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© The Rockefeller University Press,
0021-9525/1999//573 $5.00
The Journal of Cell Biology, Volume 146, Number 3,
, 1999 573-584
Original Article |
Cdc25b and Cdc25c Differ Markedly in Their Properties as Initiators of Mitosis
j.pines{at}welc.cam.ac.uk
We have used time-lapse fluorescence microscopy to study the properties of the Cdc25B and Cdc25C phosphatases that have both been implicated as initiators of mitosis in human cells. To differentiate between the functions of the two proteins, we have microinjected expression constructs encoding Cdc25B or Cdc25C or their GFP-chimeras into synchronized tissue culture cells. This assay allows us to express the proteins at defined points in the cell cycle. We have followed the microinjected cells by time-lapse microscopy, in the presence or absence of DNA synthesis inhibitors, and assayed whether they enter mitosis prematurely or at the correct time. We find that overexpressing Cdc25B alone rapidly causes S phase and G2 phase cells to enter mitosis, whether or not DNA replication is complete, whereas overexpressing Cdc25C does not cause premature mitosis. Overexpressing Cdc25C together with cyclin B1 does shorten the G2 phase and can override the unreplicated DNA checkpoint, but much less efficiently than overexpressing Cdc25B. These results suggest that Cdc25B and Cdc25C do not respond identically to the same cell cycle checkpoints. This difference may be related to the differential localization of the proteins; Cdc25C is nuclear throughout interphase, whereas Cdc25B is nuclear in the G1 phase and cytoplasmic in the S and G2 phases. We have found that the change in subcellular localization of Cdc25B is due to nuclear export and that this is dependent on cyclin B1. Our data suggest that although both Cdc25B and Cdc25C can promote mitosis, they are likely to have distinct roles in the controlling the initiation of mitosis.
Key Words: cell cycle • mitosis • green fluorescent protein • nuclear export
© 1999 The Rockefeller University Press
THE Cdc25 phosphatase has a crucial and conserved role in the initiation of mitosis in eukaryotic cells; it activates the major mitotic protein kinase (composed of cyclin B and its partner catalytic subunit cyclin-dependent kinase 1 [CDK1])1 (Nurse 1990; Dunphy 1994). Cyclin B/CDK1 causes profound changes in the cell architecture such as nuclear envelope breakdown and reorganization of the microtubule and actin filament networks in part by directly phosphorylating a number of important structural components of the cell (for review see Jackman and Pines 1997; Nigg 1993). In general, with the exception of budding yeast (Amon et al. 1992; Sorger and Murray 1992), a pool of cyclin B–CDK1 complexes accumulates through late S and G2 phase that is kept inactive by phosphorylation on residues in the ATP binding site of CDK1 (for review see Lew and Kornbluth 1996). One of these residues, tyrosine 15, is phosphorylated in all cells by the Wee1/Mik1 kinase (Russell and Nurse 1987a; Gould and Nurse 1989; Lundgren et al. 1991; Parker et al. 1992; McGowan and Russell 1993) and phosphorylation at this site appears to interfere with phosphotransfer to a bound substrate (Atherton Fessler et al. 1993). In animal cells, and in fission yeast under certain conditions (Den Haese et al. 1995), the adjacent residue, threonine 14, is phosphorylated by the membrane-bound Myt1 kinase (Kornbluth et al. 1994; Mueller et al. 1995; Booher et al. 1997; Liu et al. 1997) that interferes with ATP binding (Booher et al. 1997). The Cdc25 phosphatases are able to remove the phosphate from both Y15 and T14 and thereby activate cyclin B/CDK1 (Dunphy and Kumagai 1991; Gautier et al. 1991; Kumagai and Dunphy 1991; Millar et al. 1991b; Strausfeld et al. 1991; Sebastian et al. 1993).
Initial experiments using fission yeast showed that cdc25 is a dosage-dependent regulator of mitosis (Russell and Nurse 1986) whose level is partially regulated by proteolysis mediated by the pub1 gene product (Nefsky and Beach 1996). Cells with a defective cdc25 gene product have a pronounced G2 delay before they enter mitosis, therefore, they grow to a longer size than wild-type cells (Russell and Nurse 1986). Conversely, cells that overexpress cdc25 or have a defect in the wee1 kinase that antagonizes cdc25, enter mitosis prematurely and, therefore, divide at a smaller size than normal cells (Russell and Nurse 1987b; Enoch and Nurse 1990). Thus, the balance between the activities of cdc25 and wee1 determines when a cell enters mitosis. This control mechanism is responsive to the state of the DNA. Unreplicated or damaged DNA prevents cells from entering mitosis by altering the balance between the activities of cdc25 and wee1, such that fission yeast cells with a mutant CDK1 (cdc2) gene that cannot be phosphorylated by wee1/mik1 enter mitosis regardless of the state of the DNA (Gould and Nurse 1989; Enoch and Nurse 1990). Recently, the molecular mechanism linking damaged DNA to the activation of CDK1 has been partially elucidated. The Cds1 and Chk1 genes, which are activated by unreplicated DNA and DNA damage (Boddy et al. 1998; Blasina et al. 1999), phosphorylate a conserved site on cdc25 to create a binding site for a 14-3-3 protein that prevents cdc25 from activating cyclin B/CDK1 (Furnari et al. 1997; Peng et al. 1997; Sanchez et al. 1997; Lopez-Girona et al. 1999). In fission yeast, cds1 also phosphorylates the wee1 kinase and is required for the accumulation of the mik1 kinase in response to unreplicated DNA (Boddy et al. 1998).
