Department of Oncology, Center for Molecular Medicine, Xiangya Hospital, Central South University, Changsha 410008, China.
International Cooperation Base of Cancer Precision Therapy, Department of Science and Technology of Hunan Province, Changsha 410008, China.
Cancer Biol Med. 2020 May 15;17(2):328-342. doi: 10.20892/j.issn.2095-3941.2019.0229.
Improper activation of Wnt/β-catenin signaling has been implicated in human diseases. Beyond the well-studied glycogen synthase kinase 3β (GSK3β) and casein kinase 1 (CK1), other kinases affecting Wnt/β-catenin signaling remain to be defined. To identify the kinases that modulate Wnt/β-catenin signaling, we applied a kinase small interfering RNA (siRNA) library screen approach. Luciferase assays, immunoblotting, and real-time polymerase chain reaction (PCR) were performed to confirm the regulation of the Wnt/β-catenin signaling pathway by cyclin-dependent kinase 11 (CDK11) and to investigate the underlying mechanism. Confocal immunofluorescence, coimmunoprecipitation (co-IP), and scratch wound assays were used to demonstrate colocalization, detect protein interactions, and explore the function of CDK11. CDK11 was found to be a significant candidate kinase participating in the negative control of Wnt/β-catenin signaling. Down-regulation of CDK11 led to the accumulation of Wnt/β-catenin signaling receptor complexes, in a manner dependent on intact adenomatosis polyposis coli (APC) protein. Further analysis showed that CDK11 modulation of Wnt/β-catenin signaling engaged the endolysosomal machinery, and CDK11 knockdown enhanced the colocalization of Wnt/β-catenin signaling receptor complexes with early endosomes and decreased colocalization with lysosomes. Mechanistically, CDK11 was found to function in Wnt/β-catenin signaling by regulating microtubule stability. Depletion of CDK11 down-regulated acetyl-α-tubulin. Moreover, co-IP assays demonstrated that CDK11 interacts with the α-tubulin deacetylase SIRT2, whereas SIRT2 down-regulation in CDK11-depleted cells reversed the accumulation of Wnt/β-catenin signaling receptor complexes. CDK11 was found to suppress cell migration through altered Wnt/β-catenin signaling. CDK11 is a negative modulator of Wnt/β-catenin signaling that stabilizes microtubules, thus resulting in the dysregulation of receptor complex trafficking from early endosomes to lysosomes.
Wnt/β-catenin 信号通路的异常激活与人类疾病有关。除了研究较为深入的糖原合成酶激酶 3β（GSK3β）和酪蛋白激酶 1（CK1）外，其他影响 Wnt/β-catenin 信号通路的激酶仍有待确定。为了鉴定调节 Wnt/β-catenin 信号通路的激酶，我们采用了激酶小干扰 RNA（siRNA）文库筛选方法。通过荧光素酶检测、免疫印迹和实时聚合酶链反应（PCR）实验来确认细胞周期蛋白依赖性激酶 11（CDK11）对 Wnt/β-catenin 信号通路的调节作用，并进一步探讨其潜在机制。通过共聚焦免疫荧光、免疫共沉淀（co-IP）和划痕实验来验证 CDK11 的共定位、检测蛋白相互作用以及研究 CDK11 的功能。结果表明，CDK11 是参与 Wnt/β-catenin 信号通路负调控的重要候选激酶。下调 CDK11 导致 Wnt/β-catenin 信号受体复合物的积累，这种方式依赖于 APC 蛋白的完整性。进一步分析表明，CDK11 对 Wnt/β-catenin 信号通路的调节涉及内体溶酶体机制，CDK11 敲低增强了 Wnt/β-catenin 信号受体复合物与早期内体的共定位，减少了与溶酶体的共定位。在机制上，CDK11 通过调节微管稳定性在 Wnt/β-catenin 信号通路中发挥作用。CDK11 的缺失下调了乙酰化微管蛋白-α。此外，co-IP 实验表明 CDK11 与微管去乙酰化酶 SIRT2 相互作用，而在 CDK11 敲低的细胞中 SIRT2 的下调逆转了 Wnt/β-catenin 信号受体复合物的积累。结果表明，CDK11 通过改变 Wnt/β-catenin 信号通路抑制细胞迁移。CDK11 是 Wnt/β-catenin 信号的负调节剂，可稳定微管，从而导致受体复合物从早期内体到溶酶体的运输失调。
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