1 Laboratory of Cell and Developmental Biology, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Nagatsuta 4259, Midori-ku, Yokohama 226-8501
2 Department of Biology, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan
*Author for correspondence (e-mail: tkishimo{at}bio.titech.ac.jp)
Accepted May 30, 2001
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SUMMARY |
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Key words: Cds1, Checkpoint kinase, Cyclin B-Cdc2 kinase, Meiosis reinitiation, Xenopus oocyte
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INTRODUCTION |
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Fully grown immature oocytes of Xenopus are arrested naturally at prophase of the first meiotic cell cycle (prophase-I), which corresponds to the late G2-phase (Ferrell, 1999). The release from the arrest, meiosis reinitiation, is equivalent to the G2/M-phase transition, and is induced by exposure of oocytes to progesterone (Masui and Clarke, 1979). After oocyte maturation, fertilization initiates early cleavage cycles. Completion of the 12th cleavage marks the midblastula transition (MBT), when zygotic transcription begins and the so-called somatic cell-type cell cycle is established (Newport and Kirschner, 1982). Despite the expression of Xenopus Chk1 protein (XChk1) at approximately constant levels throughout oocyte maturation and early embryogenesis until the neurula stage (Nakajo et al., 1999; Kappas et al., 2000), Xenopus embryos lack checkpoint controls for damaged and unreplicated DNA prior to the MBT (Dasso and Newport, 1990; Clute and Masui, 1997; Kappas et al., 2000). However, checkpoints can be created in cell-free extracts derived from Xenopus eggs (Dasso and Newport, 1990; Kumagai et al., 1998). When the extracts are supplemented with a high number of sperm nuclei, XChk1 and Xenopus Cds1 (XCds1) are phosphorylated and activated in response to unreplicated or damaged DNA, leading to a delay in the entry into M-phase (Kumagai et al., 1998; Guo and Dunphy, 2000; Guo et al., 2000; Kumagai and Dunphy, 2000). These studies indicate that while the Chk1 and Cds1 pathways are functional even prior to the MBT, the DNA damage/replication checkpoints are not operational in the intact pre-MBT embryos (Kappas et al., 2000). Nonetheless, XChk1 is involved in the G2-phase arrest of immature oocytes, even though the G2-phase arrest of immature oocytes is formally distinct from the checkpoint arrest that depends on damaged or unreplicated DNA. Furthermore, in immature oocytes there is no obvious phosphorylation of Chk1, as is usually seen in other cell types in response to the activated G2 checkpoint (Nakajo et al., 1999). However, it is as yet unclear whether XChk1 and XCds1 are regulated when the checkpoints created in egg extracts are attenuated (adapted or recovered) or when progesterone releases the G2-phase arrest in immature oocytes.
In immature Xenopus oocytes, XChk1 appears to directly phosphorylate Cdc25C at Ser287 and inhibit its activity (Nakajo et al., 1999), as seen in the activated G2 checkpoint (Russell, 1998). However, it has been suggested that other Ser287-specific kinases, distinct from XChk1, could be involved in G2-phase arrest of immature oocytes (Nakajo et al., 1999). Therefore, bearing in mind the overlapping functions of Cds1 and Chk1 in the DNA damage and replication checkpoint controls, we have studied the involvement of XCds1 in the arrest of immature Xenopus oocytes. Our results show that XCds1 is active in G2-phase-arrested oocytes, but during meiosis reinitiation following progesterone treatment it is downregulated in response to the activation of cyclin B-Cdc2 kinase, but not MAP kinase activation. This is the first indication of the downregulation of Cds1 activity at entry into M-phase, regardless of the presence or absence of the activated G2-phase checkpoint.
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MATERIALS AND METHODS |
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Preparation of oocyte and egg extracts
Oocytes were washed with extraction buffer (EB: 100 mM KCl, 5 mM MgCl2, 3 mM EGTA, 20 mM Hepes-KOH, pH 7.5), and then transferred to a centrifuge tube containing EB plus 50 µg/ml cytochalasin B. The oocytes were centrifuged (1,000 g, 30 seconds, 4°C), and then excess EB was removed. After the oocytes were crushed by centrifugation (15,000 g, 30 minutes, 4°C), their cytoplasmic extracts were recovered. The extracts were further centrifuged (15,000 g, 15 minutes, 4°C), and the clear supernatants were recovered and immediately frozen in liquid nitrogen. Xenopus egg extracts were prepared as described (Nishiyama et al., 2000).