The fundamental importance of the balance between cdc25 and wee1 in preventing premature mitosis has also been demonstrated in frog egg extracts (Smythe and Newport 1992) and in hamster (Heald et al. 1993) and human tissue culture cells (Hagting et al. 1998). The complication in human cells is that there are three different Cdc25 genes: Cdc25A, B, and C (Galaktionov and Beach 1991; Nagata et al. 1991). Of these, Cdc25A is present in G1 phase cells and appears to play a part in initiating DNA replication (Hoffmann et al. 1994; Jinno et al. 1994). However, both Cdc25B and Cdc25C, which are 45% identical at the amino acid level (85% identical in the catalytic domains), are present in G2 phase cells; each appears to be necessary for cells to enter mitosis, because microinjecting either anti-Cdc25B or anti-Cdc25C antibodies, or transfecting cells with an inactive mutant of Cdc25B or Cdc25C will block cells in the G2 phase (Millar et al. 1991b; Gabrielli et al. 1996; Lammer et al. 1998). Nevertheless, there are indications that Cdc25B and Cdc25C may not have redundant roles in the initiation of mitosis. First, anti-Cdc25B immunoprecipitates have phosphatase activity from S phase onwards that increases two- to fourfold at mitosis, whereas anti-Cdc25C immunoprecipitates are inactive until late G2/M phase (Gabrielli et al. 1996; Nishijima et al. 1997; Lammer et al. 1998). In vitro, Cdc25B is also more active than Cdc25C towards cyclin B/CDK1 unless Cdc25C is activated by prior phosphorylation on its amino terminus. Second, Cdc25C is a nuclear protein in human cells (Millar et al. 1991a; Girard et al. 1992), whereas Cdc25B is reported to be cytoplasmic, but to translocate to the nucleus at mitosis or when overexpressed (Gabrielli et al. 1996, Gabrielli et al. 1997b). Third, in transient transfection experiments overexpressing Cdc25C had no effect on the cell cycle profile of cells as analyzed by flow cytometry, whereas overexpressing Cdc25B caused a slow increase in the population of G2/M cells and these cells had abnormal mini-spindles (Gabrielli et al. 1996).
There are a number of problems associated with performing cell cycle experiments in tissue culture cells. The effects of introducing mutant proteins, or overexpressing wild-type proteins, by transient transfection have to be interpreted in a heterogeneous population of cells at fixed points after transfection. Therefore, judging how a particular procedure has affected the cell cycle of any one cell is mostly a matter of informed guesswork. Furthermore, it is difficult to introduce a protein by transfection into cells at a precise stage in the cycle, and, most importantly, progress through the cell cycle, is often affected by the transfection procedure itself (Winters et al. 1998). In an effort to overcome these problems, we have begun to analyze the cell cycle by time-lapse fluorescence microscopy (Hagting et al. 1998). This allows us to follow individual cells through an entire cell cycle. We have combined this with microinjection into synchronized cells to introduce cell cycle regulators at defined points in the cell cycle. Furthermore, we introduce regulators in the form of fusion proteins with a modified form of green fluorescent protein (MmGFP) (Zernicka-Goetz et al. 1996, Zernicka-Goetz et al. 1997). This allows us to follow the subcellular localization of specific proteins in real time and gives us a dynamic view of their behavior when the cell enters mitosis. Using this experimental regime, we find that Cdc25B is a rate-limiting component of mitosis whose overexpression will overcome the unreplicated DNA checkpoint. In contrast, Cdc25C does not accelerate cells into mitosis unless it is coexpressed with cyclin B1, and even this combination does not override the S phase checkpoint. We confirm that there is a marked difference in subcellular localization between Cdc25B and Cdc25C and show that the cytoplasmic localization of Cdc25B is regulated by nuclear export that is dependent on cyclin B1.
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Materials and Methods |
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Plasmid Constructs and Protein Purification
The cyclin B1-MmGFP, cdc25C(S216G), and Wee1 constructs have been previously described (Hagting et al. 1998). Cyclin B1R42A-MmGFP was the gift of Paul Clute (Wellcome/CRC Institute). Cdc25B3 and Cdc25C were tagged at the amino terminus with MmGFP (Zernicka-Goetz et al. 1996, Zernicka-Goetz et al. 1997) by PCR using Taq polymerase and cloned into the pCMX vector (Umesono et al. 1991). The stop codon of GFP was mutated to a Hind III site to link it with the first amino acid of Cdc25B or Cdc25C creating a 3–amino acid linker (Gly-Ile-Pro). Myc-tagged cdc25B2 (Lammer et al. 1998) was cloned into the pCDNA3 vector for expression in HeLa cells. All constructs were sequenced using an Applied Biosystems DNA sequencer. Cyclin B1–cdc2K33R complexes were expressed in and purified from baculovirus-infected cells as described (Hagting et al. 1998). Cyclin B1F146A was expressed in Escherichia coli and purified as described (Hagting et al. 1999).
Microinjection and PCC Detection
Constructs expressing cDNAs under the control of the cytomegalovirus promoter were microinjected into cell nuclei using an Eppendorf semi-automatic microinjection apparatus. To assay for condensed chromatin, Hoechst 33342 was added to cells at a concentration of 1 µg/ml at the end of the experiment. Injected cells were identified by green fluorescent protein (GFP) fluorescence and those that had rounded up with abnormally condensed chromatin were scored. At least 50 cells were scored for each injected construct and experiment. Apoptotic cells were assayed using the apoptosis detection kit (R&D Systems, Inc.) and HeLa cells treated with cycloheximide plus tumor necrosis factor 
were used as positive controls.
