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.     

The Roles of Cyclin A2, B1, and B2 in Early and Late Mitotic Events

Here we have used siRNAs and time-lapse epifluorescence microscopy to examine the roles of various candidate mitotic cyclins in chromatin condensation in HeLa cells. Knocking down cyclin A2 resulted in a substantial (∼7 h) delay in chromatin condensation and histone H3 phosphorylation, and expressing an siRNA-resistant form of cyclin A2 partially rescued chromatin condensation. There was no detectable delay in DNA replication in the cyclin A2 knockdowns, arguing that the delay in chromatin condensation is not secondary to a delay in S-phase completion. Cyclin A2 is required for the activation and nuclear accumulation of cyclin B1-Cdk1, raising the possibility that cyclin B1-Cdk1 mediates the effects of cyclin A2. Consistent with this possibility, we found that chromatin condensation was tightly associated temporally with the redistribution of cyclin B1 to the nucleus. Moreover, a constitutively nuclear cyclin B1 rescued chromatin condensation in cyclin A2 knockdown cells. On the other hand, knocking down cyclin B1 delayed chromatin condensation by only about one hour. Our working hypothesis is that active, nuclear cyclin B1-Cdk1 normally cooperates with cyclin A2 to bring about early mitotic events. Because cyclin A2 is present only during the early stages of mitosis, we asked whether cyclin B knockdown might have more dramatic defects on late mitotic events. Consistent with this possibility, we found that cyclin B1- and cyclin B1/B2-knockdown cells had difficulty in maintaining a mitotic arrest in the presence of nocodazole. Taken together, these data suggest that cyclin A2 helps initiate mitosis, in part through its effects on cyclin B1, and that cyclins B1 and B2 are particularly critical for the maintenance of the mitotic state.     

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.     

The proteolysis of mitotic cyclins in mammalian cells persists from the end of mitosis until the onset of S phase.

We have studied how the cell cycle-specific oscillations of mitotic B-type cyclins are generated in mouse fibroblasts. A reporter enzyme comprising the N-terminus of a B-type cyclin fused to bacterial chloramphenicol acetyl transferase (CAT) was degraded at the end of mitosis like endogenous cyclins. Point mutations in the destruction box of this construct completely abolished its mitotic instability. When the destructible reporter was driven by the cyclin B2 promoter, CAT activity mimicked the oscillations in the level of the endogenous cyclin B2. These oscillations were largely conserved when the reporter was transcribed constitutively from the SV40 promoter. Pulse-chase experiments or addition of the proteasome inhibitors lactacystin and ALLN showed that cyclin synthesis continued after the end of mitosis. The destruction box-specific degradation of cyclins normally ceases at the onset of S phase, and is active in fibroblasts arrested in G0 and in differentiated C2 myoblasts. We were able to reproduce this proteolysis in vitro in extracts of synchronized cells. Extracts of G1 cells degraded cyclin B1 whereas p27Kip1 was stable, in contrast, cyclin B1 remained stable and p27Kip1 was degraded in extracts of S phase cells.     

Cyclin A2 regulates nuclear-envelope breakdown and the nuclear accumulation of cyclin B1

Mitosis is thought to be triggered by the activation of Cdk-cyclin complexes. Here we have used RNA interference (RNAi) to assess the roles of three mitotic cyclins, cyclins A2, B1, and B2, in the regulation of centrosome separation and nuclear-envelope breakdown (NEB) in HeLa cells. We found that the timing of NEB was affected very little by knocking down cyclins B1 and B2 alone or in combination. However, knocking down cyclin A2 markedly delayed NEB, and knocking down both cyclins A2 and B1 delayed NEB further. The timing of cyclin B1-Cdk1 activation was normal in cyclin A2 knockdown cells, and there was no delay in centrosome separation, an event apparently controlled by the activation of cytoplasmic cyclin B1-Cdk1. However, nuclear accumulation of cyclin B1-Cdk1 was markedly delayed in cyclin A2 knockdown cells. Finally, a constitutively nuclear cyclin B1, but not wild-type cyclin B1, restored normal NEB timing in cyclin A2 knockdown cells. These findings show that cyclin A2 is required for timely NEB, whereas cyclins B1 and B2 are not. Nevertheless cyclin B1 translocates to the nucleus just prior to NEB in a cyclin A2-dependent fashion and is capable of supporting NEB if rendered constitutively nuclear.     

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.     

