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Inactivation of the checkpoint kinase Cds1 is dependent on cyclin B-Cdc2 kinase activation at the meiotic G2/M-phase transition in Xenopus oocytes

Tetsuya Gotoh1, Keita Ohsumi1, Tomoko Matsui2, Haruhiko Takisawa2 and Takeo Kishimoto1,*

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



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  		Fig. 1. Specificity of anti-XCds1 antibody. (A) His6-XCds1 (1 ng/lane) was subjected to western blot analysis with control rabbit IgG, purified anti-XCds1 antibody, or anti-XCds1 antibody preabsorbed to His6-XCds1 (block). (B) Xenopus egg extracts were immunoprecipitated with control rabbit IgG or anti-XCds1 antibody. Western blot analysis confirmed that XCds1 protein was recovered in the immunoprecipitates with anti-XCds1 antibody (beads) but undetectable in the supernatant (sup).

 


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  		Fig. 2. Developmental expression and localization of XCds1. (A) Expression of XCds1 during oogenesis from stage I to VI. (B) Expression of XCds1 during oocyte maturation. Immature oocytes (IM) were treated with progesterone to induce maturation and GVBD occurred at about 2 hours. Metaphase arrest of meiosis II occurred at about 4 hours after GVBD. (C) Expression of XCds1 during embryogenesis. Embryos were staged according to Nieuwkoop and Faber (Nieuwkoop and Faber, 1956). (D) Embryos were untreated (-) or treated with hydroxyurea (HU) or aphidicolin (APH) as early as stage 8 to inhibit DNA replication and then sampled at stage 9, post-MBT. Note that mobility shift of XCds1 (upper arrow) was detectable in hydroxyurea- or aphidicolin-treated post-MBT embryos, while it was undetectable in untreated post-MBT embryos (-), as in stage-6 untreated embryos (pre-MBT). Oocytes or embryos were subjected to western blot analysis with anti-XCds1 antibody. Each lane was loaded with proteins equivalent to one oocyte or embryo. (E) Nuclear localization of XCds1 in immature Xenopus oocytes. An immature oocyte was dissected into cytoplasmic (C) and nuclear (N) fractions. Proteins were resolved by SDS-PAGE and visualized by western blot with anti-XCds1 (top) or anti-XChk1 (bottom) antibodies. Proteins equivalent to one quarter of the cytoplasmic or nuclear fraction were analyzed.

 


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  		Fig. 3. XCds1 is inactivated at meiotic G2/M-phase transition. (A) XCds1 was immunoprecipitated with anti-XCds1 antibody from oocyte extracts prepared at different times during meiosis reinitiation after progesterone treatment. The immunoprecipitates were assayed for kinase activity using GST-XCdc25C(254-316)-WT as a substrate, whose phosphorylation was detected by autoradiography (top). Note that GST-XCdc25C(254-316)-S287A was not phosphorylated by XCds1 immunoprecipitated from immature oocyte extracts (S287A). Levels of XCds1 were confirmed by western blot with anti-XCds1 antibody (bottom). (B) Changes in XCds1 kinase activity shown in A were measured and compared with the occurrence of GVBD. (C) The same extracts as in A were subjected to total histone H1 kinase assay (top) and western blot analysis with anti-MAP kinase antibody (bottom). The decrease in H1 kinase activity at 3 hours may reflect the fact that a significant number of oocytes had already passed through metaphase-I, which is observed within 1 hour after GVBD (Ohsumi et al., 1994).