Time-lapse Differential Interference Contrast (DIC) and Fluorescence Imaging
To visualize GFP-chimeras in living cells, cells were cultured on an inverted Leica DMIRB/E microscope using the 
TC3 system (Bioptechs) to maintain cells at 37°C. Images were captured with a PentaMax CCD camera (Princeton Instruments) fitted to the lateral photo port. GFP- and yellow fluorescent protein (YFP)–chimeras were detected with custom filter sets JP1 and JP2 (Chroma Technology Corp.) and two Lambda 10-2 filter wheels (Sutter Instrument) controlled by a PowerWave computer (PowerComputing). One filter wheel was used to control the wavelength of the excitation light. The other filter wheel controlled the wavelength of the emission light and also the polarizer for DIC images. To distinguish between GFP and YFP we used the JP3 filter set as described (Hagting et al. 1999). Images were collected and processed using IP Lab Spectrum software (Scanalytics Inc.) and exported to Adobe Photoshop for printing.
Immunofluorescence and Confocal Imaging
For β-tubulin and MPM2 staining, cells were fixed with 3% PFA/Triton X-100 and stained as described (Pines 1997) 3–4 h after microinjection. Tubulin was detected using an anti–β-tubulin mAb (Nycomed Amersham) and mitotic epitopes were detected using the MPM2 mAb (Upstate Biotechnology, Inc.). To detect myc-cdc25B2, pCDNA3/myc-cdc25B2 was microinjected (0.1 µg/µl) and cells were fixed with methanol/acetone (1:1) 3 h after injection and stained with the mAb 9E10 (gift of Erich Nigg, University of Geneva, Geneva). A Cy5 conjugated anti-mouse antibody (Jackson ImmunoResearch Laboratories, Inc.) was used as the secondary antibody. Cells were analyzed by confocal laser scanning microscopy using a Bio-Rad 1024 confocal microscope set on 10% laser power and Kalman averaging. Stacks of images were projected using Lasersharp software (Bio-Rad Laboratories) and exported to Adobe Photoshop for processing and printing.
Glutathione-S-transferase (GST) Pulldowns
Human cdc25B2, cdc25B3, cdc25C, and cyclin B1 were in vitro translated from pBSK/cdc25B cDNA using the TNT-coupled reticulocyte system (Promega Corp.). GST-cyclin B1, GST-Cdc25B2, GST-Cdc25B3, and GST were expressed in BL21(DE3) cells using the pGEX-4T expression vector and purified on GSH-Sepharose.
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Results |
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We found that overexpressing GFP-Cdc25B in early S phase rapidly caused cells to round up and break down their nuclear envelope (Fig. 1 A and 2 A). Staining the cells with Hoechst 33342 showed that the chromatin had condensed abnormally (Fig. 1 A) and anti–β-tubulin immunofluorescence staining revealed the presence of mini-spindles as previously described (Gabrielli et al. 1996). This suggested that the cells had entered mitosis prematurely rather than undergone apoptosis. To confirm this, we stained cells with the MPM2 mAb that recognizes mitosis-specific phospho-epitopes and for the apoptosis-specific surface marker annexin V. Cells expressing Cdc25B with abnormally condensed chromatin stained very strongly with the MPM2 antibody (Fig. 1 C) but not for annexin V (data not shown). Thus, we concluded that Cdc25B caused cells to enter mitosis prematurely (premature chromosome condensation, PCC) rather than undergo apoptosis. The GFP tag was not responsible for this effect because the untagged Cdc25B cDNA induced premature mitosis with a similar frequency to GFP-Cdc25B (not shown) and an inactive mutant of Cdc25B that lacked the catalytic cysteine residue (C488S) could not induce premature mitosis (not shown). Coinjecting a cyclin B1 expression vector with Cdc25B had only a minor effect on the frequency of premature mitosis (Fig. 2 A).
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Overexpression of cdc25C Can Only Cause Premature Mitosis at the End of S Phase, and Only When Coexpressed with Cyclin B1
The effect of overexpressing Cdc25C in HeLa cells was markedly different from that of overexpressing Cdc25B; Cdc25C could not induce PCC when overexpressed alone. Cdc25C was only able to cause a significant fraction of cells to enter mitosis prematurely when cyclin B1 was also overexpressed. Furthermore, overexpressing Cdc25C and cyclin B1 could only cause PCC after cells had entered the G2 phase (Fig. 3 A), not in the S phase cells. The unreplicated DNA and G2 phase DNA damage checkpoints prevent mitosis, at least in part, by acting on Cdc25C. Unreplicated DNA activates the Cds1 and Chk1 kinases; Chk1 is also activated by DNA damage. Both kinases phosphorylate Cdc25C on serine 216 to create a binding site for a 14-3-3 protein that inactivates Cdc25C. Therefore, to test whether Cdc25C was less potent than Cdc25B because it was downregulated by Cds1 and/or Chk1, we used a Cdc25C mutant that cannot be phosphorylated on serine 216 (S216G). Cdc25CS216G was more efficient than wild-type Cdc25C in causing PCC, but again this was only when cyclin B1 was coexpressed (Fig. 3A and Fig. B). Therefore, downregulation by Cds1 and/or Chk1 is not sufficient to explain the difference in potency between Cdc25B and Cdc25C.
