Laboratory of Pathophysiological Cell Signaling, Department of Biology, Faculty of Science and Bioengineering Sciences, Vrije Universiteit Brussel, Pleinlaan 2, 1050, Brussels, Belgium.
Biozentrum, University of Basel, Spitalstrasse 41, 4056, Basel, Switzerland.
Mol Biol Rep. 2022 May;49(5):4079-4087. doi: 10.1007/s11033-022-07279-3. Epub 2022 Apr 9.
The cell division cycle is a process that is exquisitely controlled by a complex interplay between E3 ubiquitin ligases and deubiquitinating enzymes (DUBs). We have previously reported that the DUB USP13 regulates Aurora B levels along the cell cycle. That observation prompted us to explore any possible connection between USP13 and the APC/C, the major E3 controlling Aurora B levels in cells.
We performed immunoprecipitation assays followed by western-blotting to assess the interaction between USP13 and CDH1. The cellular effects of USP13 gain or loss of function were analyzed by transfection of FLAG-tagged USP13 plasmid or small interfering RNAs and short hairpin RNAs directed against USP13. The levels of CDH1 and other proteins were quantified in cell extracts by western-blotting.
We found that USP13 binds to the APC/C adaptor CDH1. In addition, we report for the first time that USP13 controls CDH1 protein levels in cells: overexpression of USP13 increased CDH1 levels, whereas depletion of USP13 decreased CDH1 levels.
We unveil the existing interplay between USP13 and CDH1: USP13 is capable of stabilizing CDH1 levels. We previously reported that USP13 stabilizes Aurora B in cells, a known substrate of the APC/C E3 ubiquitin ligase, before their entry into mitosis. Altogether, our data identify and establish the USP13-CDH1-Aurora B axis as a new regulatory module required for flawless cell cycle progression in mammalian cells, whose misfunction may be involved in the rewiring of cell cycle pathways linked to cancer development.
细胞分裂周期是一个由 E3 泛素连接酶和去泛素化酶（DUB）之间复杂相互作用精细调控的过程。我们之前曾报道过 DUB USP13 沿细胞周期调节 Aurora B 的水平。这一观察结果促使我们探索 USP13 与 APC/C 之间可能存在的联系，APC/C 是控制细胞中 Aurora B 水平的主要 E3。
我们进行了免疫沉淀实验，随后进行了 Western blot 分析，以评估 USP13 与 CDH1 之间的相互作用。通过转染 FLAG 标记的 USP13 质粒或针对 USP13 的小干扰 RNA 和短发夹 RNA，分析 USP13 功能获得或缺失的细胞效应。通过 Western blot 从细胞提取物中定量测定 CDH1 和其他蛋白质的水平。
我们发现 USP13 与 APC/C 衔接子 CDH1 结合。此外，我们首次报道 USP13 控制细胞中 CDH1 蛋白水平：USP13 的过表达增加了 CDH1 水平，而 USP13 的耗竭则降低了 CDH1 水平。
我们揭示了 USP13 和 CDH1 之间现有的相互作用：USP13 能够稳定 CDH1 水平。我们之前曾报道过，USP13 在细胞进入有丝分裂之前稳定 APC/C E3 泛素连接酶的已知底物 Aurora B。总而言之，我们的数据确定并建立了 USP13-CDH1-Aurora B 轴作为哺乳动物细胞中细胞周期进展无缺陷所必需的新调节模块，其功能障碍可能与癌症发展相关的细胞周期途径的重新布线有关。
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