The ubiquitin ligase HUWE1 enhances WNT signaling by antagonizing destruction complex-mediated β-catenin degradation and through a mechanism independent of changes in β-catenin abundance.

IF 4 2区 生物学 Q1 GENETICS & HEREDITY
Joseph K McKenna, Yalan Wu, Praveen Sonkusre, Caleb K Sinclear, Raj Chari, Andres M Lebensohn
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引用次数: 0

Abstract

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 casein kinase 1α (CSNK1A1) and GSK3A or GSK3B. Loss of CSNK1A1 increases CTNNB1 abundance, resulting in hyperactive WNT signaling. Previously, we demonstrated that the HECT domain E3 ubiquitin ligase HUWE1 is necessary for hyperactive WNT signaling in HAP1 haploid human cells lacking CSNK1A1. Here, we investigated the mechanism underlying this requirement. In HAP1 cells lacking 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 smaller than the reduction in WNT target gene transcription. To test whether the reduction in WNT signaling caused by HUWE1 loss resulted from reduced CTNNB1 alone, we engineered the endogenous CTNNB1 locus in HAP1 cells to encode a CTNNB1 variant insensitive to destruction complex-mediated phosphorylation and degradation. HUWE1 loss in these cells did not change CTNNB1 abundance but still reduced WNT signaling, demonstrating that another mechanism was at play. Genetic interaction and overexpression analyses revealed that the reduction in WNT signaling caused by HUWE1 loss required not only GSK3A or GSK3B, but also APC and AXIN1. Therefore, in HAP1 cells lacking CSNK1A1, a residual destruction complex containing APC, AXIN1 and GSK3A or GSK3B downregulates WNT signaling by phosphorylating and targeting CTNNB1 for degradation, and HUWE1 enhances WNT signaling by antagonizing this activity. Regulation of WNT signaling by HUWE1 also requires its ubiquitin ligase activity. We conclude that HUWE1 enhances WNT/CTNNB1 signaling through two mechanisms, one that antagonizes destruction complex-mediated CTNNB1 degradation and another that is independent of changes in CTNNB1 abundance. Coordinated regulation of CTNNB1 abundance and a second signaling step by HUWE1 would be an efficient way to control WNT signaling output, enabling sensitive and robust activation of the pathway.

泛素连接酶HUWE1通过拮抗破坏复合体介导的β-连环蛋白降解,并通过独立于β-连环蛋白丰度变化的机制增强WNT信号。
WNT/β-catenin信号是由转录辅激活因子β-catenin (CTNNB1)介导的。CTNNB1丰度受磷酸化和蛋白酶体降解调控,由支架蛋白APC和AXIN1或AXIN2以及酪蛋白激酶1α (CSNK1A1)和GSK3A或GSK3B组成的破坏复合体促进。CSNK1A1缺失会增加CTNNB1丰度,导致WNT信号过度活跃。先前,我们证明了HECT结构域E3泛素连接酶HUWE1对于缺乏CSNK1A1的HAP1单倍体人类细胞中过度活跃的WNT信号传导是必要的。在这里,我们研究了这个需求背后的机制。在缺乏CSNK1A1的HAP1细胞中,GSK3A/GSK3B仍然磷酸化CTNNB1的一部分,促进其降解。HUWE1缺失增强了GSK3A/ gsk3b依赖性CTNNB1的磷酸化,进一步降低了CTNNB1的丰度。然而,HUWE1缺失导致的CTNNB1的减少要小于WNT靶基因转录的减少。为了验证HUWE1缺失引起的WNT信号的减少是否仅仅是因为CTNNB1的减少,我们在HAP1细胞中设计了内源性CTNNB1位点,编码了一个对破坏复合体介导的磷酸化和降解不敏感的CTNNB1变体。这些细胞中的HUWE1缺失并未改变CTNNB1的丰度,但仍会降低WNT信号,这表明另一种机制在起作用。遗传互作和过表达分析表明,HUWE1缺失导致的WNT信号减少不仅需要GSK3A或GSK3B,还需要APC和AXIN1。因此,在缺乏CSNK1A1的HAP1细胞中,含有APC、AXIN1和GSK3A或GSK3B的残余破坏复合物通过磷酸化并靶向CTNNB1降解来下调WNT信号,而HUWE1通过拮抗该活性来增强WNT信号。HUWE1对WNT信号的调控也需要其泛素连接酶活性。我们得出结论,HUWE1通过两种机制增强WNT/CTNNB1信号,一种是拮抗破坏复合物介导的CTNNB1降解,另一种是独立于CTNNB1丰度的变化。协调调节CTNNB1丰度和HUWE1的第二信号传导步骤将是控制WNT信号输出的有效方法,从而实现该途径的敏感和稳健激活。
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来源期刊
PLoS Genetics
PLoS Genetics GENETICS & HEREDITY-
自引率
2.20%
发文量
438
期刊介绍: PLOS Genetics is run by an international Editorial Board, headed by the Editors-in-Chief, Greg Barsh (HudsonAlpha Institute of Biotechnology, and Stanford University School of Medicine) and Greg Copenhaver (The University of North Carolina at Chapel Hill). Articles published in PLOS Genetics are archived in PubMed Central and cited in PubMed.
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