Construction of recombinant plasmids
To clone the coding sequence of Xenopus cds1 into pBluescript-RN3 vector (Lemaire et al., 1995), a BamHI site at the start codon and a NotI site at the stop codon were created by PCR using Pfu DNA polymerase (Stratagene) with Xenopus cds1 cDNA (GenBank accession number AF326574) and the primers 5'-CGCGGATCCATGATGTCTCGTGATACTAAAACAGAGTCG-3' and 5'-GGAAGAATGCGGCCGCTTATCTTTTTGCTCTCTTTTCGGCTGATG-3'. The resulting PCR fragment was digested with BamHI and NotI, and then it was ligated to pBluescript-RN3 that had been digested with BglII and NotI. A kinase-deficient XCds1 mutant was made by mutagenesis of wild-type Xenopus cds1 using QuikChange Site-Directed Mutagenesis Kit (Stratagene). The pBluescript-RN3-Xcds1-WT construct was mutagenized using 5'-GGGGTGATACATCGTGCACTGAAGCCTGAAAATGTGC-3' as a primer (in the sense strand), where the underlined codon, residue 319, now encodes Ala rather than Asp, generating pBluescript-RN3-Xcds1-KD. To clone the coding sequence of Xenopus cds1 into pTrcHisB vector (Invitrogen), a BamHI site at the start codon and an EcoRI site downstream of the stop codon were created by subcloning the PCR fragment into another vector and digesting the vector with BamHI and EcoRI. The BamHI-EcoRI fragment encoding Xcds1 was ligated to pTrcHisB that had been digested with BglII and EcoRI. To obtain pGEX-Xcdc25C(254-316)-WT construct, a DNA encoding amino acids 254-316 of wild-type Xenopus Cdc25C was prepared by PCR using Pfu DNA polymerase with wild-type Xenopus cdc25C cDNA (gift from Dr N. Sagata) as a template and the primers 5'-CGCGGATCCATGGCAATTCTTCTGTCGGGACC-3' and 5'-GGAATTCTCAACGTCTCCTTTTCACTCTGACCG-3'. The resulting PCR product was digested with BamHI and EcoRI and ligated into pGEX-4T-1 vector (Amersham Pharmacia Biotech) that had been digested with BamHI and EcoRI. To obtain the S287A mutant of GST-XCdc25C(254-316)-WT, the pGEX-Xcdc25C(254-316)-WT construct was mutagenized using 5'- CCTTTACCGCTCACCTGCGATGCCAGAGAAACTTG-3' as a primer (in the sense strand), where the underlined codon, residue 287, now encodes Ala rather than Ser, generating pGEX-XCdc25C(254-316)-S287A construct. To obtain pBluescript-RN3-Xenopus cyclin B1-myc/His6, full-length Xenopus cyclin B1 cDNA (gift from Dr T. Hunt) whose C terminus end was fused to myc and His6 genes, was cloned to pBluescript-RN3 vector (gift from Dr M. Iwabuchi).
Production of anti-XCds1 antibodies
The pTrcHisB-Xcds1 construct was introduced into the E. coli strain BL21. Expression of His6-XCds1 was induced by growing the cells at 37°C for 4 hours in the presence of 0.5 mM isopropyl-ß-D-thiogalactoside (IPTG). Insoluble His6-XCds1 was subjected to SDS-PAGE and eluted from the gels using Model 422 Electro-eluter (Bio-Rad Laboratories). XCds1 antiserum was raised in a rabbit using gel-purified His6-XCds1 as the antigen. For affinity purification of immune rabbit serum, His6-Cds1 was subjected to SDS-PAGE and transferred to the Immobilon membrane (Millipore). Blots were stained with 0.1% Ponceau S in 0.1% acetic acid and the band of His6-Cds1 was excised. The His6-Cds1 strips were neutralized in TBS (150 mM NaCl, 50 mM Tris-HCl, pH 7.5), blocked in 5% skimmed milk in TBS for 30 minutes, and incubated with 50% serum in TBS overnight at 4°C or for 1-2 hours at room temperature. The strips were washed with TBS, and the protein-bound antibodies were eluted by incubating the strips with 0.1 M glycine-HCl, pH 2.5, for three 3-minute periods. The eluates were neutralized by the addition of one twentieth volume of 1 M Tris. Another XCds1 antiserum was raised in rabbits against a synthetic peptide of the C terminus of XCds1 (CSEILPTSAEKRAKR) using the services of Sawady Technology (Tokyo). Whole serum was affinity-purified against the peptide antigen coupled to sulfolink agarose beads (Pierce).