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50% of the injected cells 6 h after injection. This would normally correspond to the middle of G1 phase (Fig. 4). Furthermore, overexpressing Cdc25C, with or without cyclin B1, or cyclin B1 alone did not force G1 phase cells into premature mitosis (Fig. 4 and data not shown).
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The Cytoplasmic Localization of cdc25B Is Dependent on Cyclin B1
Although the cytoplasmic localization of Cdc25B in S and G2 phases depended on Crm1-mediated nuclear export, Cdc25B was nuclear in the G1 phase. The cytoplasmic localization of Cdc25B correlated with the point in the cell cycle when cyclin B1 started to accumulate and cyclin B1 has been shown to be exported from nuclei in an LMB-sensitive manner (Hagting et al. 1998; Toyoshima et al. 1998; Yang et al. 1998). Taken together these observations suggested that cyclin B1 might be involved in mediating the cytoplasmic localization of Cdc25B. To test this hypothesis, we expressed GFP-cdc25B in early G1 phase or early S phase cells (i.e., cells that lack any endogenous cyclin B1), and then microinjected purified cyclin B1 or cyclin B1/CDK1K33R protein into the nucleus. GFP-cdc25B was immediately and rapidly exported from G1 (not shown) or S phase nuclei (Fig. 7 A) after injecting cyclin B1/CDK1K33R and reentered the nuclei when LMB was added to inhibit export (Fig. 7 A). This effect was observed using cyclin B1 in a complex with a kinase-dead mutant of CDK1, which strongly suggested that cyclin B1 itself and not CDK1 kinase activity was required for cdc25B export. In support of this, we found that GFP-cdc25B still accumulated in the cytoplasm of G2 phase cells in the presence of the CDK1 inhibitor, roscovitin (data not shown). Furthermore, a cyclin B1 mutant with a defective nuclear export signal, cyclin B1F146A, did not cause nuclear Cdc25B to be exported when microinjected into G1 (Fig. 7 B) or S phase nuclei (not shown). These observations suggested that Cdc25B export could be due to a direct interaction between cyclin B1/CDK1 and Cdc25B. In support of this, we were able to detect an in vitro interaction between Cdc25B and cyclin B1 that appeared to be slightly stronger than an association between cyclin B1 and Cdc25C (Fig. 7 C).
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Discussion |
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Our demonstration that overexpressing Cdc25C alone cannot overcome the unreplicated DNA checkpoint, but that Cdc25B can, suggests that Cdc25C and Cdc25B are regulated in different ways. Cdc25C appears to require a further activation step that can be partially supplied by increasing the level of cyclin B (Heald et al. 1993). Indeed, an extra copy of cyclin B (NIME) can partially suppress a mutation in cdc25 (NIMT) in Aspergillus nidulans (O'Connell et al. 1992). This activation step is most likely to be phosphorylation of the amino terminus of Cdc25C, which activates its phosphatase activity 10-fold in vitro, and cyclin B/Cdk1 is able to phosphorylate and activate Cdc25C (Izumi et al. 1992; Kumagai and Dunphy 1992; Hoffmann et al. 1993; Izumi and Maller 1995; Gabrielli et al. 1997a). More recently, members of the polo-like family of kinases have been shown to phosphorylate and activate Cdc25C (Kumagai and Dunphy 1996), but it is unclear whether they initiate Cdc25C activation at the end of G2 phase. In this regard, we were unable to activate prematurely Cdc25C by coexpressing human plk1 (data not shown), although this may be because plk1 itself needs to be activated by phosphorylation (Qian et al. 1998). Our data show that Cdc25C activation is either prevented, or rapidly reversed, in the presence of unreplicated DNA. The inhibition of Cdc25C may be partially effected through phosphorylation on S216 and the consequent binding of a 14-3-3 protein (Boddy et al. 1998; Rhind and Russell 1998). However, this cannot provide a full explanation, because we found that although an S216G mutant form of Cdc25C is able to promote premature mitosis more rapidly than the wild-type Cdc25C, it is unable to overcome the DNA replication checkpoint.
In contrast, it appears that Cdc25B either does not need an activation step or that the activator is present whenever cyclin B1 is present. Furthermore, cyclin B/Cdk1 phosphorylates Cdc25B in vitro and this correlates with a fourfold increase in Cdc25B activity (Gabrielli et al. 1996, Gabrielli et al. 1997a; Lammer et al. 1998). One explanation could be that cyclin B/CDK1 activates Cdc25B and vice versa in a positive feedback loop, and that this is more potent than the positive feedback loop between Cdc25C and cyclin B1/CDK1 because Cdc25B and cyclin B1/CDK1 are both cytoplasmic. If Cdc25B and cyclin B do activate one another, then the resulting positive feedback loop must be carefully regulated because overexpressing Cdc25B is sufficient to cause premature mitosis, regardless of whether the DNA has been replicated. One way in which Cdc25B might be regulated is by protein turnover. Cdc25B is an unstable protein, with a half-life of <30 min in hamster and HeLa cells (Nishijima et al. 1997), which can be targeted for degradation in vitro by cyclin A-CDK2 (Baldin et al. 1997a). Thus, Cdc25B may be primarily regulated by ubiquitin-mediated proteolysis in an analogous fashion to cdc25 in fission yeast (Nefsky and Beach 1996).