Specialized roles of the two mitotic cyclins in somatic cells: cyclin A as an activator of M phase-promoting factor

The role of cyclin B-CDC2 as M phase-promoting factor (MPF) is well established, but the precise functions of cyclin A remain a crucial outstanding issue. Here we show that down-regulation of cyclin A induces a G2 phase arrest through a checkpoint-independent inactivation of cyclin B-CDC2 by inhibitory phosphorylation. The phenotype is rescued by expressing cyclin A resistant to the RNA interference. In contrast, down-regulation of cyclin B disrupts mitosis without inactivating cyclin A-CDK, indicating that cyclin A-CDK acts upstream of cyclin B-CDC2. Even when ectopically expressed, cyclin A cannot replace cyclin B in driving mitosis, indicating the specific role of cyclin B as a component of MPF. Deregulation of WEE1, but not the PLK1-CDC25 axis, can override the arrest caused by cyclin A knockdown, suggesting that cyclin A-CDK may tip the balance of the cyclin B-CDC2 bistable system by initiating the inactivation of WEE1. These observations show that cyclin A cannot form MPF independent of cyclin B and underscore a critical role of cyclin A as a trigger for MPF activation.     

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.     

Unmasking the Redundancy Between Cdk1 and Cdk2 at G2 Phase in Human Cancer Cell Lines

A series of studies published in 2003 has challenged the essentiality of Cdk2. A recently published work indicates that cyclin E-Cdk1 compensates for Cdk2’s function at G1/S transition in Cdk2-/- Mefs. In this study, we uncovered a redundant mechanism between Cdk1 and Cdk2 at G2 in multiple cancer cell lines. When either Cdk2 or Cdk1 is ablated using RNAi, there were complex shifts of cyclin A towards its reciprocal partner, i.e., when Cdk2 is ablated, cyclin A redistributes to Cdk1; when Cdk1 is ablated, cyclin A forms more abundant complexes with Cdk2. Further, cyclin B redistributes to Cdk2 upon Cdk1 knockdown. These redistributions bring about increased kinase activities of corresponding complexes. Elimination of the compensatory mechanism by knockdown of both Cdk1 and Cdk2 using RNAi reveals phenotypes at G2 phase. The results suggest that the redistributed complexes contribute to the cyclin B-Cdk1 activation when either Cdk1 or Cdk2 alone is ablated and this redundancy masks Cdk2’s role when Cdk2 is singly ablated. It is also worth noting that the predominant G2 arrest described here, unlike those Cdk1-Cdk2 double ablated Mefs, raises a question of whether different Cdk activities are required for G1/S or G2/M progression in normal vs. cancer cells.     

Cyclin B1 is rate limiting but not essential for mitotic entry and progression in mammalian somatic cells

Cyclin B1 should have some rate limiting function for cell cycle progression. To test this, we measured the effect of siRNA-mediated depletion of cyclin B1 on mitotic entry and timing. We depleted cyclin B1 in HeLa and hTert-RPE1 cells to levels equivalent or below those achieved in the telophase-to-G1 window. Average cyclin B1/Cdk1 activity was measured in HeLa cells and depleted by ~99%. In both cell lines, this caused ~20% increase in the G2 and ~20% increase the M traverse time. However, co-depletion of cyclin B1 and B2 induced a profound increase in G2 cells, a dramatic reduction in mitotic cells, and an increase in a 4C cycling population. We conclude that any residual levels of cyclin B1 were not sufficient to promote stable mitotic entry and transition in absence of normal levels of cyclin B2. Therefore, we conclude that B cyclin is necessary for mitosis but cyclin B1 is not. Nocodazole treated, cyclin B1-depleted HeLa cells arrested but exited that arrest at higher rates than controls, suggesting that the duration of the spindle checkpoint was affected. In B1 depleted cells, population growth was delayed but evidence of cell death was not consistently observed. A strong phenotype of mitotic chromosomal aberration was observed in HeLa cells depleted for either cyclin but not in RPE cells. In B1 or B2 depleted cells, maloriented chromosomes at metaphase were increased 10 fold and one third of affected metaphase cells entered anaphase without congression. Lagging chromosomes at anaphase were dramatically increased. The aggregate evidence from our study and others suggests that the common effect of cyclin B1 depletion is mild cell cycle perturbation. Lack of uniformity in other phenotypes suggest that these are low penetrance effects that are exacerbated or compensated in some systems by other mechanisms.     

