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1.
Thecdc2
+ gene product (p34cdc2) is a protein kinase that regulates entry into mitosis in all eukaryotic cells. The role that p34cdc2 plays in the cell cycle has been extensively investigated in a number of organisms, including the fission yeastSchizosaccharomyces pombe. To study the degree of functional conservation among evolutionarily distant p34cdc2 proteins, we have constructed aS. pombe strain in which the yeastcdc2
+ gene has been replaced by itsDrosophila homologue CDC2Dm (theCDC2Dm strain). ThisCDC2Dm S. pombe strain is viable, capable of mating and producing four viable meiotic products, indicating that the fly p34CDC2Dm recognizes all the essentialS. pombe cdc2
+ substrates, and that it is recognized by cyclin partners and other elements required for its activity. The p34CDC2Dm protein yields a lethal phenotype in combination with the mutant B-type cyclin p56cdc13-117, suggesting that thisS. pombe cyclin might interact less efficiently with theDrosophila protein than with its native p34cdc2 counterpart. ThisCDC2Dm strain also responds to nutritional starvation and to incomplete DNA synthesis, indicating that proteins involved in these
signal transduction pathways, interact properly with p34CDC2Dm (and/or that p34cdc2-independent pathways are used). TheCDC2Dm gene produces a ‘wee’ phenotype, and it is largely insensitive to the action of theS. pombe weel
+ mitotic inhibitor, suggesting thatDrosophila weel
+ homologue might not be functionally conserved. ThisCDC2Dm strain is hypersensitive to UV irradiation, to the same degree asweel-deficient mutants. A strain which co-expresses theDrosophila and yeastcdc2+ genes shows a dominantwee phenotype, but displays a wild-type sensitivity to UV irradiation, suggesting that p34cdc2 triggers mitosis and influences the UV sensitivity by independent mechanisms.
Communicated by B. J. Kilbey 相似文献
2.
Cell cycle control in the fission yeastSchizosaccharomyces pombe involves interplay amongst a number of regulatory molecules, including thecdc2, cdc13, cdc25, weel, andmik1 gene products. Cdc2, Cdc13, and Cdc25 act as positive regulators of cell cycle progression at the G2/M boundary, while Wee1 and Mik1 play a negative regulatory role. Here, we have screened for suppressors of the lethal premature entry into mitosis, termed mitotic catastrophe, which results from simultaneous loss of function of both Wee1 and Mik1. Through such a screen, we hoped to identify additional components of the cell cycle regulatory network, and/or G2/M-specific substrates of Cdc2. Although we did not identify such molecules, we isolated a number of alleles of bothcdc2 andcdc13, including a novel wee allele ofcdc2, cdc2-5w. Here, we characterizecdc2-5w and two alleles ofcdc13, which have implications for the understanding of details of the interactions amongst Cdc2, Cdc13, and Wee1. 相似文献
3.
The p34cdc2 protein kinase plays a key role in the control of the mitotic cell cycle of fission yeast, being required for both entry into S-phase and for entry into mitosis in the mitotic cell cycle, as well as for the initiation of the second meiotic nuclear division. In recent years, structural and functional homologues of p34cdc2, as well as several of the proteins that interact with and regulate p34cdc2 function in fission yeast, have been identified in a wide range of higher eukaryotic cell types, suggesting that the control mechanisms uncovered in this simple eukaryote are likely to be well conserved across evolution. Here we describe the construction and characterisation of a fission yeast strain in which the endogenous p34cdc2 protein is entirely absent and is replaced by its human functional homologue p34CDC2, We have used this strain to analyse aspects of the function of the human p34CDC2 protein genetically. We show that the function of the human p34CDC2 protein in fission yeast cells is dependent upon the action of the protein tyrosine phosphatase p80cdc25 that it responds to altered levels of both the mitotic inhibitor p1072331 and the p34cdc2-binding protein p13suc1, and is lethal in combination with the mutant B-type cyclin p56cdc13-117. In addition, we demonstrate that the human p34CDC2 protein is proficient for fission yeast meiosis, and examine the behaviour of two mutant p34CDC2 proteins in fission yeast. 相似文献
4.
