The Wnt/β-catenin pathway is a highly conserved, frequently mutated developmental and cancer pathway. Its output is defined mainly by β-catenin’s phosphorylation- and ubiquitylation-dependent proteasomal degradation, initiated by the multi-protein β-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 β-catenin destruction complex from purified components, recapitulating complex assembly, β-catenin modification, and degradation. We reveal that AXIN1 polymerization and APC promote β-catenin capture, phosphorylation, and ubiquitylation. APC facilitates β-catenin’s flux through the complex by limiting ubiquitylation processivity and directly interacts with the SCF^β-TrCP E3 ligase complex in a β-TrCP-dependent manner. Oncogenic APC truncation variants, although part of the complex, are functionally impaired. Nonetheless, even the most severely truncated APC variant promotes β-catenin recruitment. These findings exemplify the power of biochemical reconstitution to interrogate the molecular mechanisms of Wnt/β-catenin signaling.

Wnt/β-catenin 通路是一种高度保守、经常突变的发育和癌症通路。其输出主要由 β-连环蛋白的磷酸化和泛素化依赖性蛋白酶体降解定义，由多蛋白 β-连环蛋白破坏复合物启动。破坏复合体功能的确切机制仍然未知，主要是因为缺乏合适的体外系统。这里我们描述体外从纯化的组分中重建活性人类 β-连环蛋白破坏复合物，重现复合物组装、β-连环蛋白修饰和降解。我们揭示 AXIN1 聚合和 APC 促进 β-连环蛋白捕获、磷酸化和泛素化。APC 通过限制泛素化持续合成促进 β-连环蛋白通过复合物的流动，并以依赖于 β-TrCP 的方式直接与 SCF ^β-TrCP E3 连接酶复合物相互作用。致癌 APC 截断变异虽然是复杂的一部分，但功能受损。尽管如此，即使是最严重截断的 APC 变体也会促进 β-连环蛋白的募集。这些发现证明了生化重构在探究 Wnt/β-catenin 信号传导的分子机制方面的能力。