Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA; Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA; Department of Cell Biology and Physiology, Washington University in St Louis, St Louis, Missouri, USA.
Division of Chemical Biology and Medicinal Chemistry, Structural Genomics Consortium, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.
J Biol Chem. 2022 Jun;298(6):101986. doi: 10.1016/j.jbc.2022.101986. Epub 2022 Apr 27.
Aberrant activation or suppression of WNT/β-catenin signaling contributes to cancer initiation and progression, neurodegeneration, and bone disease. However, despite great need and more than 40 years of research, targeted therapies for the WNT pathway have yet to be fully realized. Kinases are considered exceptionally druggable and occupy key nodes within the WNT signaling network, but several pathway-relevant kinases remain understudied and "dark." Here, we studied the function of the casein kinase 1γ (CSNK1γ) subfamily of human kinases and their roles in WNT signaling. miniTurbo-based proximity biotinylation and mass spectrometry analysis of CSNK1γ1, CSNK1γ2, and CSNK1γ3 revealed numerous components of the β-catenin-dependent and β-catenin-independent WNT pathways. In gain-of-function experiments, we found that CSNK1γ3 but not CSNK1γ1 or CSNK1γ2 activated β-catenin-dependent WNT signaling, with minimal effect on other signaling pathways. We also show that within the family, CSNK1γ3 expression uniquely induced low-density lipoprotein receptor-related protein 6 phosphorylation, which mediates downstream WNT signaling transduction. Conversely, siRNA-mediated silencing of CSNK1γ3 alone had no impact on WNT signaling, though cosilencing of all three family members decreased WNT pathway activity. Finally, we characterized two moderately selective and potent small-molecule inhibitors of the CSNK1γ family. We show that these inhibitors and a CSNK1γ3 kinase-dead mutant suppressed but did not eliminate WNT-driven low-density lipoprotein receptor-related protein 6 phosphorylation and β-catenin stabilization. Our data suggest that while CSNK1γ3 expression uniquely drives pathway activity, potential functional redundancy within the family necessitates loss of all three family members to suppress the WNT signaling pathway.
WNT/β-catenin 信号通路的异常激活或抑制会导致癌症的发生和发展、神经退行性变和骨疾病。然而，尽管有巨大的需求和 40 多年的研究，针对 WNT 途径的靶向治疗仍未完全实现。激酶被认为是特别适合成药的靶点，并且占据着 WNT 信号网络中的关键节点，但仍有一些与通路相关的激酶研究不足，处于“黑暗”之中。在这里，我们研究了人类激酶的酪蛋白激酶 1γ（CSNK1γ）亚家族的功能及其在 WNT 信号中的作用。基于 miniTurbo 的邻近生物素化和质谱分析 CSNK1γ1、CSNK1γ2 和 CSNK1γ3，揭示了 β-catenin 依赖性和 β-catenin 非依赖性 WNT 通路的许多组成部分。在功能获得实验中，我们发现 CSNK1γ3 而非 CSNK1γ1 或 CSNK1γ2 激活了 β-catenin 依赖性 WNT 信号通路，对其他信号通路的影响最小。我们还表明，在该家族中，CSNK1γ3 的表达独特地诱导了低密度脂蛋白受体相关蛋白 6 的磷酸化，后者介导下游的 WNT 信号转导。相反，单独用 siRNA 沉默 CSNK1γ3 对 WNT 信号没有影响，尽管同时沉默三个家族成员会降低 WNT 通路的活性。最后，我们对 CSNK1γ 家族的两种中等选择性和强效的小分子抑制剂进行了表征。我们表明，这些抑制剂和 CSNK1γ3 的激酶失活突变体抑制但不能消除 WNT 驱动的低密度脂蛋白受体相关蛋白 6 的磷酸化和 β-catenin 的稳定。我们的数据表明，虽然 CSNK1γ3 的表达独特地驱动了通路的活性，但家族内潜在的功能冗余需要同时丧失三个家族成员才能抑制 WNT 信号通路。
