Regulation of mitosis by cyclic accumulation of p80cdc25 mitotic inducer in fission yeast

The coordination of somatic cell division with cell size must be accomplished by the accumulation of mitotic inducers or the dilution, in the course of cell growth, of mitotic inhibitors. In fission yeast (Schizosaccharomyces pombe), cell size at mitosis is determined by expression of the cdc25+ and nim1+ inducer genes and of the inhibitor gene wee1+, which between them regulate the M-phase protein kinase p34cdc2. We now report that both the phosphoprotein product of cdc25+ (p80cdc25, with apparent relative molecular mass 80,000) and the corresponding messenger RNA increase in concentration as cells proceed through interphase, peaking at mitosis. We propose that the cell-cycle timing of mitosis in somatic cells is regulated by the cyclic accumulation of the cdc25 mitotic inducer, which on reaching a critical level results in activation of p34cdc2 protein kinase. Accumulation of this inducer could play a part in coordinating cell division with growth.     

S-phase feedback control in budding yeast independent of tyrosine phosphorylation of p34cdc28.

In somatic cells, entry into mitosis depends on the completion of DNA synthesis. This dependency is established by S-phase feedback controls that arrest cell division when damaged or unreplicated DNA is present. In the fission yeast Schizosaccharomyces pombe, mutations that interfere with the phosphorylation of tyrosine 15 (Y15) of p34cdc2, the protein kinase subunit of maturation promoting factor, accelerate the entry into mitosis and abolish the ability of unreplicated DNA to arrest cells in G2. Because the tyrosine phosphorylation of p34cdc2 is conserved in S. pombe, Xenopus, chicken and human cells, the regulation of p34cdc2-Y15 phosphorylation could be a universal mechanism mediating the S-phase feedback control and regulating the initiation of mitosis. We have investigated these phenomena in the budding yeast Saccharomyces cerevisiae. We report here that the CDC28 gene product (the S. cerevisiae homologue of cdc2) is phosphorylated on the equivalent tyrosine (Y19) during S phase but that mutations that prevent tyrosine phosphorylation do not lead to premature mitosis and do not abolish feedback controls. We have therefore demonstrated a mechanism that does not involve tyrosine phosphorylation of p34 by which cells arrest their division in response to the presence of unreplicated or damaged DNA. We speculate that this mechanism may not involve the inactivation of p34 catalytic activity.     

Epistatic gene interactions in the control of division in fission yeast

THERE is currently much interest in the mechanism which controls the timing of cell division. Certain features of the control have been found to be common to a variety of eukaryotes. In particular, the importance of cell size as a parameter affecting cell cycle progress has been reported for mammalian cells(1,2) and for several single-celled eukaryotes(3-6). Another feature common to several systems is that growth conditions have a direct effect on the timing of division cycle events(7-9), and on cell size(9,10). In the fission yeast Schizosaccharomyces pombe, both cell size(6) and nutritional conditions(9) have been shown to affect cycle kinetics. The organism has been used extensively as a model eukaryotic system, largely because of the ease of measuring cell size and because division occurs by binary fission(11). More recently, its genetic tractability has led to the isolation of cell division cycle (cdc) mutants(12), and also of wee mutants altered in the control coordinating growth with the division cycle(13-15). The existence of such control mutants allows a more direct approach to the investigation of the molecular basis of division control, in contrast to the indirect methods used in other systems(4,16-18). wee mutants are so far unique to S. pombe. The most conspicuous property of wee mutants is their reduced cell size(13,14). Analysis of these mutants(15,19) and other evidence(9) has shown that control over cell division timing normally acts at entry to mitosis. As the function of a number of cdc genes is specifically required for mitosis(12), interactions between wee and cdc mutants which affect mitosis might be expected. I report here that the mitotic defect caused by a defective cdc25 allele is suppressed in wee mutants. Suppression by wee1 mutants is almost complete, while the wee2.1 mutation is a less effective suppressor. The significance of these findings for genetic models of the control of mitosis is considered.     

Regulation of p34CDC28 tyrosine phosphorylation is not required for entry into mitosis in S. cerevisiae.

