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.     

Cell cycle function of a Medicago sativa A2-type cyclin interacting with a PSTAIRE-type cyclin-dependent kinase and a retinoblastoma protein

In plants multiple A-type cyclins with distinct expression patterns have been isolated and classified into three subgroups (A1-A3), while in animal somatic cells a single type of cyclin A is required for cell-cycle regulation from the S to M phases. We studied the function of an A2-type cyclin from Medicago sativa (Medsa;cycA2) which, in contrast to animal and most plant A-type cyclins, was expressed in all phases of the cell cycle. Using synchronized alfalfa cell cultures and anti-Medsa;CycA2 polyclonal antibodies, we showed that while the mRNA level increased steadily from the late G1 to the G2-M phase, the protein level after a rapid increase in S-phase reached a plateau during the G2 phase. In the yeast two-hybrid system, the Medsa;CycA2 protein interacted with the PSTAIRE-motif-containing cyclin-dependent kinase Cdc2MsA and with the maize retinoblastoma protein. Unexpectedly, the CycA2-associated kinase activity was biphasic: a first activity peak occurred in the S phase while the major one occurred during the G2/M transition, with no apparent dependence upon the actual levels of the Medsa;CycA2 and Cdc2MsA proteins. Immunohistological localization of the cyclin A2 protein by immunofluorescence and immunogold labelling revealed the presence of Medsa;CycA2 in the nucleus of the interphase and prophase cells, while it was undetectable thereafter during mitosis. Together these data suggest that Medsa;CycA2 plays a role both in the S phase and at the G2/M transition.     

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.     

A new pair of B-type cyclins from Saccharomyces cerevisiae that function early in the cell cycle.

Two new B-type cyclin genes from Saccharomyces cerevisiae, called CLB5 and CLB6, are located in a tail to tail arrangement adjacent to the G2/M phase promoting cyclins CLB2 and CLB1, respectively. These genomic cyclin arrays are flanked by tRNAs and repeated sequences of Ty elements suggesting an intrachromosomal gene duplication followed by an interchromosomal gene duplication. Based on their deduced protein sequence the CLB5 and CLB6 genes form a new pair of B-type cyclins. They are most related to each other and then to the deduced protein sequence of their adjacent genes CLB1 and CLB2. Both genes are periodically expressed, peaking early in the cell cycle. Loss of function mutants are viable, but clb5- mutants exhibit a delay in S phase whereas clb6- mutants show a delay in late G1 and/or S phase. The clb5 mutant phenotype is somewhat more pronounced in a double null mutant. Both cyclins have the potential to interact with the p34CDC28 kinase in vivo.     

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.     

The multiple roles of cyclin E1 in controlling cell cycle progression and cellular morphology of Trypanosoma brucei

Regulation of eukaryotic cell cycle progression requires sequential activation and inactivation of cyclin-dependent kinases. Previous RNA interference (RNAi) experiments in Trypanosoma brucei indicated that cyclin E1, cdc2-related kinase (CRK)1 and CRK2 are involved in regulating G1/S transition, whereas cyclin B2 and CRK3 play a pivotal role in controlling the G2/M checkpoint. To search for potential interactions between the other cyclins and CRKs that may not have been revealed by the RNAi assays, we used the yeast two-hybrid system and an in vitro glutathione-S-transferase pulldown assay and observed interactions between cyclin E1 and CRK1, CRK2 and CRK3. Cyclins E1-E4 are homologues of yeast Pho80 cyclin. But yeast complementation assays indicated that none of them possesses a Pho80-like function. Analysis of cyclin E1+CRK1 and cyclin E1+CRK2 double knockdowns in the procyclic form of T. brucei indicated that the cells were arrested more extensively in the G1 phase beyond the cumulative effect of individual knockdowns. But BrdU incorporation was impaired significantly only in cyclin E1+CRK1-depleted cells, whereas a higher percentage of cyclin E1+CRK2 knockdown cells assumed a grossly elongated posterior end morphology. A double knockdown of cyclin E1 and CRK3 arrested cells in G2/M much more efficiently than if only CRK3 was depleted. Taken together, these data suggest multiple functions of cyclin E1: it forms a complex with CRK1 in promoting G1/S phase transition; it forms a complex with CRK2 in controlling the posterior morphogenesis during G1/S transition; and it forms a complex with CRK3 in promoting passage across the G2/M checkpoint in the trypanosome.     

