Moore Jonathan D, Kornbluth Sally, Hunt Tim
Cancer Research UK London Research Institute, Clare Hall Laboratories, South Mimms, Herts, EN6 3LD, United Kingdom.
Mol Biol Cell. 2002 Dec;13(12):4388-400. doi: 10.1091/mbc.e02-07-0449.
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.
细胞周期蛋白依赖性激酶（Cdk）2/细胞周期蛋白E通过一种需要输入蛋白α和β的途径被导入非洲爪蟾卵提取物中组装的细胞核。在此，我们在非洲爪蟾细胞周期蛋白E的N端鉴定出一个碱性核定位序列（NLS）。NLS的突变消除了细胞周期蛋白E和Cdk2的核积累，并且这种细胞周期蛋白E版本无法触发DNA复制。添加来自SV40大T抗原的异源NLS恢复了Cdk2/细胞周期蛋白E的核靶向和DNA复制。我们提供的证据表明，Cdk2/细胞周期蛋白E复合物必须在细胞核内高度浓缩以支持复制，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，内源性细胞周期蛋白E的消耗增加了促进进入有丝分裂所需的细胞周期蛋白B的浓度。与其无法促进DNA复制相反，缺乏NLS的细胞周期蛋白E能够与细胞周期蛋白B协同促进有丝分裂进入。
