Phosphorylation and activation of Bub1 on unattached chromosomes facilitate the spindle checkpoint

摘要

The spindle checkpoint inhibits anaphase until all kinetochores have attached properly to spindle microtubules. The protein kinase Bub1 is an essential checkpoint component that resides at kinetochores during mitosis. It is shown herein that Xenopus Bub1 becomes hyperphosphorylated and the kinase is activated on unattached chromosomes. MAP kinase (MAPK) contributes to this phosphorylation, as inhibiting MAPK or altering MAPK consensus sites in Bub1 to alanine or valine (Bub1^5AV) abolishes the phosphorylation and activation on chromosomes. Both Bub1 and Bub1^5AV support the checkpoint under an optimal condition for spindle checkpoint activation. However, Bub1, but not Bub1^5AV, supports the checkpoint at a relatively low concentration of nuclei or the microtubule inhibitor nocodazole. Similar to Bub1^5AV, Bub1 without the kinase domain (Bub1^ΔKD) is also partially compromised in its checkpoint function and in its ability to recruit other checkpoint proteins to kinetochores. This study suggests that activation of Bub1 at kinetochores enhances the efficiency of the spindle checkpoint and is probably important in maintaining the checkpoint toward late prometaphase when the cell contains only a few or a single unattached kinetochore.