Members of the casein kinase 1 (CK1) family are important regulators of multiple signaling pathways. CK1α is a well-known negative regulator of the Wnt/β-catenin pathway, which promotes the degradation of β-catenin via its phosphorylation of Ser45. In contrast, the closest paralog of CK1α, CK1α-like, is a poorly characterized kinase of unknown function. In this study, we show that the deletion of CK1α, but not CK1α-like, resulted in a strong activation of the Wnt/β-catenin pathway. Wnt-3a treatment further enhanced the activation, which suggests there are at least two modes, a CK1α-dependent and Wnt-dependent, of β-catenin regulation. Rescue experiments showed that only two out of ten naturally occurring splice CK1α/α-like variants were able to rescue the augmented Wnt/β-catenin signaling caused by CK1α deficiency in cells. Importantly, the ability to phosphorylate β-catenin on Ser45 in the in vitro kinase assay was required but not sufficient for such rescue. Our compound CK1α and GSK3α/β KO models suggest that the additional nonredundant function of CK1α in the Wnt pathway beyond Ser45-β-catenin phosphorylation includes Axin phosphorylation. Finally, we established NanoBRET assays for the three most common CK1α splice variants as well as CK1α-like. Target engagement data revealed comparable potency of known CK1α inhibitors for all CK1α variants but not for CK1α-like. In summary, our work brings important novel insights into the biology of CK1α, including evidence for the lack of redundancy with other CK1 kinases in the negative regulation of the Wnt/β-catenin pathway at the level of β-catenin and Axin.

CEITEC Central European Institute of Technology Masaryk University Brno Czech Republic

Department of Experimental Biology Faculty of Science Masaryk University Brno Czech Republic

Department of Experimental Biology Faculty of Science Masaryk University Brno Czech Republic; CEITEC Central European Institute of Technology Masaryk University Brno Czech Republic

Department of Experimental Biology Faculty of Science Masaryk University Brno Czech Republic; Department of Cytokinetics Institute of Biophysics of the Czech Academy of Sciences Brno Czech Republic

Institute for Pharmaceutical Chemistry Johann Wolfgang Goethe University Frankfurt am Main Germany; Structural Genomics Consortium Johann Wolfgang Goethe University Frankfurt am Main Germany

Institute for Pharmaceutical Chemistry Johann Wolfgang Goethe University Frankfurt am Main Germany; Structural Genomics Consortium Johann Wolfgang Goethe University Frankfurt am Main Germany; German Cancer Consortium DKTK Site Frankfurt Mainz Heidelberg Germany

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