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.     

Identification of a G1-type cyclin puc1+ in the fission yeast Schizosaccharomyces pombe

In rapidly growing cells of the budding yeast Saccharomyces cerevisiae, the cell cycle is regulated chiefly at Start, just before the G1-S boundary, whereas in the fission yeast Schizosaccharomyces pombe, the cycle is predominantly regulated at G2-M. Both control points are present in both yeasts, and both require the p34cdc2 protein kinase. At G2-M, p34cdc2 kinase activity in S. pombe requires a B-type cyclin in a complex with p34cdc2; this complex is the same as MPF (maturation promoting factor). The p34cdc2 activity at the G1-S transition in S. cerevisiae may be regulated by a similar cyclin complex, using one of the products of a new class of cyclin genes (CLN1, CLN2 and WHI1 (DAF1/CLN3)). At least one is required for progression through the G1-S phase, and deletion of all three leads to G1 arrest. WHI1 was isolated as a dominant allele causing budding yeast cells to divide at a reduced size and was later independently identified as DAF1, a dominant allele of which rendered the cells refractory to the G1-arrest induced by the mating pheromone alpha-factor. The dominant alleles are truncations thought to yield proteins of increased stability, and the cells are accelerated through G1. Without WHI1 function, the cells are hypersensitive to alpha-factor, enlarged and delayed in G1. Heretofore, this G1-class of cyclins has not been identified in other organisms. We have isolated a G1-type cyclin gene called puc1+ from S. pombe, using a functional assay in S. cerevisiae. Expression of puc1+ in S. pombe indicates that it has a cyclin-like role in the fission yeast distinct from the role of the B-type mitotic cyclin.     

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.     

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.     

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.     

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.     

Universal control mechanism regulating onset of M-phase

The onset of M-phase is regulated by a mechanism common to all eukaryotic cells. Entry into M-phase is determined by activation of the p34cdc2 protein kinase which requires p34cdc2 dephosphorylation and association with cyclin.     

A new tropomyosin essential for cytokinesis in the fission yeast S. pombe.

Mutations in the Schizosaccharomyces pombe cdc8 gene impair cytokinesis. Here we clone cdc8+ and find that it encodes a novel tropomyosin. Gene disruption results in lethal arrest of the cell cycle, but spore germination, cell growth, DNA replication and mitosis are all unaffected. Haploid cdc8 gene disruptants are rescued by expression of a fibroblast tropomyosin complementary DNA. Immunofluorescence microscopy of wild type and cdc8 gene disruptants indicates that cdc8 tropomyosin is present in two distinct cellular distributions: in dispersed patches, and during cytokinesis as a transient medial band. Collectively these results indicate that cdc8 tropomyosin has a specialized role which, we suggest, is to form part of the F-actin contractile ring at cytokinesis. These results establish the basis for further genetic studies of cytokinesis and of contractile protein function in S. pombe.     

Kinesin-related cut7 protein associates with mitotic and meiotic spindles in fission yeast.

Several mitotic and meiotic gene products are related to the microtubule motor kinesin, providing insight into the molecular basis of the complex motile events responsible for spindle formation and function. Of these genes, three have been shown to affect spindle structure when mutated. The most severe phenotype is seen in Aspergillus nidulans bimC and Schizosaccharomyces pombe cut7 mutants. In both fungi the intranuclear spindle is bipolar, with microtubules that emanate from spindle pole bodies at either pole, interdigitating in a central overlap zone. In bimC and cut7 mutants, microtubule interdigitation does not appear to take place, instead two unconnected half spindles form and chromosome separation fails. Here we report that cut7 protein concentrates on or near the spindle pole bodies throughout mitotic and meiotic nuclear division and associates with mitotic spindle microtubules in a stage-specific manner, associating with the mid-anaphase B midzone. In cut7ts mutants, spindle pole bodies stain but mitotic microtubules do not.     

A MAP kinase-dependent actin checkpoint ensures proper spindle orientation in fission yeast.

