A genome–wide screen to identify genes controlling the rate of entry into mitosis in fission yeast

We have carried out a haploinsufficiency (HI) screen in fission yeast using heterozygous deletion diploid mutants of a genome-wide set of cell cycle genes to identify genes encoding products whose level determines the rate of progression through the cell cycle. Cell size at division was used as a measure of advancement or delay of the G2-M transition of rod-shaped fission yeast cells. We found that 13 mutants were significantly longer or shorter (greater than 10%) than control cells at cell division. These included mutants of the cdc2, cdc25, wee1 and pom1 genes, which have previously been shown to play a role in the timing of entry into mitosis, and which validate this approach. Seven of these genes are involved in regulation of the G2-M transition, 5 for nuclear transport and one for nucleotide metabolism. In addition we identified 4 more genes that were 8–10% longer or shorter than the control that also had roles in regulation of the G2-M transition or in nuclear transport. The genes identified here are all conserved in human cells, suggesting that this dataset will be useful as a basis for further studies to identify rate-limiting steps for progression through the cell cycle in other eukaryotes.     

Securin regulates entry into M-phase by modulating the stability of cyclin B

Timely progression into mitosis is necessary for normal cell division. This transition is sensitive to the levels of cyclin B, the regulatory subunit of the master mitotic kinase, Cdk1. Cyclin B accumulates during G2 and prophase when its rate of destruction by the anaphase promoting complex (APC) is low. Securin is also an APC substrate and is known for its role in inactivating the cohesin-cleaving enzyme, separase, until the metaphase to anaphase transition. Here we show that securin has an additional role in cell-cycle regulation, that of modulating the timing of entry into M-phase. In mouse oocytes, excess securin caused stabilization of cyclin B and precocious entry into M-phase. Depletion of securin increased cyclin B degradation, resulting in delayed progression into M-phase. This effect required APC activity and was reversed by expression of wild-type securin. These data reveal a role for securin at the G2-M transition and suggest a more general mechanism whereby physiological levels of co-competing APC substrates function in modulating the timing of cell-cycle transitions.     

Segregation of unreplicated chromosomes in Saccharomyces cerevisiae reveals a novel G1/M-phase checkpoint.

Saccharomyces cerevisiae dbf4 and cdc7 cell cycle mutants block initiation of DNA synthesis (i.e., are iDS mutants) at 37 degrees C and arrest the cell cycle with a 1C DNA content. Surprisingly, certain dbf4 and cdc7 strains divide their chromatin at 37 degrees C. We found that the activation of the Cdc28 mitotic protein kinase and the Dbf2 kinase occurred with the correct relative timing with respect to each other and the observed division of the unreplicated chromatin. Furthermore, the division of unreplicated chromatin depended on a functional spindle. Therefore, the observed nuclear division resembled a normal mitosis, suggesting that S. cerevisiae commits to M phase in late G1 independently of S phase. Genetic analysis of dbf4 and cdc7 strains showed that the ability to restrain mitosis during a late G1 block depended on the genetic background of the strain concerned, since the dbf4 and cdc7 alleles examined showed the expected mitotic restraint in other backgrounds. This restraint was genetically dominant to lack of restraint, indicating that an active arrest mechanism, or checkpoint, was involved. However, none of the previously described mitotic checkpoint pathways were defective in the iDS strains that carry out mitosis without replicated DNA, therefore indicating that the checkpoint pathway that arrests mitosis in iDS mutants is novel. Thus, spontaneous strain differences have revealed that S. cerevisiae commits itself to mitosis in late G1 independently of entry into S phase and that a novel checkpoint mechanism can restrain mitosis if cells are blocked in late G1. We refer to this as the G1/M-phase checkpoint since it acts in G1 to restrain mitosis.     

The function of the chicken p34CDC2 protein kinase in fission yeast is cold sensitive for cell cycle progression through the G1 phase and temperature sensitive for traversal of mitosis

The protein kinase p34^cdc2 is required at the onset of DNA replication and for entry into mitosis. The catalytic subunit and its regulatory proteins, notably the cyclins, are conserved from yeast to man. This suggests that the control mechanisms necessary for progression through the cell cycle in fission yeast are conserved throughout evolution. This work describes the characterization of a fission yeast strain that is dependent for cell cycle progression on the activity of the p34^CDC2 protein kinase from chicken. The response of the chicken p34^CDC2 protein kinase to cell cycle components of fission yeast was examined. Cells expressing the chicken p34^CDC2 protein divide at reduced size at 31°?C. Cells are temperature sensitive at 35.5°?C and die as a result of mitotic catastrophe. This phenotype can be rescued by delaying cell cycle progression at the G1-S transition by adding low concentrations of hydroxyurea. Schizosaccharomyces pombe cells that are dependent on chicken p34^CDC2 are cold sensitive. At 19°?C to 25°?C cells arrest in the G1 phase, while traversal of the G2-M transition is not blocked at low temperature. Expression of chicken p34^CDC2 in the cold-sensitive G2-M mutant cdc2A21 suppresses the G1 arrest.     

