Centrosome-targeting region of CG-NAP causes centrosome amplification by recruiting cyclin E-cdk2 complex
Centrosome-targeting region of CG-NAP causes centrosome amplification by recruiting cyclin E-cdk2 complex
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DOI：
10.1111/j.1365-2443.2005.00816.x
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发表时间：
2005-01-01
期刊：
影响因子：
2.1
通讯作者：
Ono, Y
中科院分区：
文献类型：
--
作者：
Nishimura, T;Takahashi, M;Ono, Y
中心体复制在每个细胞周期发生一次，被认为是由细胞周期蛋白E-cdk 2触发的。然而，在很大程度上不知道如何管理重复。在这里，我们发现，CG-NAP（中心体和高尔基体定位PKN相关蛋白）的中心体靶向区域的表达，我们指定为CG-NAP/D，增加中国仓鼠卵巢（CHO）-K1细胞中的中心体的数量。扩增的中心体与中心体标记物γ-微管蛋白、中心蛋白-2和kendrin以及内源性CG-NAP共定位。当通过删除中心体靶向结构域或通过与膜靶向序列融合将CG-NAP/D从中心体中脱位时，中心体扩增被抑制。CG-NAP/D与共定位于中心体的外源表达的cyclin E相互作用。CG-NAP/D的免疫沉淀物显示组蛋白H1激酶活性，提示活性cyclin-dk复合物的免疫共沉淀。此外，从表达CG-NAP/D的细胞制备的中心体组分与来自对照细胞的那些相比含有增加量的cdk 2。CG-NAP/ D的中心体扩增被不能与cdk 2相互作用的突变细胞周期蛋白E的共表达抑制。这些结果表明，CG-NAP/ D通过锚定过量的cyclin E-cdk 2到中心体而引起中心体扩增，并且可能通过在正常细胞周期中募集cyclin E-cdk 2到中心体而参与中心体复制。
Centrosome duplication occurs once per cell cycle and is thought to be triggered by cyclin E-cdk2. However, it is largely unknown how the duplication is regulated. Here, we found that the expression of the centrosome-targeting region of CG-NAP ( centrosome and Golgi-localized PKN-associated protein), which we designate as CG-NAP/D, increased the number of centrosomes in Chinese hamster ovary (CHO)-K1 cells. The amplified centrosomes co-localized with centrosome markers gamma-tubulin, centrin- 2 and kendrin as well as endogenous CG-NAP. When CG-NAP/D was dislocated from centrosomes by deleting the centrosome-targeting domain or by fusing with a membrane-targeting sequence, centrosome amplification was suppressed. CG-NAP/D interacted with exogenously expressed cyclin E, which co-localized at centrosomes. The immunoprecipitates of CG-NAP/D exhibited histone H1 kinase activity, suggesting the co-immunoprecipitation of active cyclinc-dk complexes. Furthermore, centrosome fractions prepared from cells expressing CG-NAP/D contained increased amount of cdk2 compared with those from control cells. Centrosome amplification by CG-NAP/ D was suppressed by co-expression of a mutant cyclin E unable to interact with cdk2. These results suggest that CG-NAP/ D causes centrosome amplification by anchoring excess amount of cyclin E-cdk2 to centrosomes and, possibly, CG-NAP participates in centrosome duplication by recruiting cyclin E-cdk2 to centrosomes in normal cell cycle.