spo12 is a multicopy suppressor of mcs3 that is periodically expressed in fission yeast mitosis

Hyperactivation of Cdc2 in fission yeast causes cells to undergo a lethal premature mitosis, a phenomenon called mitotic catastrophe. This phenotype is observed in cdc2-3w wee1-50 cells at high temperature and is suppressed by a single recessive mutant, mcs3-12. Mcs3 acts independently of the Wee1 kinase and Cdc25 phosphatase, two major regulators of Cdc2. We have isolated multicopy suppressors of the cell cycle arrest phenotype of mcs3-12 wee1-50 cdc25-22 cells, but did not identify the mcs3 gene itself. Instead several known mitotic regulators were isolated, including the Cdc25 phosphatase, Wis2 cyclophilin, Cek1 kinase, and an Hsp90 homologue, Swo1. We also isolated clones encoding non-functional, truncated forms of the Wee1 kinase and Dis2 type 1 phosphatase. In addition we identified a multicopy suppressor that encodes a structural homologue of the budding yeast SPO12 gene. We find that overexpression of fission yeast spo12 not only suppresses the phenotype of the mcs3-12 wee1-50 cdc25-22 strain, but also that of a win1-1 wee1-50 cdc25-22 strain at high temperature, indicating that the function of spo12 is not directly related to mcs3. We show that spo12 mRNA is periodically expressed during the fission yeast cell cycle, peaking at the G2/M transition coincidently with cdc15. Deletion of spo12, however, has no overt effect on either the mitotic or meiotic cell cycles, except when the function of the major B type cyclin, Cdc13, is compromised.     

cdc2MsB, a cognate cdc2 gene from alfalfa, complements the G1/S but not the G2/M transition of budding yeast cdc28 mutants

The product of the cdc2 gene encodes the p34^cdc2 protein kinase that controls entry of yeast cells into S phase and mitosis. In higher eukaryotes, at least two cdc2 -like genes appear to be involved in these processes. A cdc2 homologous gene has previously been isolated from alfalfa and shown to complement a fission yeast cdc2 ^ts mutant. Here the isolation of cdc2MsB , a cognate cdc2 gene from alfalfa ( Medicago sativa ) is reported. Southern blot analysis shows that cdc2MsA and cdc2MsB are present as single copy genes in different tetraploid Medicago species. cdc2MsB encodes a slightly larger mRNA (1.5 kb) than cdc2MsA (1.4 kb). Both genes were found to be expressed at similar steady state levels in different alfalfa organs. Expression levels of both cdc2Ms genes correlate with the proliferative state of the organs. Complementation studies revealed that in contrast to cdc2MsA, cdc2MsB was not able to rescue a cdc2 ^ts fission yeast mutant. cdc2MsB was also unable to rescue a G2/M-arrested cdc28 ^ts budding yeast mutant which could be rescued by expression of the cdc2MsA gene. Conversely, cdc2MsB but not cdc2MsA was found to complement the G1/S block of another cdc28 ^ts budding yeast mutant. These results suggest that cdc2MsA and cdc2MsB function at different control points in the cell cycle.     

Specific activation of cdc25 tyrosine phosphatases by B-type cyclins: evidence for multiple roles of mitotic cyclins.

Two previously unidentified human cdc25 genes have been isolated, cdc25A and cdc25B. Both genes rescue a cdc25ts mutant of fission yeast. Microinjection of anti-cdc25A antibodies into HeLa cells causes their arrest in mitosis. cdc25A and cdc25B display endogenous tyrosine phosphatase activity that is stimulated several-fold, in the absence of cdc2, by stoichiometric addition of either cyclin B1 or B2 but not A or D1. Association between cdc25A and cyclin B1/cdc2 was detected in the HeLa cells. These findings indicate that B-type cyclins are multifunctional proteins that not only act as M phase regulatory subunits of the cdc2 protein kinase, but also activate the cdc25 tyrosine phosphatase, of which cdc2 is the physiological substrate. A region of amino acid similarity between cyclins and tyrosine PTPases has been detected. This region is absent in cdc25 phosphatases. The motif may represent an activating domain that has to be provided to cdc25 by intermolecular interaction with cyclin B.     

