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

Abstract

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 ...

Comprehensive Proteomics Analysis Reveals New Substrates and Regulators of the Fission Yeast Clp1/Cdc14 Phosphatase

“…Lastly, the 14 -3-3 proteins Rad24 and Rad25 were preferentially recovered with wild-type Clp1 compared with Clp1-C286S (supplemental Table S7). Phosphorylation of Clp1 by Sid2 or Cds1 provides docking sites for 14 -3-3 proteins (RXXS), and 14 -3-3 binding antagonizes Clp1 localization to the nucleolus (62)(63)(64). It is possible that because Clp1-C286S is constitutively bound to substrates, its association with Rad24 or Rad25 is partially inhibited.…”

“…Alternatively, Mid1 may not inhibit Clp1, but simply be its tether to the cytokinetic ring. On activation of the SIN or the DNA damage checkpoint, Clp1 is released from the nucleolus and phosphorylated at RXXS sites, which facilitates Clp1's interaction with Rad24 and Rad25 and retention in the nucleoplasm (62)(63)(64)80). Thus, 14 -3-3 proteins may regulate Clp1 localization, but may also prevent its function (i.e.…”

Human Cdc14A becomes a cell cycle gene in controlling Cdk1 activity at the G2/M transition

“…[28][29][30] Additional functions of Flp1/Clp1 are its contribution to chromosome segregation, the regulation of spindle midzone functions, and full activation of the checkpoint response to replicative stress. [31][32][33] The mammal genomes encode two Cdc14 isoforms, Cdc14A and Cdc14B, and in hominids, a third isoform, Cdc14Bretro/Cdc14C, which is very similar to Cdc14B, is originated from a retrogene. 34 Given the important roles of Cdc14 phosphatase in yeast, human Cdc14 homologs have been addressed in many functional studies aimed at shedding light on their molecular functions.…”

Centrosome signaling pathways consult on their decision

“…Casein kinase 1δ binds to Sid4 phosphorylated on T584 and phosphorylates the scaffold protein on two other residues. These residues can then bind to a third kinase, Chk2 Cds1 , that is known to phosphorylate the protein phosphatase Flp1 and displace it from SPBs (4). Flp1, a member of the Cdc14 family of phosphatases, opposes Cdk1–Cyclin B activation and dephosphorylates many of its targets.…”

“…Moreover, these pathways can be influenced by many other inputs; Sid4 is involved in a pathway that arrests cells in response to spindle assembly defects (5), whereas Chk2 Cds1 forms part of the DNA replication checkpoint (4). Polo activation by Cut12, meanwhile, has been linked to nutrient sensing pathways (6, 7).…”