Clarke P R, Klebe C, Wittinghofer A, Karsenti E
Cell Biology Programme, European Molecular Biology Laboratory, Heidelberg, Germany.
J Cell Sci. 1995 Mar;108 ( Pt 3):1217-25. doi: 10.1242/jcs.108.3.1217.
During the cell cycle, a checkpoint prevents the initiation of mitosis until S-phase is completed. The molecular mechanism may involve the RCC1 protein, which catalyses guanine nucleotide exchange on the Ras-related nuclear protein, Ran (or TC4). Genetic studies have suggested that RCC1 may be involved in sensing the replication state of DNA and controlling the activation of Cdc2/cyclin B protein kinase through Ran. In this report, we present direct biochemical evidence for the post-translational control of Cdc2/cyclin B activation by Ran. In a cell-free system of concentrated Xenopus egg extracts supplemented with nuclei, a mutant form of Ran (T24N) analogous to dominant inactive mutants of other Ras-related GTPases inhibits Cdc2/cyclin B activation in the presence of replicating nuclear DNA. This role for Ran is mediated through control of the tyrosine phosphorylation state of Cdc2 and appears to be distinct from other effects on nuclear import, nuclear formation and DNA replication. When extracts were supplemented with RCC1 protein prior to addition of Ran T24N, inhibition of Cdc2/cyclin B by Ran T24N was relieved. This suggests that Ran T24N may act in a dominant manner by sequestering RCC1 in an inactive form. In contrast to Ran T24N, a mutant of Ran (Q69L) defective in GTPase activity and hence locked in the GTP-bound state has no inhibitory effect on Cdc2/cyclin B activation. In the light of these results, we propose that generation of the GTP-bound form of Ran is required for Cdc2/cyclin B activation and entry into mitosis when this process is coupled to the progression of S-phase.
在细胞周期中，一个检查点会阻止有丝分裂的启动，直到S期完成。分子机制可能涉及RCC1蛋白，它催化与Ras相关的核蛋白Ran（或TC4）上的鸟嘌呤核苷酸交换。遗传学研究表明，RCC1可能参与感知DNA的复制状态，并通过Ran控制Cdc2/细胞周期蛋白B蛋白激酶的激活。在本报告中，我们提供了Ran对Cdc2/细胞周期蛋白B激活进行翻译后控制的直接生化证据。在补充了细胞核的浓缩非洲爪蟾卵提取物的无细胞系统中，一种类似于其他Ras相关GTP酶的显性失活突变体的Ran突变形式（T24N），在存在复制性核DNA的情况下抑制Cdc2/细胞周期蛋白B的激活。Ran的这一作用是通过控制Cdc2的酪氨酸磷酸化状态介导的，并且似乎与对核输入、核形成和DNA复制的其他影响不同。当在添加Ran T24N之前向提取物中补充RCC1蛋白时，Ran T24N对Cdc2/细胞周期蛋白B的抑制作用得到缓解。这表明Ran T24N可能通过将RCC1隔离在无活性形式中以显性方式起作用。与Ran T24N相反，一种GTP酶活性有缺陷并因此锁定在GTP结合状态的Ran突变体（Q69L）对Cdc2/细胞周期蛋白B的激活没有抑制作用。根据这些结果，我们提出，当这个过程与S期的进展相关联时，Ran的GTP结合形式的产生是Cdc2/细胞周期蛋白B激活和进入有丝分裂所必需的。