Production of GST-XCdc25C(254-316)-WT and GST-XCdc25C(254-316)-S287A
The pGEX-Xcdc25C(254-316)-WT or -S287A constructs were introduced into the E. coli strain BL21. Expression of GST-XCdc25C(254-316) was induced by growing the cells at 37°C for 3 hours in the presence of 0.5 mM IPTG. GST-XCdc25C(254-316) was purified by glutathione-Sepharose 4B (Amersham Pharmacia Biotech). The resulting eluate was dialyzed against kinase buffer (20 mM MgCl2, 1 mM dithiothreitol (DTT), 40 mM Hepes-KOH, pH 7.3), concentrated to approximately 10 mg/ml using a Centricon 30 (Amicon) and stored in portions at -80°C.
Immunoprecipitation and kinase assay for XCds1
For immunoprecipitation, oocyte extracts (2 µl) were diluted in 300 µl of immunoprecipitation buffer (80 mM ß-glycerophosphate, 20 mM EGTA, 15 mM MgCl2, 50 mM NaF, 1 mM Na3VO4, 0.5 mM phenylmethylsulfonyl fluoride (PMSF), 0.5% Nonidet P40 (NP-40), 20 mM Hepes-NaOH, pH 7.3). The diluted extracts were incubated with 20 µl of Protein A-Sepharose CL-4B (Amersham Pharmacia Biotech) and 2 µg of control rabbit IgG (Zymed) for 30 minutes at 4°C with constant rotation. After removal of the beads by centrifugation, 20 µl of Protein A-Sepharose CL-4B and 2 µg of affinity-purified XCds1 antibodies was added to the supernatant. After a 2-hour incubation with constant rotation at 4°C, the beads were washed four times with beads wash buffer (5 mM NaF, 250 mM NaCl, 5 mM EGTA, 5 mM EDTA, 0.5 mM PMSF, 0.1% NP-40, 50 mM Tris-HCl, pH 7.5). The immunoprecipitates were dissolved in SDS-sample buffer, and boiled for 3 minutes, followed by separation with SDS-PAGE. For measuring XCds1 kinase activity, XCds1 immunoprecipitates were washed four times with bead wash buffer, twice more with kinase buffer and incubated with 30 µl of kinase buffer containing 10 µM ATP, 5 µCi [-32P]ATP and 5 µg GST-XCdc25C(254-316)-WT as a substrate (Guo and Dunphy, 2000). After incubation at 22°C for 30 minutes, the reactions were stopped by the addition of SDS-sample buffer, boiled for 3 minutes, and separated by SDS-PAGE. Phosphorylated forms of GST-XCdc25C(254-316)-WT were detected on gels by autoradiography and quantitated by Cherenkov counting, or by immunoblots with anti-phospho human Cdc25C (Ser-216) antibody. This antibody was able to recognize phosphorylation of Ser-287 on Xenopus Cdc25C as well, but not phosphorylations by cyclin B-Cdc2 kinase (data not shown).
Treatment of XCds1 with cyclin B-Cdc2 kinase
To phosphorylate XCds1 with cyclin B-Cdc2 kinase, XCds1 immnoprecipitates were mixed with 30 µl of kinase buffer containing 10 µM ATP and 5 µCi [-32P]ATP with or without active cyclin B-Cdc2 kinase purified from starfish oocytes (Okumura et al., 1996), and then incubated at 22°C for 30 minutes. The reactions were stopped by the addition of SDS-sample buffer, subjected to SDS-PAGE, and autoradiographed on X-ray film. For XCds1 kinase assay, XCds1 immnoprecipitates were mixed with 30 µl of kinase buffer containing 600 µM ATP with or without starfish cyclin B-Cdc2 kinase. After incubation at 22°C for 30 minutes, the reaction mixture was added with 5 µg of GST-XCdc25C(254-316)-WT and incubated at 22°C for 30 minutes. The reaction was stopped by the addition of SDS-sample buffer, and subjected to western blot analysis.