Remarkably, we found that when Cdc25B and cyclin B1 were coexpressed in G1 phase cells the cells attempted to enter mitosis, showing that cell division and DNA replication can be uncoupled in human cells. This may also be relevant to how the cell causes two sequential M phases in meiosis and it will be interesting to determine whether Cdc25B is required for gametogenesis.
Despite the differences in their regulation, both Cdc25B and Cdc25C are antagonized by Wee1. Wee1 and Cdc25C are both nuclear proteins and, therefore, their relative activities will determine whether the nucleus will activate or inhibit cyclin B/CDK1 when it is imported into the nucleus. However, Cdc25B is primarily a cytoplasmic protein in the G2 phase and so might act on the predominantly cytoplasmic pool of cyclin B/CDK1. Therefore, Wee1 may only counteract the effects of Cdc25B when active cyclin B1/Cdk1 shuttles into the nucleus (Hagting et al. 1998).
Human Cdc25B was originally described as being a cytoplasmic protein that translocated to the nucleus at mitosis in concert with cyclin B (Gabrielli et al. 1996, Gabrielli et al. 1997b). However, these studies were performed by immunofluorescence in which the exact cell cycle stage of an individual cell was often difficult to judge, except by the appearance of condensed chromosomes. We have shown here that Cdc25B is nuclear in G1 cells, but gradually accumulates in the cytoplasm as cells progress through the S and G2 phases. Furthermore, Cdc25B will accumulate in the nucleus when S or G2 phase cells are treated with LMB, a specific inhibitor of crm1/exportin1–mediated nuclear export (Nishi et al. 1994; Fornerod et al. 1997; Wolff et al. 1997). This suggests that the localization of Cdc25B, like that of cyclin B1 (Hagting et al. 1998; Toyoshima et al. 1998; Yang et al. 1998), is primarily determined by nuclear export. However, Cdc25B does not have a recognizable nuclear export signal (Bogerd et al. 1996) and is nuclear in the G1 phase. When we microinjected wild-type cyclin B1 into the nuclei of G1 cells, we observed that cdc25B was immediately exported, but not when we microinjected an export-defective cyclin B1. Furthermore, we found that cyclin B1 binds to Cdc25B in an in vitro binding assay. These results suggest that the cytoplasmic localization of cdc25B depends upon cyclin B1, and that the two proteins are likely to be exported together from the nucleus. This is reminiscent of recent results showing that fission yeast cdc25C is exported from the nucleus after DNA damage by binding to the rad24 14-3-3 protein (Lopez-Girona et al. 1999). In both cases, the Cdc25 protein does not have a nuclear export signal itself, but is exported by virtue of its association with another export-capable protein.
In conclusion, we have demonstrated marked differences in the abilities of Cdc25B and Cdc25C to promote mitosis and in their cell cycle behavior. Our results suggest that controlling the level of Cdc25B is crucial to prevent premature mitosis, whereas Cdc25C can be regulated at a subsequent step, most likely by phosphorylation. The activity of Wee1 in the nucleus is very important in counteracting the effects of both the nuclear Cdc25C and the primarily cytoplasmic Cdc25B, emphasizing the dynamic nature of the interactions between cell cycle components that shuttle between the nucleus and the cytoplasm.
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Acknowledgments |
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C. Karlsson and A. Hagting are supported by Marie Curie fellowships from the European Community. This work was supported by a European Union Training and Mobility Research network grant to I. Hoffmann and J. Pines, a program grant to J. Pines from the Cancer Research Campaign and made possible by a Medical Research Council project grant to J. Pines.
Submitted: 4 June 1999
Revised: 21 June 1999
Accepted: 24 June 1999
1.used in this paper: CDK1, cyclin-dependent kinase 1; DIC, differential interference contrast; GFP; green fluorescent protein; LMB, leptomycin B; MmGFP, modified green fluorescent protein; PCC, premature chromosome condensation; YFP, yellow fluorescent protein
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References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Amon A., Surana U., Muroff I. & Nasmyth K.. Regulation of p34CDC28 tyrosine phosphorylation is not required for entry into mitosis in S. cerevisiae, Nature, 355, 1992, 368–371.[Medline]
Amon A., Irniger S. & Nasmyth K.. Closing the cell cycle circle in yeastG2 cyclin proteolysis initiated at mitosis persists until the activation of G1 cyclins in the next cycle, Cell, 77, 1994, 1037–1050.[Medline]
Atherton Fessler S., Parker L.L., Geahlen R.L. & Piwnica-Worms H.. Mechanisms of p34cdc2 regulation, Mol. Cell Biol, 13, 1993, 1675–1685.
Baldin V., Cans C., Knibiehler M. & Ducommun B.. Phosphorylation of human CDC25B phosphatase by CDK1-cyclin A triggers its proteasome-dependent degradation, J. Biol. Chem, 272, 1997, 32731–32734a.
Baldin V., Cans C., Superti Furga G. & Ducommun B.. Alternative splicing of the human CDC25B tyrosine phosphatase. Possible implications for growth control?, Oncogene, 14, 1997, 2485–2495b.[Medline]
Blasina A., de Weyer I.V., Laus M.C., Luyten W., Parker A.E. & McGowan C.H.. A human homologue of the checkpoint kinase cds1 directly inhibits cdc25 phosphatase, Curr. Biol, 9, 1999, 1–10.[Medline]
Boddy M.N., Furnari B., Mondesert O. & Russell P.. Replication checkpoint enforced by kinases Cds1 and Chk1, Science, 280, 1998, 909–912.
