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.     

APC(ste9/srw1) promotes degradation of mitotic cyclins in G(1) and is inhibited by cdc2 phosphorylation

Fission yeast ste9/srw1 is a WD-repeat protein highly homologous to budding yeast Hct1/Cdh1 and DROSOPHILA: Fizzy-related that are involved in activating APC/C (anaphase-promoting complex/cyclosome). We show that APC(ste9/srw1) specifically promotes the degradation of mitotic cyclins cdc13 and cig1 but not the S-phase cyclin cig2. APC(ste9/srw1) is not necessary for the proteolysis of cdc13 and cig1 that occurs at the metaphase-anaphase transition but it is absolutely required for their degradation in G(1). Therefore, we propose that the main role of APC(ste9/srw1) is to promote degradation of mitotic cyclins when cells need to delay or arrest the cell cycle in G(1). We also show that ste9/srw1 is negatively regulated by cdc2-dependent protein phosphorylation. In G(1), when cdc2-cyclin kinase activity is low, unphosphorylated ste9/srw1 interacts with APC/C. In the rest of the cell cycle, phosphorylation of ste9/srw1 by cdc2-cyclin complexes both triggers proteolysis of ste9/srw1 and causes its dissociation from the APC/C. This mechanism provides a molecular switch to prevent inactivation of cdc2 in G(2) and early mitosis and to allow its inactivation in G(1).     

Human cyclins B1 and B2 are localized to strikingly different structures: B1 to microtubules, B2 primarily to the Golgi apparatus.

We have raised and characterized antibodies specific for human cyclin B2 and have compared the properties of cyclins B1 and B2 in human tissue culture cells. Cyclin B1 and B2 levels are very low in G1 phase, increase in S and G2 phases and peak at mitosis. Both B-type cyclins associate with p34cdc2; their associated kinase activities appear when cells enter mitosis and disappear as the cyclins are destroyed in anaphase. However, human cyclins B1 and B2 differ dramatically in their subcellular localization. Cyclin B1 co-localizes with microtubules, whereas cyclin B2 is primarily associated with the Golgi region. In contrast to cyclin B1, cyclin B2 does not relocate to the nucleus at prophase, but becomes uniformly distributed throughout the cell. The different subcellular locations of human cyclins B1 and B2 implicate them in the reorganization of different aspects of the cellular architecture at mitosis and indicate that different mitotic cyclin-cyclin-dependent kinase complexes may have distinct roles in the cell cycle.     

A fission yeast B-type cyclin functioning early in the cell cycle.

We have cloned a fission yeast gene, cig1+, encoding a 48 kd product that is most similar to cyclin B proteins. The cig1+ protein has a "cyclin box" approximately 40% identical to B-type cyclins of other species, but lacks the "destruction box" required for proteolysis of mitotic cyclins. Deletion of cig1+ had no observable effect on cell viability or progression through G2 or M phase, but instead caused a marked lag in the progression from G1 to S phase. G1 constituted approximately 70% of the cell cycle in cig1 deletion strains, as compared with less than 10% in cig1+ strains. Constitutive cig1+ overexpression was lethal, causing cessation of growth and arrest in G1. Expression of cig1+ failed to rescue an S. cerevisiae strain lacking CLN Start cyclins. Thus, cig1+ identifies a new class of B-type cyclin acting in G1 or S phase that appears to be functionally distinct from all previously described cyclin proteins.     

Regulation of p34cdc2 protein kinase during mitosis

The cell-cycle timing of mitosis in fission yeast is determined by the cdc25+ gene product activating the p34cdc2 protein kinase leading to mitotic initiation. Protein kinase activity remains high in metaphase and then declines during anaphase. Activation of the protein kinase also requires the cyclin homolog p56cdc13, which also functions post activation at a later stage of mitosis. The continuing function of p56cdc13 during mitosis is consistent with its high level until the metaphase/anaphase transition. At anaphase the p56cdc13 level falls dramatically just before the decline in p34cdc2 protein kinase activity. The behavior of p56cdc13 is similar to that observed for cyclins in oocytes. p13suc1 interacts closely with p34cdc2; it is required during the process of mitosis and may play a role in the inactivation of the p34cdc2 protein kinase. Therefore, the cdc25+, cdc13+, and suc1+ gene products are important for regulating p34cdc2 protein kinase activity during entry into, progress through, and exit from mitosis.     

