Felix Hoppe-Seyler Lecture 1999. Cyclin dependent kinases and regulation of the fission yeast cell cycle.

The cyclin dependent kinases (CDKs), formed by complexes between Cdc2p and the B-cyclins Cig2p and Cdc13p, have a central role in regulating the fission yeast cell cycle and maintaining genomic stability. The CDK Cig2p/Cdc2p controls the onset of S-phase and the CDK Cdc13p/Cdc2p controls the onset of mitosis and ensures that there is only one S-phase in each cell. Cdc13p/Cdc2p can replace Cig2p/Cdc2p forthe onset of S-phase, suggesting that the increasing activity of a single CDK during the cell cycle is sufficient to drive a cell in an orderly fashion into S-phase and into mitosis. If S-phase is incomplete, then inhibition of Cdc13p/ Cdc2p prevents cells with unreplicated DNA from undergoing a catastrophic entry into mitosis. Control of CDK activity is also important to allow cells to exit the cell cycle and accumulate in G1 in response to nutritional deprivation and the presence of pheromone.     

G(1)/S CDK is inhibited to restrain mitotic onset when DNA replication is blocked in fission yeast

Cyclin-dependent kinase (CDK) Tyr15 phosphorylation plays a major role in regulating G(2)/M CDKs, but the role of this phosphorylation in regulating G(1)/S CDKs is less clear. We have studied the regulation and function of Cdc2-Tyr15 phosphorylation in the fission yeast Schizosaccharomyces pombe G(1)/S CDK Cig2/Cdc2. This complex is subject to high level Cdc2-Tyr15 phosphorylation inhibiting its kinase activity in hydroxyurea-treated cells blocked in S-phase. We show that this Tyr15 phosphorylation is required to maintain efficient mitotic checkpoint arrest, because Cig2 accumulates during the block and this accumulation can advance mitotic onset. This mitotic induction operates, at least in part, through activation of the normal G(2)/M CDK complex Cdc13/Cdc2. Thus, Tyr15 phosphorylation of G(1)/S CDK complexes is important in the checkpoint control blocking mitotic onset when DNA replication is inhibited.     

Two fission yeast B-type cyclins, cig2 and Cdc13, have different functions in mitosis.

Cyclin B interacts with Cdc2 kinase to induce cell cycle events, particularly those of mitosis. The existence of cyclin B subtypes in several species has been known for some time, leading to speculation that key events of mitosis may be carried out by distinct functional classes of Cdc2/cyclin B. We report the discovery of cig2, a third B-type cyclin gene in Schizosaccharomyces pombe. Disruption of cig2 delays the onset of mitosis, to the degree that a cig2 null allele rescues mitotic catastrophe mutants, including those that are unable to carry out the inhibitory tyrosyl phosphorylation of Cdc2 kinase. Consistent with this, a cig2 null allele exhibits synthetic lethal interactions with cdc25ts and cdc2ts mutations. Mitotic phenotypes caused by disruption of cig2 are not reversed by increased production of Cdc13, the other fission yeast B-type cyclin that functions in mitosis. Likewise, a cdc13ts mutation is not rescued by increased gene dosage of cig2+. These data indicate that Cdc13 and Cig2 interact with Cdc2 to carry out different functions in mitosis. We suggest that some cyclin B subtypes found in other species, including humans, are also likely to have distinct, nonoverlapping functions in mitosis.     

Cig2, a B-type cyclin, promotes the onset of S in Schizosaccharomyces pombe.

Cdc2, a catalytic subunit of cyclin-dependent kinases, is required for both the G1-to-S and G2-to-M transitions in the fission yeast Schizosaccharomyces pombe. Cdc13, a B-type cyclin, is required for the M-phase induction function of Cd2. Two additional B-type cyclins, Cig1 and Cig2, have been identified in S. pombe, but none of the B-type cyclins are individually required for the onset of S. We report that Cdc13 is important for DNA replication in a strain lacking Cig2. Unlike deltacdc13 cells, double-mutant deltacdc13 deltacig2 cells are defective in undergoing multiple rounds of DNA replication. The conclusion that Cig2 promotes S is further supported by the finding that Cig2 protein and Cig2-associated kinase activity appear soon after the completion of M and peak during S, as well as the observation that S is delayed in deltacig2 cells as they recover from a G1 arrest induced by nitrogen starvation. These studies indicate that Cig2 is the primary S-phase-promoting cyclin in S. pombe but that Cdc13 can effectively substitute for Cig2 in deltacig2 cells. These observations also suggest that the gradual increase in the activity of Cdc2-Cdc13 kinase can be sufficient for the correct temporal ordering of S and M phases in deltacig2 cells.     

