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 R CC1 protein, which catalyses guanine nucleotide exchange on the Ras-re lated nuclear protein, Ran (or TC4), Genetic studies have suggested th at RCC1 may be involved in sensing the replication state of DNA and co ntrolling the activation of Cdc2/cyclin B protein kinase through Ran, In this report, we present direct biochemical evidence for the post-tr anslational 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 pr esence of replicating nuclear DNA. This role for Ran is mediated throu gh control of the tyrosine phosphorylation state of Cdc2 and appears t o 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 T24 N was relieved, This suggests that Ran T24N may act in a dominant mann er by sequestering RCC1 in an inactive form, In contrast to Ran T24N, a mutant of Ran (Q69L) defective in GTPase activity and hence locked i n the GTP-bound state has no inhibitory effect on Cdc2/cyclin B activa tion, 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 en try into mitosis when this process is coupled to the progression of S- phase.