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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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Models for the interaction of spindle checkpoint proteins at the kinetochore. (A) Bub1 interacts directly with CENP-E and Mad1–Mad2 on unattached kinetochores. Binding of CENP-E to microtubules may induce a conformational change in Bub1 that disrupts its interaction with the Mad1–Mad2 complex. The anti–CENP-E antibody may produce a similar effect on Bub1, thus dissociating Mad1–Mad2 from kinetochores (Abrieu et al. 2000). (B) Bub1 interacts with Mad1–Mad2 through CENP-E. Loss of Mad1–Mad2 on kinetochores in Bub1-depleted extracts may be a result of a lack of CENP-E on kinetochores.
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Figure 9: Models for the interaction of spindle checkpoint proteins at the kinetochore. (A) Bub1 interacts directly with CENP-E and Mad1–Mad2 on unattached kinetochores. Binding of CENP-E to microtubules may induce a conformational change in Bub1 that disrupts its interaction with the Mad1–Mad2 complex. The anti–CENP-E antibody may produce a similar effect on Bub1, thus dissociating Mad1–Mad2 from kinetochores (Abrieu et al. 2000). (B) Bub1 interacts with Mad1–Mad2 through CENP-E. Loss of Mad1–Mad2 on kinetochores in Bub1-depleted extracts may be a result of a lack of CENP-E on kinetochores.

Mentions: One of the possible roles for Bub1 is to physically provide a platform on the kinetochore to which checkpoint proteins, including Mad1–Mad2 complex and CENP-E, can bind (Fig. 9). In the absence of Bub1, other checkpoint proteins are unable to assemble onto the kinetochore. Consistent with this notion, human Bub1 has been found to assemble onto the kinetochore before CENP-F, BubR1, and CENP-E (Jablonski et al. 1998). The order of kinetochore binding between CENP-E and the Mad1–Mad2 complex is not clear. However, a potential interaction among these proteins is indicated by the observation that anti–CENP-E antibodies dissociate Mad1 and Mad2 from kinetochores during checkpoint maintenance (Abrieu et al. 2000). Mad1–Mad2 may interact directly with Bub1 (Fig. 9 A) or indirectly with Bub1 through CENP-E (Fig. 9 B). Furthermore, CENP-E is known to bind BubR1 in human cells (Chan et al. 1998). We did not detect CENP-E in a Bub1 immunoprecipitate prepared from CSF-arrested extract (Fig. 4). It remains a possibility that CENP-E may interact with Bub1 only at the kinetochore, or that our anti-Bub1 antibody may disrupt any weak interaction between CENP-E and Bub1. The loss of CENP-E staining at the kinetochore in Bub1-depleted extract suggests that BubR1 alone is not sufficient to localize CENP-E or that Bub1 may also be required for BubR1 to bind kinetochores.


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)

Models for the interaction of spindle checkpoint proteins at the kinetochore. (A) Bub1 interacts directly with CENP-E and Mad1–Mad2 on unattached kinetochores. Binding of CENP-E to microtubules may induce a conformational change in Bub1 that disrupts its interaction with the Mad1–Mad2 complex. The anti–CENP-E antibody may produce a similar effect on Bub1, thus dissociating Mad1–Mad2 from kinetochores (Abrieu et al. 2000). (B) Bub1 interacts with Mad1–Mad2 through CENP-E. Loss of Mad1–Mad2 on kinetochores in Bub1-depleted extracts may be a result of a lack of CENP-E on kinetochores.
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Related In: Results  -  Collection

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Figure 9: Models for the interaction of spindle checkpoint proteins at the kinetochore. (A) Bub1 interacts directly with CENP-E and Mad1–Mad2 on unattached kinetochores. Binding of CENP-E to microtubules may induce a conformational change in Bub1 that disrupts its interaction with the Mad1–Mad2 complex. The anti–CENP-E antibody may produce a similar effect on Bub1, thus dissociating Mad1–Mad2 from kinetochores (Abrieu et al. 2000). (B) Bub1 interacts with Mad1–Mad2 through CENP-E. Loss of Mad1–Mad2 on kinetochores in Bub1-depleted extracts may be a result of a lack of CENP-E on kinetochores.
Mentions: One of the possible roles for Bub1 is to physically provide a platform on the kinetochore to which checkpoint proteins, including Mad1–Mad2 complex and CENP-E, can bind (Fig. 9). In the absence of Bub1, other checkpoint proteins are unable to assemble onto the kinetochore. Consistent with this notion, human Bub1 has been found to assemble onto the kinetochore before CENP-F, BubR1, and CENP-E (Jablonski et al. 1998). The order of kinetochore binding between CENP-E and the Mad1–Mad2 complex is not clear. However, a potential interaction among these proteins is indicated by the observation that anti–CENP-E antibodies dissociate Mad1 and Mad2 from kinetochores during checkpoint maintenance (Abrieu et al. 2000). Mad1–Mad2 may interact directly with Bub1 (Fig. 9 A) or indirectly with Bub1 through CENP-E (Fig. 9 B). Furthermore, CENP-E is known to bind BubR1 in human cells (Chan et al. 1998). We did not detect CENP-E in a Bub1 immunoprecipitate prepared from CSF-arrested extract (Fig. 4). It remains a possibility that CENP-E may interact with Bub1 only at the kinetochore, or that our anti-Bub1 antibody may disrupt any weak interaction between CENP-E and Bub1. The loss of CENP-E staining at the kinetochore in Bub1-depleted extract suggests that BubR1 alone is not sufficient to localize CENP-E or that Bub1 may also be required for BubR1 to bind kinetochores.

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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