Caffeine-mediated override of checkpoint controls. A requirement for rhp6 (Schizosaccharomyces pombe)

Cells exposed to inhibitors of DNA synthesis or suffering DNA damage are arrested or delayed in interphase through the action of checkpoint controls. If the arrested cell is exposed to caffeine, relatively normal cell cycle progression is resumed and, as observed in checkpoint control mutants, loss of checkpoint control activity is associated with a reduction in cell viability. To address the mechanism of caffeine's action on cell progression, fission yeast mutants that take up caffeine but are not sensitized to hydroxyurea (HU) by caffeine were selected. Mutants 788 and 1176 are point mutants of rhp6, the fission yeast homolog of the budding yeast RAD6 gene. Mutant rhp6-788 is slightly HU sensitive, radiosensitive, and exhibits normal checkpoint responses to HU, radiation, or inactivation of DNA ligase. However, the addition of caffeine does not override the associated cell cycle blocks. Both point and deletion mutations show synthetic lethality at room temperature with temperature-sensitive mutations in cyclin B (cdc13-117) or the phosphatase cdc25 (cdc25-22). These observations suggest that the rhp6 gene product, a ubiquitin-conjugating enzyme required for DNA damage repair, promotes entry to mitosis in response to caffeine treatment.     

Regulation of p34cdc2 protein kinase during mitosis

The cell-cycle timing of mitosis in fission yeast is determined by the cdc25+ gene product activating the p34cdc2 protein kinase leading to mitotic initiation. Protein kinase activity remains high in metaphase and then declines during anaphase. Activation of the protein kinase also requires the cyclin homolog p56cdc13, which also functions post activation at a later stage of mitosis. The continuing function of p56cdc13 during mitosis is consistent with its high level until the metaphase/anaphase transition. At anaphase the p56cdc13 level falls dramatically just before the decline in p34cdc2 protein kinase activity. The behavior of p56cdc13 is similar to that observed for cyclins in oocytes. p13suc1 interacts closely with p34cdc2; it is required during the process of mitosis and may play a role in the inactivation of the p34cdc2 protein kinase. Therefore, the cdc25+, cdc13+, and suc1+ gene products are important for regulating p34cdc2 protein kinase activity during entry into, progress through, and exit from mitosis.     

Fission yeast p13 blocks mitotic activation and tyrosine dephosphorylation of the Xenopus cdc2 protein kinase

It has been demonstrated that the Xenopus homolog of the fission yeast cdc2 protein is a component of M phase promoting factor (MPF). We show that the Xenopus cdc2 protein is phosphorylated on tyrosine in vivo, and that this tyrosine phosphorylation varies markedly with the stage of the cell cycle. Tyrosine phosphorylation is high during interphase (in Xenopus oocytes and activated eggs) but absent during M phase (in unfertilized eggs). In vitro activation of pre-MPF from Xenopus oocytes results in tyrosine dephosphorylation of the cdc2 protein and switching-on of its kinase activity. The product of the fission yeast suc1 gene (p13), which inhibits the entry into mitosis in Xenopus extracts, completely blocks tyrosine dephosphorylation and kinase activation. However, p13 has no effect on the activated form of the cdc2 kinase. These findings suggest that p13 controls the activation of the cdc2 kinase, and that tyrosine dephosphorylation is an important step in this process.     

