Carboxy-terminal phosphorylation sites in Cdc25 contribute to enforcement of the DNA damage and replication checkpoints in fission yeast.
In fission yeast and vertebrate cells, Cdc25 phosphatase is the target of checkpoint-mediated response to DNA replication blocks, DNA damage, and extracellular stress. As such, it is a key regulator of cell cycle progress and genomic stability. In fission yeast, phosphorylation of Cdc25 by the checkpoint kinases Cds1 and Chk1 and also Srk1 during stress creates a binding site for the 14-3-3 homolog Rad24; the complex is then exported from the nucleus. Cdc25 contains 12 potential serine/threonine phosphorylation sites that are phosphorylated in vitro by Cds1; 9 reside in the amino terminal half of the protein with the remaining sites are located in the extreme C-terminus. We have previously shown that deletion of the nine amino terminal sites results in degradation of the mutant protein while the checkpoint is enforced by the Mik1 kinase acting on Cdc2 tyrosine-15. Here, we examine the influence of the three C-terminal sites on the negative regulation of Cdc25. These sites are conserved in vertebrates and have been shown to be phosphorylated following DNA damage and replication blocks. We show that these three sites have a role in the negative regulation of Cdc25 following replication arrest, but perhaps more importantly they appear to particularly contribute to regulating the duration, and thus the effectiveness of the arrested state.
Department of Biomedical and Molecular Sciences, Queen's University, Kingston, ON, K7L 3N6, Canada.
Corey
C
Frazer
Paul G
PG
Young
D000595:Amino Acid Sequence; D000818:Animals; D059447:Cell Cycle Checkpoints; D004249:DNA Damage; D004261:DNA Replication; D004271:DNA, Fungal / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D010766:Phosphorylation; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D012568:Schizosaccharomyces / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D020687:cdc25 Phosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D004271:DNA, Fungal; D020687:cdc25 Phosphatases; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease