The β-catenin destruction complex (BDC) is a central node in WNT/β-catenin signaling, governing embryonic development and adult tissue homeostasis. Although recognized as a prime therapeutic target in colorectal cancer (CRC) for three decades, its dynamic architecture and biochemical complexity have hindered mechanistic understanding. Here, we systematically mapped the sequence-function landscape of the BDC using tiled base editor screens across four endogenous components—CTNNB1, AXIN1, APC, and GSK3B. Validation studies identified ∼150 previously unreported mutations across these genes that affected WNT/β-catenin signaling. In addition to known cancer-associated mutations, we discovered rare gain-of-function and separation-of-function alleles of AXIN1 and CTNNB1 that provide mechanistic insights into complex assembly and regulation. We describe a region in β-catenin that regulates its binding to TCF/LEF transcription factors and demonstrate that the AXIN1–β-catenin interface is critical for controlling signaling flux through the oncogenic BDC. Mechanistic studies revealed that assembly of the oncogenic BDC is scaffolded by its own substrate β-catenin, establishing an autoregulatory mechanism that represents an unexploited vulnerability in cancers harboring common APC truncations. Our comprehensive mutational resource provides a foundation for understanding WNT/β-catenin signaling mechanisms in health and disease, while revealing strategies for therapeutic intervention in WNT-driven cancers.

Summary 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 SCFβ-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.