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.     

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.     

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.     

Control of Swe1p degradation by the morphogenesis checkpoint.

In the budding yeast Saccharomyces cerevisiae, a cell cycle checkpoint coordinates mitosis with bud formation. Perturbations that transiently depolarize the actin cytoskeleton cause delays in bud formation, and a 'morphogenesis checkpoint' detects the actin perturbation and imposes a G2 delay through inhibition of the cyclin-dependent kinase, Cdc28p. The tyrosine kinase Swe1p, homologous to wee1 in fission yeast, is required for the checkpoint-mediated G2 delay. In this report, we show that Swe1p stability is regulated both during the normal cell cycle and in response to the checkpoint. Swe1p is stable during G1 and accumulates to a peak at the end of S phase or in early G2, when it becomes unstable and is degraded rapidly. Destabilization of Swe1p in G2 and M phase depends on the activity of Cdc28p in complexes with B-type cyclins. Several different perturbations of actin organization all prevent Swe1p degradation, leading to the persistence or further accumulation of Swe1p, and cell cycle delay in G2.     

The Stress-activated Protein Kinase Hog1 Mediates S Phase Delay in Response to Osmostress

Control of cell cycle progression by stress-activated protein kinases (SAPKs) is essential for cell adaptation to extracellular stimuli. Exposure of yeast to osmostress activates the Hog1 SAPK, which modulates cell cycle progression at G1 and G2 by the phosphorylation of elements of the cell cycle machinery, such as Sic1 and Hsl1, and by down-regulation of G1 and G2 cyclins. Here, we show that upon stress, Hog1 also modulates S phase progression. The control of S phase is independent of the S phase DNA damage checkpoint and of the previously characterized Hog1 cell cycle targets Sic1 and Hsl1. Hog1 uses at least two distinct mechanisms in its control over S phase progression. At early S phase, the SAPK prevents firing of replication origins by delaying the accumulation of the S phase cyclins Clb5 and Clb6. In addition, Hog1 prevents S phase progression when activated later in S phase or cells containing a genetic bypass for cyclin-dependent kinase activity. Hog1 interacts with components of the replication complex and delays phosphorylation of the Dpb2 subunit of the DNA polymerase. The two mechanisms of Hog1 action lead to delayed firing of origins and prolonged replication, respectively. The Hog1-dependent delay of replication could be important to allow Hog1 to induce gene expression before replication.     

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.     

Conservation of function and regulation within the Cdc28/cdc2 protein kinase family: characterization of the human Cdc2Hs protein kinase in Saccharomyces cerevisiae.

Whereas the Cdc28 protein kinase of the budding yeast Saccharomyces cerevisiae plays an essential role in cell cycle progression during the G1 interval, a function in the progression from the G2 interval into M phase has been inferred for its homologs, including the Cdc2Hs protein kinase of humans. To better understand these apparently disparate roles, we constructed a yeast strain in which the resident CDC28 gene was replaced by its human homolog, CDC2Hs. This transgenic yeast strain was able to perform the G1 functions attributed to the Cdc28 protein kinase, including the ability to grow and divide normally, to respond to environmental signals that induce G1 arrest, and to regulate the Cdc2Hs protein kinase appropriately in response to these signals.     

Clb5-associated Kinase Activity is Required Early in the Spindle Pathway for Correct Preanaphase Nuclear Positioning in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, a single cyclin-dependent kinase, Cdc28, regulates both G1/S and G2/M phase transitions by associating with stage-specific cyclins. During progression through S phase and G2/M, Cdc28 is activated by the B-type cyclins Clb1–6. Because of functional redundancy, specific roles for individual Clbs have been difficult to assign. To help genetically define such roles, strains carrying a cdc28^ts allele, combined with single CLB deletions were studied. We assumed that by limiting the activity of the kinase, these strains would be rendered more sensitive to loss of individual Clbs.