 


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  		Fig. 4. Overexpression of XCds1 delays meiosis reinitiation. (A) Immature oocytes were uninjected or injected with 10 ng of mRNA for XCds1-WT, XCds1-KD or LacZ and cultured for 12 hours. Western blots show the expression levels of endogenous XCds1 (uninjected and LacZ), introduced XCds1-WT or -KD proteins in addition to endogenous XCds1, and ß-galactosidase (ßgal) derived from control LacZ mRNA. (B) 12 hours after the injection, immature oocytes of A were treated with progesterone to induce maturation and then scored for the percentage of GVBD at the indicated times. Oocytes were uninjected (open triangles), or injected with mRNA for XCds1-WT (solid circles), XCds1-KD (open circles) or control LacZ (open squares). (C) XCds1-overexpressed immature oocytes were dissected into cytoplasmic (C) and nuclear (N) fractions, followed by western blot analysis with anti-XCds1 antibody. (D) Before or after overexpression of XCds1-WT, the GV was removed from some of the immature oocytes. Then, XCds1 was recovered with immunoprecipitation from enucleated (-) or nucleated (+) oocytes (100 each), and was assayed for kinase activity using GST-XCdc25C(254-316)-WT as a substrate (top). Phosphorylation at Ser287 was detected by immunoblots with anti-phospho human Cdc25C (Ser-216) antibody. Immunoblots with anti-XCds1 indicate protein amounts of XCds1 (bottom).

 


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  		Fig. 5. XCds1 inactivation depends on the activation of cyclin B-Cdc2 kinase but not MAP kinase. (A) Immature oocytes were incubated for 3 hours in the presence of U0126, a MEK inhibitor, and then added with progesterone. Oocytes were collected when GVBD occurred in progesterone-treated oocytes. (B) Immature oocytes were incubated overnight in the presence of olomoucine, a Cdk inhibitor, and then incubated with progesterone. Olomoucine and progesterone-treated oocytes with intact GV were collected when 100% of the control oocytes, which were treated with progesterone alone, had undergone GVBD. XCds1 was immunoprecipitated with anti-XCds1 antibody and assayed for its kinase activity using GST-XCdc25C(254-316)-WT as a substrate (top). Western blot analysis confirmed that equivalent amounts of XCds1 were recovered in the immunoprecipitates with anti-XCds1 antibody (A, middle upper; B, middle). The same extracts were subjected to total histone H1 kinase assay (A, middle lower; B, bottom) and western blot analysis with anti-MAP kinase antibody (A, bottom).

 


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  		Fig. 6. XCds1 is indirectly inactivated by cyclin B-Cdc2 kinase. (A) Extracts prepared from immature oocytes were immunoprecipitated with control rabbit IgG (open circles) or anti-XCds1 antibody (closed circles). The immunoprecipitates were incubated with starfish cyclin B-Cdc2 kinase. Phosphorylation was measured as a function of incubation time. (B) After 30 minutes incubation with cyclin B-Cdc2 kinase in A, phosphorylated proteins were resolved by SDS-PAGE and visualized by autoradiography (top). The arrow indicates the position corresponding to XCds1. Western blot analysis confirmed that equivalent amounts of XCds1 were recovered in the immunoprecipitates with anti-XCds1 antibody (bottom). The immunoprecipitates were subjected to XCds1 kinase assay, using GST-XCdc25C(254-316)-WT as a substrate (middle). Phosphorylation at Ser-287 on XCdc25C was visualized by western blot with anti-phospho human Cdc25C (Ser-216) antibody. (C) Immature oocytes were injected with 10 ng of mRNA for LacZ or Xenopus cyclin B1. Cyclin B1 mRNA-injected oocytes and progesterone-treated oocytes were collected when they underwent GVBD. XCds1 was immunoprecipitated with anti-XCds1 antibody and assayed for its kinase activity (top). Western blot analysis confirmed that equivalent amounts of XCds1 were recovered in the immunoprecipitates with anti-XCds1 antibody (middle). The same extracts were subjected to total histone H1 kinase assay (bottom).

 


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  		Fig. 7. Model for mechanism of XCds1 inactivation at meiotic G2/M-phase transition in Xenopus oocytes. Although it remains unclear whether XCds1 is actually involved in G2-phase arrest of immature oocytes (dashed line), progesterone might overcome or bypass its effect to induce the initial activation of cyclin B-Cdc2 kinase, leading to the downregulation of XCds1. The inactivation of XCds1 would presumably be accomplished through phosphorylation of Xenopus functional homolog of BRCT protein by cyclin B-Cdc2 kinase.

 

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