A single fission yeast mitotic cyclin B p34cdc2 kinase promotes both S-phase and mitosis in the absence of G1 cyclins.

Deletion of the fission yeast mitotic B-type cyclin gene cdc13 causes cells to undergo successive rounds of DNA replication. We have used a strain which expresses cdc13 conditionally to investigate re-replication. Activity of Start genes cdc2 and cdc10 is necessary and p34cdc2 kinase is active in re-replicating cells. We tested to see whether other cyclins were required for re-replication using cdc13delta. Further deletion of cig1 and puc1 had no effect, but deletion of cig2/cyc17 caused a severe delay in re-replication. Deletion of cig1 and cig2/cyc17 together abolished re-replication completely and cells arrested in G1. This, and analysis of the temperature sensitive cdc13-117 mutant, suggests that cdc13 can effectively substitute for the G1 cyclin activity of cig2/cyc17. We have characterized p56cdc13 activity and find evidence that in the absence of G1 cyclins, S-phase is delayed until the mitotic p34cdc2-p56cdc13 kinase is sufficiently active. These data suggest that a single oscillation of p34cdc2 kinase activity provided by a single B-type cyclin can promote ordered progression into both DNA replication and mitosis, and that the level of cyclin-dependent kinase activity may act as a master regulator dictating whether cells undergo S-phase or mitosis.     

Functional homology among human and fission yeast Cdc14 phosphatases

Budding and fission yeast Cdc14 homologues, a conserved family of serine-threonine phosphatases, play a role in the inactivation of mitotic cyclin-dependent kinases (CDKs) by molecularly distinct mechanisms. Saccharomyces cerevisiae Cdc14 protein phosphatase inactivates CDKs by promoting mitotic cyclin degradation and the accumulation of a CDK inhibitor to allow budding yeast cells to exit from mitosis. Schizosaccharomyces pombe Flp1 phosphatase down-regulates CDK/cyclin activity, controlling the degradation of the Cdc25 tyrosine phosphatase for fission yeast cells to undergo cytokinesis. In the present work, we show that human Cdc14 homologues (hCdc14A and hCdc14B) rescued flp1-deficient fission yeast strains, indicating functional homology. We also show that hCdc14A and B interacted in vivo with S. pombe Cdc25 and that hCdc14A dephosphorylated this mitotic inducer both in vitro and in vivo. Our results support a Cdc14 conserved inhibitory mechanism acting on S. pombe Cdc25 protein and suggest that human cells may regulate Cdc25 in a similar manner to inactivate Cdk1-mitotic cyclin complexes.     

Stable association of mitotic cyclin B/Cdc2 to replication origins prevents endoreduplication

We show that in fission yeast the mitotic B type cyclin Cdc13/Cdc2 kinase associates with replication origins in vivo. This association is dependent on the origin recognition complex (ORC), is established as chromosomes are replicated, and is maintained during G2 and early mitosis. Cells expressing an orp2 (ORC2) allele that reduces binding of Cdc13 to replication origins are acutely prone to chromosomal reduplication. In synchronized endoreduplicating cells, following Cdc13 ablation, replication origins are coordinately licensed prior to each successive round of S phase with the same periodicity as in a normal cell cycle. Thus, ORC bound mitotic Cyclin B/Cdc2 kinase imposes the dependency of S phase on an intervening mitosis but not the temporal licensing of replication origins between each S phase.     