Histone H1 kinase assay
Oocyte extracts (1 µl) were diluted in ßGP-EB (80 mM ß-glycerophosphate, 5 mM MgCl2, 20 mM EGTA, 20 mM Hepes-KOH, pH 7.4) to a final volume of 10 µl. The diluted extracts were mixed with 20 µl of reaction buffer (80 mM ß-glycerophosphate, 20 mM MgCl2, 0.6 µg/ml leupeptin, 0.6 µg/ml aprotinin, 600 µM ATP) containing 12 µg histone H1 and 2 µCi [-32P]ATP, and incubated for 30 minutes at 22°C. The reactions were stopped by the addition of SDS-sample buffer, boiled for 3 minutes, and subjected to SDS-PAGE, and autoradiographed on X-ray film.
Microinjection of mRNA into oocytes
The pBluescript-RN3-Xcds1-WT, -Xcds1-KD, -LacZ or -Xenopus cyclin B1-myc/His6 constructs were cut singly and in vitro transcribed into 5'-capped mRNAs by using the mCAP RNA Capping Kit (Stratagene). The in vitro synthesized mRNA was purified by Sephadex G-50 (Amersham Pharmacia Biotech) and dissolved in distilled water. Microinjection of mRNA into immature Xenopus oocyte was performed using Nanoject (Drummond Scientific Co.). Except for Xenopus cyclin B1 mRNA, the oocytes injected with mRNA were cultured overnight to overexpress the encoded protein, and then treated with progesterone.
Western blot analysis
Oocytes, eggs and embryos were homogenized with ten volumes of ßGP-EB. The homogenates were centrifuged (10,000 g, 10 minutes, 4°C) and their cytoplasmic extracts were recovered. The extracts were mixed with SDS-sample buffer, and boiled for 3 minutes. The samples were subjected to SDS-PAGE and blotted onto an Immobilon membrane according to Towbin et al. (Towbin et al., 1979). After blocking with TBS containing 5% skimmed milk for 30 minutes, the membrane was incubated with primary antibodies overnight at 4°C or for 1-2 hours at room temperature. The primary antibodies used in this study were anti-XCds1, anti-XChk1 (gift from Dr N. Sagata), anti-MAP kinase (Upstate), anti-ß-galactosidase (Promega), and anti-phospho human Cdc25C (Ser-216) (New England Biolabs). The membrane was then incubated with alkaline phosphatase- or horseradish peroxidase-conjugated secondary antibodies for 1-2 hours at room temperature. Signals were visualized by the BCIP/NBT phosphatase substrate system (KPL) or the ECL system (Amersham Pharmacia Biotech).
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RESULTS |
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XCds1 is inactivated at G2/M-phase transition after progesterone treatment
We examined whether endogenous XCds1 is active in immature oocytes and whether its kinase activity changes during meiosis reinitiation. For this purpose, XCds1 was immunoprecipitated from oocyte extracts that were prepared at different times after progesterone treatment, and the immunoprecipitates were assayed for kinase activity using a GST fusion protein containing a fragment of Cdc25C that includes the 14-3-3 binding site (GST-XCdc25C[254-316]-WT) as a substrate. XCds1 recovered from immature oocytes highly phosphorylated GST-XCdc25C(254-316)-WT, but not its mutant form containing Ala instead of Ser at position 287 (GST-XCdc25C[254-316]-S287A) (Fig. 3A,B), indicating that the phosphorylation occurs on Ser-287 in the Cdc25C fragment. After the addition of progesterone into immature oocytes, GVBD started at 2 hours, and 100% GVBD was reached at 3.5 hours (Fig. 3B) (in this batch, GVBD occurred earlier than usual; see Fig. 4C). XCds1 kinase activity decreased in parallel with the progression of GVBD and reached background levels shortly after 100% of the oocytes had undergone GVBD, while XCds1 protein levels remained constant (Fig. 3A,B). A slight but significant retardation in the electrophoretic mobility of XCds1 was detected along with the decrease in XCds1 kinase activity (Fig. 3A; see also Fig. 6C), unlike the remarkable shift to a slower mobility seen in response to the activated DNA replication checkpoints after the MBT (compare with Fig. 2D). The small shift-up of XCds1 at GVBD was not cancelled after treatment with alkaline- or -phosphatase (data not shown). These observations indicate that endogenous XCds1 is active in G2-phase-arrested oocytes but is inactivated through some modification at the meiotic G2/M-phase transition after progesterone treatment.
