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Spindle checkpoint protein Bub1 is required for kinetochore localization of Mad1, Mad2, Bub3, and CENP-E, independently of its kinase activity.

Sharp-Baker H, Chen RH - J. Cell Biol. (2001)

Bottom Line: Antibodies raised against Bub1 recognize a 150-kD phosphoprotein at both interphase and mitosis, but the molecular mass is reduced to 140 upon dephosphorylation in vitro.Interestingly, reintroducing either wild-type or kinase-deficient Bub1 protein restores the checkpoint and the kinetochore localization of these proteins.Our studies demonstrate that Bub1 plays a central role in triggering the spindle checkpoint signal from the kinetochore, and that its kinase activity is not necessary for the spindle checkpoint in Xenopus egg extracts.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York 14853, USA.

ABSTRACT
The spindle checkpoint inhibits the metaphase to anaphase transition until all the chromosomes are properly attached to the mitotic spindle. We have isolated a Xenopus homologue of the spindle checkpoint component Bub1, and investigated its role in the spindle checkpoint in Xenopus egg extracts. Antibodies raised against Bub1 recognize a 150-kD phosphoprotein at both interphase and mitosis, but the molecular mass is reduced to 140 upon dephosphorylation in vitro. Bub1 is essential for the establishment and maintenance of the checkpoint and is localized to kinetochores, similar to the spindle checkpoint complex Mad1-Mad2. However, Bub1 differs from Mad1-Mad2 in that Bub1 remains on kinetochores that have attached to microtubules; the protein eventually dissociates from the kinetochore during anaphase. Immunodepletion of Bub1 abolishes the spindle checkpoint and the kinetochore binding of the checkpoint proteins Mad1, Mad2, Bub3, and CENP-E. Interestingly, reintroducing either wild-type or kinase-deficient Bub1 protein restores the checkpoint and the kinetochore localization of these proteins. Our studies demonstrate that Bub1 plays a central role in triggering the spindle checkpoint signal from the kinetochore, and that its kinase activity is not necessary for the spindle checkpoint in Xenopus egg extracts.

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Dependency of kinetochore binding between various checkpoint proteins. Mitotic chromosomes were assembled in mock-depleted, Bub1-depleted, or Mad1-depleted extracts in the presence of nocodazole. The isolated chromosomes were then stained with mouse anti-Mad1 antibody along with rabbit anti-Mad2, anti-Bub1, anti-Bub3, or anti–CENP-E antibodies. The use of secondary antibodies and DNA staining were as described in the legend to Fig. 3. For space conservation, pictures of Mad1 staining are shown only once for each type of extract. Photographs for each antibody staining were taken for the same exposure time to reflect a difference in the staining level. Bar, 10 μm.
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Figure 5: Dependency of kinetochore binding between various checkpoint proteins. Mitotic chromosomes were assembled in mock-depleted, Bub1-depleted, or Mad1-depleted extracts in the presence of nocodazole. The isolated chromosomes were then stained with mouse anti-Mad1 antibody along with rabbit anti-Mad2, anti-Bub1, anti-Bub3, or anti–CENP-E antibodies. The use of secondary antibodies and DNA staining were as described in the legend to Fig. 3. For space conservation, pictures of Mad1 staining are shown only once for each type of extract. Photographs for each antibody staining were taken for the same exposure time to reflect a difference in the staining level. Bar, 10 μm.

Mentions: In Bub1-depleted extract, kinetochore staining of Bub1, Bub3, Mad1, Mad2, and CENP-E was diminished (Fig. 5), indicating that Bub1 is required for these checkpoint proteins to bind to kinetochores. Besides kinetochore staining, anti-Bub3 antibody gave an overall chromosomal staining that was not affected by the Bub1 depletion (Fig. 5). Anti-Bub3 antibodies preblocked with recombinant Bub3 protein did not stain kinetochores (data not shown), indicating its specificity to Bub3. Immunodepletion of Mad1 abolished kinetochore localization of Mad2, whereas the levels of Bub1 and Bub3 at kinetochores were the same as in mock-depleted extract (Fig. 5). Thus, kinetochore binding of Bub1–Bub3 complex is independent of Mad1–Mad2. Interestingly, CENP-E staining at kinetochores was reduced in Mad1-depleted extract (Fig. 5).