The p34cdc2 protein kinase plays a key role in the control of the mitotic cell cycle of fission yeast, being required for both entry into S-phase and for entry into mitosis in the mitotic cell cycle, as well as for the initiation of the second meiotic nuclear division. In recent years, structural and functional homologues of p34cdc2, as well as several of the proteins that interact with and regulate p34cdc2 function in fission yeast, have been identified in a wide range of higher eukaryotic cell types, suggesting that the control mechanisms uncovered in this simple eukaryote are likely to be well conserved across evolution. Here we describe the construction and characterisation of a fission yeast strain in which the endogenous p34cdc2 protein is entirely absent and is replaced by its human functional homologue p34CDC2, We have used this strain to analyse aspects of the function of the human p34CDC2 protein genetically. We show that the function of the human p34CDC2 protein in fission yeast cells is dependent upon the action of the protein tyrosine phosphatase p80cdc25 that it responds to altered levels of both the mitotic inhibitor p1072331 and the p34cdc2-binding protein p13suc1, and is lethal in combination with the mutant B-type cyclin p56cdc13-117. In addition, we demonstrate that the human p34CDC2 protein is proficient for fission yeast meiosis, and examine the behaviour of two mutant p34CDC2 proteins in fission yeast. 相似文献
5.
N. Schmitz 《Molecular genetics and genomics : MGG》1999,261(4-5):716-724
The protein kinase p34cdc2 is required at the onset of DNA replication and for entry into mitosis. The catalytic subunit and its regulatory proteins, notably the cyclins, are conserved from yeast to man. This suggests that the control mechanisms necessary for progression through the cell cycle in fission yeast are conserved throughout evolution. This work describes the characterization of a fission yeast strain that is dependent for cell cycle progression on the activity of the p34CDC2 protein kinase from chicken. The response of the chicken p34CDC2 protein kinase to cell cycle components of fission yeast was examined. Cells expressing the chicken p34CDC2 protein divide at reduced size at 31°?C. Cells are temperature sensitive at 35.5°?C and die as a result of mitotic catastrophe. This phenotype can be rescued by delaying cell cycle progression at the G1-S transition by adding low concentrations of hydroxyurea. Schizosaccharomyces pombe cells that are dependent on chicken p34CDC2 are cold sensitive. At 19°?C to 25°?C cells arrest in the G1 phase, while traversal of the G2-M transition is not blocked at low temperature. Expression of chicken p34CDC2 in the cold-sensitive G2-M mutant cdc2A21 suppresses the G1 arrest. 相似文献
6.
7.
Muriel Audit Michle Barbier Marie-Odile Soyer-Gobillard Marie Albert Marie-Line Graud Gisle Nicolas Guy Lenaers 《Biology of the cell / under the auspices of the European Cell Biology Organization》1996,86(1):1-10
Summary— The eucaryote cell cycle is driven by a set of cyclin dependent kinases (CDKs) associated to cyclins, which confer not only the activity but also the substrate specificity and the proper localization of the kinase activity. In the fission yeast Schizosaccharomyces pombe, only one cyclin, the product of the cdc13 gene (p56cdc13), is required to be associated with p34cdc2, to control the complete cell cycle. Earlier studies have localized this complex mainly in the nucleus and its periphery. Using new improved electron microscopy (EM) technologies, based on high pressure freezing fixation, we refined previous studies, evidencing cytoplasmic localization of p56cdc13, in addition to the nuclear localization previously observed. Further immunofluorescence studies, performed on aldehydically fixed cells, confirmed our EM results, emphasizing the major cytoplasmic localization of p56cdc13 in interphase cells and the relocalization towards the nucleus in mitotic cells, suggesting that the S pombe cyclin B localization is cell cycle-regulated. 相似文献
8.
A WD Repeat Protein Controls the Cell Cycle and Differentiation
by Negatively Regulating Cdc2/B-Type Cyclin Complexes 总被引:12,自引:2,他引:10
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Satoko Yamaguchi Hiroshi Murakami Hiroto Okayama 《Molecular biology of the cell》1997,8(12):2475-2486
In the fission yeast Schizosaccharomyces pombe, p34cdc2 plays a central role controlling the cell cycle. We recently isolated a new gene named srw1+, capable of encoding a WD repeat protein, as a multicopy suppressor of hyperactivated p34cdc2. Cells lacking srw1+ are sterile and defective in cell cycle controls. When starved for nitrogen source, they fail to effectively arrest in G1 and die of accelerated mitotic catastrophe if regulation of p34cdc2/Cdc13 by inhibitory tyrosine phosphorylation is compromised by partial inactivation of Wee1 kinase. Fertility is restored to the disruptant by deletion of Cig2 B-type cyclin or slight inactivation of p34cdc2. srw1+ shares functional similarity with rum1+, having abilities to induce endoreplication and restore fertility to rum1 disruptants. In the srw1 disruptant, Cdc13 fails to be degraded when cells are starved for nitrogen. We conclude that Srw1 controls differentiation and cell cycling at least by negatively regulating Cig2- and Cdc13-associated p34cdc2 and that one of its roles is to down-regulate the level of the mitotic cyclin particularly in nitrogen-poor environments. 相似文献
9.