Overexpression of wild-type XCds1 delays entry into M-phase after progesterone treatment
To address whether the downregulation of XCds1 is implicated in the release from the G2-phase arrest of immature Xenopus oocytes, we injected immature oocytes with 10 ng of mRNA for wild-type XCds1 (XCds1-WT), a kinase-deficient XCds1 mutant (XCds1-KD) or LacZ (as a control for mRNA injection). Immunoblots with anti-XCds1 antibody revealed that XCds1-WT and XCds1-KD were expressed at similar levels, which were approximately tenfold higher than those of endogenous XCds1 (Fig. 4A). The electrophoretic mobility of overexpressed XCds1-WT was partially retarded compared to that of overexpressed XCds1-KD, indicating that this modification was dependent on the presence of catalytically active XCds1. After progesterone treatment, oocytes that had been injected with either XCds1-KD or LacZ mRNA underwent GVBD essentially with the same kinetics as uninjected oocytes (Fig. 4B). In contrast, GVBD in oocytes injected with XCds1-WT mRNA occurred about 2 hours later than that in control oocytes (Fig. 4B). Thus, an increased amount of XCds1-WT, but not of XCds1-KD, could significantly delay the meiotic G2/M-phase transition. At present we do not know the reason why overexpression of XCds1-KD did not exert a dominant-negative effect (i.e. acceleration of GVBD), and accordingly it is unclear whether XCds1 is involved in the G2-phase arrest of immature oocytes. But the downregulation of XCds1 activity might be implicated in the progression of the meiotic G2/M-phase transition.
Unlike endogenous XCds1, overexpressed XCds1 was both cytoplasmic and nuclear (Fig. 4C). After the overexpression of XCds1-WT, the GV was removed from immature oocytes (Ford and Gurdon, 1977), and then XCds1 kinase activity was compared between enucleated and nucleated oocytes. Although significant XCds1 kinase activity was detected in the enucleated oocytes, its level was less than that in the nucleated oocytes and was as low as that in the control (uninjected and nucleated) immature oocytes (Fig. 4D). Considering the volume ratio of the GV to the cytoplasm (approx. 3%), these observations support the theory that relative XCds1 activity is much less in the cytoplasm of overexpressed oocytes than in the GV of overexpressed or uninjected oocytes. It is plausible that an increased activity of XCds1 in the GV might be a major cause for delaying GVBD after progesterone treatment, although cytoplasmic XCds1 may also be involved in the delay.
XCds1 inactivation is dependent on cyclin B-Cdc2 kinase activation
To understand how XCds1 is inactivated after progesterone treatment, we compared the timing of XCds1 inactivation with the activation of cyclin B-Cdc2 kinase and MAP kinase, since both kinases are concurrently activated shortly before progesterone-treated oocytes undergo GVBD (Gotoh et al., 1991). The activity of cyclin B-Cdc2 kinase was measured by total histone H1 kinase activity, and the MAP kinase activation was estimated by its electrophoretic retardation due to its phosphorylation. The inactivation of XCds1 coincided with the activation of both kinases (Fig. 3C, compare with Fig. 3A,B). To distinguish the effects of each kinase, we first examined whether XCds1 inactivation was dependent on MAP kinase activation. For this purpose, immature oocytes were incubated for 3 hours in the presence of U0126, a MEK inhibitor, before progesterone addition (Gross et al., 2000). GVBD in U0126-treated oocytes occurred 1-2 hours later than that in control oocytes treated with progesterone alone, and approximately 80% of U0126-treated oocytes underwent GVBD. Immunoblots for MAP kinase showed that U0126 treatment completely suppressed the electrophoretic retardation of MAP kinase in oocytes which underwent GVBD (Fig. 5A, bottom), whereas the levels of histone H1 kinase activity in the U0126-treated oocytes were almost as high as those in progesterone-treated control oocytes (Fig. 5A, middle lower). These observations confirm the previous report by Gross et al. (Gross et al., 2000), indicating that after progesterone treatment, cyclin B-Cdc2 kinase is activated even if the activation of MAP kinase is prevented by U0126 treatment. Under these conditions, the level of XCds1 kinase activity in the inhibitor-treated oocytes that had undergone GVBD was as low as that in the control oocytes (Fig. 5A, top and midde upper). Thus, XCds1 was inactivated even in the absence of MAP kinase activation after meiosis reinitiation, indicating that XCds1 inactivation occurs independently of MAP kinase activity.
