Distinct nuclear and cytoplasmic functions of the S. pombe Cdc14-like phosphatase Clp1p/Flp1p and a role for nuclear shuttling in its regulation

Cdc14-like phosphatases regulate a variety of cell cycle events by dephosphorylating CDK sites. Their cell cycle-dependent changes in localization may be important to carry out distinct functions. Work in budding and fission yeast suggested that Cdc14-like phosphatases are inhibited by nucleolar sequestration. In S. cerevisiae, Cdc14p is released from the nucleolus by the FEAR network and Cdk1, whereas the S. pombe CDC14-like phosphatase Clp1p (also known as Flp1p) is released at mitotic entry by an unknown mechanism. The mitotic exit network (MEN) in S. cerevisiae and its homologous network, the septation initiation network (SIN), in S. pombe act through an unknown mechanism to keep the phosphatase out of the nucleolus in late mitosis. SIN-dependent cytoplasmic maintenance of Clp1p is thought to be essential for the cytokinesis checkpoint, which blocks further rounds of nuclear division until cytokinesis is completed. By targeting Clp1p to the nucleus or the cytoplasm, we demonstrate distinct functions for these pools of Clp1p in chromosome segregation and cytokinesis, respectively. Our results further suggest that the SIN does not keep Clp1p out of the nucleolus by regulating nucleolar affinity, as proposed for S. cerevisiae Cdc14p, but instead, Clp1p may be regulated by nuclear import/export.     

Fission yeast Clp1p phosphatase regulates G2/M transition and coordination of cytokinesis with cell cycle progression.

Background: In Saccharomyces cerevisiae the mitotic-exit network (MEN) functions in anaphase to promote the release of the Cdc14p phosphatase from the nucleolus. This release causes mitotic exit via inactivation of the cyclin-dependent kinase (Cdk). Cdc14p-like proteins are highly conserved; however, it is unclear if these proteins regulate mitotic exit as in S. cerevisiae. In Schizosaccharomyces pombe a signaling pathway homologous to the MEN and termed the septation initiation network (SIN) is required not for mitotic exit, but for initiation of cytokinesis and for a cytokinesis checkpoint that inhibits further cell cycle progression until cytokinesis is complete.Results: We have identified the S. pombe Cdc14p homolog, Clp1p, and show that it is not required for mitotic exit but rather functions together with the SIN in coordinating cytokinesis with the nuclear-division cycle. As cells enter mitosis, Clp1p relocalizes from the nucleolus to the spindle and site of cell division. Clp1p exit from the nucleolus does not depend on the SIN, but the SIN is required for keeping Clp1p out of the nucleolus until completion of cytokinesis. Clp1p, in turn, may promote the activation of the SIN by antagonizing Cdk activity until cytokinesis is complete and thus ensuring that cytokinesis is completed prior to the initiation of the next cell cycle. In addition to its roles in anaphase, Clp1p regulates the G2/M transition since cells deleted for clp1 enter mitosis precociously and cells overexpressing Clp1p delay mitotic entry. Unlike Cdc14p, Clp1p appears to antagonize Cdk activity by preventing dephosphorylation of Cdc2p on tyrosine.Conclusions: S. pombe Clp1p affects cell cycle progression in a markedly different manner than its S. cerevisiae homolog, Cdc14p. This finding raises the possibility that related phosphatases in animal cells will prove to have important roles in coordinating the onset of cytokinesis with the events of mitosis.     