The protein kinase cdc2p is a key regulator of the G1-S and G2-M cell cycle transitions in the yeast Schizosaccharomyces pombe. Activation of cdc2p is regulated by its phosphorylation state and by interaction with other proteins. We have analyzed the consequences for cell cycle progression of altering the conserved threonine phosphorylation site, within the activation loop of cdc2p, to glutamic acid. This mutant, T167 E, promotes entry into mitosis, as judged by the accumulation of mitotic spindles and condensed chromosomes, despite the fact that it lacks demonstrable kinase activity both in vitro and in vivo. However, T167 E cannot promote the metaphase-anaphase transition. Since a component of the anaphase-promoting complex (APC) in S. pombe, cut9p, remains hypophosphorylated at the T167 E arrest point, the cell cycle block might be due to the inability of T167 E to activate the APC. T167 E is lethal when overexpressed, and overproduction also causes a mitotic arrest. Multicopy suppressors of the dominant negative phenotype were isolated, and identified as cdc13 + and suc1 + . Overexpression of suc1 + suppresses the effects of T167 E overproduction by restoring sufficient amounts of suc1p to the cell to allow passage through mitosis. 相似文献
10.
A Dual-Specificity Phosphatase Cdc25B Is an Unstable Protein and Triggers p34cdc2/Cyclin B Activation in Hamster BHK21 Cells Arrested with Hydroxyurea 总被引:4,自引:0,他引:4
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Hitoshi Nishijima Hideo Nishitani Takashi Seki Takeharu Nishimoto 《The Journal of cell biology》1997,138(5):1105-1116
By incubating at 30°C in the presence of an energy source, p34cdc2/cyclin B was activated in the extract prepared from a temperature-sensitive mutant, tsBN2, which prematurely enters mitosis at 40°C, the nonpermissive temperature (Nishimoto, T., E. Eilen, and C. Basilico. 1978. Cell. 15:475–483), and wild-type cells of the hamster BHK21 cell line arrested in S phase, without protein synthesis. Such an in vitro activation of p34cdc2/cyclin B, however, did not occur in the extract prepared from cells pretreated with protein synthesis inhibitor cycloheximide, although this extract still retained the ability to inhibit p34cdc2/cyclin B activation. When tsBN2 cells arrested in S phase were incubated at 40°C in the presence of cycloheximide, Cdc25B, but not Cdc25A and C, among a family of dual-specificity phosphatases, Cdc25, was lost coincidentally with the lack of the activation of p34cdc2/cyclin B. Consistently, the immunodepletion of Cdc25B from the extract inhibited the activation of p34cdc2/cyclin B. Cdc25B was found to be unstable (half-life < 30 min). Cdc25B, but not Cdc25C, immunoprecipitated from the extract directly activated the p34cdc2/cyclin B of cycloheximide-treated cells as well as that of nontreated cells, although Cdc25C immunoprecipitated from the extract of mitotic cells activated the p34cdc2/cyclin B within the extract of cycloheximide-treated cells. Our data suggest that Cdc25B made an initial activation of p34cdc2/cyclin B, which initiates mitosis through the activation of Cdc25C. 相似文献
11.
Cell cycle control in the fission yeastSchizosaccharomyces pombe involves interplay amongst a number of regulatory molecules, including thecdc2, cdc13, cdc25, weel, andmik1 gene products. Cdc2, Cdc13, and Cdc25 act as positive regulators of cell cycle progression at the G2/M boundary, while Wee1 and Mik1 play a negative regulatory role. Here, we have screened for suppressors of the lethal premature entry into mitosis, termed mitotic catastrophe, which results from simultaneous loss of function of both Wee1 and Mik1. Through such a screen, we hoped to identify additional components of the cell cycle regulatory network, and/or G2/M-specific substrates of Cdc2. Although we did not identify such molecules, we isolated a number of alleles of bothcdc2 andcdc13, including a novel wee allele ofcdc2, cdc2-5w. Here, we characterizecdc2-5w and two alleles ofcdc13, which have implications for the understanding of details of the interactions amongst Cdc2, Cdc13, and Wee1. 相似文献
12.
