Identification of the nuclear localization signal in Xenopus cyclin E and analysis of its role in replication and mitosis
Identification of the nuclear localization signal in Xenopus cyclin E and analysis of its role in replication and mitosis
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DOI：
10.1091/mbc.e02-07-0449
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发表时间：
2002-12-01
影响因子：
3.3
通讯作者：
Hunt, T
中科院分区：
文献类型：
--
作者：
Moore, JD;Kornbluth, S;Hunt, T
细胞周期蛋白依赖性激酶(CDK)2/细胞周期蛋白E通过一条需要输入素-α和-β的途径进入非洲爪哇卵提取液组装的细胞核中。在这里，我们确定了非洲爪哇Cyclin E N端的一个基本核定位序列(NLS)。NLS突变消除了Cyclin E和CDK2的核积累，并且Cyclin E的这些版本不能触发DNA复制。添加了一个异源NLS。SV40大T抗原恢复了CDK2/Cyclin E的核靶向性和DNA复制。我们提出的证据表明，CDK2/细胞周期蛋白E复合体必须在核内高度集中才能支持复制，并发现细胞周期蛋白A可以在低得多的核内浓度下触发复制。我们证实，内源性Cyclin E的耗尽增加了促进进入有丝分裂所必需的Cyclin B的浓度。与其不能促进DNA复制相反，缺乏NLS的Cyclin E能够与Cyclin B协同促进有丝分裂进入。
Cyclin-dependent kinase (Cdk)2/cyclin E is imported into nuclei assembled in Xenopus egg extracts by a pathway that requires importin-alpha and -beta. Here, we identify a basic nuclear localization sequence (NLS) in the N-terminus of Xenopus cyclin E. Mutation of the NLS eliminated nuclear accumulation of both cyclin E and Cdk2, and such versions of cyclin E were unable to trigger DNA replication. Addition of a heterologous NLS from. SV40 large T antigen restored both nuclear targeting of Cdk2/cyclin E and DNA replication. We present evidence indicating that Cdk2/cyclin E complexes must become highly concentrated within nuclei to support replication and find that cyclin A can trigger replication at much lower intranuclear concentrations. We confirmed that depletion of endogenous cyclin E increases the concentration of cyclin B necessary to promote entry into mitosis. In contrast to its inability to promote DNA replication, cyclin E lacking its NLS was able to cooperate with cyclin B in promoting mitotic entry.