Cyclins of the fission yeast Schizosaccharomyces pombe

Five cyclin-like genes, cig1, cig2/cyc17, mcs2, puc1 and cdc13, have been discovered in S. pombe to date. It is not yet clear what their functions are or even whether they are all involved with control of the cell cycle. Conflicting data for cig1 and cig2/cyc17 have obscured analysis of their function and cig1 remains largely uncharacterized, although clues to the role of cig2/cyc17 have emerged. There is genetic data available for the more distant cyclin homologue mcs2, which has an essential although as yet unspecified role. Puc1 may be involved in regulation of exit from the cell cycle. The first cyclin to be discovered, and the best understood, is cdc13 which with cdc2 promotes mitosis. Studies of the roles of cdc2 and cdc13 in the overall ordering of the cell cycle suggest that cdc13 and probably other cyclins are key regulators, maintaining the order of S phase and mitosis during the cell cycle.     

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.     

Mitotic destruction of the cell cycle regulated NIMA protein kinase of Aspergillus nidulans is required for mitotic exit.

NIMA is a cell cycle regulated protein kinase required, in addition to p34cdc2/cyclin B, for initiation of mitosis in Aspergillus nidulans. Like cyclin B, NIMA accumulates when cells are arrested in G2 and is degraded as cells traverse mitosis. However, it is stable in cells arrested in mitosis. NIMA, and related kinases, have an N-terminal kinase domain and a C-terminal extension. Deletion of the C-terminus does not completely inactivate NIMA kinase activity but does prevent functional complementation of a temperature sensitive mutation of nimA, showing it to be essential for function. Partial C-terminal deletion of NIMA generates a highly toxic kinase although the kinase domain alone is not toxic. Transient induction experiments demonstrate that the partially truncated NIMA is far more stable than the full length NIMA protein which likely accounts for its toxicity. Unlike full length NIMA, the truncated NIMA is not degraded during mitosis and this affects normal mitotic progression. Cells arrested in mitosis with non-degradable NIMA are able to destroy cyclin B, demonstrating that the arrest is not due to stabilization of p34cdc2/cyclin B activity. The data establish that NIMA degradation during mitosis is required for correct mitotic progression in A. nidulans.     

Inhibition of membrane fusion in vitro via cyclin B but not cyclin A.

It is now clear that complexes of cdc2 kinase with "mitotic" cyclins regulate the transition between the G2 phase of the cell cycle and mitosis and that membrane traffic in mammalian cells is arrested during mitosis. Using a cell-free assay, we have previously reported that the fusion of early endosomes is, in fact, inhibited via the cdc2 kinase (Tuomikoski, T., Felix, M.-A., Dorée, M., and Gruenberg, J. (1989) Nature 342, 942-945). In the present paper, we show that this in vitro inhibition occurs efficiently only when the kinase activity is specifically evoked by a cyclin of the B-type but not by cyclins of the A-type. In addition, high resolution two-dimensional gel analysis revealed that the kinases associated with A- and B-type cyclins exhibit different substrate preferences. These data suggest that the complexes of the cdc2 kinase with different cyclins may control specific events of the cell cycle.     

Evidence that the G1-S and G2-M transitions are controlled by different cdc2 proteins in higher eukaryotes.

Xenopus eggs contain two distinct cdc2 homologs of 34 and 32 kd. We show that the 32 kd cdc2 protein, like the 34 kd protein, is a kinase. However, unlike the 34 kd homolog, the 32 kd cdc2 kinase activity does not decrease dramatically at the end of mitosis. The 32 kd protein does not associate with mitotic cyclins B1 and B2 but does associate with cyclin A and a novel doublet of proteins of 54 kd that may regulate its activity. We also show that depletion of the 32 kd cdc2 homolog from a Xenopus extract blocks DNA replication, but does not inhibit entry into mitosis. By contrast, depletion of the 34 kd cdc2 homolog or absence of mitotic cyclins from an extract does not inhibit replication, but does block entry into mitosis. Our results indicate that in higher eukaryotes, DNA replication (G1-S) and mitosis (G2-M) may be controlled by distinctly different cdc2 proteins.     

rum1: a CDK inhibitor regulating G1 progression in fission yeast

In all eukaryotes, entry into mitosis from G2 phase is initiated by a complex of the cdc2 kinase and a B-type cyclin. It has now been shown that, in fission yeast, B-type cyclins also activate cdc2 in G1, thus governing cell-cycle commitment, as well as the onset of S phase. In this article, Karim Labib and Sergio Moreno review the evidence that ruml inhibits the kinase activity of cdc2 associated with B-type cyclins and is an important regulator o f G1 progression in fission yeast.     

