McKenna Joseph K, Wu Yalan, Sonkusre Praveen, Chari Raj, Lebensohn Andres M
Laboratory of Cellular and Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, United States of America.
Genome Modification Core, Laboratory Animal Sciences Program, Frederick National Lab for Cancer Research, Frederick, Maryland, United States of America.
bioRxiv. 2024 Mar 17:2024.02.02.578552. doi: 10.1101/2024.02.02.578552.
WNT/β-catenin signaling is mediated by the transcriptional coactivator β-catenin (CTNNB1). CTNNB1 abundance is regulated by phosphorylation and proteasomal degradation promoted by a destruction complex composed of the scaffold proteins APC and AXIN1 or AXIN2, and the kinases CSNK1A1 and GSK3A or GSK3B. Loss of CSNK1A1 increases CTNNB1 abundance, resulting in hyperactive WNT signaling. Previously, we demonstrated that the HECT domain ubiquitin ligase HUWE1 is necessary for hyperactive WNT signaling in HAP1 haploid human cells lacking CSNK1A1. Here, we investigate the mechanism underlying this requirement. In the absence of CSNK1A1, GSK3A/GSK3B still phosphorylated a fraction of CTNNB1, promoting its degradation. HUWE1 loss enhanced GSK3A/GSK3B-dependent CTNNB1 phosphorylation, further reducing CTNNB1 abundance. However, the reduction in CTNNB1 caused by HUWE1 loss was disproportionately smaller than the reduction in WNT target gene transcription. To test if the reduction in WNT signaling resulted from reduced CTNNB1 abundance alone, we engineered the endogenous locus in HAP1 cells to encode a CTNNB1 variant insensitive to destruction complex-mediated phosphorylation and degradation. HUWE1 loss in these cells reduced WNT signaling with no change in CTNNB1 abundance. Genetic interaction and overexpression analyses revealed that the effects of HUWE1 on WNT signaling were not only mediated by GSK3A/GSK3B, but also by APC and AXIN1. Regulation of WNT signaling by HUWE1 required its ubiquitin ligase activity. These results suggest that in cells lacking CSNK1A1, a destruction complex containing APC, AXIN1 and GSK3A/GSK3B downregulates WNT signaling by phosphorylating and targeting CTNNB1 for degradation. HUWE1 enhances WNT signaling by antagonizing this activity. Therefore, HUWE1 enhances WNT/CTNNB1 signaling through two mechanisms, one that regulates CTNNB1 abundance and another that is independent of CTNNB1 stability. Coordinated regulation of CTNNB1 abundance and an independent signaling step by HUWE1 would be an efficient way to control WNT signaling output, enabling sensitive and robust activation of the pathway.
WNT/β-连环蛋白信号传导由转录共激活因子β-连环蛋白（CTNNB1）介导。CTNNB1的丰度受磷酸化以及由支架蛋白APC和AXIN1或AXIN2，激酶CSNK1A1和GSK3A或GSK3B组成的破坏复合物所促进的蛋白酶体降解的调控。CSNK1A1缺失会增加CTNNB1的丰度，导致WNT信号过度激活。此前，我们证明在缺乏CSNK1A1的单倍体人类细胞HAP1中，HECT结构域泛素连接酶HUWE1对于WNT信号过度激活是必需的。在此，我们研究这种需求背后的机制。在缺乏CSNK1A1的情况下，GSK3A/GSK3B仍会磷酸化一部分CTNNB1，促进其降解。HUWE1缺失增强了GSK3A/GSK3B依赖的CTNNB1磷酸化，进一步降低了CTNNB1的丰度。然而，由HUWE1缺失导致的CTNNB1减少比WNT靶基因转录的减少程度要小得多。为了测试WNT信号减少是否仅由CTNNB1丰度降低引起，我们对HAP1细胞中的内源性位点进行改造，使其编码一种对破坏复合物介导的磷酸化和降解不敏感的CTNNB1变体。这些细胞中HUWE1缺失降低了WNT信号，而CTNNB1丰度没有变化。遗传相互作用和过表达分析表明，HUWE1对WNT信号的影响不仅由GSK3A/GSK3B介导，还由APC和AXIN1介导。HUWE1对WNT信号的调控需要其泛素连接酶活性。这些结果表明，在缺乏CSNK1A1的细胞中，包含APC、AXIN1和GSK3A/GSK3B的破坏复合物通过磷酸化并靶向CTNNB1进行降解来下调WNT信号。HUWE1通过拮抗这种活性来增强WNT信号。因此，HUWE1通过两种机制增强WNT/CTNNB1信号，一种机制调节CTNNB1丰度，另一种机制独立于CTNNB1稳定性。HUWE1对CTNNB1丰度和一个独立信号步骤的协同调控将是控制WNT信号输出的有效方式，能够实现该信号通路的灵敏且稳健的激活。
