scholarly journals p34cdc2 acts as a lamin kinase in fission yeast.

1991 ◽  
Vol 112 (5) ◽  
pp. 797-807 ◽  
Author(s):  
T Enoch ◽  
M Peter ◽  
P Nurse ◽  
E A Nigg
Keyword(s):  
Fission Yeast ◽  
Kinase Activity ◽  
Nuclear Lamina ◽  
Yeast Cells ◽  
Eukaryotic Cells ◽  
Temperature Sensitive ◽  
Nuclear Lamins ◽  
Cdc2 Gene ◽  
Genetic Techniques ◽  
Higher Eukaryotes

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.

Site-specific mutagenesis of cdc2+, a cell cycle control gene of the fission yeast Schizosaccharomyces pombe

1986 ◽  
Vol 6 (10) ◽  
pp. 3523-3530
Author(s):  
R Booher ◽  
D Beach
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Fission Yeast ◽  
Cell Cycle Control ◽  
Temperature Sensitive ◽  
Protein Kinase Activity ◽  
Atp Binding Site ◽  
Leu2 Gene ◽  
Cdc2 Gene ◽  
A Site

The cdc2+ gene of Schizosaccharomyces pombe is homologous to the CDC28 gene of Saccharomyces cerevisiae. Both genes share limited homology with vertebrate protein kinases and have protein kinase activity. cdc2+ has been subjected to mutagenesis in vitro. A null allele of the gene, constructed by insertion of the S. cerevisiae LEU2 gene into a site within the gene, has a phenotype similar to that of many temperature-sensitive alleles of cdc2. Mutations within the predicted ATP-binding site and in a region which may be a site of phosphorylation result in loss of cdc2+ activity. A single substitution of Gly-146 to Asp-146 has been identified in cdc2-1w, a dominant activated allele of the gene. The four introns within the cdc2+ gene have been deleted. The resulting gene not only functions in fission yeast but also rescues cdc28(Ts) strains of S. cerevisiae, a property which is not shared by the genomic cdc2+ gene.

scholarly journals Recombination-Based Telomere Maintenance Is Dependent on Tel1-MRN and Rap1 and Inhibited by Telomerase, Taz1, and Ku in Fission Yeast

2007 ◽  
Vol 28 (5) ◽  
pp. 1443-1455 ◽  
Author(s):  
Lakxmi Subramanian ◽  
Bettina A. Moser ◽  
Toru M. Nakamura
Keyword(s):  
Dna Repair ◽  
Fission Yeast ◽  
Catalytic Subunit ◽  
Telomere Maintenance ◽  
Yeast Cells ◽  
Eukaryotic Cells ◽  
Checkpoint Kinase ◽  
Telomere Binding Protein ◽  
Evolutionarily Conserved ◽  
Linear Chromosomes

ABSTRACT Fission yeast cells survive loss of the telomerase catalytic subunit Trt1 (TERT) through recombination-based telomere maintenance or through chromosome circularization. Although trt1Δ survivors with linear chromosomes can be obtained, they often spontaneously circularize their chromosomes. Therefore, it was difficult to establish genetic requirements for telomerase-independent telomere maintenance. In contrast, when the telomere-binding protein Taz1 is also deleted, taz1Δ trt1Δ cells are able to stably maintain telomeres. Thus, taz1Δ trt1Δ cells can serve as a valuable tool in understanding the regulation of telomerase-independent telomere maintenance. In this study, we show that the checkpoint kinase Tel1 (ATM) and the DNA repair complex Rad32-Rad50-Nbs1 (MRN) are required for telomere maintenance in taz1Δ trt1Δ cells. Surprisingly, Rap1 is also essential for telomere maintenance in taz1Δ trt1Δ cells, even though recruitment of Rap1 to telomeres depends on Taz1. Expression of catalytically inactive Trt1 can efficiently inhibit recombination-based telomere maintenance, but the inhibition requires both Est1 and Ku70. While Est1 is essential for recruitment of Trt1 to telomeres, Ku70 is dispensable. Thus, we conclude that Taz1, TERT-Est1, and Ku70-Ku80 prevent telomere recombination, whereas MRN-Tel1 and Rap1 promote recombination-based telomere maintenance. Evolutionarily conserved proteins in higher eukaryotic cells might similarly contribute to telomere recombination.