Sub-cellular localisation of GFP-tagged tobacco mitotic cyclins during the cell cycle and after spindle checkpoint activation

We have previously shown that the tobacco cyclin B1;1 protein accumulates during the G2 phase of the cell cycle and is subsequently destroyed during mitosis. Here, we investigated the sub-cellular localisation of two different B1-types and one A3-type cyclin during the cell cycle by using confocal imaging and differential interference contrast (DIC) microscopy. The cyclins were visualised as GFP-tagged fusion proteins in living tobacco cells. Both B1-type cyclins were found in the cytoplasm and in the nucleus during G2 but when cells entered into prophase, both cyclins became associated with condensing chromatin and remained on chromosomes until metaphase. As cells exited metaphase, the B1-type cyclins became degraded, as shown by time-lapse images. A stable variant of cyclin B1;1-GFP fusion protein, in which the destruction box had been mutated, maintained its association with the nuclear material at later phases of mitosis such as anaphase and telophase. Furthermore, we demonstrated that cyclin B1;1 protein is stabilised in metaphase-arrested cells after microtubule destabilising drug treatments. In contrast to the B1-type cyclins, the cyclin A3;1 was found exclusively in the nucleus in interphase cells and disappeared earlier than the cyclin B1 proteins during mitosis.     

Cyclin levels during TNF-alpha-induced apoptosis in HIV-infected cells.

Cyclins are cell cycle regulatory proteins. We compared the concurrent kinetics of apoptosis and cyclin expression between HIV-infected cells (J1.1), and uninfected Jurkat cells. Cells were cultured with TNF-alpha and harvested at 24, 48 and 72 hr to examine cyclin expression and DNA content. We found a decline in the levels of the mitotic B cyclin in Jurkat cells (16 to 2%, 48 hr), while in J1.1 cells it was observed in cyclin E (60 to 37%, 72 hr). Because cyclin B is mitotic, results suggest that Jurkat cells undergo apoptosis at G2, while J1.1 cells enter mitosis and then die by apoptosis, as no changes in cyclin B or DNA content at G2M were observed. G1 cyclin E decline in J1.1 cells also suggests that they die after entering mitosis. Based on differences in the cyclins involved, it seems that HIV-1 manipulates the cell cycle to protect J1.1 cells from apoptosis induction at G2, a critical cell cycle phase for HIV replication. Thus, cyclins are useful to characterize points in the cell cycle at which apoptosis is induced, and could become excellent tools to evaluate mechanisms of action of antiretroviral drugs in the cell cycle of HIV-infected cells.     

Loss of p53 induces M-phase retardation following G2 DNA damage checkpoint abrogation

Most cell lines that lack functional p53 protein are arrested in the G2 phase of the cell cycle due to DNA damage. When the G2 checkpoint is abrogated, these cells are forced into mitotic catastrophe. A549 lung adenocarcinoma cells, in which p53 was eliminated with the HPV16 E6 gene, exhibited efficient arrest in the G2 phase when treated with adriamycin. Administration of caffeine to G2-arrested cells induced a drastic change in cell phenotype, the nature of which depended on the status of p53. Flow cytometric and microscopic observations revealed that cells that either contained or lacked p53 resumed their cell cycles and entered mitosis upon caffeine treatment. However, transit to the M phase was slower in p53-negative cells than in p53-positive cells. Consistent with these observations, CDK1 activity was maintained at high levels, along with stable cyclin B1, in p53-negative cells. The addition of butyrolactone I, which is an inhibitor of CDK1 and CDK2, to the p53-negative cells reduced the floating round cell population and induced the disappearance of cyclin B1. These results suggest a relationship between the p53 pathway and the ubiquitin-mediated degradation of mitotic cyclins and possible cross-talk between the G2-DNA damage checkpoint and the mitotic checkpoint.     

Overexpression of the cytoplasmic retention signal region of cyclin B2, but not of cyclin B1, inhibits bipolar spindle formation in Xenopus oocytes

Cyclin B, a regulatory subunit of maturation/M-phase promoting factor (MPF), has several subtypes in many vertebrate species. However, it is not known whether the different B-type cyclins have any different functions in vertebrate cells, although their subcellular localizations seem to differ largely from each other. To examine the roles of two major B-type cyclins, B1 and B2, in spindle formation in M phase, we overexpressed their N-termini in Xenopus oocytes; the N-termini of cyclins B1 and B2 contained a cytoplasmic retention signal (CRS), and hence their overexpressions were expected to competitively inhibit the subcellular localizations of the endogenous cyclins B1 and B2, respectively. Upon entry into meiosis I, oocytes overexpressing the cyclin B1 N-terminus formed an apparently normal bipolar spindle, but those oocytes overexpressing the cyclin B1 N-terminus formed a monopolar (or monoastral) spindle. This defect in bipolar spindle formation was observed only when the cyclin B2 N-terminus contained its own CRS sequence, and was able to be rescued by overexpression of full-length cyclin B2. These results suggest, for the first time, that the correct subcellular localization of cyclin B2, but not of cyclin B1, is essential for (the initiation of) bipolar spindle formation in Xenopus oocytes.     

