Regulation of Cdc2/cyclin B activation by Ran, a Ras-related GTPase

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.