Isolation and characterization of fission yeast mutants defective in the assembly and placement of the contractile actin ring

1996 ◽  
Vol 109 (1) ◽  
pp. 131-142 ◽  
Author(s):  
F. Chang ◽  
A. Woollard ◽  
P. Nurse
Keyword(s):  
Fission Yeast ◽  
Ring Formation ◽  
Cell Cycle Checkpoint ◽  
Yeast Cells ◽  
Temperature Sensitive ◽  
Actin Ring ◽  
Division Site ◽  
Isolation And Characterization ◽  
Cycle Checkpoint ◽  
Actin Cables

Fission yeast cells divide by medial cleavage using an actin-based contractile ring. We have conducted a genetic screen for temperature-sensitive mutants defective in the assembly and placement of this actin ring. Six genes necessary for actin ring formation and one gene necessary for placement of the actin ring have now been identified. The genes can be further organized into different phenotypic groups, suggesting that the gene products may have different functions in actin ring formation. Mutants of cdc3 and cdc8, which encode profilin and tropomyosin respectively, display disorganized actin patches in all cells. cdc12 and cdc15 mutants display disorganized actin patches during mitosis, but normal interphase actin patterns. cdc4 and rng2 mutants display disorganized actin cables during mitosis, but normal interphase actin patterns. In mid1 mutants, the actin ring and septum are positioned at random locations and angles on the cell surface, although the nucleus is positioned normally, indicating that the mid1 gene product is required to couple the division site to the position of the nucleus. mid1 mutant cells may reveal a new cell cycle checkpoint in telophase that coordinates cell division and the proper distribution of nuclei. The actin ring forms medially in a beta-tubulin mutant, showing that actin ring formation and placement are not dependent on the mitotic spindle.

scholarly journals Lamin B constitutes an intermediate filament attachment site at the nuclear envelope.

1987 ◽  
Vol 105 (1) ◽  
pp. 117-125 ◽  
Author(s):  
S D Georgatos ◽  
G Blobel
Keyword(s):  
Plasma Membrane ◽  
Specific Binding ◽  
Attachment Site ◽  
Nuclear Lamina ◽  
Membrane Skeleton ◽  
Eukaryotic Cells ◽  
Lamin A ◽  
Lamin B ◽  
Nuclear Lamins

We found that urea extraction of turkey erythrocyte nuclear envelopes abolished their ability to bind exogenous 125I-vimentin, while, at the same time, it removed the nuclear lamins from the membranes. After purification of the lamins from such urea extracts, a specific binding between isolated vimentin and lamin B, or a lamin A + B hetero-oligomer, was detected by affinity chromatography. Similar analysis revealed that the 6.6-kD vimentin tail piece was involved in this interaction. By other approaches (quantitative immunoprecipitation, rate zonal sedimentation, turbidometric assays) a substoichiometric lamin B-vimentin binding was determined under in vitro conditions. It was also observed that anti-lamin B antibodies but not other sera (anti-lamin A, anti-ankyrin, preimmune) were able to block 70% of the binding of 125I-vimentin to native, vimentin-depleted, nuclear envelopes. These data, which were confirmed by using rat liver nuclear lamins, indicate that intermediate filaments may be anchored directly to the nuclear lamina, providing a continuous network connecting the plasma membrane skeleton with the karyoskeleton of eukaryotic cells.

scholarly journals Fission Yeast dim1+ Encodes a Functionally Conserved Polypeptide Essential for Mitosis

1997 ◽  
Vol 137 (6) ◽  
pp. 1337-1354 ◽  
Author(s):  
Lynne D. Berry ◽  
Kathleen L. Gould
Keyword(s):  
Fission Yeast ◽  
Chromosome Segregation ◽  
Mitotic Spindle ◽  
Kinase Activity ◽  
Nuclear Division ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Sensitive Mutant ◽  
Evolutionarily Conserved ◽  
Mutant Cells

