Regulation of the Cdc25 mitotic inducer following replication arrest and DNA damage
Frazer, Corey Thomas
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Dephosphorylation of the Cdc2 kinase by the Cdc25 tyrosine phosphatase is the universally conserved trigger for mitotic entry. Cdc25 is also the point of convergence for checkpoint signaling pathways which monitor the genome for damaged DNA and incomplete replication. In addition, Cdc25 is inhibited by a MAP kinase cascade in the event of osmotic, oxidative and/or heat stress. These pathways inhibit cell cycle progression by phosphorylating Cdc25 resulting in its association with 14-3-3 and nuclear export. Although Cdc25 can be observed leaving the nucleus following inhibitory signals it is controversial whether phosphorylation, 14-3-3 binding or export itself is required for checkpoint proficiency. In fission yeast, Cdc25 is phosphorylated in vitro on 12 serine and threonine residues by the effector kinase of the DNA replication checkpoint, Cds1. Nine of these residues reside in the N-terminal regulatory region, while three are found in the extreme C-terminus of the protein. We show here that phosphorylation the nine N-terminal residues, nor any of the 12 in vitro sites, are required for enforcement of the DNA replication checkpoint. In lieu of Cdc25 phosphorylation the phosphatase is rapidly degraded and mitotic entry prevented by the action of the Mik1 kinase, targeting Cdc2. Thus, multiple mechanisms exist for preventing mitotic entry when S-phase progression is inhibited. The three C-terminal in vitro phosphorylation sites have not previously been examined in fission yeast. However, homology exists between the S. pombe protein and the Cdc25 orthologues in humans, Xenopus and Drosophila in this region. We report here that in S. pombe these sites are required to prevent mitotic entry following replication arrest in the absence of Mik1, and in the maintenance, but not establishment, of arrest following DNA damage. Our previous work showed that Cdc25 nuclear import requires the Sal3 importin-β but at the time we were unable to show a direct interaction between these two proteins. The final chapter of this thesis proves physical interaction by co-immunoprecipitation. Cdc25 mutants lacking all twelve putative Cds1 sites show nuclear localization during mitosis in a sal3- background, effectively reversing the cell cycle regulated pattern of accumulation of the phosphatase.