The Multifunctional Kinase Bub1 Acts as a Signaling Hub for the Spindle Checkpoint

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2015-11-12

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Jia, Luying

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Abstract

The spindle checkpoint is an essential mechanism to ensure accurate chromosome segregation during mitosis. The checkpoint signal originates from the kinetochore, which is a huge protein assembly on centromeric chromatin. Kinetochore is also the receptor for spindle microtubules, which enables it to translate microtubule attachment status into spindle checkpoint signal. The separation of the sister chromatids and the progression from metaphase to anaphase requires the activation of an ubiquitin E3 ligase, anaphase-promoting complex or cyclosome (APC/C). Cdc20 is the mitosis-specific APC/C activator. The spindle checkpoint prevents premature sister chromatids separation by preventing Cdc20 from activating APC/C. Bub1 is a highly conserved spindle checkpoint protein that plays multiple roles in checkpoint signaling. On the kinetochore, Bub1 recruits other important checkpoint proteins like BubR1, Mad1 and Cdc20. We found phosphorylation on Bub1 serine 459 is essential for spindle checkpoint and for Bub1-Mad1 interaction. However, the majority of Mad1 still localize to the kinetochore in cells expressing Bub1-S459A mutant. These results suggest that the direct binding between Bub1 and Mad1 through Bub1-S459 may not be responsible for the localization of Mad1 to the kinetochore region. Instead, this interaction enables Mad1 to function in the checkpoint signaling pathway, possibly through regulating its interaction with Bub1-bound BubR1 and Cdc20. Bub1 is also a serine/threonine kinase. The only two identified substrates are histone H2A and Cdc20. Bub1 phosphorylates histone H2A threonine 120, which is important in recruiting Sgo1 and Aurora B kinase to the kinetochore. Bub1 also phosphorylates Cdc20 serine 153. It was shown in vitro that phosphorylation by Bub1 can inhibit APC/CCdc20. However, mouse embryonic fibroblasts (MEFs) expressing Bub1 kinase dead mutant only display mild checkpoint defect due to abnormal Aurora B localization. In addition, over-expression of Bub1 kinase dead mutant in HeLa cells can rescue the checkpoint defect caused by Bub1 depletion using siRNA. These results challenged the importance of Cdc20 phosphorylation by Bub1 in the spindle checkpoint. Here I show that Bub1 binds another kinase Plk1, forming a kinase complex. Phosphorylation of Cdc20 by Bub1-Plk1 not only inhibits APC/CCdc20 in vitro, but also is required for proper spindle checkpoint function in HeLa cells.

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