Spindle checkpoint protein Bub1 is required for kinetochore localization of Mad1, Mad2, Bub3, and CENP-E, independently of its kinase activity.

Sharp-Baker H, Chen RH - J. Cell Biol. (2001)

Dependency of kinetochore binding between various checkpoint proteins. Mitotic chromosomes were assembled in mock-depleted, Bub1-depleted, or Mad1-depleted extracts in the presence of nocodazole. The isolated chromosomes were then stained with mouse anti-Mad1 antibody along with rabbit anti-Mad2, anti-Bub1, anti-Bub3, or anti–CENP-E antibodies. The use of secondary antibodies and DNA staining were as described in the legend to Fig. 3. For space conservation, pictures of Mad1 staining are shown only once for each type of extract. Photographs for each antibody staining were taken for the same exposure time to reflect a difference in the staining level. Bar, 10 μm.
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Related In: Results  -  Collection

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Figure 5: Dependency of kinetochore binding between various checkpoint proteins. Mitotic chromosomes were assembled in mock-depleted, Bub1-depleted, or Mad1-depleted extracts in the presence of nocodazole. The isolated chromosomes were then stained with mouse anti-Mad1 antibody along with rabbit anti-Mad2, anti-Bub1, anti-Bub3, or anti–CENP-E antibodies. The use of secondary antibodies and DNA staining were as described in the legend to Fig. 3. For space conservation, pictures of Mad1 staining are shown only once for each type of extract. Photographs for each antibody staining were taken for the same exposure time to reflect a difference in the staining level. Bar, 10 μm.
Mentions: In Bub1-depleted extract, kinetochore staining of Bub1, Bub3, Mad1, Mad2, and CENP-E was diminished (Fig. 5), indicating that Bub1 is required for these checkpoint proteins to bind to kinetochores. Besides kinetochore staining, anti-Bub3 antibody gave an overall chromosomal staining that was not affected by the Bub1 depletion (Fig. 5). Anti-Bub3 antibodies preblocked with recombinant Bub3 protein did not stain kinetochores (data not shown), indicating its specificity to Bub3. Immunodepletion of Mad1 abolished kinetochore localization of Mad2, whereas the levels of Bub1 and Bub3 at kinetochores were the same as in mock-depleted extract (Fig. 5). Thus, kinetochore binding of Bub1–Bub3 complex is independent of Mad1–Mad2. Interestingly, CENP-E staining at kinetochores was reduced in Mad1-depleted extract (Fig. 5).

Bottom Line: Antibodies raised against Bub1 recognize a 150-kD phosphoprotein at both interphase and mitosis, but the molecular mass is reduced to 140 upon dephosphorylation in vitro.Interestingly, reintroducing either wild-type or kinase-deficient Bub1 protein restores the checkpoint and the kinetochore localization of these proteins.Our studies demonstrate that Bub1 plays a central role in triggering the spindle checkpoint signal from the kinetochore, and that its kinase activity is not necessary for the spindle checkpoint in Xenopus egg extracts.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York 14853, USA.

ABSTRACT
The spindle checkpoint inhibits the metaphase to anaphase transition until all the chromosomes are properly attached to the mitotic spindle. We have isolated a Xenopus homologue of the spindle checkpoint component Bub1, and investigated its role in the spindle checkpoint in Xenopus egg extracts. Antibodies raised against Bub1 recognize a 150-kD phosphoprotein at both interphase and mitosis, but the molecular mass is reduced to 140 upon dephosphorylation in vitro. Bub1 is essential for the establishment and maintenance of the checkpoint and is localized to kinetochores, similar to the spindle checkpoint complex Mad1-Mad2. However, Bub1 differs from Mad1-Mad2 in that Bub1 remains on kinetochores that have attached to microtubules; the protein eventually dissociates from the kinetochore during anaphase. Immunodepletion of Bub1 abolishes the spindle checkpoint and the kinetochore binding of the checkpoint proteins Mad1, Mad2, Bub3, and CENP-E. Interestingly, reintroducing either wild-type or kinase-deficient Bub1 protein restores the checkpoint and the kinetochore localization of these proteins. Our studies demonstrate that Bub1 plays a central role in triggering the spindle checkpoint signal from the kinetochore, and that its kinase activity is not necessary for the spindle checkpoint in Xenopus egg extracts.

Show MeSH
Related in: MedlinePlus