a Department of Pathology , Dalhousie University , Halifax , Canada.
b Department of Biochemistry & Molecular Biology , Dalhousie University , Halifax , Canada.
Cell Cycle. 2019 May;18(10):1135-1153. doi: 10.1080/15384101.2019.1612696. Epub 2019 May 5.
The cullin-RING E3 ubiquitin ligases (CRLs) play crucial roles in modulating the stability of proteins in the cell and are, in turn, regulated by post-translational modification by the ubiquitin-like (Ubl) protein NEDD8. This process, termed neddylation, is reversible through the action of the COP9 signalosome (CSN); a multi-subunit metalloprotease conserved among eukaryotes that plays direct or indirect roles in DNA repair, cell signaling and cell cycle regulation in part through modulating the activity of the CRLs. Previously, inhibition of CRL neddylation by MLN4924, a small molecule inhibitor of the NEDD8-activating enzyme 1 (NAE1), was shown to induce interphase cell cycle arrest and cell death. Using fixed and living cell microscopy, we re-evaluated the cell cycle effects of inhibition of neddylation by MLN4924 in both asynchronous and mitotic cell populations. Consistent with previous studies, treatment of asynchronous cells with MLN4924 increased CDT1 expression levels, induced G2 arrest and increased nuclear size. However, in synchronized cells treated in mitosis, mitotic defects were observed including lagging chromosomes and binucleated daughter cells. Consistent with neddylation and deneddylation playing a role in cytokinesis, NEDD8, as well as subunits of the CSN, could be localized at the midbody and cleavage furrow. Finally, treatment of mitotic cells with MLN4924 induced the premature accumulation of MKLP1 at the cleavage furrow, a key regulator of cytokinesis, which was concomitant with increased abscission delay and failure. Thus, these studies uncover an uncharacterized mitotic effect of MLN4924 on MKLP1 accumulation at the midbody and support a role for neddylation during cytokinesis. : CSN, COP9 Signalosome; MKLP1, mitotic kinesin-like protein 1; NEDD8, Neural precursor cell Expressed, Developmentally Down-regulated 8.
Cullin-RING E3 泛素连接酶（CRLs）在调节细胞内蛋白质稳定性方面发挥着关键作用，并且反过来又受到泛素样（Ubl）蛋白 NEDD8 的翻译后修饰的调节。这个过程被称为 neddylation，通过 COP9 信号体（CSN）的作用是可逆的；CSN 是一种在真核生物中保守的多亚基金属蛋白酶，通过调节 CRLs 的活性，在 DNA 修复、细胞信号转导和细胞周期调控中发挥直接或间接作用。先前，小分子抑制剂 MLN4924 抑制 NEDD8 激活酶 1（NAE1）的 CRL neddylation，导致间期细胞周期停滞和细胞死亡。使用固定和活细胞显微镜，我们重新评估了 MLN4924 抑制 neddylation 在异步和有丝分裂细胞群体中的细胞周期效应。与先前的研究一致，用 MLN4924 处理异步细胞增加了 CDT1 表达水平，诱导 G2 期阻滞和核增大。然而，在有丝分裂中处理的同步细胞中，观察到有丝分裂缺陷，包括滞后染色体和双核子细胞。与 neddylation 和 deneddylation 在胞质分裂中发挥作用一致，NEDD8 以及 CSN 的亚基可以定位在中体和分裂沟处。最后，用 MLN4924 处理有丝分裂细胞诱导 MKLP1 在分裂沟处过早积累，MKLP1 是胞质分裂的关键调节因子，这与后期延迟和失败增加有关。因此，这些研究揭示了 MLN4924 在中体处对 MKLP1 积累的未被描述的有丝分裂作用，并支持 neddylation 在胞质分裂中的作用。CSN，COP9 信号体；MKLP1，有丝分裂驱动蛋白样蛋白 1；NEDD8，神经前体细胞表达、发育下调 8。
