The fission yeast S-phase cyclin Cig2 can drive mitosis

Commitment to mitosis is regulated by cyclin-dependent kinase (CDK) activity. In the fission yeast Schizosaccharomyces pombe, the major B-type cyclin, Cdc13, is necessary and sufficient to drive mitotic entry. Furthermore, Cdc13 is also sufficient to drive S phase, demonstrating that a single cyclin can regulate alternating rounds of replication and mitosis, and providing the foundation of the quantitative model of CDK function. It has been assumed that Cig2, a B-type cyclin expressed only during S phase and incapable of driving mitosis in wild-type cells, was specialized for S-phase regulation. Here, we show that Cig2 is capable of driving mitosis. Cig2/CDK activity drives mitotic catastrophe—lethal mitosis in inviably small cells—in cells that lack CDK inhibition by tyrosine-phosphorylation. Moreover, Cig2/CDK can drive mitosis in the absence of Cdc13/CDK activity and constitutive expression of Cig2 can rescue loss of Cdc13 activity. These results demonstrate that in fission yeast, not only can the presumptive M-phase cyclin drive S phase, but the presumptive S-phase cyclin can drive M phase, further supporting the quantitative model of CDK function. Furthermore, these results provide an explanation, previously proposed on the basis of computational analyses, for the surprising observation that cells expressing a single-chain Cdc13-Cdc2 CDK do not require Y15 phosphorylation for viability. Their viability is due to the fact that in such cells, which lack Cig2/CDK complexes, Cdc13/CDK activity is unable to drive mitotic catastrophe.     

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.     

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.     

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.     

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.     

Negative regulation of G1 and G2 by S-phase cyclins of Saccharomyces cerevisiae.

Cell cycle progression in the budding yeast Saccharomyces cerevisiae is controlled by the Cdc28 protein kinase, which is sequentially activated by different sets of cyclins. Previous genetic analysis has revealed that two B-type cyclins, Clb5 and Clb6, have a positive role in DNA replication. In the present study, we show, in addition, that these cyclins negatively regulate G1- and G2-specific functions. The consequences of this negative regulation were most apparent in clb6 mutants, which had a shorter pre-Start G1 phase as well as a shorter G2 phase than congenic wild-type cells. As a consequence, clb6 mutants grew and proliferated more rapidly than wild-type cells. It was more difficult to assess the role of Clb5 in G1 and G2 by genetic analysis because of the extreme prolongation of S phase in clb5 mutants. Nevertheless, both Clb5 and Clb6 were shown to be responsible for down-regulation of the protein kinase activities associated with Cln2, a G1 cyclin, and Clb2, a mitotic cyclin, in vivo. These observations are consistent with the observed cell cycle phase accelerations associated with the clb6 mutant and are suggestive of similar functions for Clb5. Genetic evidence suggested that the inhibition of mitotic cyclin-dependent kinase activities was dependent on and possibly mediated through the CDC6 gene product. Thus, Clb5 and Clb6 may stabilize S phase by promoting DNA replication while inhibiting other cell cycle activities.     

Distinct roles for cyclins E and A during DNA replication complex assembly and activation

Initiation of DNA replication is regulated by cyclin-dependent protein kinase 2 (Cdk2) in association with two different regulatory subunits, cyclin A and cyclin E (reviewed in ref. 1). But why two different cyclins are required and why their order of activation is tightly regulated are unknown. Using a cell-free system for initiation of DNA replication that is based on G1 nuclei, G1 cytosol and recombinant proteins, we find that cyclins E and A have specialized roles during the transition from G0 to S phase. Cyclin E stimulates replication complex assembly by cooperating with Cdc6, to make G1 nuclei competent to replicate in vitro. Cyclin A has two separable functions: it activates DNA synthesis by replication complexes that are already assembled, and it inhibits the assembly of new complexes. Thus, cyclin E opens a 'window of opportunity' for replication complex assembly that is closed by cyclin A. The dual functions of cyclin A ensure that the assembly phase (G1) ends before DNA synthesis (S) begins, thereby preventing re-initiation until the next cell cycle.     

Regulation of Cdc2p and Cdc13p is required for cell cycle arrest induced by defective RNA splicing in fission yeast

Screening of cdc mutants of fission yeast for those whose cell cycle arrest is independent of the DNA damage checkpoint identified the RNA splicing-deficient cdc28 mutant. A search for mutants of cdc28 cells that enter mitosis with unspliced RNA resulted in the identification of an orb5 point mutant. The orb5+ gene, which encodes a catalytic subunit of casein kinase II, was found to be required for cell cycle arrest in other mutants with defective RNA metabolism but not for operation of the DNA replication or DNA damage checkpoints. Loss of function of wee1+ or rad24+ also suppressed the arrest of several splicing mutants. Overexpression of the major B-type cyclin Cdc13p induced cdc28 cells to enter mitosis. The abundance of Cdc13p was reduced, and the phosphorylation of Cdc2p on tyrosine 15 was maintained in splicing-defective cells. These results suggest that regulation of Cdc13p and Cdc2p is required for G2 arrest in splicing mutants.     

