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. INTRODUCTIONAdequate genome replication and transfer to daughter cells is compromised in the course of each cell cycle by both intraand extracellular factors that perturb the stability of the genome. Molecular mechanisms to ensure genome integrity and to cope with DNA harassment were developed in eukaryotes likely at early stages of evolution, and they are consequently conserved. Therefore, a replication checkpoint ensures genomic integrity and cell survival when cells are not able to correctly replicate their genetic material, due to limitation in nucleotide pools or to deleterious alterations in template DNA.Serine/threonine phosphatase Flp1p (cdc fourteen-like phosphatase, also called Clp1p; hereafter referred as Flp1p) has been proved to control rapid degradation of Cdc25p at the end of mitosis (Esteban et al., 2004;Wolfe and Gould, 2004), which results in enhanced inhibitory Y15 phosphorylation of Cdc2p and in the corresponding loss of kinase activity necessary for mitotic exit. Flp1p is required for the ubiquitination of Cdc25p by the anaphase promoting complex/cyclosome at this cell cycle stage, and cells deleted for flp1 ϩ present higher basal levels of Cdc25p. Interestingly, this role may be conserved in higher eukaryotes as hCdc14Ap is involved in the cell cycle regulation of hCdc25Ap stability through dephosphorylation of Serines 115 and 329 of the mitotic inducer in human cells (Esteban et al., 2006). In fission yeast, ⌬flp1 cells enter mitosis at a reduced cell size, presenting a wee phenotype (Cueille et al., 2001;Trautmann et al., 2001). Overexpression of Flp1p arrests ...
Reactive oxygen species (ROS) are an important source of cellular damage. When ROS intracellular levels increase, oxidative stress takes place affecting DNA stability and metabolic functions. To prevent these effects, stress-activated protein kinases (SAPKs) delay cell cycle progression and induce a transcriptional response that activates antioxidant mechanisms ensuring cell adaptation and survival. Fission yeast Cdc14-like phosphatase Flp1 (also known as Clp1) has a well-established role in cell cycle regulation. Moreover, Flp1 contributes to checkpoint activation during replication stress. Here, we show that Flp1 has a role in fine-tuning the cellular oxidative stress response. Rad24-dependent nucleolar release of Flp1 in response to oxidative stress conditions plays a role in the cellular transcriptional response. Thus, Flp1 ablation increases the transcriptional response to oxidative stress, in both intensity and duration, upregulating Atf1/Pcr1 transcription factors and, consequently, genes such as ctt1 + , hsp9 + , gpd1 + and pyp2 +. Remarkably, we found that Flp1 interacts with the Atf1/Pcr1 complex with Pcr1 acting as a direct substrate. Our results provide evidence that Flp1 modulates the oxidative stress response by limiting the Atf1/Pcr1-mediated transcription.
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