Splice variants of CK1a and CK1a-like: Comparative analysis of subcellular localization, kinase activity, and function in the Wnt signaling pathway

Members of the casein kinase 1 (CK1) family are important regulators of multiple signaling pathways. CK1a is a wellknown negative regulator of the Wnt/b-catenin pathway, which promotes the degradation of b-catenin via its phosphorylation of Ser45. In contrast, the closest paralog of CK1a, CK1a-like, is a poorly characterized kinase of unknown function. In this study, we show that the deletion of CK1a, but not CK1a-like, resulted in a strong activation of the Wnt/b-catenin pathway. Wnt-3a treatment further enhanced the activation, which suggests there are at least two modes, a CK1a-dependent and Wnt-dependent, of b-catenin regulation. Rescue experiments showed that only two out of ten naturally occurring splice CK1a/a-like variants were able to rescue the augmented Wnt/b-catenin signaling caused by CK1a deficiency in cells. Importantly, the ability to phosphorylate b-catenin on Ser45 in the in vitro kinase assay was required but not sufficient for such rescue. Our compound CK1a and GSK3a/b KO models suggest that the additional nonredundant function of CK1a in the Wnt pathway beyond Ser45-b-catenin phosphorylation includes Axin phosphorylation. Finally, we established NanoBRET assays for the three most common CK1a splice variants as well as CK1a-like. Target engagement data revealed comparable potency of known CK1a inhibitors for all CK1a variants but not for CK1a-like. In summary, our work brings important novel insights into the biology of CK1a, including evidence for the lack of redundancy with other CK1 kinases in the negative regulation of the Wnt/b-catenin pathway at the level of b- catenin and Axin.