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Since XCds1 contains in its kinase domain a consensus sequence, 359TP, for phosphorylation by cyclin B-Cdc2 kinase, it is plausible that XCds1 could be directly inactivated via its phosphorylation by cyclin B-Cdc2 kinase. To examine this possibility, XCds1 that was immunoprecipitated from extracts of immature Xenopus oocytes was incubated in vitro with active cyclin B-Cdc2 kinase purified from starfish oocytes (Okumura et al., 1996). As shown in Fig. 6A, XCds1 was indeed phosphorylated in vitro by cyclin B-Cdc2 kinase sufficiently within 30 minutes in the present condition. However, the phosphorylation did not cause the inactivation of XCds1 (Fig. 6B).
Finally, we verified that cyclin B-Cdc2 kinase activation could induce XCds1 inactivation in Xenopus oocytes. To this end, immature oocytes were injected with 10 ng of mRNA for Xenopus cyclin B1 or control LacZ. Overexpression of cyclin B1, but not that of ß-galactosidase (the LacZ gene products), caused cyclin B-Cdc2 kinase activation and GVBD without progesterone treatment (Fig. 6C, bottom), as reported previously (Freeman et al., 1991). The levels of XCds1 kinase activity in the cyclin B1-overexpressing oocytes that had undergone GVBD were almost as low as those in progesterone-treated oocytes (Fig. 6C, top and middle), indicating that cyclin B-Cdc2 kinase activation leads to XCds1 inactivation. Taken together, these observations support the theory that XCds1 is indirectly inactivated by cyclin B-Cdc2 kinase during meiosis reinitiation in Xenopus oocytes.
An indirect effect of cyclin B-Cdc2 kinase could possibly be mediated by polo-like kinase, Plx1, because at meiosis reinititation in Xenopus oocytes Plx1 is concurrently activated with cyclin B-Cdc2 kinase, and a positive feedback regulation is reported between both kinases (Abrieu et al., 1998; Qian et al., 1998). However, recombinant mouse Plk (gift from Dr N. Watanabe), which is able to phosphorylate -casein as a control, neither phosphorylated XCds1 recovered from immature Xenopus oocytes nor affected its kinase activity (data not shown), excluding the direct effect of Plx1 on XCds1 inactivation.
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DISCUSSION |
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Cds1 activity in G2-phase
Originally, both Chk1 and Cds1 were identified as effector kinases that respond to the G2 checkpoint activated by damaged or unreplicated DNA (Elledge, 1996; Murakami and Nurse, 2000; OConnell et al., 2000; Zhou and Elledge, 2000). Accordingly, unless the G2 checkpoint is activated, both Chk1 and Cds1 should be nonessential, as seen in fission yeast (Walworth et al., 1993; Murakami and Okayama, 1995). In mice, nonetheless, targeted gene disruption of Chk1 or ATR results in embryonic lethality as early as the blastocyst stage, or causes a severe proliferation defect in embryonic stem (ES) cells, indicating that ATR-Chk1 is essential in early embryonic development (Brown and Baltimore, 2000; Liu et al., 2000; Takai et al., 2000). Similarly, Grapes, a Drosophila homolog of Chk1, has an essential developmental function during the late stage of syncytial blastoderm divisions and for the MBT that marks the transition from maternal to zygotic control of embryogenesis, since loss of Grapes fails to terminate syncytial division after mitosis 13 leading to embryonic degeneration (Fogarty et al., 1997; Sibon et al., 1997; Yu et al., 2000). It has been suggested that during early embryonic development Chk1/Grapes might be required to sense incomplete DNA replication and prevent premature entry into mitosis. It remains unclear, however, whether the embryonic lethality is due to the failure of a cell cycle checkpoint function that requires Chk1 (Liu et al., 2000). In fact, it has not yet been verified that Chk1 is phosphorylated and activated when it is required in the embryo, as is ordinarily seen at the G2 checkpoint in response to damaged or incompletely replicated DNA. Rather, in normal human fibroblasts (MJ90) Chk1 is expressed from S- to M-phase without obvious phosphorylation, and is an active kinase at this time in the absence of DNA damage (Kaneko et al., 1999). Furthermore, in immature Xenopus oocytes, which apparently lack damaged or unreplicated DNA, Chk1 is involved in the G2-phase arrest through a form that is apparently distinct from that seen at the activated G2 checkpoint after the MBT (Nakajo et al., 1999; Oe et al., 2001) (compare with Kappas et al., 2000). Together, these observations make it plausible that Chk1 might be implicated in both the G2-phase control that is dependent on the activated G2 checkpoint and in one that is independent of a G2 checkpoint.