The accurate segregation of chromosomes at mitosis depends on a correctly assembled bipolar spindle that exerts balanced forces on each sister chromatid. The integrity of mitotic chromosome segregation is ensured by the spindle assembly checkpoint (SAC) that delays mitosis in response to defective spindle organisation or failure of chromosome attachment. Here we describe a distinct mitotic checkpoint in the fission yeast, Schizosaccharomyces pombe, that monitors the integrity of the actin cytoskeleton and delays sister chromatid separation, spindle elongation and cytokinesis until spindle poles have been properly oriented. This mitotic delay is imposed by a stress-activated mitogen-activated protein (MAP) kinase pathway but is independent of the anaphase-promoting complex (APC).     

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.     

Phosphorylation of non-muscle caldesmon by p34cdc2 kinase during mitosis

One of the profound changes in cellular morphology which occurs during mitosis is a massive alteration in the organization of the microfilament cytoskeleton. This change, together with other mitotic events including nuclear membrane breakdown, chromosome condensation and formation of mitotic spindles, is induced by a molecular complex called maturation promoting factor. This consists of at least two subunits, a polypeptide of relative molecular mass 45,000-62,000 (Mr 45-62K) known as cyclin, and a 34K catalytic subunit which has serine/threonine kinase activity and is known as cdc2 kinase. Non-muscle caldesmon, an 83K actin- and calmodulin-binding protein, is dissociated from microfilaments during mitosis, apparently as a consequence of mitosis-specific phosphorylation. We now report that cdc2 kinase phosphorylates caldesmon in vitro principally at the same sites as those phosphorylated in vivo during mitosis, and that phosphorylation reduces the binding affinity of caldesmon for both actin and calmodulin. Because caldesmon inhibits actomyosin ATPase, our results suggest that cdc2 kinase directly causes microfilament reorganization during mitosis.     

Emi1 is required for cytostatic factor arrest in vertebrate eggs

Vertebrate eggs are arrested at metaphase of meiosis II with stable cyclin B and high cyclin B/Cdc2 kinase activity. The ability of the anaphase-promoting complex/cyclosome (APC), an E3 ubiquitin ligase, to trigger cyclin B destruction and metaphase exit is blocked in eggs by the activity of cytostatic factor (CSF) (reviewed in ref. 1). CSF was defined as an activity in mature oocytes that caused mitotic arrest when injected into dividing embryos. Fertilization causes a transient increase in cytoplasmic calcium concentration leading to CSF inactivation, APC activation, cyclin B destruction and mitotic exit. The APC activator Cdc20 is required for APC activation after fertilization. We show here that the APC(cdc20) inhibitor Emi1 (ref. 6) is necessary and sufficient to inhibit the APC and to prevent mitotic exit in CSF-arrested eggs. CSF extracts immunodepleted of Emi1 degrade cyclin B, and exit from mitosis prematurely in the absence of calcium. Addition of Emi1 to these Emi1-depleted extracts blocks premature inactivation of the CSF-arrested state. Emi1 is required to arrest unfertilized eggs at metaphase of meiosis II and seems to be the long-sought mediator of CSF activity.     

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.     

A novel cyclin encoded by a bcl1-linked candidate oncogene

We have previously identified a candidate oncogene (PRAD1 or D11S287E) on chromosome 11q13 which is clonally rearranged with the parathyroid hormone locus in a subset of benign parathyroid tumours. We now report that a cloned human placental PRAD1 complementary DNA encodes a protein of 295 amino acids with sequence similarities to the cyclins. Cyclins can form a complex with and activate p34cdc2 protein kinase, thereby regulating progress through the cell cycle. PRAD 1 messenger RNA levels vary dramatically across the cell cycle in HeLa cells. Addition of the PRAD1 protein to interphase clam embryo lysates containing inactive p34cdc2 kinase and lacking endogenous cyclins allows it to be isolated using beads bearing p13suc1, a yeast protein that binds cdc2 and related kinases with high affinity and coprecipitates kinase-associated proteins. Addition of PRAD1 also induces phosphorylation of histone H1, a preferred substrate of cdc2. These data suggest that PRAD1 encodes a novel cyclin whose overexpression may play an important part in the development of various tumours with abnormalities in 11q13.