Molecular mechanisms behind the functional (non)redundancy of CK1 paralogs in the Wnt pathway

The casein kinase 1 (CK1) family of serine/threonine protein kinases consists of seven isoforms in humans. CK1 family members are important regulators of the Wnt/β-catenin signaling pathway. Using a comprehensive panel of CRISPR/Cas9-generated knockout cell lines, we demonstrated the opposing roles of endogenous CK1α (negative, via phosphorylation of β-catenin in the destruction complex) and CK1δ/ε (positive, via phosphorylation of DVL in the signalosome), while no phenotype was observed for CK1γ1/2/3 triple knockout cells. Using in vitro kinase assays and TurboID-based interactomics, we revealed that this functional divergence between CK1α and CK1ε is not due to the intrinsically different capacity to phosphorylate β-catenin or DVL but rather due to different affinities for the destruction complex and signalosome in the cellular environment. Through functional analysis of CK1α–CK1ε chimeras containing both N-terminal and C-terminal domain swaps, we identified the N-terminal lobe of CK1α and the C-terminus of CK1ε as determinants mediating increased affinity towards the degradasome and signalosome, respectively. We further show that the CK1α N-lobe not only drives cellular activity toward β-catenin but also underlies the CK1α-specific interaction with the scaffolding protein SACK1G (also known as FAM83G and PAWS1). Additionally, despite clearly distinct physiological roles of CK1α and CK1δ/ε, we provide evidence that, in the absence of CK1α, CK1δ and CK1ε can physically and functionally substitute for CK1α in the β-catenin destruction complex. This rewires CK1δ and CK1ε as negative regulators acting via phosphorylation of β-catenin in the destruction complex. These findings resolve prior contradictions by (i) clarifying context-dependent CK1 roles, (ii) identifying mechanistic determinants navigating CK1α and CK1ε to different substrates, and (iii) defining the limitations of these affinity-based subcellular distributions that become apparent especially in the physical absence of the physiological, high-affinity kinase. An important implication of our findings is the identification of a mechanism that changes the ultimate outcome of CK1δ/ε inhibitor treatment from Wnt/β-catenin pathway inhibition to its robust activation.