Molecular mechanisms behind the functional (non)redundancy of CK1 paralogs in the Wnt pathway
Year: 2026
Type: preprint
Abstract: ABSTRACT 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.
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
Awards: Molecular and functional analysis of casein kinase 1 biology, G1241006667, G1761812275, G176604203, G1869016439, National institute for cancer research, G1935710221, G208869049, G2450917069, G2478170884, G2552582303, G2609894497, G291036901, G3267687010, G3490171961, G3503563290, G3735356012, G3850027771, G4280972364, G4459401953, G5288176631, G5641063287, G5652504370, G5916014575, National Infrastructure for Chemical Biology, G6761430300, G6807155651, G7534432959, G7662032182, G8140083606, Czech Infrastructure for Integrative Structural Biology, G8693535379
OpenAlex ID: W7212218051