Date of Award
Doctor of Philosophy (PhD)
Aurora B, the kinase subunit of the chromosomal passenger complex (CPC), promotes accurate cell division by destabilizing erroneous kinetochoremicrotubule attachments and activating the spindle assembly checkpoint (SAC). Both functions require Aurora B activation, which is promoted by its binding to the C terminus of the CPC subunit INCENP, and by localization to the inner centromere, which requires the N terminus of INCENP. Inner centromere localization also requires phosphorylation of histone 3 threonine 3 (H3T3ph). While H3T3ph is required for CPC localization and the SAC during early mitosis, it must be dephosphorylated at anaphase to facilitate mitotic exit. However, the H3T3 kinase Haspin is predicted to be constitutively active, raising questions as to how H3T3ph is regulated with the cell cycle. Additionally, a single alpha helix (SAH) domain in INCENP is required to maintain the SAC elicited by the microtubule stabilizing drug taxol. While the SAH domain binds microtubules and is required for targeting the CPC to the spindle-midzone during anaphase, its role in the SAC during early mitosis remains unknown. Here we uncover the molecular mechanism that couples H3T3ph to the cell cycle and elaborate how the INCENP SAH domain supports the SAC. Using iv Xenopus egg extract and human tissue culture cells, we show that the kinase domain of Haspin is autoinhibited during interphase by a previously unidentified Haspin basic inhibitory segment (HBIS) downstream of the kinase domain. Upon entry into mitosis, Cdk1- and Polo-dependent multisite phosphorylation of the Haspin N terminus displace the HBIS from the kinase domain, resulting in Haspin activation and H3T3ph during mitosis. We also demonstrate that the INCENP SAH domain binds chromatin and supports CPC localization and stability at the centromere. Deleting the SAH domain reduces Aurora B-dependent phosphorylation at the kinetochore and attenuates the taxol-mediated SAC in human cells. We show that the microtubule-binding capacity of the SAH domain, rather than its role in localization, is required for the SAC in taxol. The microtubule-binding affinity of the SAH domain is also regulated by Cdk1- dependent phosphorylation in a phospho-regulatory domain (PRD) adjacent to the SAH domain, which we demonstrate also contributes to the checkpoint. Finally, while targeting INCENP to the centromere/kinetochore or microtubules supports the SAC in taxol, neither activity alone is sufficient for a robust checkpoint arrest. We propose a model wherein INCENP interacts with chromatin and microtubules during early mitosis to support local activation of Aurora B and the SAC. This work has implications for understanding how the CPC promotes the checkpoint and how Aurora B activity is regulated to ensure accurate chromosome segregation.
Wheelock, Michael, "Regulated Recruitment of the Chromosomal Passenger Complex to Chromatin and Microtubules Promotes Accurate Cell Division" (2017). Student Theses and Dissertations. 399.