Hiromi Maekawa Tomoko Nakagawa Yoko Uno Kenji Kitamura Chikashi Shimoda 《Molecular genetics and genomics : MGG》1994,244(5):456-464
When the fission yeastSchizosaccharomyces pombe is starved for nitrogen, the cells are arrested in the G1 phase, enter the G0 phase and initiate sexual development. Theste13 mutant, however, fails to undergo a G1 arrest when starved for nitrogen and since this mutant phenotype is not suppressed by a mutation in adenylyl cyclase (cyr1), it would appear thatste13 + either acts independently of the decrease in the cellular cAMP level induced by starvation for nitrogen, or functions downstream of this controlling event. We have used functional complementation to clone theste13 + gene from anS. pombe genomic library and show that its disruption is not lethal, indicating that, while the gene is required for sexual development, it is not essential for cell growth. Nucleotide sequencing predicts thatste13 + should encode a protein of 485 amino acids in which the consensus motifs of ATP-dependent RNA helicases of the DEAD box family are completely conserved. Point mutations introduced into these consensus motifs abolished theste13 + functions. The predicted Ste13 protein is 72% identical to theDrosophila melanogaster Me31B protein over a stretch of 391 amino acids. ME31B is a developmentally regulated gene that is expressed preferentially in the female germline and may be required for oogenesis. Expression of ME31B cDNA inS. pombe suppresses theste13 mutation. These two evolutionarily conserved genes encoding putative RNA helicases may play a pivotal role in sexual development. 相似文献
13.
Although Cks proteins were the first identified binding partners of cyclin-dependent protein kinases (cdks), their cell cycle functions have remained unclear. To help elucidate the function of Cks proteins, we examined whether their binding to p34cdc2 (the mitotic cdk) varies during the cell cycle in Xenopus egg extracts. We observed that binding of human CksHs2 to p34cdc2 was stimulated by cyclin B. This stimulation was dependent on the activating phosphorylation of p34cdc2 on Thr-161, which follows cyclin binding and is mediated by the cdk-activating kinase. Neither the inhibitory phosphorylations of p34cdc2 nor the catalytic activity of p34cdc2 was required for this stimulation. Stimulated binding of CksHs2 to another cdk, p33cdk2, required both cyclin A and activating phosphorylation. Our findings support recent models that suggest that Cks proteins target active forms of p34cdc2 to substrates. 相似文献
14.
N. Schmitz 《Molecular & general genetics : MGG》1999,261(4-5):716-724
The protein kinase p34cdc2 is required at the onset of DNA replication and for entry into mitosis. The catalytic subunit and its regulatory proteins,
notably the cyclins, are conserved from yeast to man. This suggests that the control mechanisms necessary for progression
through the cell cycle in fission yeast are conserved throughout evolution. This work describes the characterization of a
fission yeast strain that is dependent for cell cycle progression on the activity of the p34CDC2 protein kinase from chicken. The response of the chicken p34CDC2 protein kinase to cell cycle components of fission yeast was examined. Cells expressing the chicken p34CDC2 protein divide at reduced size at 31° C. Cells are temperature sensitive at 35.5° C and die as a result of mitotic catastrophe.
This phenotype can be rescued by delaying cell cycle progression at the G1-S transition by adding low concentrations of hydroxyurea.
Schizosaccharomyces pombe cells that are dependent on chicken p34CDC2 are cold sensitive. At 19° C to 25° C cells arrest in the G1 phase, while traversal of the G2-M transition is not blocked
at low temperature. Expression of chicken p34CDC2 in the cold-sensitive G2-M mutant cdc2A21 suppresses the G1 arrest.
Received: 14 October 1998 / Accepted: 15 March 1999 相似文献
15.