The role of proteolysis in cell cycle progression in Schizosaccharomyces pombe.

A cell-free system derived from Xenopus eggs was used to identify the 'destruction box' of the Schizosaccharomyces pombe B-type cyclin, Cdc13, as residues 59-67: RHALDDVSN. Expression of indestructible Cdc13 from a regulated promoter in S.pombe blocked cells in anaphase and inhibited septation, showing that destruction of Cdc13 is necessary for exit from mitosis, but not for sister chromatid separation. In contrast, strong expression of a polypeptide comprising the N-terminal 70 residues of Cdc13, which acts as a competitive inhibitor of destruction box-mediated proteolysis, inhibited both sister chromatid separation and the destruction of Cdc13, whereas an equivalent construct with a mutated destruction box did not. Appropriately timed expression of this N-terminal fragment of Cdc13 overcame the G1 arrest seen in cdc10 mutant strains, suggesting that proteins required for the initiation of S phase are subject to destruction by the same proteolytic machinery as cyclin.     

Human cyclin B3. mRNA expression during the cell cycle and identification of three novel nonclassical nuclear localization signals

Cyclins form complexes with cyclin-dependent kinases. By controlling activity of the enzymes, cyclins regulate progression through the cell cycle. A- and B-type cyclins were discovered due to their distinct appearance in S and G(2) phases and their rapid proteolytic destruction during mitosis. Transition from G(2) to mitosis is basically controlled by B-type cyclins. In mammals, two cyclin B proteins are well characterized, cyclin B1 and cyclin B2. Recently, a human cyclin B3 gene was described. In contrast to the expression pattern of other B-type cyclins, we find cyclin B3 mRNA expressed not only in S and G(2)/M cells but also in G(0) and G(1). Human cyclin B3 is expressed in different variants. We show that one isoform remains in the cytoplasm, whereas the other variant is translocated to the nucleus. Transport to the nucleus is dependent on three autonomous nonclassical nuclear localization signals that where previously not implicated in nuclear translocation. It had been shown that cyclin B3 coimmunoprecipitates with cdk2; but this complex does not exhibit any kinase activity. Furthermore, a degradation-resistant version of cyclin B3 can arrest cells in G(1) and G(2). Taken together with the finding that cyclin B3 mRNA is not only expressed in G(2)/M but is also detected in significant amounts in resting cells and in G(1) cells. This may suggest a dominant-negative function of human cyclin B3 in competition with activating cyclins in G(0) and the G(1) phase of the cell cycle.     

Loss of meiotic rereplication block in Saccharomyces cerevisiae cells defective in Cdc28p regulation

Cdc28p is the major cyclin-dependent kinase in Saccharomyces cerevisiae. Its activity is required for blocking the reinitiation of DNA replication during mitosis. Here, we show that under conditions where Cdc28p activity is improperly regulated--either through the loss of function of the Schizosaccharomyces pombe wee1 ortholog Swe1p or through the expression of a dominant CDC28 allele, CDC28AF--diploid yeast cells are able to complete several rounds of premeiotic DNA replication within a single meiotic cell cycle. Moreover, a percentage of mutant cells exhibit a "multispore" phenotype, possessing the ability to package more than four spores within a single ascus. These multispored asci contain both even and odd numbers of viable spores. In order for meiotic rereplication and multispore formation to occur, cells must initiate homologous recombination and maintain proper chromosome cohesion during meiosis I. Rad9p- or Rad17p-dependent checkpoint mechanisms are not required for multispore formation and neither are the B-type cyclin Clb6p and the cyclin-dependent kinase inhibitor Sic1p. Finally, we present evidence of a possible role for a Cdc55p-dependent protein phosphatase 2A in initiating meiotic replication.     

Dephosphorylation of threonine 169 of Cdc28 is not required for exit from mitosis but may be necessary for start in Saccharomyces cerevisiae.

Entry into mitosis requires activation of cdc2 kinase brought on by its association with cyclin B, phosphorylation of the conserved threonine (Thr-167 in Schizosaccharomyces pombe) in the T loop, and dephosphorylation of the tyrosine residue at position 15. Exit from mitosis, on the other hand, is induced by inactivation of cdc2 activity via cyclin destruction. It has been suggested that in addition to cyclin degradation, dephosphorylation of Thr-167 may also be required for exit from the M phase. Here we show that Saccharomyces cerevisiae cells expressing cdc28-E169 (a CDC28 allele in which the equivalent threonine, Thr-169, has been replaced by glutamic acid) are able to degrade mitotic cyclin Clb2, inactivate the Cdc28/Clb2 kinase, and disassemble the anaphase spindles, suggesting that they exit mitosis normally. The cdc28-E169 allele is active with respect to its mitotic functions, since it complements the mitosis-defective cdc28-1N allele. Whereas replacement of Thr-169 with serine affects neither Start nor the mitotic activity of Cdc28, replacement with glutamic acid or alanine renders Cdc28 inactive for Start-related functions. Coimmunoprecipitation experiments show that although Cdc28-E169 associates with mitotic cyclin Clb2, it fails to associate with the G1 cyclin Cln2. Thus, an unmodified threonine at position 169 in Cdc28 is important for interaction with G1 cyclins. We propose that in S. cerevisiae, dephosphorylation of Thr-169 is not required for exit from mitosis but may be necessary for commitment to the subsequent division cycle.     