Promotion of mitosis by activated protein kinase B after DNA damage involves polo-like kinase 1 and checkpoint protein CHFR

The role of the protein kinase B (PKB/Akt) in the regulation of cell survival and proliferation is well established. PKB is a key effector in the phosphatidylinositol 3-kinase pathway and plays a role in the initiation of S phase and in the G(2)-M transition. I report here that activated PKB shortens the G(2) arrest induced by DNA damage and promotes early entry into mitosis. Activated PKB supports high levels of expression and activity of the polo-like kinase 1 (Plk1) after DNA damage as cells accumulate in G(2). The checkpoint protein CHFR implicated in degradation of Plk1 is involved in the regulation of Plk1 by PKB. PKB phosphorylates CHFR in vitro and in vivo. Expression of a mutant form of CHFR that cannot be phosphorylated by PKB results in reduction of levels of Plk1 and inhibition of mitotic entry under normal conditions and after DNA damage. Results of this study support a model in which PKB facilitates mitotic resolution of DNA damage-induced G(2) arrest by inhibiting the checkpoint function of CHFR. The deregulated activation of PKB that occurs frequently in tumors might inhibit CHFR activity after DNA damage and therefore promote Plk1 accumulation leading to the disruption of the DNA damage checkpoint.     

The function of the chicken p34CDC2 protein kinase in fission yeast is cold sensitive for cell cycle progression through the G1 phase and temperature sensitive for traversal of mitosis.

The protein kinase p34^cdc2 is required at the onset of DNA replication and for entry into mitosis. The catalytic subunit and its regulatory proteins, notably the cyclins, are conserved from yeast to man. This suggests that the control mechanisms necessary for progression through the cell cycle in fission yeast are conserved throughout evolution. This work describes the characterization of a fission yeast strain that is dependent for cell cycle progression on the activity of the p34^CDC2 protein kinase from chicken. The response of the chicken p34^CDC2 protein kinase to cell cycle components of fission yeast was examined. Cells expressing the chicken p34^CDC2 protein divide at reduced size at 31° C. Cells are temperature sensitive at 35.5° C and die as a result of mitotic catastrophe. This phenotype can be rescued by delaying cell cycle progression at the G1-S transition by adding low concentrations of hydroxyurea. Schizosaccharomyces pombe cells that are dependent on chicken p34^CDC2 are cold sensitive. At 19° C to 25° C cells arrest in the G1 phase, while traversal of the G2-M transition is not blocked at low temperature. Expression of chicken p34^CDC2 in the cold-sensitive G2-M mutant cdc2A21 suppresses the G1 arrest. Received: 14 October 1998 / Accepted: 15 March 1999     

mei-41 and bub1 block mitosis at two distinct steps in response to incomplete DNA replication in Drosophila embryos.

Drosophila double park encodes a homolog of Cdt1 that functions in initiation of DNA replication in fission yeast and Xenopus. dup mutants complete the first 15 embryonic cell cycles, presumably via maternal dup products, and show defects in the 16(th) S phase (S16). Cells carrying dup(a1) allele forgo S16 altogether but enter mitosis 16 (M16). We find that the timing of entry into M16 is similar in dup(a1) and heterozygous or wild-type (wt) controls. In contrast, we find that mutant cells carrying another allele, dup(a3), undergo a partial S16 and delay the entry into M16. Thus, initiation of S16 appears necessary for delaying M16. This delay is absent in double mutants of dup(a3) and mei-41 (Drosophila ATR), indicating that a mei-41-dependent checkpoint acts to delay the entry into mitosis in response to incomplete DNA replication. dup(a3) and dup(a1) mutant cells that enter M16 become arrested in M16. We find that mitotic cyclins are stabilized and that a spindle checkpoint protein, Bub1, localizes onto chromosomes during mitotic arrest in dup mutants. These features suggest an arrest prior to metaphase-anaphase transition. dup(a3) bub1 double mutant cells exit M16, indicating that a bub1-mediated checkpoint acts to block mitotic exit in dup mutants. To our knowledge, this is the first report of (1) incomplete DNA replication affecting both the entry into and the exit from mitosis in a single cell cycle via different mechanisms and (2) the role of bub1 in regulating mitotic exit in response to incomplete DNA replication.     