Identification of p34 and p13, human homologs of the cell cycle regulators of fission yeast encoded by cdc2+ and suc1+ 总被引:41,自引:0,他引:41
cdc2+ and CDC28 play central roles in the cell division cycles of the widely divergent yeasts Schizosaccharomyces pombe and Saccharomyces cerevisiae, respectively. The genes encode protein kinases that show 62% protein sequence identity and are capable of cross-complementation. Monoclonal antibodies were raised against p34cdc2, and a subset recognize p36cdc28. The cross-reacting antibodies detected a 34 kd homolog of the p34cdc2/p36CDC28, protein in HeLa cells. Human p34 was also recognized by an affinity-purified polyclonal anti-p34cdc2 serum. Peptide mapping of p34cdc2, p36CDC28, and human p34 revealed complete conservation of four tryptophan residues in the three proteins. p34 thus appears to be closely related to the two yeast proteins. In addition, a p34 immune complex showed protein kinase activity in vitro, and HeLa cell p34 interacts with p13, the human homolog of the suc1+ gene product of S. pombe. 相似文献
16.
17.
18.
Identification of two cell-cycle-controlling cdc2 gene homologs in Arabidopsis thaliana 总被引:21,自引:0,他引:21
The cdc2 gene product (p34cdc2) has been thought to play a central role in control of the mitotic cell cycle of yeasts and animals. To approach an understanding of the cell-cycle-control system in higher plants, we isolated, from an Arabidopsis thaliana cDNA library, two clones (CDC2a and CDC2b) similar to the Schizosaccharomyces pombe cdc2 gene. Genomic Southern-blot analysis with the CDC2a and CDC2b cDNA probes suggested that the A. thaliana genome contains several additional cdc2-like genes, which together with the CDC2a and CDC2b genes may constitute a CDC2 gene family. The CDC2a cDNA expressed in Sc. pombe corrected the elongated morphology, caused by the temperature-sensitive cdc2-33 mutation, to the normal shapes, indicating that the A. thaliana CDC2a gene product resembles Sc. pombe p34cdc2 functionally as well as structurally. These results support the view that the cell cycle of higher plants is controlled by an analogue of a p34cdc2-centered regulatory system like that of yeasts and animals. 相似文献
19.
Summary The cdc2 gene of the fission yeast Schizosaccharomyces pombe encodes a 34 kDa phosphoprotein with serine/threonine protein kinase activity that acts as the key component in regulation of the eukaryotic cell cycle. We used a repressible promoter fused to the cdc2 cDNA to isolate conditionally dominant negative mutants of cdc2. One of these mutants, DL5, is described in this paper. Overexpression of the mutant protein in a wild-type cdc2 background is lethal and confers cell cycle arrest with a typical cdc phenotype. Sequencing of the mutant cdc2 gene revealed a single amino acid substitution in a region highly conserved in cdc2-like proteins. The mutant protein exhibits no protein kinase activity, but is able to bind a component(s) required for an active protein kinase complex and thereby prevents binding of this component(s) to the co-existing wild-type cdc2 protein. We also demonstrate that S. pombe p34cdc2 contains no phosphoserine. 相似文献
20.
There`se David-Pfeuty Yolande Nouvian-Dooghe Dany Rouillard 《Biology of the cell / under the auspices of the European Cell Biology Organization》1997,89(3):179-197
An affinity-purified antibody (anti-Cdc2C) raised against the carboxy terminal sequence LDNQIKKM of p34cdc2 uncovered in NIH 3T3 cells a protein subpopulation, the location and the level of accumulation of which evolve during progression through the cell cycle: it first emerges inside the nucleus in late G1/early S phase and continues to build up principally in this location throughout S phase; a cytoplasmic expression then becomes apparent near the end of S phase, develops during G2 and sometimes prevails over the nuclear expression; it finally relocates to the nucleus in early prophase. We propose that a major part of this subpopulation would represent p34cdc2 molecules existing inside a complex with cyclin B1. NIH 3T3 cells arrested in early S phase with aphidicolin do not commit prematurely to mitosis which indicates that the regulatory pathway involved in preserving the temporal order of S and M phases is functioning in these conditions. Conjugated Western blot analysis and immunofluorescence microscopy showed that cyclin A, cyclin B1 and tyrosine-phosphorylated p34cdc2 continue to build up predominantly in the nucleus of the arrested cells. After release from the block, the cells rapidly reenter S and G2 phases and, concomitantly, cyclin B1 and tyrosine-phosphorylated p34cdc2 relocate to the cytoplasm before redistributing again in the nucleus in early prophase. These data would suggest that delaying the onset of M phase in NIH 3T3 cells in which the rate of DNA replication is reduced, is first ensured by a mechanism that prevents the cytoplasmic relocation of inactive p34cdc2/cyclin B1 complexes continually forming in the nucleus once the G1 period of mitotic cyclin instability is over. 相似文献