Progression from G2 to M phase in eukaryotes requires activation of a protein kinase composed of p34cdc2/CDC28 associated with G1-specific cyclins. In some organisms the activation of the kinase at the G2/M boundary is due to dephosphorylation of a highly conserved tyrosine residue at position 15 (Y15) of the cdc2 protein. Here we report that in the budding yeast Saccharomyces cerevisiae, p34CDC28 also undergoes cell-cycle regulated dephosphorylation on an equivalent tyrosine residue (Y19). However, in contrast to previous observations in S. pombe, Xenopus and mammalian cells, dephosphorylation of Y19 is not required for the activation of the CDC28/cyclin kinase. Furthermore, mutation of this tyrosine residue does not affect dependence of mitosis on DNA synthesis nor does it abolish G2 arrest induced by DNA damage. Our data imply that regulated phosphorylation of this tyrosine residue is not the 'universal' means by which the onset of mitosis is determined. We propose that there are other unidentified controls that regulate entry into mitosis.     

Dephosphorylation and activation of a p34cdc2/cyclin B complex in vitro by human CDC25 protein.

Oocytes arrested in the G2 phase of the cell cycle contain a p34cdc2/cyclin B complex which is kept in an inactive form by phosphorylation of its p34cdc2 subunit on tyrosine, threonine and perhaps serine residues. The phosphatase(s) involved in p34cdc2 dephosphorylation is unknown, but the product of the fission yeast cdc25+ gene, and its homologues in budding yeast and Drosophila are probably positive regulators of the transition from G2 to M phase. We have purified the inactive p34cdc2/cyclin B complex from G2-arrested starfish oocytes. Addition of the purified bacterially expressed product of the human homologue of the fission yeast cdc25+ gene (p54CDC25H) triggers p34cdc2 dephosphorylation and activates H1 histone kinase activity in this preparation. We propose that the cdc25+ gene product directly activates the p34cdc2-cyclin B complex.     

Wee1(+)-like gene in human cells.

The wee1+ gene is a mitotic inhibitor controlling the G2 to M transition of the fission yeast Schizosaccharomyces pombe and encodes a protein kinase with both serine- and tyrosine-phosphorylating activities. We have cloned a human gene (WEE1Hu) similar to wee1+ by transcomplementation of a yeast mutant. WEE1Hu encodes a protein homologous to the S. pombe wee1+ and mik1+ (a functionally redundant sibling of wee1+) kinases and effectively rescues a wee1 mutation. We report here that overexpression of WEE1Hu in fission yeast generates very elongated cells as a result of inhibition of the G2-M transition in the cell cycle. In addition, we detected a 3-kilobase-long WEE1Hu messenger RNA in all the human cell lines we examined. We conclude that a wee1(+)-like gene exists and is expressed in human cells.     

Triggering of cyclin degradation in interphase extracts of amphibian eggs by cdc2 kinase

The cell cycles of early Xenopus embryos consist of a rapid succession of alternating S and M phases. These cycles are controlled by the activity of a protein kinase complex (cdc2 kinase) which contains two subunits. One subunit is encoded by the frog homologue of the fission yeast cdc2+ gene, p34cdc2 and the other is a cyclin. The concentration of cyclins follows a sawtooth oscillation because they accumulate in interphase and are destroyed abruptly during mitosis. The association of cyclin and p34cdc2 is not sufficient for activation of cdc2 kinase, however; dephosphorylation of key tyrosine and threonine residues of p34cdc2 is necessary to turn on its kinase activity. The activity of cdc2 kinase is thus regulated by a combination of translational and post-translational mechanisms. The loss of cdc2 kinase activity at the end of mitosis depends on the destruction of the cyclin subunits. It has been suggested that this destruction is induced by cdc2 kinase itself, thereby providing a negative feedback loop to terminate mitosis. Here we report direct experimental evidence for this idea by showing that cyclin proteolysis can be triggered by adding cdc2 kinase to a cell-free extract of interphase Xenopus eggs.     