In a screen for second site mutations capable of reducing the restrictive temperature of the fission yeast mutant cdc2-D217N, we have isolated a novel temperature-sensitive mutant, dim1-35. When shifted to restrictive temperature, dim1-35 mutant cells arrest before entry into mitosis or proceed through mitosis in the absence of nuclear division, demonstrating an uncoupling of proper DNA segregation from other cell cycle events. Deletion of dim1 from the Schizosaccharomyces pombe genome produces a lethal G2 arrest phenotype. Lethality is rescued by overexpression of the mouse dim1 homolog, mdim1. Likewise, deletion of the Saccharomyces cerevisiae dim1 homolog, CDH1, is lethal. Both mdim1 and dim1+ are capable of rescuing lethality in the cdh1::HIS3 mutant. Although dim1-35 displays no striking genetic interactions with various other G2/M or mitotic mutants, dim1-35 cells incubated at restrictive temperature arrest with low histone H1 kinase activity. Morevoer, dim1-35 displays sensitivity to the microtubule destabilizing drug, thiabendazole (TBZ). We conclude that Dim1p plays a fundamental, evolutionarily conserved role as a protein essential for entry into mitosis as well as for chromosome segregation during mitosis. Based on TBZ sensitivity and failed chromosome segregation in dim1-35, we further speculate that Dim1p may play a role in mitotic spindle formation and/or function.

scholarly journals Site-specific mutagenesis of cdc2+, a cell cycle control gene of the fission yeast Schizosaccharomyces pombe.

1986 ◽  
Vol 6 (10) ◽  
pp. 3523-3530 ◽  
Author(s):  
R Booher ◽  
D Beach
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Fission Yeast ◽  
Cell Cycle Control ◽  
Temperature Sensitive ◽  
Protein Kinase Activity ◽  
Atp Binding Site ◽  
Leu2 Gene ◽  
Cdc2 Gene ◽  
A Site

The cdc2+ gene of Schizosaccharomyces pombe is homologous to the CDC28 gene of Saccharomyces cerevisiae. Both genes share limited homology with vertebrate protein kinases and have protein kinase activity. cdc2+ has been subjected to mutagenesis in vitro. A null allele of the gene, constructed by insertion of the S. cerevisiae LEU2 gene into a site within the gene, has a phenotype similar to that of many temperature-sensitive alleles of cdc2. Mutations within the predicted ATP-binding site and in a region which may be a site of phosphorylation result in loss of cdc2+ activity. A single substitution of Gly-146 to Asp-146 has been identified in cdc2-1w, a dominant activated allele of the gene. The four introns within the cdc2+ gene have been deleted. The resulting gene not only functions in fission yeast but also rescues cdc28(Ts) strains of S. cerevisiae, a property which is not shared by the genomic cdc2+ gene.

scholarly journals Bub3p Facilitates Spindle Checkpoint Silencing in Fission Yeast

2009 ◽  
Vol 20 (24) ◽  
pp. 5096-5105 ◽  
Author(s):  
Vincent Vanoosthuyse ◽  
John C. Meadows ◽  
Sjaak J.A. van der Sar ◽  
Jonathan B.A. Millar ◽  
Kevin G. Hardwick
Keyword(s):  
Fission Yeast ◽  
Kinase Activity ◽  
Spindle Checkpoint ◽  
Aurora Kinase ◽  
Yeast Cells ◽  
Anaphase Promoting Complex ◽  
Checkpoint Signaling ◽  
Checkpoint Proteins ◽  
Anaphase Onset ◽  
Checkpoint Recovery

Although critical for spindle checkpoint signaling, the role kinetochores play in anaphase promoting complex (APC) inhibition remains unclear. Here we show that spindle checkpoint proteins are severely depleted from unattached kinetochores in fission yeast cells lacking Bub3p. Surprisingly, a robust mitotic arrest is maintained in the majority of bub3Δ cells, yet they die, suggesting that Bub3p is essential for successful checkpoint recovery. During recovery, two defects are observed: (1) cells mis-segregate chromosomes and (2) anaphase onset is significantly delayed. We show that Bub3p is required to activate the APC upon inhibition of Aurora kinase activity in checkpoint-arrested cells, suggesting that Bub3p is required for efficient checkpoint silencing downstream of Aurora kinase. Together, these results suggest that spindle checkpoint signals can be amplified in the nucleoplasm, yet kinetochore localization of spindle checkpoint components is required for proper recovery from a spindle checkpoint-dependent arrest.