The cell cycle control gene cdc2+ of fission yeast encodes a protein kinase potentially regulated by phosphorylation

The cdc2+ gene function has an important role in controlling the commitment of the fission yeast cell to the mitotic cycle and the timing of mitosis. We have raised antibodies against the cdc2+ protein using synthetic peptides and have demonstrated that it is a 34 kd phosphoprotein with protein kinase activity. The protein level and phosphorylation state remain unchanged during the mitotic cycle of rapidly growing cells. When cells cease to proliferate and arrest in G1 the protein becomes dephosphorylated and loses protein kinase activity. Exit from the mitotic cycle and entry into stationary phase may be controlled in part by modulation of the cdc2 protein kinase activity by changes in its phosphorylation state.     

Signaling Pathways that Regulate Cell Division

Cell division requires careful orchestration of three major events: entry into mitosis, chromosomal segregation, and cytokinesis. Signaling within and between the molecules that control these events allows for their coordination via checkpoints, a specific class of signaling pathways that ensure the dependency of cell-cycle events on the successful completion of preceding events. Multiple positive- and negative-feedback loops ensure that a cell is fully committed to division and that the events occur in the proper order. Unlike other signaling pathways, which integrate external inputs to decide whether to execute a given process, signaling at cell division is largely dedicated to completing a decision made in G1 phase—to initiate and complete a round of mitotic cell division. Instead of deciding if the events of cell division will take place, these signaling pathways entrain these events to the activation of the cell-cycle kinase cyclin-dependent kinase 1 (CDK1) and provide the opportunity for checkpoint proteins to arrest cell division if things go wrong.     

The GADD45 inhibition of Cdc2 kinase correlates with GADD45-mediated growth suppression

Cell cycle growth arrest is an important cellular response to genotoxic stress. Gadd45, a p53-regulated stress protein, plays an important role in the cell cycle G(2)-M checkpoint following exposure to certain types of DNA-damaging agents such as UV radiation and methylmethane sulfonate. Recent findings indicate that Gadd45 interacts with Cdc2 protein and inhibits Cdc2 kinase activity. In the present study, a series of Myc-tagged Gadd45 deletion mutants and a Gadd45 overlapping peptide library were used to define the Gadd45 domains that are involved in the interaction of Gadd45 with Cdc2. Both in vitro and in vivo studies indicate that the interaction of Gadd45 with Cdc2 involves a central region of the Gadd45 protein (amino acids 65-84). The Cdc2-binding domain of Gadd45 is also required for Gadd45 inhibition of Cdc2 kinase activity. Sequence analysis of the central Gadd45 region reveals no homology to inhibitory motifs of known cyclin-dependent kinase inhibitors, indicating that the Cdc2-binding and -inhibitory domains on Gadd45 are a novel motif. The peptide containing the Cdc2-binding domain (amino acids 65-84) disrupted the Cdc2-cyclin B1 protein complex, suggesting that dissociation of this complex results from a direct interaction between the Gadd45 and Cdc2 proteins. GADD45-induced cell cycle G(2)-M arrest was abolished when its Cdc2 binding motif was disrupted. Importantly, a short term survival assay demonstrated that GADD45-induced cell cycle G(2)-M arrest correlates with GADD45-mediated growth suppression. These findings indicate that the cell cycle G(2)-M growth arrest mediated by GADD45 is one of the major mechanisms by which GADD45 suppresses cell growth.     

A phosphorylation site mutant of Schizosaccharomyces pombe cdc2p fails to promote the metaphase to anaphase transition

The protein kinase cdc2p is a key regulator of the G1-S and G2-M cell cycle transitions in the yeast Schizosaccharomyces pombe. Activation of cdc2p is regulated by its phosphorylation state and by interaction with other proteins. We have analyzed the consequences for cell cycle progression of altering the conserved threonine phosphorylation site, within the activation loop of cdc2p, to glutamic acid. This mutant, T167 E, promotes entry into mitosis, as judged by the accumulation of mitotic spindles and condensed chromosomes, despite the fact that it lacks demonstrable kinase activity both in vitro and in vivo. However, T167 E cannot promote the metaphase-anaphase transition. Since a component of the anaphase-promoting complex (APC) in S. pombe, cut9p, remains hypophosphorylated at the T167 E arrest point, the cell cycle block might be due to the inability of T167 E to activate the APC. T167 E is lethal when overexpressed, and overproduction also causes a mitotic arrest. Multicopy suppressors of the dominant negative phenotype were isolated, and identified as cdc13 ⁺ and suc1 ⁺ . Overexpression of suc1 ⁺ suppresses the effects of T167 E overproduction by restoring sufficient amounts of suc1p to the cell to allow passage through mitosis.     