Isolation of a human cyclin cDNA: evidence for cyclin mRNA and protein regulation in the cell cycle and for interaction with p34cdc2

This paper reports the nucleotide and predicted amino acid sequence of a human B-type cyclin. The predicted protein sequence shows strong homology to the other known cyclins in the central third of the protein. We show that the level of cyclin mRNA is regulated during the cell cycle, increasing during G2 phase to four time that present in G1. The protein accumulates steadily during G2 to at least 20 times its level in G1 and is abruptly destroyed at mitosis. In G2/M phase, cyclin is associated with p34cdc2, the human homolog of the fission yeast gene cdc2+, and this complex has histone H1 kinase activity.     

Differential function and expression of Saccharomyces cerevisiae B-type cyclins in mitosis and meiosis.

We have studied the patterns of expression of four B-type cyclins (Clbs), Clb1, Clb2, Clb3, and Clb4, and their ability to activate p34cdc28 during the mitotic and meiotic cell cycles of Saccharomyces cerevisiae. During the mitotic cell cycle, Clb3 and Clb4 were expressed and induced a kinase activity in association with p34cdc28 from early S phase up to mitosis. On the other hand, Clb1 and Clb2 were expressed and activated p34cdc28 later in the mitotic cell cycle, starting in late S phase and continuing up to mitosis. The pattern of expression of Clb3 and Clb4 suggests a possible role in the regulation of DNA replication as well as mitosis. Clb1 and Clb2, whose pattern of expression is similar to that of other known Clbs, are likely to have a role predominantly in the regulation of M phase. During the meiotic cell cycle, Clb1, Clb3, and Clb4 were expressed and induced a p34cdc28-associated kinase activity just before the first meiotic division. The fact that Clb3 and Clb4 were not synthesized earlier, in S phase, suggests that these cyclins, which probably have a role in S phase during the mitotic cell cycle, are not implicated in premeiotic S phase. Clb2, the primary mitotic cyclin in S. cerevisiae, was not detectable during meiosis. Sporulation experiments on strains deleted for one, two, or three Clbs indicate, in agreement with the biochemical data, that Clb1 is the primary cyclin for the regulation of meiosis, while Clb2 is not involved at all.     

Members of the NAP/SET family of proteins interact specifically with B- type cyclins

Cyclin-dependent kinase complexes that contain the same catalytic subunit are able to induce different events at different times during the cell cycle, but the mechanisms by which they do so remain largely unknown. To address this problem, we have used affinity chromatography to identify proteins that bind specifically to mitotic cyclins, with the goal of finding proteins that interact with mitotic cyclins to carry out the events of mitosis. This approach has led to the identification of a 60-kD protein called NAP1 that interacts specifically with members of the cyclin B family. This interaction has been highly conserved during evolution: NAP1 in the Xenopus embryo interacts with cyclins B1 and B2, but not with cyclin A, and the S. cerevisiae homolog of NAP1 interacts with Clb2 but not with Clb3. Genetic experiments in budding yeast indicate that NAP1 plays an important role in the function of Clb2, while biochemical experiments demonstrate that purified NAP1 can be phosphorylated by cyclin B/p34cdc2 kinase complexes, but not by cyclin A/p34cdc2 kinase complexes. These results suggest that NAP1 is a protein involved in the specific functions of cyclin B/p34cdc2 kinase complexes. In addition to NAP1, we found a 43-kD protein in Xenopus that is homologous to NAP1 and also interacts specifically with B-type cyclins. This protein is the Xenopus homolog of the human SET protein, which was previously identified as part of a putative oncogenic fusion protein (Von Lindern et al., 1992).     

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.     

p25rum1 promotes proteolysis of the mitotic B-cyclin p56cdc13 during G1 of the fission yeast cell cycle.

The fission yeast Schizosaccharomyces pombe CDK inhibitor p25rum1 plays a major role in regulating cell cycle progression during G1. Here we show that p25rum1 associates with the CDK p34cdc2/p56cdc13 during G1 in normally cycling cells and is required for the rapid proteolysis of p56cdc13. In vitro binding data indicate that p25rum1 has specificity for the B-cyclin p56cdc13 component of the CDK and can bind the cyclin even in the absence of the cyclin destruction box. At the G1-S-phase transition, p25rum1 levels decrease and p56cd13 levels increase. We also show that on release from a G1 block, the rapid disappearance of p25rum1 requires the activity of the CDK p34cdc2/cig1p and that this same CDK phosphorylates p25rum1 in vitro. We propose that the binding of p25rum1 to p56cdc13 promotes cyclin proteolysis during G1, with p25rum1 possibly acting as an adaptor protein, promoting transfer of p56cdc13 to the proteolytic machinery. At the G1-S-phase transition, p25rum1 becomes targeted for proteolysis by a mechanism which may involve p34cdc2/cig1p phosphorylation. As a consequence, at this point in the cell cycle p56cdc13 proteolysis is inhibited, leading to a rise of p56cdc13 levels in preparation for 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.