Degradation of Cdc25A by beta-TrCP during S phase and in response to DNA damage

The Cdc25A phosphatase is essential for cell-cycle progression because of its function in dephosphorylating cyclin-dependent kinases. In response to DNA damage or stalled replication, the ATM and ATR protein kinases activate the checkpoint kinases Chk1 and Chk2, which leads to hyperphosphorylation of Cdc25A. These events stimulate the ubiquitin-mediated proteolysis of Cdc25A and contribute to delaying cell-cycle progression, thereby preventing genomic instability. Here we report that beta-TrCP is the F-box protein that targets phosphorylated Cdc25A for degradation by the Skp1/Cul1/F-box protein complex. Downregulation of beta-TrCP1 and beta-TrCP2 expression by short interfering RNAs causes an accumulation of Cdc25A in cells progressing through S phase and prevents the degradation of Cdc25A induced by ionizing radiation, indicating that beta-TrCP may function in the intra-S-phase checkpoint. Consistent with this hypothesis, suppression of beta-TrCP expression results in radioresistant DNA synthesis in response to DNA damage--a phenotype indicative of a defect in the intra-S-phase checkpoint that is associated with an inability to regulate Cdc25A properly. Our results show that beta-TrCP has a crucial role in mediating the response to DNA damage through Cdc25A degradation.     

Fission yeast p107wee1 mitotic inhibitor is a tyrosine/serine kinase.

The fission yeast wee1+ gene product is a dose-dependent, negative regulator of entry into mitosis. wee1+ encodes a protein of relative molecular mass 107,000 (Mr 107K), the C-terminal third of which has strong similarities with the serine/threonine protein kinase family. Here we report that p107wee1 immune complexes phosphorylate p107wee1 equally on serine and tyrosine residues, and also phosphorylate an exogenous substrate, angiotensin II, on tyrosine. Both kinase activities are attributable to p107wee1 because they are also observed when wee1+ is expressed in heterologous systems; both are abolished by a point mutation in the ATP-binding domain, and both behave like an asymmetric monomer of Mr114K on gel filtration and density-gradient centrifugation. Thus the wee1+ gene product is representative of a novel class of protein kinase that phosphorylates both serine and tyrosine residues.     

14-3-3Sigma is required to prevent mitotic catastrophe after DNA damage.

14-3-3Sigma is a member of a family of proteins that regulate cellular activity by binding and sequestering phosphorylated proteins. It has been suggested that 14-3-3sigma promotes pre-mitotic cell-cycle arrest following DNA damage, and that its expression can be controlled by the p53 tumour suppressor gene. Here we describe an improved approach to the generation of human somatic-cell knockouts, which we have used to generate human colorectal cancer cells in which both 14-3-3sigma alleles are inactivated. After DNA damage, these cells initially arrested in the G2 phase of the cell cycle, but, unlike cells containing 14-3-3sigma, the 14-3-3sigma-/- cells were unable to maintain cell-cycle arrest. The 14-3-3sigma-/- cells died ('mitotic catastrophe') as they entered mitosis. This process was associated with a failure of the 14-3-3sigma-deficient cells to sequester the proteins (cyclin B1 and cdc2) that initiate mitosis and prevent them from entering the nucleus. These results may indicate a mechanism for maintaining the G2 checkpoint and preventing mitotic death.     

Activation at M-phase of a protein kinase encoded by a starfish homologue of the cell cycle control gene cdc2+

In both starfish and amphibian oocytes, the activity of a major protein kinase which is independent of Ca2+ and cyclic nucleotides increases dramatically at meiotic and mitotic nuclear divisions. The in vivo substrates of this kinase are unknown, but phosphorylation of H1 histone can be used as an in vitro assay. We have purified this kinase from starfish oocytes. The major band in the most highly purified preparation contained a polypeptide of relative molecular mass (Mr) 34,000 (34K). This is the same size as the protein kinase encoded by cdc2+, which regulates entry into mitosis in fission yeast and is a component of MPF purified from Xenopus. Here, we show that antibodies against p34 recognize the starfish 34K protein and propose that entry into meiotic and mitotic nuclear divisions involves activation of the protein kinase encoded by a homologue of cdc2+. Given the wide occurrence of cdc2+ homologues from budding yeast to Xenopus and human cells, this activation may act as a common mechanism controlling entry into mitosis in eukaryotic cells.     

