Replication checkpoint requires phosphorylation of the phosphatase Cdc25 by Cds1 or Chk1.
Checkpoints maintain the order and fidelity of events of the cell cycle by blocking mitosis in response to unreplicated or damaged DNA. In most species this is accomplished by preventing activation of the cell-division kinase Cdc2, which regulates entry into mitosis. The Chk1 kinase, an effector of the DNA-damage checkpoint, phosphorylates Cdc25, an activator of Cdc2. Phosphorylation of Cdc25 promotes its binding to 14-3-3 proteins, preventing it from activating Cdc2. Here we propose that a similar pathway is required for mitotic arrest in the presence of unreplicated DNA (that is, in the replication checkpoint) in fission yeast. We show by mutagenesis that Chk1 functions redundantly with the kinase Cds1 at the replication checkpoint and that both kinases phosphorylate Cdc25 on the same sites, which include serine residues at positions 99, 192 and 359. Mutation of these residues reduces binding of 14-3-3 proteins to Cdc25 in vitro and disrupts the replication checkpoint in vivo. We conclude that both Cds1 and Chk1 regulate the binding of Cdc25 to 14-3-3 proteins as part of the checkpoint response to unreplicated DNA.
Department of Cell Biology and Physiology, Washington University School of Medicine, St Louis, Missouri 63110, USA.
Y
Y
Zeng
K C
KC
Forbes
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Z
Wu
S
S
Moreno
H
H
Piwnica-Worms
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T
Enoch
D048948:14-3-3 Proteins; D018797:Cell Cycle Proteins / Q000378:metabolism*; D000071877:Checkpoint Kinase 1; D064447:Checkpoint Kinase 2; D004261:DNA Replication; D004271:DNA, Fungal / Q000096:biosynthesis / Q000235:genetics; D006918:Hydroxyurea / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins; D016296:Mutagenesis; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D010766:Phosphorylation; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases; D011506:Proteins / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012568:Schizosaccharomyces / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D029702:Schizosaccharomyces pombe Proteins; D012694:Serine / Q000378:metabolism; D014446:Tyrosine 3-Monooxygenase; D020687:cdc25 Phosphatases
D048948:14-3-3 Proteins; D018797:Cell Cycle Proteins; D004271:DNA, Fungal; D047908:Intracellular Signaling Peptides and Proteins; D011506:Proteins; D011993:Recombinant Fusion Proteins; D029702:Schizosaccharomyces pombe Proteins; C501114:rad24 protein, S pombe; D012694:Serine; D014446:Tyrosine 3-Monooxygenase; D011494:Protein Kinases; D064447:Checkpoint Kinase 2; C578636:Cds1 protein, S pombe; D000071877:Checkpoint Kinase 1; C000605873:Chk1 protein, S pombe; D017346:Protein Serine-Threonine Kinases; D010749:Phosphoprotein Phosphatases; D020687:cdc25 Phosphatases; D006918:Hydroxyurea