The Golgi mitotic checkpoint is controlled by BARS-dependent fission of the Golgi ribbon into separate stacks in G2

The Golgi ribbon is a complex structure of many stacks interconnected by tubules that undergo fragmentation during mitosis through a multistage process that allows correct Golgi inheritance. The fissioning protein CtBP1-S/BARS (BARS) is essential for this, and is itself required for mitotic entry: a block in Golgi fragmentation results in cell-cycle arrest in G2, defining the 'Golgi mitotic checkpoint'. Here, we clarify the precise stage of Golgi fragmentation required for mitotic entry and the role of BARS in this process. Thus, during G2, the Golgi ribbon is converted into isolated stacks by fission of interstack connecting tubules. This requires BARS and is sufficient for G2/M transition. Cells without a Golgi ribbon are independent of BARS for Golgi fragmentation and mitotic entrance. Remarkably, fibroblasts from BARS-knockout embryos have their Golgi complex divided into isolated stacks at all cell-cycle stages, bypassing the need for BARS for Golgi fragmentation. This identifies the precise stage of Golgi fragmentation and the role of BARS in the Golgi mitotic checkpoint, setting the stage for molecular analysis of this process.     

Fission yeast receptor of activated C kinase (RACK1) ortholog Cpc2 regulates mitotic commitment through Wee1 kinase

In the fission yeast Schizosaccharomyces pombe, Wee1-dependent inhibitory phosphorylation of the highly conserved Cdc2/Cdk1 kinase determines the mitotic onset when cells have reached a defined size. The receptor of activated C kinase (RACK1) is a scaffolding protein strongly conserved among eukaryotes which binds to other proteins to regulate multiple processes in mammalian cells, including the modulation of cell cycle progression during G(1)/S transition. We have recently described that Cpc2, the fission yeast ortholog to RACK1, controls from the ribosome the activation of MAPK cascades and the cellular defense against oxidative stress by positively regulating the translation of specific genes whose products participate in the above processes. Intriguingly, mutants lacking Cpc2 display an increased cell size at division, suggesting the existence of a specific cell cycle defect at the G(2)/M transition. In this work we show that protein levels of Wee1 mitotic inhibitor are increased in cells devoid of Cpc2, whereas the levels of Cdr2, a Wee1 inhibitor, are down-regulated in the above mutant. On the contrary, the kinetics of G(1)/S transition was virtually identical both in control and Cpc2-less strains. Thus, our results suggest that in fission yeast Cpc2/RACK1 positively regulates from the ribosome the mitotic onset by modulating both the protein levels and the activity of Wee1. This novel mechanism of translational control of cell cycle progression might be conserved in higher eukaryotes.     

Differential occurrence of CSF-like activity and transforming activity of Mos during the cell cycle in fibroblasts.

The Xenopus c-mos proto-oncogene product, Mosxe, possesses cytostatic factor (CSF) activity to arrest maturing oocytes in metaphase II and has weak transforming activity in mouse NIH3T3 cells. We show that Mosxe mutants bearing 'stabilizing' penultimate N-terminal amino acids are strongly transforming and can retard progression through the G2-M phases in Mosxe-transformed cells, probably via their CSF activity. On the other hand, a cyclin-Mosxe fusion protein, which undergoes abrupt degradation at the end of mitosis and is restored to its normal levels only after the G1 phase, transforms cells much less efficiently than a mutated cyclin-Mosxe fusion protein that is stable during M-G1 transition. Moreover, in low-serum medium, cells transformed by the unstable cyclin-Mosxe require a long period to enter the S phase, in contrast with the rapid entry into the S phase of cells transformed by the stable cyclin-Mosxe. These results provide strong evidence that unlike the physiological CSF activity, the transforming activity of Mos is exerted in the G1 phase of the cell cycle.     

Cdk inhibitor ste9p/srw1p is involved in response to protein synthesis inhibition in fission yeast

It remains unknown whether the cell cycle system responds properly to protein synthesis inhibition. In this paper I report finding in Schizosaccharomyces pombe that partially deleted elongation factor 3 genes rescue various mitotic catastrophe mutants depending on deltaste9 in a dominant-negative manner. In response to protein synthesis inhibitors, deltaste9 and some other mutants delay halting the cell cycle at G2-M and the combined cdc2-M26 deltaste9 mutant greatly loses viability. It is suggested that cell cycle be positively controlled in an ste9-dependent manner before essential factors for viability and other important functions are exhausted when protein synthesis is inhibited.