Cds1 controls the release of Cdc14-like phosphatase Flp1 from the nucleolus to drive full activation of the checkpoint response to replication stress in fission yeast.
The Cdc14p-like phosphatase Flp1p (also known as Clp1p) is regulated by cell cycle-dependent changes in its subcellular localization. Flp1p is restricted to the nucleolus and spindle pole body until prophase, when it is dispersed throughout the nucleus, mitotic spindle, and medial ring. Once released, Flp1p antagonizes Cdc2p/cyclin activity by reverting Cdc2p-phosphorylation sites on Cdc25p. On replication stress, ataxia-telangiectasia mutated/ATM/Rad3-related kinase Rad3p activates Cds1p, which phosphorylates key proteins ensuring the stability of stalled DNA replication forks. Here, we show that replication stress induces changes in the subcellular localization of Flp1p in a checkpoint-dependent manner. Active Cds1p checkpoint kinase is required to release Flp1p into the nucleus. Consistently, a Flp1p mutant (flp1-9A) lacking all potential Cds1p phosphorylation sites fails to relocate in response to replication blocks and, similarly to cells lacking flp1 (Deltaflp1), presents defects in checkpoint response to replication stress. Deltaflp1 cells accumulate reduced levels of a less active Cds1p kinase in hydroxyurea (HU), indicating that nuclear Flp1p regulates Cds1p full activation. Consistently, Deltaflp1 and flp1-9A have an increased percentage of Rad22p-recombination foci during HU treatment. Together, our data show that by releasing Flp1p into the nucleus Cds1p checkpoint kinase modulates its own full activation during replication stress.
Díaz-Cuervo
Helena
H
Instituto de Biología Molecular y Celular del Cáncer, Departamento de Microbiología y Genética, Campus Miguel de Unamuno, Universidad de Salamanca/Consejo Superior de Investigaciones Científicas, 37007 Salamanca, Spain.
Bueno
Avelino
A
D048948:14-3-3 Proteins / Q000378:metabolism; D016203:CDC2 Protein Kinase / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002466:Cell Nucleolus / Q000187:drug effects / Q000201:enzymology*; D064447:Checkpoint Kinase 2; D004261:DNA Replication / Q000187:drug effects; D004268:DNA-Binding Proteins / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006918:Hydroxyurea / Q000494:pharmacology; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D007700:Kinetics; D009154:Mutation / Q000235:genetics; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011995:Recombination, Genetic / Q000235:genetics; D016196:S Phase / Q000187:drug effects; D012568:Schizosaccharomyces / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology*; D029702:Schizosaccharomyces pombe Proteins / Q000378:metabolism*
D048948:14-3-3 Proteins; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D047908:Intracellular Signaling Peptides and Proteins; D011993:Recombinant Fusion Proteins; D029702:Schizosaccharomyces pombe Proteins; C501114:rad24 protein, S pombe; C085412:rad52 protein, S pombe; D049452:Green Fluorescent Proteins; D064447:Checkpoint Kinase 2; C578636:Cds1 protein, S pombe; D017346:Protein Serine-Threonine Kinases; D016203:CDC2 Protein Kinase; C503885:CDC14 protein, S pombe; D010749:Phosphoprotein Phosphatases; C439269:Clp1 protein, S. pombe; D017027:Protein Tyrosine Phosphatases; D006918:Hydroxyurea