Chk1 prevents abnormal mitosis of S-phase HeLa cells containing DNA damage

To explore effects of DNA damage on cell-cycle progression in p53-deficient tumor cells, synchronized HeLa cells at G1, S and G2/M phases were treated with methyl methanesulfnate （MMS）. The results showed that the MMS treatment resulted in the cell-cycle arrest or delay in all 3 phases, while the S-phase cells were the most sensitive to MMS. Further studies demonstrated that ATM-Chk2 and p38 MAPK signaling pathways were activated in all 3 phases when the cells were treated with MMS; whereas Chk1 was activated only in S phase under the drug treatment, indicating that Chk1 specifically participated in S-phase checkpoints. To analyze the role of Chk1 in S-phase checkpoints, we administered a specific Chk1 inhibitor, UCN-01, to the S-phase cells. The results showed that the S-phase cells treated with MMS＋UCN-01 could enter aberrant mitosis without finishing DNA replication, indicating that Chk1 mainly functions in the DNA damage checkpoint rather than in the replication checkpoint. In addition, MMS treatment alone inhibited the accumulation of cyclin B1, a key component of M-phase CDK-cyclin complex, in the S-phase cells, whereas the inhibition of Chk1 activation resulted in the accumulation of cyclin B1 in the MMS-treated S-phase cells. This observation further supports the view that DNA-damaged S-phase cells enter abnormal mitosis when Chk1 activation is inhibited. Our results demonstrate that Chk1 is a specific kinase that plays an important role in the MMS-induced S-phase DNA damage checkpoint. As p53 is not involved in this process, Chk1 may be a potential target for p53-deficient tumor therapy.     

Dephosphorylation and activation of Xenopus p34cdc2 protein kinase during the cell cycle

Genetic studies in the fission yeast Schizosaccharomyces pombe have established that a critical element required for the G2----M-phase transition in the cell cycle is encoded by the cdc2+ gene. The product of this gene is a serine/threonine protein kinase, designated p34cdc, that is highly conserved functionally from yeast to man2 and has a relative molecular mass of 34,000 (34 K). Purified maturation-promoting factor (MPF) is a complex of p34cdc2 and a 45K substrate that appears in late G2 phase and is sufficient to drive cells into mitosis. This factor has been identified in all eukaryotic cells, and in vitro histone H1 is the preferred substrate for phosphorylation. The increase in the activity of H1 kinase in M-phase is associated with a large increase in total cell protein phosphorylation which is believed to be a consequence of MPF activation. We show here that the H1 kinase activity of p34cdc2 oscillates during the cell cycle in Xenopus, and maximal activity correlates with the dephosphorylated state of p34cdc2. Direct inactivation of MPF in vitro is accompanied by phosphorylation of p34cdc2 and reduction of its protein kinase activity.     

cdc2 links the Drosophila cell cycle and asymmetric division machineries

Asymmetric cell divisions can be mediated by the preferential segregation of cell-fate determinants into one of two sibling daughters. In Drosophila neural progenitors, Inscuteable, Partner of Inscuteable and Bazooka localize as an apical cortical complex at interphase, which directs the apical-basal orientation of the mitotic spindle as well as the basal/cortical localization of the cell-fate determinants Numb and/or Prospero during mitosis. Although localization of these proteins shows dependence on the cell cycle, the involvement of cell-cycle components in asymmetric divisions has not been demonstrated. Here we show that neural progenitor asymmetric divisions require the cell-cycle regulator cdc2. By attenuating Drosophila cdc2 function without blocking mitosis, normally asymmetric progenitor divisions become defective, failing to correctly localize asymmetric components during mitosis and/or to resolve distinct sibling fates. cdc2 is not necessary for initiating apical complex formation during interphase; however, maintaining the asymmetric localization of the apical components during mitosis requires Cdc2/B-type cyclin complexes. Our findings link cdc2 with asymmetric divisions, and explain why the asymmetric localization of molecules like Inscuteable show cell-cycle dependence.     

Complementation used to clone a human homologue of the fission yeast cell cycle control gene cdc2

A human homologue of the cdc2 gene has been cloned by expressing a human cDNA library in fission yeast and selecting for clones that can complement a mutant of cdc2. The predicted protein sequence of the human homologue is very similar to that of the yeast cdc2 gene. These data indicate that elements of the mechanism by which the cell cycle is controlled are likely to be conserved between yeast and humans.