Bogerd H.P., Fridell R.A., Benson R.E., Hua J. & Cullen B.R.. Protein sequence requirements for function of the human T-cell leukemia virus type 1 Rex nuclear export signal delineated by a novel in vivo randomization-selection assay, Mol. Cell Biol, 16, 1996, 4207–4214.
Booher R.N., Holman P.S. & Fattaey A.. Human Myt1 is a cell cycle-regulated kinase that inhibits Cdc2 but not Cdk2 activity, J. Biol. Chem, 272, 1997, 22300–22306.
Brandeis M. & Hunt T.. The proteolysis of mitotic cyclins in mammalian cells persists from the end of mitosis until the onset of S phase, EMBO (Eur. Mol. Biol. Organ.) J., 15, 1996, 5280–5289.[Medline]
Den Haese G.J., Walworth N., Carr A.M. & Gould K.L.. The Wee1 protein kinase regulates T14 phosphorylation of fission yeast Cdc2, Mol. Cell Biol, 6, 1995, 371–385.
Dunphy W.G.. The decision to enter mitosis, Trends Cell Biol, 4, 1994, 202–207.[Medline]
Dunphy W.G. & Kumagai A.. The cdc25 protein contains an intrinsic phosphatase activity, Cell, 67, 1991, 189–196.[Medline]
Enoch T. & Nurse P.. Mutation of fission yeast cell cycle control genes abolishes dependence of mitosis on DNA replication, Cell, 60, 1990, 665–673.[Medline]
Fornerod M., Ohno M., Yoshida M. & Mattaj I.. CRM1 is an export receptor for leucine-rich nuclear export signals, Cell, 90, 1997, 1051–1060.[Medline]
Furnari B., Rhind N. & Russell P.. Cdc25 mitotic inducer targeted by chk1 DNA damage checkpoint kinase, Science, 277, 1997, 1495–1497.
Gabrielli B.G., De Souza C.P., Tonks I.D., Clark J.M., Hayward N.K. & Ellem K.A.. Cytoplasmic accumulation of cdc25B phosphatase in mitosis triggers centrosomal microtubule nucleation in HeLa cells, J. Cell Sci, 109, 1996, 1081–1093.[Abstract]
Gabrielli B.G., Clark J.M., McCormack A.K. & Ellem K.A.. Hyperphosphorylation of the N-terminal domain of Cdc25 regulates activity toward cyclin B1/Cdc2 but not cyclin A/Cdk2, J. Biol. Chem, 272, 1997, 28607–28614a.
Gabrielli B.G., Clark J.M., McCormack A.K. & Ellem K.A.. Ultraviolet light-induced G2 phase cell cycle checkpoint blocks cdc25-dependent progression into mitosis, Oncogene, 15, 1997, 749–758b.[Medline]
Galaktionov K. & Beach D.. Specific activation of cdc25 tyrosine phosphatases by B-type cyclinsevidence for mutiple roles of mitotic cyclins, Cell, 67, 1991, 1181–1194.[Medline]
Gautier J., Solomon M.J., Booher R.N., Bazan J.F. & Kirschner M.W.. cdc25 is a specific tyrosine phosphatase that directly activates p34cdc2, Cell, 67, 1991, 197–211.[Medline]
Girard F., Strausfeld U., Cavadore J.C., Russell P., Fernandez A. & Lamb N.J.. cdc25 is a nuclear protein expressed constitutively throughout the cell cycle in nontransformed mammalian cells, J. Cell Biol, 118, 1992, 785–794.
Gould K.L. & Nurse P.. Tyrosine phosphorylation of the fission yeast cdc2+ protein kinase regulates entry into mitosis, Nature, 342, 1989, 39–45.[Medline]
Hagting A., Karlsson C., Clute P., Jackman M. & Pines J.. MPF localisation is controlled by nuclear export, EMBO (Eur. Mol. Biol. Organ.) J., 17, 1998, 4127–4138.[Medline]
Hagting A., Jackman M., Simpson K. & Pines J.. The translocation of cyclin B1 to the nucleus at prophase requires a phosphorylation-dependent nuclear import signal, Curr. Biol, 9, 1999, 680–689.[Medline]
Heald R., McLoughlin M. & McKeon F.. Human Wee1 maintains mitotic timing by protecting the nucleus from cytoplasmically activated Cdc2 kinase, Cell, 74, 1993, 463–474.[Medline]
Hoffmann I., Clarke P.R., Marcote M.J., Karsenti E. & Draetta G.. Phosphorylation and activation of human cdc25-C by cdc2-cyclin B and its involvement in the self-amplification of MPF at mitosis, EMBO (Eur. Mol. Biol. Organ.) J., 12, 1993, 53–63.[Medline]
Hoffmann I., Draetta G. & Karsenti E.. Activation of the phosphatase activity of human cdc25A by a cdk2-cyclin E dependent phosphorylation at the G1/S transition, EMBO (Eur. Mol. Biol. Organ.) J., 13, 1994, 4302–4310.[Medline]
Izumi T. & Maller J.L.. Phosphorylation and activation of the Xenopus Cdc25 phosphatase in the absence of Cdc2 and Cdk2 kinase activity, Mol. Biol. Cell, 6, 1995, 215–226.[Abstract]
Izumi T., Walker D.H. & Maller J.L.. Periodic changes in phosphorylation of the Xenopus cdc25 phosphatase regulate its activity, Mol. Biol. Cell, 3, 1992, 927–939.[Abstract]
Jackman M.R. & Pines J.N.. Cyclins and the G2/M transition, Kastan M.B.. Checkpoint Controls and Cancer. Vol. 29, 1997, 47–73, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.