Both cyclin A delta 60 and B delta 97 are stable and arrest cells in M-phase, but only cyclin B delta 97 turns on cyclin destruction.

Previous work has established that destruction of cyclin B is necessary for exit from mitosis and entry into the next interphase. Sea urchin cyclin B lacking an N-terminal domain is stable, permanently activates cdc2 kinase, resulting in mitotic arrest, and permanently activates the destruction pathway acting on full length cyclin B. Here we have compared the properties of clam cyclins A and B lacking related N-terminal domains. Both cyclin A delta 60 and B delta 97 bind to cdc2 kinase, keep it hyperactivated and block the completion of mitosis. By adding purified delta cyclin proteins to a cell-free system at different cell cycle times, we find that when the cell-free system reaches the cyclin destruction point in the presence of either A delta 60 or B delta 97, the cyclin destruction pathway acting on full length cyclins fails to be turned off. However, the two cyclins differ dramatically in their ability to turn on cyclin destruction. When added to emetine-arrested interphase lysates devoid of endogenous cyclins, only cyclin B delta 97 activates the cyclin destruction system; cyclin A delta 60 does not. This functional difference between the two cyclin types, the first to be described, provides strong support for the idea that the two cyclins have different roles in the cell cycle and suggests that one specialized role of the cyclin B-cdc2 complex is to activate the cyclin destruction pathway and drive cells into interphase of the next cell cycle.     

The schedule of destruction of three mitotic cyclins can dictate the timing of events during exit from mitosis

BACKGROUND: Degradation of the mitotic cyclins is a hallmark of the exit from mitosis. Induction of stable versions of each of the three mitotic cyclins of Drosophila, cyclins A, B, and B3, arrests mitosis with different phenotypes. We tested a recent proposal that the destruction of the different cyclins guides progress through mitosis. RESULTS: Real-time imaging revealed that arrest phenotypes differ because each stable cyclin affects specific mitotic events differently. Stable cyclin A prolonged or blocked chromosome disjunction, leading to metaphase arrest. Stable cyclin B allowed the transition to anaphase, but anaphase A chromosome movements were slowed, anaphase B spindle elongation did not occur, and the monooriented disjoined chromosomes began to oscillate between the spindle poles. Stable cyclin B3 prevented normal spindle maturation and blocked major mitotic exit events such as chromosome decondensation but nonetheless allowed chromosome disjunction, anaphase B, and formation of a cytokinetic furrow, which split the spindle. CONCLUSIONS: We conclude that degradation of distinct mitotic cyclins is required to transit specific steps of mitosis: cyclin A degradation facilitates chromosome disjunction, cyclin B destruction is required for anaphase B and cytokinesis and for directional stability of univalent chromosome movements, and cyclin B3 degradation is required for proper spindle reorganization and restoration of the interphase nucleus. We suggest that the schedule of degradation of cyclin A, cyclin B, and then cyclin B3 contributes to the temporal coordination of mitotic events.     

The yeast cell cycle engine

The yeast cell cycle is regulated by a number of different cyclin-Cdc28 complexes, some of which orchestrate G1 events, and some of which orchestrate G2/M events. G1 cyclins lead to expression of G2 cyclins; the G2 cyclins then repress the G1 cyclins. G2 cyclin expression eventually leads to mitosis, which causes loss of the G2 cyclins, allowing derepression and reappearance of the G1 cyclins. These interactions between different classes of cyclins push the yeast cell cycle forward. Nutrients act through the G1 cyclins to stimulate division, while mating pheromones act through G1 cyclins to inhibit division.     

Cdc2 protein kinase is complexed with both cyclin A and B: evidence for proteolytic inactivation of MPF

In the clam, Spisula, two previously described proteins known as cyclin A and B display the unusual property of selective proteolytic degradation at the end of each mitosis. We show here that clam oocytes and embryos contain a cdc2 protein kinase. This protein kinase is a component of the M phase promoting factor (MPF) in frog eggs and the M phase-specific histone H1 kinase in starfish. Clam cdc2 is found in association with both cyclin A and B, probably not as a trimolecular association, but as separate cdc2/cyclin A and cdc2/cyclin B complexes. Clam cdc2 and the associated cyclins bind to p13suc1-Sepharose. The p13-bound complex, and also anti-cyclin A or B immunoprecipitates, each display cell cycle-dependent histone H1 kinase activity. We suggest that in addition to the cdc2 protein kinase, the cyclins are further components of the M phase promoting factor and that cyclin proteolysis provides the mechanism of MPF inactivation and thus exit from mitosis.