dishevelled (DVL). During canonical (β-catenin dependent) signalling, Dishevelled assembles signalosomes via dynamic head-to-tail polymerization of its DIX domain, which are cross-linked by its DEP domain through a conformational switch from monomer to domain-swapped dimer. The domain-swapped conformation of DEP masks the site through which Dishevelled binds to Frizzled, implying that DEP domain swapping results in the detachment of Dishevelled from Frizzled. This would be incompatible with noncanonical Wnt signalling, which relies on long-term association between Dishevelled and Frizzled. It is therefore likely that DEP domain swapping is differentially regulated during canonical and noncanonical Wnt signalling.
In the first chapter of this thesis work I used biophysical techniques, nuclear magnetic resonance spectroscopy (NMR) and cell-based assays to uncover intermolecular contacts in the DEP dimer that are essential for its stability and for Dishevelled function in relaying canonical Wnt signals. These contacts are mediated by an intrinsically structured sequence spanning a conserved serine phosphorylation site upstream of the DEP domain (S418 in human DVL2) that serves to clamp down the swapped N-terminal α-helix onto the structural core of a reciprocal DEP molecule in the domain-swapped configuration. I also characterized the human DVL2 S435 conserved phosphorylation site located in the DEP hinge loop, a sensitive region for DEP domain swapping. Mutations of S418 and its cognate surface on the reciprocal DEP core or in S435 attenuate DEP-dependent dimerization of Dishevelled and its canonical signalling activity in cells, without impeding its binding to Frizzled. DVL2 S418 and S435 are crucial residues that could be employed to switch off canonical Wnt signalling.
In the second chapter I focused on the study of Diversin. The protein Diversin plays a positive role in noncanonical Wnt signalling such as planar cell polarity and has been reported to play an inhibitory role on canonical Wnt signalling. Reports indicate that Dishevelled DEP domain and Diversin ANK domain interact and that this interaction is crucial for the role of Diversin on noncanonical outputs. Using cell-bases assays, I concluded that overexpression of Diversin inhibits canonical Wnt signalling, consistent with previous results. Moreover, I observed that Dishevelled and Diversin interact and that DEP and ANK are required for this interaction. However, I could not observe a direct interaction between monomeric DEP and ANK, suggesting that the interaction between Dishevelled and Diversin depends on extra regions, different protein conformation or that accessory factors are required for their interaction.