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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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Analysis of Bub1 and Bub3 from Xenopus egg extracts. (A) Characterization of affinity-purified anti-Bub1 and anti-Bub3 antibodies. CSF-arrested egg extracts (lanes 1, 2, 7, and 8), or immunoprecipitates generated with control IgG (lanes 3, 5, 9, and 11), with anti-Bub1 (lanes 4 and 10) or anti-Bub3 antibodies (lanes 6 and 12) were probed with affinity-purified anti-Bub1 antibodies (lanes 1, 3–6), anti-Bub1 antibodies preblocked with recombinant Bub1 protein (lane 2), anti-Bub3 antibodies (lanes 7, 9–12), or anti-Bub3 preblocked with recombinant Bub3 protein (lane 8). The migration of molecular weight standards is indicated on the left. The prominent 55-kD protein is the IgG heavy chain. (B) Bub1 is a phosphoprotein. Bub1 was immunoprecipitated from CSF-arrested extract and treated with LPP (PPase) in the presence or absence of phosphatase inhibitors (PPase Inh.) as indicated on the top. (C) Bub1 is phosphorylated at interphase and mitosis, and under the checkpoint-active condition. Bub1 and Bub3 were coimmunoprecipitated with anti-Bub1 antibodies from interphase (lanes 1 and 4), CSF-arrested (lanes 2 and 5, mitosis), or checkpoint-active extracts (lanes 3 and 6, checkpoint), and left untreated (lanes 1–3) or treated with LPP (lanes 4–6). The upper panel was probed with anti-Bub1 antibody and the lower panel was probed with anti-Bub3 antibody.
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Figure 1: Analysis of Bub1 and Bub3 from Xenopus egg extracts. (A) Characterization of affinity-purified anti-Bub1 and anti-Bub3 antibodies. CSF-arrested egg extracts (lanes 1, 2, 7, and 8), or immunoprecipitates generated with control IgG (lanes 3, 5, 9, and 11), with anti-Bub1 (lanes 4 and 10) or anti-Bub3 antibodies (lanes 6 and 12) were probed with affinity-purified anti-Bub1 antibodies (lanes 1, 3–6), anti-Bub1 antibodies preblocked with recombinant Bub1 protein (lane 2), anti-Bub3 antibodies (lanes 7, 9–12), or anti-Bub3 preblocked with recombinant Bub3 protein (lane 8). The migration of molecular weight standards is indicated on the left. The prominent 55-kD protein is the IgG heavy chain. (B) Bub1 is a phosphoprotein. Bub1 was immunoprecipitated from CSF-arrested extract and treated with LPP (PPase) in the presence or absence of phosphatase inhibitors (PPase Inh.) as indicated on the top. (C) Bub1 is phosphorylated at interphase and mitosis, and under the checkpoint-active condition. Bub1 and Bub3 were coimmunoprecipitated with anti-Bub1 antibodies from interphase (lanes 1 and 4), CSF-arrested (lanes 2 and 5, mitosis), or checkpoint-active extracts (lanes 3 and 6, checkpoint), and left untreated (lanes 1–3) or treated with LPP (lanes 4–6). The upper panel was probed with anti-Bub1 antibody and the lower panel was probed with anti-Bub3 antibody.

Mentions: By immunoblot analysis, the anti-Bub1 antibody generated against amino acids 274–467 of Bub1 specifically recognized a protein of ∼150 kD in mitotic frog egg extracts (Fig. 1 A, lane 1). Antibodies made against a known Xenopus Bub3 sequence (Goto and Kinoshita 1999) specifically recognized a 45-kD doublet (Fig. 1 A, lane 7). Immunoprecipitation with anti-Bub1 antibodies isolated the 150-kD protein (Fig. 1 A, lane 4) that was also present in the anti-Bub3 immunoprecipitate (Fig. 1 A, lane 6). Similarly, both 45-kD polypeptides that were recognized by the anti-Bub3 antibodies were present in the anti-Bub1 immunoprecipitates (Fig. 1 A, lane 10). Interestingly, only the slower-migrating polypeptide in the 45-kD doublet was immunoprecipitated by the anti-Bub3 antibodies (Fig. 1 A, lane 12). It appears that the anti-Bub3 antibodies can recognize both polypeptides that have been denatured, as in the case of immunoblot analysis (Fig. 1 A, lanes 7 and 10). However, the antibodies cannot recognize the faster migrating species under native conditions, as in the case of immunoprecipitation (Fig. 1 A, lane 12). We do not know whether these two polypeptides represent two closely related gene products, or whether they are isoforms of the same gene product. Nevertheless, these results demonstrate that Xenopus Bub1 and Bub3 associate with each other, and that the Bub1 we have isolated is an authentic Bub1 homologue.


