Reduced O-GlcNAcase expression promotes mitotic errors and spindle defects
Reduced O-GlcNAcase expression promotes mitotic errors and spindle defects
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DOI：
10.1080/15384101.2016.1167297
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发表时间：
2016-01-01
期刊：
影响因子：
4.3
通讯作者：
Slawson, Chad
中科院分区：
文献类型：
--
作者：
Lanza, Chris;Tan, Ee Phie;Slawson, Chad
O-GlcNAc循环的改变，O-GlcNAc的添加和去除，导致有丝分裂缺陷和非整倍体增加。在此，我们生成了稳定的O-GlcNAcase （OGA，一种去除O-GlcNAc的酶）敲除HeLa细胞系，并表征了OGA活性降低对细胞周期进程的影响。从G(1)/S释放后，OGA敲低细胞在S期正常进展，但表现出有丝分裂退出缺陷。Cyclin A在敲低细胞中表达升高，而Cyclin B和D表达降低。与对照组相比，视网膜母细胞瘤蛋白（RB）磷酸化在基因敲除中也有所增加。在M期，敲除细胞的纺锤体染色单体比对照细胞更致密，多极纺锤体细胞的比例更高。此外，在OGA敲低的细胞中，细胞周期蛋白依赖性激酶1 （CDK1）的抑制性酪氨酸磷酸化的时间发生了改变。虽然染色体乘客蛋白复合物（CPC）的表达和定位不变，但在其中一个OGA敲除细胞系中，组蛋白H3苏氨酸3的磷酸化水平降低。尤因肉瘤断点区1蛋白（EWS）参与纺锤体CPC的组织，是O-GlcNAc转移酶（OGT，添加O-GlcNAc的酶）的已知底物。EWS o - glcn酰化在OGA敲低的细胞中显著增加，促进有丝分裂中间区定位不均匀。我们的数据表明，O-GlcNAc循环是有丝分裂信号传导和纺锤体形成的重要机制，而O-GlcNAc循环速率的改变会产生异常纺锤体并促进非整倍性。
Alterations in O-GlcNAc cycling, the addition and removal of O-GlcNAc, lead to mitotic defects and increased aneuploidy. Herein, we generated stable O-GlcNAcase (OGA, the enzyme that removes O-GlcNAc) knockdown HeLa cell lines and characterized the effect of the reduction in OGA activity on cell cycle progression. After release from G(1)/S, the OGA knockdown cells progressed normally through S phase but demonstrated mitotic exit defects. Cyclin A was increased in the knockdown cells while Cyclin B and D expression was reduced. Retinoblastoma protein (RB) phosphorylation was also increased in the knockdown compared to control. At M phase, the knockdown cells showed more compact spindle chromatids than control cells and had a greater percentage of cells with multipolar spindles. Furthermore, the timing of the inhibitory tyrosine phosphorylation of Cyclin Dependent Kinase 1 (CDK1) was altered in the OGA knockdown cells. Although expression and localization of the chromosomal passenger protein complex (CPC) was unchanged, histone H3 threonine 3 phosphorylation was decreased in one of the OGA knockdown cell lines. The Ewing Sarcoma Breakpoint Region 1 Protein (EWS) participates in organizing the CPC at the spindle and is a known substrate for O-GlcNAc transferase (OGT, the enzyme that adds O-GlcNAc). EWS O-GlcNAcylation was significantly increased in the OGA knockdown cells promoting uneven localization of the mitotic midzone. Our data suggests that O-GlcNAc cycling is an essential mechanism for proper mitotic signaling and spindle formation, and alterations in the rate of O-GlcNAc cycling produces aberrant spindles and promotes aneuploidy.