The Clp1/Cdc14 phosphatase contributes to the robustness of cytokinesis by association with anillin-related Mid1

Cdc14 phosphatases antagonize cyclin-dependent kinase-directed phosphorylation events and are involved in several facets of cell cycle control. We investigate the role of the fission yeast Cdc14 homologue Clp1/Flp1 in cytokinesis. We find that Clp1/Flp1 is tethered at the contractile ring (CR) through its association with anillin-related Mid1. Fluorescent recovery after photobleaching analyses indicate that Mid1, unlike other tested CR components, is anchored at the cell midzone, and this physical property is likely to account for its scaffolding role. By generating a mutation in mid1 that selectively disrupts Clp1/Flp1 tethering, we reveal the specific functional consequences of Clp1/Flp1 activity at the CR, including dephosphorylation of the essential CR component Cdc15, reductions in CR protein mobility, and CR resistance to mild perturbation. Our evidence indicates that Clp1/Flp1 must interact with the Mid1 scaffold to ensure the fidelity of Schizosaccharomyces pombe cytokinesis.     

The SIN kinase Sid2 regulates cytoplasmic retention of the S. pombe Cdc14-like phosphatase Clp1

Cdc14-family phosphatases play a conserved role in promoting mitotic exit and cytokinesis by dephosphorylating substrates of cyclin-dependent kinase (Cdk). Cdc14-family phosphatases have been best studied in yeast (for review, see [1, 2]), where budding yeast Cdc14 and its fission yeast homolog Clp1 are regulated partly by their localization; both proteins are thought to be sequestered in the nucleolus in interphase. Cdc14 and Clp1 are released from the nucleolus in mitosis, and in late mitosis conserved signaling pathways termed the mitotic exit network (MEN) and the septation initiation network (SIN) keeps Cdc14 and Clp1, respectively, out of the nucleolus through an unknown mechanism [3-6]. Here we show that the most downstream SIN component, the Ndr-family kinase Sid2, maintains Clp1 in the cytoplasm in late mitosis by phosphorylating Clp1 directly and thereby creating binding sites for the 14-3-3 protein Rad24. Mutation of the Sid2 phosphorylation sites on Clp1 disrupts the Clp1-Rad24 interaction and causes Clp1 to return prematurely to the nucleolus during cytokinesis. Loss of Clp1 from the cytoplasm in telophase renders cells sensitive to perturbation of the actomyosin ring but does not affect other Clp1 functions. Because all components of this pathway are conserved, this might be a broadly conserved mechanism for regulation of Cdc14-family phosphatases.     

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

Cdc14 belongs to a dual-specificity phosphatase family highly conserved through evolution that preferentially reverses CDK (Cyclin dependent kinases) –dependent phosphorylation events. In the yeast Saccharomyces cerevisiae, Cdc14 is an essential regulator of late mitotic events and exit from mitosis by counteracting CDK activity at the end of mitosis. However, many studies have shown that Cdc14 is dispensable for exiting mitosis in all other model systems analyzed. In fission yeast, the Cdc14 homologue Flp1/Clp1 regulates the stability of the mitotic inducer Cdc25 at the end of mitosis to ensure Cdk1 inactivation before cytokinesis. We have recently reported that human Cdc14A, the Cdc14 isoform located at the centrosomes during interphase, down-regulates Cdc25 activity at the G2/M transition to prevent premature activation of Cdk1-Cyclin B1 complexes and untimely entry into mitosis. Here we speculate about new molecular mechanisms for Cdc14A and discuss the current evidence suggesting that Cdc14 phosphatase plays a role in cell cycle control in higher eukaryotes.     

Phospho-regulation of the Cdc14/Clp1 phosphatase delays late mitotic events in S. pombe

In eukaryotes, exit from mitosis occurs through the inactivation of the Cdk1-cyclin B kinase complex and the reversal of its phosphorylation events. These late mitotic events are tightly regulated to occur only after the onset of anaphase and prior to cytokinesis. Central to this regulation is the conserved Cdc14 family of protein phosphatases, whose activity reverses Cdk-dependent phosphorylation events. S. cerevisiae Cdc14 activity is restrained from dephosphorylating Cdk substrates and inactivating Cdk1 through its nucleolar sequestration prior to anaphase. Here, we describe a unique mode of Cdc14 regulation that operates prior to anaphase in fission yeast. Cdk1 phosphorylates and inhibits the catalytic activity of the Cdc14 family member, Clp1/Flp1. As Cdk1 activity declines during anaphase progression, Clp1/Flp1 autocatalytically reverses these phosphorylation events to stimulate its own activity. These findings point to a simple regulatory circuit that couples Cdk1 activation with its inactivation mediated through phosphorylation-dependent regulation of Clp1/Flp1 phosphatase activity.     