Jinno S., Suto K., Nagata A., Igarashi M., Kanaoka Y., Nojima H. & Okayama H.. Cdc25A is a novel phosphatase functioning early in the cell cycle, EMBO (Eur. Mol. Biol. Organ.) J., 13, 1994, 1549–1556.[Medline]
Kornbluth S., Sebastian B., Hunter T. & Newport J.. Membrane localization of the kinase which phosphorylates p34cdc2 on threonine 14, Mol. Biol. Cell, 5, 1994, 273–282.[Abstract]
Kudo N., Khochbin S., Nishi K., Kitano K., Yanagida M., Yoshida M. & Horinouchi S.. Molecular cloning and cell cycle-dependent expression of mammalian CRM1, a protein involved in nuclear export of proteins, J. Biol. Chem, 272, 1997, 29742–29751.
Kumagai A. & Dunphy W.G.. The cdc25 protein controls tyrosine dephosphorylation of the cdc2 protein in a cell-free system, Cell, 64, 1991, 903–914.[Medline]
Kumagai A. & Dunphy W.G.. Regulation of the cdc25 protein during the cell cycle in Xenopus extracts, Cell, 70, 1992, 139–151.[Medline]
Kumagai A. & Dunphy W.G.. Purification and molecular cloning of Plx1, a Cdc25-regulatory kinase from Xenopus egg extracts, Science, 273, 1996, 1377–1380.[Abstract]
Lammer C., Wagerer S., Saffrich R., Mertens D., Ansorge W. & Hoffmann I.. The cdc25B phosphatase is essential for the G2/M phase transition in human cells, J. Cell Sci, 111, 1998, 2445–2453.[Abstract]
Lew D.J. & Kornbluth S.. Regulatory roles of cyclin dependent kinase phosphorylation in cell cycle control, Curr. Opin. Cell Biol, 8, 1996, 795–804.[Medline]
Liu F., Stanton J.J., Wu Z. & Piwnica-Worms H.. The human Myt1 kinase preferentially phosphorylates Cdc2 on threonine 14 and localizes to the endoplasmic reticulum and Golgi complex, Mol. Cell Biol, 17, 1997, 571–583.
Lopez-Girona A., Furnari B., Mondesert O. & Russell P.. Nuclear localization of Cdc25 regulated by DNA damage and 14-3-3 protein, Nature, 397, 1999, 172–175.[Medline]
Lundgren K., Walworth N., Booher R., Dembski M., Kirschner M. & Beach D.. mik1 and wee1 cooperate in the inhibitory tyrosine phosphorylation of cdc2, Cell, 64, 1991, 1111–1122.[Medline]
McGowan C.H. & Russell P.. Human Wee1 kinase inhibits cell division by phosphorylating p34cdc2 exclusively on Tyr15, EMBO (Eur. Mol. Biol. Organ.) J., 12, 1993, 75–85.[Medline]
Millar J.B., Blevitt J., Gerace L., Sadhu K., Featherstone C. & Russell P.. p55CDC25 is a nuclear protein required for the initiation of mitosis in human cells, Proc. Natl. Acad. Sci. USA, 88, 1991, 10500–10504a.
Millar J.B.A., McGowan C.H., Lenaers G., Jones R. & Russell P.. p80cdc25 mitotic inducer is the tyrosine phosphatase that activates p34cdc2 kinase in fission yeast, EMBO (Eur. Mol. Biol. Organ.) J., 10, 1991, 4301–4309b.[Medline]
Mueller P.R., Coleman T.R., Kumagai A. & Dunphy W.G.. Myt1a membrane-associated inhibitory kinase that phosphorylates Cdc2 on both threonine-14 and tyrosine-15, Science, 270, 1995, 86–90.
Nagata A., Igarashi M., Jinno S., Suto K. & Okayama H.. An additional homolog of the fission yeast cdc25+ gene occurs in humans and is highly expressed in some cancer cells, New Biol, 3, 1991, 959–968.[Medline]
Nefsky B. & Beach D.. Pub1 acts as an E6-AP-like protein ubiquitin ligase in the degradation of cdc25, EMBO (Eur. Mol. Biol. Organ.) J., 15, 1996, 1301–1312.[Medline]
Nigg E.A.. Cellular substrates of p34cdc2 and its companion cyclin-dependent kinases, Trends Cell Biol., 3, 1993, 296–301.[Medline]
Nishi K., Yoshida M., Fujiwara D., Nishikawa M., Horinouchi S. & Beppu T.. Leptomycin B targets a regulatory cascade of crm1, a fission yeast nuclear protein, involved in control of higher order chromosome structure and gene expression, J. Biol. Chem, 269, 1994, 6320–6324.
Nishijima H., Nishitani H., Seki T. & Nishimoto T.. A dual-specificity phosphatase Cdc25B is an unstable protein and triggers p34(cdc2)/cyclin B activation in hamster BHK21 cells arrested with hydroxyurea, J. Cell Biol, 138, 1997, 1105–1116.
