Abstract：
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.
摘要：
酪蛋白激酶1(CK1)家族成员是多种信号通路的重要调节因子。CK1α是众所周知的Wnt/β-catenin通路的负调节因子，通过Ser45的磷酸化促进β-连环蛋白的降解。相比之下,CK1α的最接近的模拟，CK1α样,是一种功能不明的激酶。在这项研究中，我们表明CK1α的缺失，但不是CK1α样，导致Wnt/β-catenin途径的强烈激活。Wnt-3a处理进一步增强了活化，这表明至少有两种模式，CK1α依赖性和Wnt依赖性，β-连环蛋白的调节。挽救实验表明，10个天然存在的剪接CK1α/α样变体中只有2个能够挽救由细胞中CK1α缺陷引起的增强的Wnt/β-catenin信号传导。重要的是,在体外激酶测定中需要使Ser45上的β-catenin磷酸化的能力,但不足以进行这种挽救.我们的化合物CK1α和GSK3α/β敲除模型表明，除了Ser45-β-catenin磷酸化之外，CK1α在Wnt途径中的其他非冗余功能包括Axin磷酸化。最后,我们建立了针对三种最常见的CK1α剪接变体以及CK1α样的NanoBRET测定法。目标参与数据显示，已知CK1α抑制剂对所有CK1α变体的效力相当，但对CK1α样却没有。总之,我们的工作为CK1α的生物学带来了重要的新见解，包括在β-catenin和Axin水平上对Wnt/β-catenin途径的负调节中缺乏其他CK1激酶冗余的证据。