The 14-3-3 protein rad24p modulates function of the cdc14p family phosphatase clp1p/flp1p in fission yeast

Schizosaccharomyces pombe cells divide through the use of an actomyosin-based contractile ring. In response to perturbation of the actomyosin ring, S. pombe cells delay in a "cytokinesis-competent" state characterized by continuous repair and maintenance of the actomyosin ring and a G2 delay. This checkpoint mechanism requires the function of the Cdc14p-family phosphatase Clp1p/Flp1p and the septation initiation network (SIN). In response to cytokinetic defects, Clp1p, normally nucleolar in interphase, is retained in the cytoplasm until completion of cell division in a SIN-dependent manner. Here, we show that a phosphorylated form of Clp1p binds the 14-3-3 protein Rad24p and is retained in the cytoplasm in a Rad24p-dependent manner in response to cytokinesis defects. This physical interaction depends on the function of the SIN component, Sid2p. In the absence of Rad24p, cells are unable to maintain SIN signaling and lose viability upon mild cytokinetic stress. The requirement of Rad24p in this checkpoint is bypassed by ectopic activation of the SIN. Furthermore, SIN-dependent nuclear exclusion of Clp1p is dependent on Rad24p function. We conclude that Rad24p-mediated cytoplasmic retention of Clp1p/Flp1p is important for cell viability upon stress to the division apparatus.     

Human Cdc14A phosphatase modulates the G2/M transition through Cdc25A and Cdc25B

The Cdc14 family of serine-threonine phosphatases antagonizes CDK activity by reversing CDK-dependent phosphorylation events. It is well established that the yeast members of this family bring about the M/G1 transition. Budding yeast Cdc14 is essential for CDK inactivation at the end of mitosis and fission yeast Cdc14 homologue Flp1/Clp1 down-regulates Cdc25 to ensure the inactivation of mitotic CDK complexes to trigger cell division. However, the functions of human Cdc14 homologues remain poorly understood. Here we have tested the hypothesis that Cdc14A might regulate Cdc25 mitotic inducers in human cells. We found that increasing levels of Cdc14A delay entry into mitosis by inhibiting Cdk1-cyclin B1 activity. By contrast, lowering the levels of Cdc14A accelerates mitotic entry. Biochemical analyses revealed that Cdc14A acts through key Cdk1-cyclin B1 regulators. We observed that Cdc14A directly bound to and dephosphorylated Cdc25B, inhibiting its catalytic activity. Cdc14A also regulated the activity of Cdc25A at the G2/M transition. Our results indicate that Cdc14A phosphatase prevents premature activation of Cdk1 regulating Cdc25A and Cdc25B at the entry into mitosis.     

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.     

Crm1-Mediated Nuclear Export of Cdc14 is Required for the Completion of Cytokinesis in Budding Yeast

The mitotic exit network (MEN) controls the exit from mitosis in budding yeast. The proline-directed phosphatase, Cdc14p, is a key component of MEN and promotes mitotic exit by activating the degradation of Clb2p and by reversing Cdk-mediated mitotic phosphorylation. Cdc14p is sequestered in the nucleolus during much of the cell cycle and is released in anaphase from the nucleolus to the nucleoplasm and cytoplasm to perform its functions. Release of Cdc14p from the nucleolus during anaphase is well understood. In contrast, less is known about the mechanism by which Cdc14p is released from the nucleus to the cytoplasm. Here we show that Cdc14p contains a leucine-rich nuclear export signal (NES) that interacts with Crm1p physically. Mutations in the NES of Cdc14p allow Clb2p degradation and mitotic exit, but cause abnormal morphology and cytokinesis defects at non-permissive temperatures. Cdc14p localizes to the bud neck, among other cytoplasmic structures, following its release from the nucleolus in late anaphase. This bud neck localization of Cdc14p is disrupted by mutations in its NES and by the leptomycin B-mediated inhibition of Crm1p. Our results suggest a requirement for Crm1p-dependent nuclear export of Cdc14p in coordinating mitotic exit and cytokinesis in budding yeast.     

