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.     

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.     

Distinct nuclear and spindle pole body population of cyclin-cdc2 in fission yeast.

Cyclins, as subunits of the protein kinase encoded by the cdc2 gene are major controlling elements of the eukaryotic cell cycle. The fission yeast Schizosaccharomyces pombe has a B-type cyclin, which is a nuclear protein encoded by the cdc13 gene. Here we demonstrate the presence of two spatially distinct cdc13 cyclin populations in the nucleus of S. pombe, one of which is associated with the mitotic spindle poles. Both populations colocalize with the product of the cdc2 gene (p34cdc2). Treatment of cells with the antimicrotubule drug thiabendazole prevents cyclin degradation and blocks the tyrosine dephosphorylation and activation of cdc2. These results suggest a key regulatory role of the cdc2-cyclin complex in the initiation of mitotic spindle formation and also that mitotic microtubule function is required for cdc2 activation.     

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.     

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.     

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 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.     

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.     

Polo-like kinase 1 phosphorylates cyclin B1 and targets it to the nucleus during prophase

In vertebrate cells, the nuclear entry of Cdc2-cyclin B1 (MPF) during prophase is thought to be essential for the induction and coordination of M-phase events. Phosphorylation of cyclin B1 is central to its nuclear translocation, but the kinases that are responsible remain unknown. Here we have purified a protein kinase from Xenopus M-phase extracts that phosphorylates a crucial serine residue (S147) in the middle of the nuclear export signal sequence of cyclin B1. We have identified this kinase as Plx1 (ref. 16), a Xenopus homologue of Polo-like kinase (Plk)-1. During cell-cycle progression in HeLa cells, a change in the kinase activity of endogenous Plk1 toward S147 and/or S133 correlates with a kinase activity in the cell extracts. An anti-Plk1 antibody depletes the M-phase extracts of the kinase activity toward S147 and/or S133. An anti-phospho-S147 antibody reacts specifically with cyclin B1 only during G2/M phase. A mutant cyclin B1 in which S133 and S147 are replaced by alanines remains in the cytoplasm, whereas wild-type cyclin B1 accumulates in the nucleus during prophase. Co-expression of constitutively active Plk1 stimulates nuclear entry of cyclin B1. Our results indicate that Plk1 may be involved in targeting MPF to the nucleus during prophase.     

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.     

In vitro effects on microtubule dynamics of purified Xenopus M phase-activated MAP kinase.

The protein kinase MAP kinase, also called MAP2 kinase, is a serine/threonine kinase whose activation and phosphorylation are induced by a variety of mitogens, and which is thought to have a critical role in a network of protein kinases in mitogenic signal transduction. A burst in kinase activation and protein phosphorylation may also be important in triggering the dramatic reorganization of the cell during the transition from interphase to mitosis. The interphase-metaphase transition of microtubule arrays is under the control of p34cdc2 kinase, a central control element in the G2-M transition of the cell cycle. Here we show that a Xenopus kinase, closely related to the mitogen-activated mammalian MAP kinase, is phosphorylated and activated during M phase of meiotic and mitotic cell cycles, and that the interphase-metaphase transition of microtubule arrays can be induced by the addition of purified Xenopus M phase-activated MAP kinase or mammalian mitogen-activated MAP kinase to interphase extracts in vitro.     

Cell cycle oscillation of phosphatase inhibitor-2 in rat fibroblasts coincident with p34cdc2 restriction

Attention has focused on the regulation of the eucaryotic cell division cycle since the protein kinase p34cdc2 was identified as a key enzyme in mitotic induction. The level of this kinase remains constant throughout the cell cycle but its activity alters, particularly before M phase. Although the factors regulating cdc2 activity are still unknown, there is increasing evidence that it is influenced by p34cdc2 dephosphorylation. Protein phosphatase inhibitor-2 (I2) is a specific inhibitor of phosphatase type-1, which with type-2A is one of the two principal Ser(P) and Thr(P) phosphatases. Here we show that the level of I2, assayed by immunofluorescence staining, activity measurements, western immunoblotting and metabolic labelling, oscillates during the cell cycle in rat fibroblasts, peaking at S phase and mitosis. Moreover, when we inhibited I2 in vivo by microinjection of anti-I2 antibodies in S-phase cells, the pseudo-mitotic cellular response to injected p34cdc2 was restored, indicating that I2 might have a role in the modulation of p34cdc2 activity.     

The wee1 protein kinase is required for radiation-induced mitotic delay.

Cellular feedback or 'checkpoint' mechanisms maintain the order of completion of essential, cell-cycle related functions. In the budding yeast, for example, the RAD9 gene product is required to delay progression into mitosis in response to DNA damage. Similarly, in fission yeast, the cdc25 and cdc2 gene products influence the ability of cells to delay mitosis in response to the inhibition of DNA synthesis. Because these two checkpoint controls regulate the same event, mitosis, we observed the effect of gamma-irradiation on cell cycle progression in fission yeast, to test whether the two controls require the same cell-cycle regulatory elements. We show that gamma-radiation-induced mitotic delay requires functional wee1 protein kinase but does not seem to involve the cdc25 pathway. Mitotic delay in response to DNA damage is thus distinct from the delay induced by inhibition of DNA synthesis, which involves cdc25 but is not dependent on wee1.