In contrast to Chk1, there has been no report that implicates Cds1 in the control of G2-phase when the G2 checkpoint is not activated. In this context, our present study provides the first example that Cds1 is active in G2-phase in the apparent absence of the G2 checkpoint activation, because the electrophoretic mobility of endogenous XCds1 in immature oocytes was clearly different from that seen at the activated G2 checkpoint after the MBT (see Fig. 2D). At present, however, we do not know how XCds1 becomes an active kinase in the absence of the G2 checkpoint activation, although ataxia telangiectasia mutated (ATM), which normally directly phosphorylates and activates Cds1 upon the activation of G2 checkpoint (Matsuoka et al., 2000; Melchionna et al., 2000), is localized with XCds1 to the GV of immature Xenopus oocytes (Robertson et al., 1999). Alternatively, Cds1 might have an intrinsic or basal kinase activity, which would be independent of the activated G2 checkpoint. Presumably, when the G2 checkpoint is activated in response to DNA damage or incomplete DNA replication, Cds1 might be hyperphosphorylated and hyperactivated.
Downregulation of Cds1 activity
Our present study is the first demonstration of the downregulation of Cds1 activity at the entry into M-phase. The results indicate that the inactivation of XCds1 depends on the activation of cyclin B-Cdc2 kinase (Fig. 5, Fig. 6). Conversely, it is well understood that the ultimate function of Cds1 is to prevent the activation of cyclin B-Cdc2 kinase (Elledge, 1996; Murakami and Nurse, 2000; OConnell et al., 2000; Zhou and Elledge, 2000). In accordance with the latter function, overexpression of wild-type XCds1 in immature oocytes caused the delay in the occurrence of M-phase (Fig. 4B). The apparent contradiction between these observations would be reconciled, however, if the intracellular localization of the cyclin B-Cdc2 complex and XCds1 in immature Xenopus oocytes were taken into consideration. Nuclear XCds1 (see Fig. 2E) might be involved in preventing the activation of the small amounts of inactive cyclin B-Cdc2 kinase that enter the germinal vesicle from the cytoplasm, where the majority of cyclin B-Cdc2 kinase is found in immature oocytes (Gautier and Maller, 1991). Progesterone stimulation leads to the initial activation of cyclin B-Cdc2 kinase that occurs in the cytoplasm (Oe et al., 2001). Once activated, cyclin B-Cdc2 kinase may accumulate in the nucleus just before GVBD ((Pines, 1999); see also Ookata et al. (Ookata et al., 1992) in starfish oocytes), and the increase in the amount of active nuclear cyclin B-Cdc2 kinase could cause the inactivation of nuclear XCds1. Thus, once the activity of XCds1 begins to decrease, the activation of cyclin B-Cdc2 kinase might be enhanced through a feedback control that results in abrogation of the XCds1 activity (Fig. 7).
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In conclusion, Cds1 has so far been studied only in the context of the checkpoint response that is activated by damaged or unreplicated DNA. By contrast, the present study demonstrates that Cds1 is also active in immature Xenopus oocytes that are physiologically arrested at G2-phase, apparently in the absence of checkpoint activation, and that Cds1 is inactivated at entry into M-phase by an indirect mechanism that depends on the activation of cyclin B-Cdc2 kinase. The implication is that Cds1 might be involved in the negative regulation of the G2/M-phase transition through two mechanisms, one that is dependent upon the activated G2 checkpoint and the other that is independent of it. In both, the cyclin B-Cdc2 kinase-dependent inactivation of Cds1 might enhance the activation of cyclin B-Cdc2 kinase for entry into M-phase.
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ACKNOWLEDGMENTS |
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