A Highlights from MBoC Selection: Multiple protein kinases influence the redistribution of fission yeast Clp1/Cdc14 phosphatase upon genotoxic stress

The Cdc14 phosphatase family antagonizes Cdk1 phosphorylation and is important for mitotic exit. To access their substrates, Cdc14 phosphatases are released from nucleolar sequestration during mitosis. Clp1/Flp1, the Schizosaccharomyces pombe Cdc14 orthologue, and Cdc14B, a mammalian orthologue, also exit the nucleolus during interphase upon DNA replication stress or damage, respectively, implicating Cdc14 phosphatases in the response to genotoxic insults. However, a mechanistic understanding of Cdc14 phosphatase nucleolar release under these conditions is incomplete. We show here that relocalization of Clp1 during genotoxic stress is governed by complex phosphoregulation. Specifically, the Rad3 checkpoint effector kinases Cds1 and/or Chk1, the cell wall integrity mitogen-activated protein kinase Pmk1, and the cell cycle kinase Cdk1 directly phosphorylate Clp1 to promote genotoxic stress–induced nucleoplasmic accumulation. However, Cds1 and/or Chk1 phosphorylate RxxS sites preferentially upon hydroxyurea treatment, whereas Pmk1 and Cdk1 preferentially phosphorylate Clp1 TP sites upon H₂O₂ treatment. Abolishing both Clp1 RxxS and TP phosphosites eliminates any genotoxic stress–induced redistribution. Reciprocally, preventing dephosphorylation of Clp1 TP sites shifts the distribution of the enzyme to the nucleoplasm constitutively. This work advances our understanding of pathways influencing Clp1 localization and may provide insight into mechanisms controlling Cdc14B phosphatases in higher eukaryotes.     

Cell cycle-dependent roles for the FCH-domain protein Cdc15p in formation of the actomyosin ring in Schizosaccharomyces pombe

Cell division in the fission yeast Schizosaccharomyces pombe requires the formation and constriction of an actomyosin ring at the division site. The actomyosin ring is assembled in metaphase and anaphase A, is maintained throughout mitosis, and constricts after completion of anaphase. Maintenance of the actomyosin ring during late stages of mitosis depends on the septation initiation network (SIN), a signaling cascade that also regulates the deposition of the division septum. However, SIN is not active in metaphase and is not required for the initial assembly of the actomyosin ring early in mitosis. The FER/CIP4-homology (FCH) domain protein Cdc15p is a component of the actomyosin ring. Mutations in cdc15 lead to failure in cytokinesis and result in the formation of elongated, multinucleate cells without a division septum. Here we present evidence that the requirement of Cdc15p for actomyosin ring formation is dependent on the stage of mitosis. Although cdc15 mutants are competent to assemble actomyosin rings in metaphase, they are unable to maintain actomyosin rings late in mitosis when SIN is active. In the absence of functional Cdc15p, ring formation upon metaphase arrest depends on the anillin-like Mid1p. Interestingly, when cytokinesis is delayed due to perturbations to the division machinery, Cdc15p is maintained in a hypophosphorylated form. The dephosphorylation of Cdc15p, which occurs transiently in unperturbed cytokinesis, is partially dependent on the phosphatase Clp1p/Flp1p. This suggests a mechanism where both SIN and Clp1p/Flp1p contribute to maintenance of the actomyosin ring in late mitosis through Cdc15p, possibly by regulating its phosphorylation status.     