CDK inactivation is the only essential function of the APC/C and the mitotic exit network proteins for origin resetting during mitosis

Passage through mitosis is required to reset replication origins for the subsequent S phase. During mitosis, a series of biochemical reactions involving cyclin-dependent kinases (CDKs), the anaphase promoting complex or cyclosome (APC/C), and a mitotic exit network including Cdc5, 14, and 15 coordinates the proper separation and segregation of sister chromatids. Here we show that cyclin B/CDK inactivation can drive origin resetting in either early S phase or mitosis. This origin resetting occurs efficiently in the absence of APC/C function and mitotic exit network function. We conclude that CDK inactivation is the single essential event in mitosis required to allow pre-RC assembly for the next cell cycle.     

The cyclin-dependent kinase inhibitor Roughex is involved in mitotic exit in Drosophila

Background: Exit from mitosis is a tightly regulated event. This process has been studied in greatest detail in budding yeast, where several activities have been identified that cooperate to downregulate activity of the cyclin-dependent kinase (CDK) Cdc28 and force an exit from mitosis. Cdc28 is inactivated through proteolysis of B-type cyclins by the multisubunit ubiquitin ligase termed the anaphase promoting complex/cyclosome (APC/C) and inhibition by the cyclin-dependent kinase inhibitor (CKI) Sic1. In contrast, the only mechanism known to be essential for CDK inactivation during mitosis in higher eukaryotes is cyclin destruction.Results: We now present evidence that the Drosophila CKI Roughex (Rux) contributes to exit from mitosis. Observations of fixed and living embryos show that metaphase is significantly longer in rux mutants than in wild-type embryos. In addition, Rux overexpression is sufficient to drive cells experimentally arrested in metaphase into interphase. Furthermore, rux mutant embryos are impaired in their ability to overcome a transient metaphase arrest induced by expression of a stable cyclin A. Rux has numerous functional similarities with Sic1. While these proteins share no sequence similarity, we show that Sic1 inhibits mitotic Cdk1-cyclin complexes from Drosophila in vitro and in vivo.Conclusions: Rux inhibits Cdk1-cyclin A kinase activity during metaphase, thereby contributing to exit from mitosis. To our knowledge, this is the first mitotic function ascribed to a CKI in a multicellular organism and indicates the existence of a novel regulatory mechanism for the metaphase to anaphase transition during development.     

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.     

B-type cyclins regulate G1 progression in fission yeast in opposition to the p25rum1 cdk inhibitor.

The onset of S phase in fission yeast is regulated at Start, the point of commitment to the mitotic cell cycle. The p34cdc2 kinase is essential for G1 progression past Start, but until now its regulation has been poorly understood. Here we show that the cig2/cyc17 B-type cyclin has an important role in G1 progression, and demonstrate that p34cdc2 kinase activity is periodically associated with cig2 in G1. Cells lacking cig2 are defective in G1 progression, and this is particularly clear in small cells that must regulate Start with respect to cell size. We also find that the cig1 B-type cyclin can promote G1 progression. Whilst p25rum1 can inhibit cig2/cdc2 activity in vitro, and may transiently inhibit this complex in vivo, cig1 is regulated independently of p25rum1. Since cig1/cdc2 kinase activity peaks in mitotic cells, and decreases after mitosis with similar kinetics to cdc13-associated kinase activity, we suggest that cig2 is likely to be the principal fission yeast G1 cyclin. cig2 protein levels accumulate in G1 cells, and we propose that p25rum1 may transiently inhibit cig2-associated p34cdc2 activity until the critical cell size required for Start is reached.     

Negative Regulation of Cdc18 DNA Replication Protein by Cdc2

Fission yeast Cdc18, a homologue of Cdc6 in budding yeast and metazoans, is periodically expressed during the S phase and required for activation of replication origins. Cdc18 overexpression induces DNA rereplication without mitosis, as does elimination of Cdc2-Cdc13 kinase during G₂ phase. These findings suggest that illegitimate activation of origins may be prevented through inhibition of Cdc18 by Cdc2. Consistent with this hypothesis, we report that Cdc18 interacts with Cdc2 in association with Cdc13 and Cig2 B-type cyclins in vivo. Cdc18 is phosphorylated by the associated Cdc2 in vitro. Mutation of a single phosphorylation site, T104A, activates Cdc18 in the rereplication assay. The cdc18-K9 mutation is suppressed by a cig2 mutation, providing genetic evidence that Cdc2-Cig2 kinase inhibits Cdc18. Moreover, constitutive expression of Cig2 prevents rereplication in cells lacking Cdc13. These findings identify Cdc18 as a key target of Cdc2-Cdc13 and Cdc2-Cig2 kinases in the mechanism that limits chromosomal DNA replication to once per cell cycle.     