p34cdc2 acts as a lamin kinase in fission yeast

The nuclear lamina is an intermediate filament network that underlies the nuclear membrane in higher eukaryotic cells. During mitosis in higher eukaryotes, nuclear lamins are phosphorylated by a mitosis-specific kinase and this induces disassembly of the lamina structure. Recently, p34cdc2 protein kinase purified from starfish has been shown to induce phosphorylation of lamin proteins and disassembly of the nuclear lamina when incubated with isolated chick nuclei suggesting that p34cdc2 is likely to be the mitotic lamin kinase (Peter, M., J. Nakagawa, M. Dorée, J.C. Labbe, and E.A. Nigg. 1990b. Cell. 45:145-153). To confirm and extend these studies using genetic techniques, we have investigated the role of p34cdc2 in lamin phosphorylation in the fission yeast. As fission yeast lamins have not been identified, we have introduced a cDNA encoding the chicken lamin B2 protein into fission yeast. We report here that the chicken lamin B2 protein expressed in fission yeast is assembled into a structure that associates with the nucleus during interphase and becomes dispersed throughout the cytoplasm when cells enter mitosis. Mitotic reorganization correlates with phosphorylation of the chicken lamin B2 protein by a mitosis-specific yeast lamin kinase with similarities to the mitotic lamin kinase of higher eukaryotes. We show that a lamin kinase activity can be detected in cell-free yeast extracts and in p34cdc2 immunoprecipitates prepared from yeast cells arrested in mitosis. The fission yeast lamin kinase activity is temperature sensitive in extracts and immunoprecipitates prepared from strains bearing temperature-sensitive mutations in the cdc2 gene. These results in conjunction with the previously reported biochemical studies strongly suggest that disassembly of the nuclear lamina at mitosis in higher eukaryotic cells is a consequence of direct phosphorylation of nuclear lamins by p34cdc2.     

Tracking nucleolar dynamics with GFP-Nopp140 during Drosophila oogenesis and embryogenesis

We expressed two green fluorescent protein (GFP)-tagged Nopp140 isoforms in transgenic Drosophila melanogaster to study nucleolar dynamics during oogenesis and early embryogenesis. Specifically, we wanted to test whether the quiescent oocyte nucleus stored maternal Nopp140 and then to determine precisely when nucleoli formed during embryogenesis. During oogenesis nurse cell nucleoli accumulated GFP-Nopp140 gradually such that posterior nurse cell nucleoli in egg chambers at stage 10 were usually brighter than the more anterior nurse cell nucleoli. Nucleoli within apoptotic nurse cells disassembled in stages 12 and 13, but not all GFP-Nopp140 entered the oocyte through inter-connecting cytoplasmic bridges. Oocytes, on the other hand, lost their nucleoli by stage 3, but GFP-Nopp140 gradually accumulated in oocyte nuclei during stages 8–13. Most oocyte nuclei at stage 10 stored GFP-Nopp140 uniformly, but many stage 10 oocytes accumulated GFP-Nopp140 in presumed endobodies or in multiple smaller spheres. All oocyte nuclei at stages 11-12 were uniformly labeled, and GFP-Nopp140 diffused to the cytoplasm upon nuclear disassembly in stage 13. GFP-Nopp140 reappeared during embryogenesis; initial nucleologenesis occurred in peripheral somatic nuclei during embryonic stage 13, one stage earlier than reported previously. These GFP-Nopp140-containing foci disassembled at the 13th syncytial mitosis, and a second nucleologenesis occurred in early stage 14. The resulting nucleoli occupied nuclear regions closest to the periphery of the embryos. Pole cells contained GFP-Nopp140 during the syncytial embryonic stages, but their nucleologenesis started at gastrulation. This work was supported by the National Science Foundation (grant MCB-0234245). O'Keith Dellafosse was supported by the Louisiana Alliance for Minority Participation (LAMP).     

Structure and developmental expression of the chicken CDC2 kinase.

The cdc2 protein kinase plays a key role in controlling the eukaryotic cell cycle. We have isolated a cDNA clone for the chicken homolog of the cdc2 gene, raised antibodies against the corresponding protein, and studied the expression of cdc2 mRNA and protein during chicken embryonic development. The protein encoded by the chicken cdc2 cDNA shares extensive structural homology with cdc2 gene products from other species. Moreover, when expressed in fission yeast, the chicken cdc2 kinase is able to rescue a temperature-sensitive (ts) cdc2 mutant, demonstrating that it is functional as a cell cycle regulator. By Northern analysis and immunoblotting, we found that in total embryos both cdc2 mRNA and protein levels decreased substantially between day 3 and day 11 after egg laying, and no significant amounts of either cdc2 mRNA or protein were detected in adult liver, brain, heart or skeletal muscle. These data indicate the existence of a coarse correlation between the abundance of cdc2 mRNA and the proliferative state of a given tissue. Interestingly, however, when examining individual embryonic tissues, no correlation was observed between levels of cdc2 mRNA and protein, suggesting that cdc2 expression in developing chicken may be regulated at multiple levels.