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)

Analysis of Bub1 and Bub3 from Xenopus egg extracts. (A) Characterization of affinity-purified anti-Bub1 and anti-Bub3 antibodies. CSF-arrested egg extracts (lanes 1, 2, 7, and 8), or immunoprecipitates generated with control IgG (lanes 3, 5, 9, and 11), with anti-Bub1 (lanes 4 and 10) or anti-Bub3 antibodies (lanes 6 and 12) were probed with affinity-purified anti-Bub1 antibodies (lanes 1, 3–6), anti-Bub1 antibodies preblocked with recombinant Bub1 protein (lane 2), anti-Bub3 antibodies (lanes 7, 9–12), or anti-Bub3 preblocked with recombinant Bub3 protein (lane 8). The migration of molecular weight standards is indicated on the left. The prominent 55-kD protein is the IgG heavy chain. (B) Bub1 is a phosphoprotein. Bub1 was immunoprecipitated from CSF-arrested extract and treated with LPP (PPase) in the presence or absence of phosphatase inhibitors (PPase Inh.) as indicated on the top. (C) Bub1 is phosphorylated at interphase and mitosis, and under the checkpoint-active condition. Bub1 and Bub3 were coimmunoprecipitated with anti-Bub1 antibodies from interphase (lanes 1 and 4), CSF-arrested (lanes 2 and 5, mitosis), or checkpoint-active extracts (lanes 3 and 6, checkpoint), and left untreated (lanes 1–3) or treated with LPP (lanes 4–6). The upper panel was probed with anti-Bub1 antibody and the lower panel was probed with anti-Bub3 antibody.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2192030&req=5

Figure 1: Analysis of Bub1 and Bub3 from Xenopus egg extracts. (A) Characterization of affinity-purified anti-Bub1 and anti-Bub3 antibodies. CSF-arrested egg extracts (lanes 1, 2, 7, and 8), or immunoprecipitates generated with control IgG (lanes 3, 5, 9, and 11), with anti-Bub1 (lanes 4 and 10) or anti-Bub3 antibodies (lanes 6 and 12) were probed with affinity-purified anti-Bub1 antibodies (lanes 1, 3–6), anti-Bub1 antibodies preblocked with recombinant Bub1 protein (lane 2), anti-Bub3 antibodies (lanes 7, 9–12), or anti-Bub3 preblocked with recombinant Bub3 protein (lane 8). The migration of molecular weight standards is indicated on the left. The prominent 55-kD protein is the IgG heavy chain. (B) Bub1 is a phosphoprotein. Bub1 was immunoprecipitated from CSF-arrested extract and treated with LPP (PPase) in the presence or absence of phosphatase inhibitors (PPase Inh.) as indicated on the top. (C) Bub1 is phosphorylated at interphase and mitosis, and under the checkpoint-active condition. Bub1 and Bub3 were coimmunoprecipitated with anti-Bub1 antibodies from interphase (lanes 1 and 4), CSF-arrested (lanes 2 and 5, mitosis), or checkpoint-active extracts (lanes 3 and 6, checkpoint), and left untreated (lanes 1–3) or treated with LPP (lanes 4–6). The upper panel was probed with anti-Bub1 antibody and the lower panel was probed with anti-Bub3 antibody.
Mentions: By immunoblot analysis, the anti-Bub1 antibody generated against amino acids 274–467 of Bub1 specifically recognized a protein of ∼150 kD in mitotic frog egg extracts (Fig. 1 A, lane 1). Antibodies made against a known Xenopus Bub3 sequence (Goto and Kinoshita 1999) specifically recognized a 45-kD doublet (Fig. 1 A, lane 7). Immunoprecipitation with anti-Bub1 antibodies isolated the 150-kD protein (Fig. 1 A, lane 4) that was also present in the anti-Bub3 immunoprecipitate (Fig. 1 A, lane 6). Similarly, both 45-kD polypeptides that were recognized by the anti-Bub3 antibodies were present in the anti-Bub1 immunoprecipitates (Fig. 1 A, lane 10). Interestingly, only the slower-migrating polypeptide in the 45-kD doublet was immunoprecipitated by the anti-Bub3 antibodies (Fig. 1 A, lane 12). It appears that the anti-Bub3 antibodies can recognize both polypeptides that have been denatured, as in the case of immunoblot analysis (Fig. 1 A, lanes 7 and 10). However, the antibodies cannot recognize the faster migrating species under native conditions, as in the case of immunoprecipitation (Fig. 1 A, lane 12). We do not know whether these two polypeptides represent two closely related gene products, or whether they are isoforms of the same gene product. Nevertheless, these results demonstrate that Xenopus Bub1 and Bub3 associate with each other, and that the Bub1 we have isolated is an authentic Bub1 homologue.

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