Safeguard of Mitosis: The Spindle Checkpoint

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2016-11-18

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Ji, Zhejian

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Abstract

In mitosis, the kinetochore-microtubule attachment is under surveillance by the spindle checkpoint to ensure the fidelity of chromosome segregation. Defects in the checkpoint could lead to aneuploidy, which has been implicated in cancers, birth defects, and other human diseases. In presence of kinetochores that are not attached or improperly attached to microtubules, the checkpoint signals to assemble the mitotic checkpoint complex (MCC), which consists of BubR1-Bub3, Mad2, and Cdc20. The diffusible MCC inhibits the ubiquitin ligase activity of the anaphase-promoting complex or cyclosome bound to its co-activator Cdc20 (APC/C-Cdc20) to arrest cells in mitosis. Nevertheless, it remains unknown how the checkpoint monitors the status of the kinetochore-microtubule attachment. Neither is clear how MCC is assembled in an active checkpoint signaling. My graduate work has answered these two questions by revealing the critical functions of a checkpoint kinase, monopolar spindle 1 (Mps1), in both attachment sensing and checkpoint signaling. Of kinetochore proteins, the KMN network acts as both a critical microtubule receptor and a signaling platform for the spindle checkpoint. The human KMN contains the kinetochore null 1 complex (Knl1C), the minichromosome instability 12 complex (Mis12C), and the nuclear division cycle 80 complex (Ndc80C). In my first project, I have shown that the non-kinase domain of Mps1 directly binds to Ndc80C through two independent interactions. Both interactions involve the microtubule-binding surfaces of Ndc80C and are directly inhibited by microtubules. Elimination of one such interaction in human cells causes checkpoint defects expected from a failure in detecting unattached kinetochores. This competition between Mps1 and microtubules for Ndc80C binding thus constitutes a direct mechanism for unattached kinetochore detection. The next question is how the kinetochore-associated Mps1 kinase rules the checkpoint signaling. At kinetochore, Mps1 phosphorylates the scaffolding protein Knl1. Phosphorylated Knl1 (pKnl1) recruits checkpoint complexes budding uninhibited by benomyl 1-3 (Bub1-Bub3) and Bub1-related protein in complex with Bub3 (BubR1-Bub3) to kinetochores. My following work has demonstrated that Mps1 promotes the inhibition of APC/CCdc20 by MCC components in vitro through phosphorylating Bub1 and mitosis arrest deficiency 1 (Mad1). Phosphorylated Bub1 (pBub1) binds with Mad1-Mad2. Phosphorylated Mad1 (pMad1) directly interacts with Cdc20. Mutations of Mps1 phosphorylation sites in Bub1 or Mad1 abrogate the spindle checkpoint in human cells. Therefore, Mps1 promotes checkpoint activation through a pKnl1-pBub1-pMad1 phosphorylation cascade, in which phosphorylation of upstream components enables binding of downstream ones. We propose that this sequential multi-target phosphorylation cascade allows Mps1 to amplify checkpoint signals and makes the checkpoint highly responsive to Mps1, which itself is regulated by kinetochore-microtubule attachment. Taken together, my graduate work has solved two long-standing questions in spindle checkpoint regulation. Accordingly, Mps1 recognizes the unattached kinetochores via its non-kinase domain, while activates the checkpoint signaling through its kinase activity. The dual function of Mps1 couples checkpoint activation with unattached kinetochore detection, making checkpoint under the control of kinetochore-microtubule attachment.

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