A Mammalian In Vitro Centriole Duplication System: Evidence for Involvement of CDK2/Cyclin E and Nucleophosmin/B23 in Centrosome Duplication
A Mammalian In Vitro Centriole Duplication System: Evidence for Involvement of CDK2/Cyclin E and Nucleophosmin/B23 in Centrosome Duplication
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DOI：
10.4161/cc.1.1.103
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发表时间：
2002-01-01
期刊：
影响因子：
4.3
通讯作者：
Fukasawa, Kenji
中科院分区：
文献类型：
--
作者：
Tarapore, Pheruza;Okuda, Masaru;Fukasawa, Kenji
哺乳动物细胞中的中心体复制是一个高度调节的过程，与其他细胞周期事件协调发生。然而，这一重要的细胞过程的分子探索一直是困难的，由于一个简单的检测系统的不可用。在这里，使用松散的中心体与从培养细胞中分离的细胞核，我们开发了一个无细胞的中心粒（中心体的复制单位）复制系统：未复制的中心体绑定到细胞核能够进行复制的存在下，G1/S提取物。我们发现，G1/S提取物在体外诱导中心粒复制的能力取决于活性CDK 2/细胞周期蛋白E的存在。已经显示，由CDK 2/细胞周期蛋白E介导的磷酸化触发的中心体核磷酸蛋白（NPM）/B23的解离是中心体复制起始所必需的。我们发现，当细胞核与抗NPM/B23抗体预孵育时，中心粒复制被阻断，该抗体可防止NPM/B23被CDK 2/细胞周期蛋白E磷酸化。这些研究不仅为中心体启动复制需要CDK 2/cyclin E和NPM/B23磷酸化提供了直接证据，而且为进一步探索高等动物体细胞中心体复制的分子调控提供了有价值的实验体系。
Centrosome duplication in mammalian cells is a highly regulated process, occurs in coordination of other cell cycle events. However, molecular exploration of this important cellular process had been difficult due to unavailability of a simple assay system. Here, using centrosomes loosely associated with nuclei isolated from cultured cells, we developed a cell-free centriole (duplication unit of the centrosome) duplication system: unduplicated centrosomes bound to the nuclei are able to undergo duplication in the presence of G1/S extracts. We show that the ability of G1/S extracts to induce centriole duplication in vitro depends on the presence of active CDK2/cyclin E. It has been shown that dissociation of centrosomal nucleophosmin (NPM)/B23 triggered by CDK2/cyclin E-mediated phosphorylation is required for initiation of centrosome duplication. We show that centriole duplication is blocked when nuclei were preincubated with the anti-NPM/B23 antibody that prevents phosphorylation of NPM/B23 by CDK2/cyclin E. These studies provide not only direct evidence for the requirement of CDK2/cyclin E and phosphorylation of NPM/B23 for centrosomes to initiate duplication, but a valuable experimental system for further exploration of the molecular regulation of centrosome duplication in somatic cells of higher animals.