Rereplication phenomenon in fission yeast requires MCM proteins and other S phase genes.

The fission yeast Schizosaccharomyces pombe can be induced to perform multiple rounds of DNA replication without intervening mitoses by manipulating the activity of the cyclin-dependent kinase p34(cdc2). We have examined the role in this abnormal rereplication of a large panel of genes known to be involved in normal S phase. The genes analyzed can be grouped into four classes: (1) those that have no effect on rereplication, (2) others that delay DNA accumulation, (3) several that allow a gradual increase in DNA content but not in genome equivalents, and finally, (4) mutations that completely block rereplication. The rereplication induced by overexpression of the CDK inhibitor Rum1p or depletion of the Cdc13p cyclin is essentially the same and requires the activity of two minor B-type cyclins, cig1(+) and cig2(+). In particular, the level, composition, and localization of the MCM protein complex does not alter during rereplication. Thus rereplication in fission yeast mimics the DNA synthesis of normal S phase, and the inability to rereplicate provides an excellent assay for novel S-phase mutants.     

Human cyclin A is required for mitosis until mid prophase.

We have used microinjection and time-lapse video microscopy to study the role of cyclin A in mitosis. We have injected purified, active cyclin A/cyclin-dependent kinase 2 (CDK2) into synchronized cells at specific points in the cell cycle and assayed its effect on cell division. We find that cyclin A/CDK2 will drive G2 phase cells into mitosis within 30 min of microinjection, up to 4 h before control cells enter mitosis. Often this premature mitosis is abnormal; the chromosomes do not completely condense and daughter cells fuse. Remarkably, microinjecting cyclin A/CDK2 into S phase cells has no effect on progress through the following G2 phase or mitosis. In complementary experiments we have microinjected the amino terminus of p21(Cip1/Waf1/Sdi1) (p21N) into cells to inhibit cyclin A/CDK2 activity. We find that p21N will prevent S phase or G2 phase cells from entering mitosis, and will cause early prophase cells to return to interphase. These results suggest that cyclin A/CDK2 is a rate-limiting component required for entry into mitosis, and for progress through mitosis until late prophase. They also suggest that cyclin A/CDK2 may be the target of the recently described prophase checkpoint.     

A WD Repeat Protein Controls the Cell Cycle and Differentiation by Negatively Regulating Cdc2/B-Type Cyclin Complexes

In the fission yeast Schizosaccharomyces pombe, p34^cdc2 plays a central role controlling the cell cycle. We recently isolated a new gene named srw1⁺, capable of encoding a WD repeat protein, as a multicopy suppressor of hyperactivated p34^cdc2. Cells lacking srw1⁺ are sterile and defective in cell cycle controls. When starved for nitrogen source, they fail to effectively arrest in G₁ and die of accelerated mitotic catastrophe if regulation of p34^cdc2/Cdc13 by inhibitory tyrosine phosphorylation is compromised by partial inactivation of Wee1 kinase. Fertility is restored to the disruptant by deletion of Cig2 B-type cyclin or slight inactivation of p34^cdc2. srw1⁺ shares functional similarity with rum1⁺, having abilities to induce endoreplication and restore fertility to rum1 disruptants. In the srw1 disruptant, Cdc13 fails to be degraded when cells are starved for nitrogen. We conclude that Srw1 controls differentiation and cell cycling at least by negatively regulating Cig2- and Cdc13-associated p34^cdc2 and that one of its roles is to down-regulate the level of the mitotic cyclin particularly in nitrogen-poor environments.     

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.