Nsk1 ensures accurate chromosome segregation by promoting association of kinetochores to spindle poles during anaphase B

Type 1 phosphatase (PP1) antagonizes Aurora B kinase to stabilize kinetochore-microtubule attachments and to silence the spindle checkpoint. We screened for factors that exacerbate the growth defect of Δdis2 cells, which lack one of two catalytic subunits of PP1 in fission yeast, and identified Nsk1, a novel protein required for accurate chromosome segregation. During interphase, Nsk1 resides in the nucleolus but spreads throughout the nucleoplasm as cells enter mitosis. Following dephosphorylation by Clp1 (Cdc14-like) phosphatase and at least one other phosphatase, Nsk1 localizes to the interface between kinetochores and the inner face of the spindle pole body during anaphase. In the absence of Nsk1, some kinetochores become detached from spindle poles during anaphase B. If this occurs late in anaphase B, then the sister chromatids of unclustered kinetochores segregate to the correct daughter cell. These unclustered kinetochores are efficiently captured, retrieved, bioriented, and segregated during the following mitosis, as long as Dis2 is present. However, if kinetochores are detached from a spindle pole early in anaphase B, then these sister chromatids become missegregated. These data suggest Nsk1 ensures accurate chromosome segregation by promoting the tethering of kinetochores to spindle poles during anaphase B.     

SIN and the art of splitting the fission yeast cell

The septation initiation network (SIN) triggers the onset of cytokinesis in the fission yeast Schizosaccharomyces pombe by promoting contraction of the medially placed F-actin ring. SIN signaling is regulated by the polo-like kinase plo1p and by cdc2p, the initiator of mitosis, and its activation is co-ordinated with other events in mitosis to ensure that cytokinesis does not begin until chromosomes have been separated. Though the SIN controls the contractile ring, the signal originates from the poles of the mitotic spindle. Recent studies suggest that the spindle pole body may act as a dynamic assembly site for active SIN signaling complexes. In the budding yeast Saccharomyces cerevisiae the counterpart of the SIN, called the MEN, mediates both mitotic exit and cytokinesis, in part through regulating activation of the phosphoprotein phosphatase Cdc14p. Flp1p, the S. pombe ortholog of Cdc14p, is not essential for mitotic exit, but may contribute to an orderly mitosis-G1 transition by regulating the destruction of the mitotic inducer cdc25p.     

Regulation of multiple cell cycle events by Cdc14 homologues in vertebrates

Whereas early cytokinesis events have been relatively well studied, little is known about its final stage, abscission. The Cdc14 phosphatase is involved in the regulation of multiple cell cycle events, and in all systems studied Cdc14 misexpression leads to cytokinesis defects. In this work, we have cloned two CDC14 cDNA from Xenopus, including a previously unreported CDC14B homologue. We use Xenopus and human cell lines and demonstrate that localization of Cdc14 proteins is independent of both cell-type and species specificity. Ectopically expressed XCdc14A is centrosomal in interphase and localizes to the midbody in cytokinesis. By using XCdc14A misregulation, we confirm its control over different cell cycle events and unravel new functions during abscission. XCdc14A regulates the G1/S and G2/M transitions. We show that Cdc25 is an in vitro substrate for XCdc14A and might be its target at the G2/M transition. Upregulated wild-type or phosphatase-dead XCdc14A arrest cells in a late stage of cytokinesis, connected by thin cytoplasmic bridges. It does not interfere with central spindle formation, nor with the relocalization of passenger protein and centralspindlin complexes to the midbody. We demonstrate that XCdc14A upregulation prevents targeting of exocyst and SNARE complexes to the midbody, both essential for abscission to occur.     

