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Vol. 19, Issue 6, 2488-2499, June 2008

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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

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

Submitted August 2, 2007; Revised March 19, 2008; Accepted March 20, 2008
Monitoring Editor: Fred Chang

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 (flp1), presents defects in checkpoint response to replication stress. flp1 cells accumulate reduced levels of a less active Cds1p kinase in hydroxyurea (HU), indicating that nuclear Flp1p regulates Cds1p full activation. Consistently, flp1 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.

Address correspondence to: Avelino Bueno (abn{at}usal.es).

Abbreviations used: DAPI, 4',6-diamidino-2-phenylindole; DiFMUP, 6,8-difluoro-4-methylumbelliferyl phosphate; GFP, green fluorescent protein; GST, glutathione transferase; Ha, hemagglutinin; HU, hydroxyurea; MBP, myelin basic protein; MMS, methyl methanesulfonate; SIN, septation initiation network; SPB, spindle pole body.