Stockpiling of Cdc25 during a DNA replication checkpoint arrest in Schizosaccharomyces pombe.

The DNA replication checkpoint couples the onset of mitosis with the completion of S phase. It is clear that in the fission yeast Schizosaccharomyces pombe, operation of this checkpoint requires maintenance of the inhibitory tyrosyl phosphorylation of Cdc2. Cdc25 phosphatase induces mitosis by dephosphorylating tyrosine 15 of Cdc2. In this report, Cdc25 is shown to accumulate to a very high level in cells arrested in S. This shows that mechanisms which modulate the abundance of Cdc25 are unconnected to the DNA replication checkpoint. Using a Cdc2/cyclin B activation assay, we found that Cdc25 activity increased approximately 10-fold during transit through M phase. Cdc25 was activated by phosphorylations that were dependent on Cdc2 activity in vivo. Cdc25 activation was suppressed in cells arrested in G1 and S. However, Cdc25 was more highly modified and appeared to be somewhat more active in S than in G1. This finding might be connected to the fact that progression from G1 to S increases the likelihood that constitutive Cdc25 overproduction will cause inappropriate mitosis.     

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.     

A role for cyclin A1 in the activation of MPF and G2-M transition during meiosis of male germ cells in mice

Cell-cycle transition at G2-M is controlled by MPF (M-phase-promoting factor), a complex consisting of the Cdc2 kinase and a B-type cyclin. We have shown that in mice, targeted disruption of an A-type cyclin gene, cyclin A1, results in a block of spermatogenesis prior to the entry into metaphase I. The meiotic arrest is accompanied by a defect in Cdc2 kinase activation at the G2--M transition, raising the possibility that a cyclin A1-dependent process dictates the activation of MPF. Here we show that like Cdc2, the expression of B-type cyclins is retained in cyclin A1-deficient spermatocytes, while their associated kinases are kept at inactive states. Treatment of arrested germ cells with the protein phosphatase type-1 and -2A inhibitor okadaic acid restores the MPF activity and induces entry into M phase and the formation of normally condensed chromosome bivalents, concomitant with hyperphosphorylation of Cdc25 proteins. Conversely, inhibition of tyrosine phosphatases, including Cdc25s, by vanadate suppresses the okadaic acid-induced metaphase induction. The highest levels of Cdc25A and Cdc25C expression and their subcellular localization during meiotic prophase coincide with that of cyclin A1, and when overexpressed in HeLa cells, cyclin A1 coimmunoprecipitates with Cdc25A. Furthermore, the protein kinase complexes consisting of cyclin A1 and either Cdc2 or Cdk2 phosphorylate both Cdc25A and Cdc25C in vitro. These results suggest that in normal meiotic male germ cells, cyclin A1 participates in the regulation of other protein kinases or phosphatases critical for the G2-M transition. In particular, it may be directly involved in the initial amplification of MPF through the activating phosphorylation on Cdc25 phosphatases.     

Identification of a novel vertebrate cyclin: cyclin B3 shares properties with both A- and B-type cyclins.

Cyclins play a key role in controlling progression through the cell cycle. They act as regulatory subunits of p34cdc2/CDC28 and related cyclin-dependent protein kinases (cdks). In vertebrates, cyclins B1 and B2 function during M phase, whereas cyclin A is required for S phase as well as the G2 to M phase transition. Here, we describe the identification and characterization of a novel vertebrate cyclin, termed cyclin B3. The assignment of this cyclin to the B-type subfamily is based on its cDNA-derived sequence and its pattern of expression in synchronized cells, both suggesting a distant relationship to other B-type cyclins. Interestingly, however, cyclin B3 also displays properties that resemble those of A- rather than B-type cyclins. Specifically, cyclin B3 localizes to the cell nucleus throughout the cell cycle, and is able to associate in vivo with at least two kinase subunits, p34cdc2 and p33cdk2. Furthermore, deletion of 26 amino acids from the C-terminus of cyclin B3 impairs both its interaction with kinase catalytic subunits and its nuclear localization, reminiscent of recent results obtained with cyclin A. Based on these observations, we conclude that cyclin B3 may share functional properties with both A- and B-type cyclins.