Reconstitution of the destruction complex defines roles of AXIN polymers and APC in beta-catenin capture, phosphorylation, and ubiquitylation

Unprocessed, original data for Ranes et al., 2021
https://doi.org/10.1016/j.molcel.2021.07.013

Reconstitution of the destruction complex defines roles of AXIN polymers and APC in beta-catenin capture, phosphorylation, and ubiquitylation
Michael Ranes, Mariola Zaleska, Saira Sakalas, Ruth Knight, Sebastian Guettler
Divisions of Structural Biology and Cancer Biology, The Institute of Cancer Research (ICR), London, UK

Summary:
The Wnt/beta-catenin pathway is a highly conserved, frequently mutated developmental and cancer pathway. Its output is defined mainly by beta-catenin’s phosphorylation- and ubiquitylation-dependent proteasomal degradation, initiated by the multi-protein beta-catenin destruction complex. The precise mechanisms underlying destruction complex function have remained unknown, largely because of the lack of suitable in vitro systems. Here we describe the in vitro reconstitution of an active human beta-catenin destruction complex from purified components, recapitulating complex assembly, beta-catenin modification, and degradation. We reveal that AXIN1 polymerisation and APC promote beta-catenin capture, phosphorylation, and ubiquitylation. APC facilitates beta-catenin’s flux through the complex by limiting ubiquitylation processivity and directly interacts with the SCF beta-TrCP E3 ligase complex in a beta-TrCP-dependent manner. Oncogenic APC truncation variants, although part of the complex, are functionally impaired. Nonetheless, even the most severely truncated APC variant promotes beta-catenin recruitment, potentially explaining why APC loss is selected against during carcinogenesis. These findings exemplify the power of biochemical reconstitution to interrogate the molecular mechanisms of Wnt/beta-catenin signalling.