The Flp1/Clp1 phosphatase cooperates with HECT-type Pub1/2 protein-ubiquitin ligases in Schizosaccharomyces pombe

The Schizosaccharomyces pombe Flp1p serine-threonine phosphatase is required for the degradation of the mitotic inducer Cdc25p at the end of mitosis. Cdc25p degradation prevents Cdc2p-tyrosine 15 dephosphorylation and, thus, contributes to the timely inactivation of mitotic CDK-associated kinase activity. Both RING- and HECT-type protein-ubiquitin ligases are involved in Cdc25p destabilization. Flp1p function is required for Cdc25p ubiquitination via anaphase-promoting complex/cyclosome or APC/C (RING-type) and the absence of Pub1p (HECT-type) stabilizes the mitotic inducer. In the present report, we study the functional relationship of Flp1p with Pub1p and Pub2p HECT-type-protein ubiquitin ligases. We show that Flp1p is required for the rapid degradation of Cdc25p while Pub1p is responsible for the long-term destabilization of the mitotic inducer. Accordingly, flp1 and pub1 mutants have a strong genetic interaction, correlating defects in the coordination of mitosis and cytokinesis with the stabilization of hyperactive Cdc25p. However, we also show that Flp1 and Pub2p proteins functionally interact in vivo suggesting that both proteins belong to the same regulatory network in S. pombe cells. Thus Flp1p appears to have an important role in integrating HECT- and RING-type ubiquitin ligases in cell cycle control.     

Elevating the level of Cdc34/Ubc3 ubiquitin-conjugating enzyme in mitosis inhibits association of CENP-E with kinetochores and blocks the metaphase alignment of chromosomes

Cdc34/Ubc3 is a ubiquitin-conjugating enzyme that functions in targeting proteins for proteasome-mediated degradation at the G1 to S cell cycle transition. Elevation of Cdc34 protein levels by microinjection of bacterially expressed Cdc34 into mammalian cells at prophase inhibited chromosome congression to the metaphase plate with many chromosomes remaining near the spindle poles. Chromosome condensation and nuclear envelope breakdown occurred normally, and chromosomes showed oscillatory movements along mitotic spindle microtubules. Most injected cells arrested in a prometaphase-like state. Kinetochores, even those of chromosomes that failed to congress, possessed the normal trilaminar plate ultrastructure. The elevation of Cdc34 protein levels in early mitosis selectively blocked centromere protein E (CENP-E), a mitotic kinesin, from associating with kinetochores. Other proteins, including two CENP-E-associated proteins, BubR1 and phospho-p42/p44 mitogen-activated protein kinase, and mitotic centromere-associated kinesin, cytoplasmic dynein, Cdc20, and Mad2, all exhibited normal localization to kinetochores. Proteasome inhibitors did not affect the prometaphase arrest induced by Cdc34 injection. These studies suggest that CENP-E targeting to kinetochores is regulated by ubiquitylation not involving proteasome-mediated degradation.     

Phosphorylation and spindle pole body localization of the Cdc15p mitotic regulatory protein kinase in budding yeast

Cdc15p is an essential protein kinase and functions with a group of late mitotic proteins that includes Lte1p, Tem1p, Cdc14p and Dbf2p/Dbf20p to inactivate Cdc28p-Clb2p at the end of mitosis in budding yeast [1] [2]. Cdc14p is activated and released from the nucleolus at late anaphase/telophase to dephosphorylate important regulators of Cdc28p-Clb2p such as Hct1p/Cdh1p, Sic1p and Swi5p in a CDC15-dependent manner [3] [4] [5] [6] [7]. How Cdc15p itself is regulated is not known. Here, we report that both the phosphorylation and localization of Cdc15p are cell cycle regulated. The extent of phosphorylation of Cdc15p gradually increases during cell-cycle progression until some point during late anaphase/telophase when it is rapidly dephosphorylated. We provide evidence suggesting that Cdc14p is the phosphatase responsible for the dephosphorylation of Cdc15p. Using a Cdc15p fusion protein coupled at its carboxyl terminus to green fluorescent protein (GFP), we found that Cdc15p, like its homologue Cdc7p [8] in fission yeast, localizes to the spindle pole bodies (SPBs) during mitosis. At the end of telophase, a portion of Cdc15p is located at the mother-bud neck, suggesting a possible role for Cdc15p in cytokinesis.