Entry into mitosis without Cdc2 kinase activation

1998 ◽  
Vol 111 (22) ◽  
pp. 3401-3410
Author(s):  
P.M. Gowdy ◽  
H.J. Anderson ◽  
M. Roberge
Keyword(s):  
Protein Kinase ◽  
Okadaic Acid ◽  
Structural Changes ◽  
Kinase Activity ◽  
Kinase Inhibitor ◽  
Tyrosine Phosphatase ◽  
Nuclear Lamina ◽  
Temperature Sensitive ◽  
Cdc2 Kinase ◽  
Phosphatase Inhibitors

Mouse FT210 cells at 39 degreesC cannot enter mitosis but arrest in G2 phase, because they lack Cdc2 kinase activity as a result of a temperature-sensitive lesion in the cdc2 gene. Incubation of arrested cells with the protein phosphatase 1 and 2A inhibitor okadaic acid induces morphologically normal chromosome condensation. We now show that okadaic acid also induces two other landmark events of early mitosis, nuclear lamina depolymerization and centrosome separation, in the absence of Cdc2 kinase activity. Okadaic acid-induced entry into mitosis is accompanied by partial activation of Cdc25C and may be prevented by tyrosine phosphatase inhibitors and by the protein kinase inhibitor staurosporine, suggesting that Cdc25C and kinases distinct from Cdc2 are required for these mitotic events. Using in-gel assays, we show that a 45-kDa protein kinase normally activated at mitosis is also activated by okadaic acid independently of Cdc2 kinase. The 45-kDa kinase can utilize GTP, is stimulated by spermine and is inhibited by heparin. These properties are characteristic of the kinase CK2, but immunoprecipitation studies indicate that it is not CK2. The data underline the importance of a tyrosine phosphatase, possibly Cdc25C, and of kinases other than Cdc2 in the structural changes the cell undergoes at mitosis, and indicate that entry into mitosis involves the activation of multiple kinases working in concert with Cdc2 kinase.

scholarly journals TheSchizosaccharomyces pombe spo20+Gene Encoding a Homologue ofSaccharomyces cerevisiaeSec14 Plays an Important Role in Forespore Membrane Formation

2001 ◽  
Vol 12 (4) ◽  
pp. 901-917 ◽  
Author(s):  
Yukiko Nakase ◽  
Taro Nakamura ◽  
Aiko Hirata ◽  
Sheri M. Routt ◽  
Henry B. Skinner ◽  
...  
Keyword(s):  
Fission Yeast ◽  
Yeast Cells ◽  
Temperature Sensitive ◽  
Secretory Function ◽  
Membrane Structures ◽  
Direct Role ◽  
Haploid Nucleus ◽  
Transfer Protein ◽  
Golgi Secretory Function ◽  
Phosphatidylinositol Transfer

The Schizosaccharomyces pombe spo20-KC104 mutation was originally isolated in a screen for sporulation-deficient mutants, and the spo20-KC104 mutant exhibits temperature-sensitive growth. Herein, we report that S. pombe, spo20+is essential for fission yeast cell viability and is constitutively expressed throughout the life cycle. We also demonstrate that thespo20+gene product is structurally homologous to Saccharomyces cerevisiae Sec14, the major phosphatidylinositol transfer protein of budding yeast. This structural homology translates to a significant degree of functional relatedness because reciprocal complementation experiments demonstrate that each protein is able to fulfill the essential function of the other. Moreover, biochemical experiments show that, like Sec14, Spo20 is a phosphatidylinositol/phosphatidylcholine-transfer protein. That Spo20 is required for Golgi secretory function in vegetative cells is indicated by our demonstration that the spo20-KC104mutant accumulates aberrant Golgi cisternae at restrictive temperatures. However, a second phenotype observed in Spo20-deficient fission yeast is arrest of cell division before completion of cell separation. Consistent with a direct role for Spo20 in controlling cell septation in vegetatively growing cells, localization experiments reveal that Spo20 preferentially localizes to the cell poles and to sites of septation of fission yeast cells. We also report that, when fission yeasts are challenged with nitrogen starvation, Spo20 translocates to the nucleus. This nuclear localization persists during conjugation and meiosis. On completion of meiosis, Spo20 translocates to forespore membranes, and it is the assembly of forespore membranes that is abnormal in spo20-KC104 cells. In such mutants, a considerable fraction of forming prespores fail to encapsulate the haploid nucleus. Our results indicate that Spo20 regulates the formation of specialized membrane structures in addition to its recognized role in regulating Golgi secretory function.

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