Nurse P.. Universal control mechanism regulating onset of M-phase, Nature, 344, 1990, 503–508.[Medline]
O'Connell M.J., Osmani A.H., Morris N.R. & Osmani S.A.. An extra copy of nimEcyclinB elevates pre-MPF levels and partially suppresses mutation of nimTcdc25 in Aspergillus nidulans, EMBO (Eur. Mol. Biol. Organ.) J, 11, 1992, 2139–2149.[Medline]
Parker L.L., Atherton Fessler S. & Piwnica-Worms H.. p107wee1 is a dual-specificity kinase that phosphorylates p34cdc2 on tyrosine 15, Proc. Natl. Acad. Sci. USA, 89, 1992, 2917–2921.
Peng C.Y., Graves P.R., Thoma R.S., Wu Z., Shaw A.S. & Piwnica-Worms H.. Mitotic and G2 checkpoint controlregulation of 14-3-3 protein binding by phosphorylation of Cdc25C on serine-216, Science, 277, 1997, 1501–1505.
Pines J.. Localization of cell cycle regulators by immunofluorescence, Methods Enzymol., 283, 1997, 99–113.[Medline]
Pines J. & Hunter T.. Isolation of a human cyclin cDNAevidence for cyclin mRNA and protein regulation in the cell cycle and for interaction with p34cdc2, Cell, 58, 1989, 833–846.[Medline]
Qian Y.W., Erikson E. & Maller J.L.. Purification and cloning of a protein kinase that phosphorylates and activates the polo-like kinase Plx1, Science, 282, 1998, 1701–1704.
Rhind N. & Russell P.. Tyrosine phosphorylation of cdc2 is required for the replication checkpoint in Schizosaccharomyces pombe, Mol. Cell Biol, 18, 1998, 3782–3787.
Russell P. & Nurse P.. cdc25+ functions as an inducer in the mitotic control of fission yeast, Cell, 45, 1986, 145–153.[Medline]
Russell P. & Nurse P.. The mitotic inducer nim1+ functions in a regulatory network of protein kinase homologs controlling the initiation of mitosis, Cell, 49, 1987, 569–576a.[Medline]
Russell P. & Nurse P.. Negative regulation of mitosis by wee1+, a gene encoding a protein kinase homolog, Cell, 49, 1987, 559–567b.[Medline]
Sanchez Y., Wong C., Thoma R.S., Richman R., Wu Z., Piwnica-Worms H. & Elledge S.J.. Conservation of the Chk1 checkpoint pathway in mammalslinkage of DNA damage to Cdk regulation through Cdc25, Science, 277, 1997, 1497–1501.
Sebastian B., Kakizuka A. & Hunter T.. Cdc25M2 activation of cyclin-dependent kinases by dephosphorylation of threonine-14 and tyrosine-15, Proc. Natl. Acad. Sci. USA, 90, 1993, 3521–3524.
Smythe C. & Newport J.W.. Coupling of mitosis to the completion of S phase in Xenopus occurs via modulation of the tyrosine kinase that phosphorylates p34cdc2, Cell, 68, 1992, 787–797.[Medline]
Sorger P.K. & Murray A.W.. S-phase feedback control in budding yeast independent of tyrosine phosphorylation of p34cdc28, Nature, 355, 1992, 365–368.[Medline]
Strausfeld U., Labbé J.C., Fesquet D., Cavadore J.C., Picard A., Sadhu K., Russell P. & Dorée M.. Dephosphorylation and activation of a p34cdc2/cyclin B complex in vitro by human cdc25 protein, Nature, 351, 1991, 242–245.[Medline]
Toyoshima F., Moriguchi T., Wada A., Fukuda M. & Nishida E.. Nuclear export of cyclin B1 and its possible role in the DNA damage-induced G2 checkpoint, EMBO (Eur. Mol. Biol. Organ.) J., 17, 1998, 2728–2735.[Medline]
Umesono K., Murakami K.K., Thompson C.C. & Evans R.M.. Direct repeats as selective response elements for the thyroid hormone, retinoic acid, and vitamin D3 receptors, Cell, 65, 1991, 1255–1266.[Medline]
Winters Z.E., Ongkeko W.M., Harris A.L. & Norbury C.J.. p53 regulates Cdc2 independently of inhibitory phosphorylation to reinforce radiation-induced G2 arrest in human cells, Oncogene, 17, 1998, 673–684.[Medline]
Wolff B., Sanglier J. & Wang Y.. Leptomycin B is an inhibitor of nuclear exportInhibition of nucleo-cytoplasmic translocation of the human immunodeficiency virus type 1 (HIV-1) Rev protein and Rev-dependent mRNA, Chem. Biol, 4, 1997, 139–147.[Medline]
Yang J., Bardes E.S., Moore J.D., Brennan J., Powers M.A. & Kornbluth S.. Control of cyclin B1 localization through regulated binding of the nuclear export factor CRM1, Genes Dev, 12, 1998, 2131–2143.
Zernicka-Goetz M., Pines J., Ryan K., Siemering K.R., Haseloff J. & Gurdon J.B.. An indelible lineage marker for Xenopus using a mutated green fluorescent protein, Development, 122, 1996, 3719–3724.[Abstract]
Zernicka-Goetz M., Pines J., Dixon J., Hunter S., Siemering K.R., Haseloff J. & Evans M.J.. Following cell fate in the living mouse embryo, Development, 124, 1997, 1133–1137.[Abstract]
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