Davezac N, Baldin V, Gabrielli B, Forrest A, Theis-Febvre N, Yashida M, Ducommun B
LBCMCP-CNRS UMR5088, Université Paul Sabatier, Toulouse, France.
Oncogene. 2000 Apr 27;19(18):2179-85. doi: 10.1038/sj.onc.1203545.
The CDC25B dual specificity phosphatase is involved in the control of the G2/M transition of the cell cycle. Subcellular localization might represent an important aspect of the regulation of its activity. We have examined in transiently transfected asynchronous HeLa cells the localization of HA-tagged CDC25B proteins and found that they are nuclear or cytoplasmic suggesting the existence of an active shuttling. Accordingly, localization analysis of deletion and truncation proteins indicates that CDC25B contains a putative nuclear localization signal located between residues 335 and 354. We also demonstrated that a short 58 residues deletion of the amino-terminus end of CDC25B is sufficient to retain it to the nucleus. Mutational analysis indicates that a nuclear export sequence is located between residues 28 and 40. In addition, treatment of the cells with the exportin inhibitor, Leptomycin B, has the same effect. The mutation of Ser-323, a residue that is essential for the interaction with 14-3-3 proteins, also abolishes cytoplasmic staining. The subcellular localization of CDC25B is therefore dependent on the combined effects of a nuclear localization signal, a nuclear export signal and on the interaction with 14-3-3 proteins.
细胞周期蛋白依赖性激酶25B双特异性磷酸酶参与细胞周期G2/M期转换的调控。亚细胞定位可能是其活性调控的一个重要方面。我们在瞬时转染的非同步化HeLa细胞中检测了HA标签的细胞周期蛋白依赖性激酶25B蛋白的定位，发现它们定位于细胞核或细胞质，提示存在活跃的穿梭机制。相应地，对缺失和截短蛋白的定位分析表明，细胞周期蛋白依赖性激酶25B含有一个假定的核定位信号，位于第335至354位氨基酸残基之间。我们还证明，细胞周期蛋白依赖性激酶25B氨基末端缺失58个氨基酸残基就足以使其保留在细胞核内。突变分析表明，一个核输出序列位于第28至40位氨基酸残基之间。此外，用核输出蛋白抑制剂 leptomycin B处理细胞也有同样的效果。丝氨酸323（与14-3-3蛋白相互作用所必需的一个残基）的突变也消除了细胞质染色。因此，细胞周期蛋白依赖性激酶25B的亚细胞定位取决于核定位信号、核输出信号以及与14-3-3蛋白相互作用的综合效应。
