Browsing by Subject "M Phase Cell Cycle Checkpoints"
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Item The Multifunctional Kinase Bub1 Acts as a Signaling Hub for the Spindle Checkpoint(2015-11-12) Jia, Luying; De Martino, George; Yu, Hongtao; Cobb, Melanie H.; White, Michael A.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.Item Spindle Checkpoint at Kinetochores(2014-07-24) Kim, Soonjoung; Sternweis, Paul C.; Cobb, Melanie H.; Rice, Luke M.; Yu, HongtaoThe kinetochore—a large protein assembly on centromeric chromatin—functions as the docking site for spindle microtubules and as a signaling hub for the spindle checkpoint. The Constitutive Centromere-Associated Network (CCAN) at the inner kinetochore nucleates the formation of the mature outer kinetochore during mitosis, including the recruitment of the KMN network that consists of Knl1, the Mis12 complex (Mis12C), and the Ndc80 complex (Ndc80C). The KMN is a critical receptor for microtubules, and provides a landing pad for various spindle checkpoint proteins and regulatory factors. The spindle checkpoint protein Mad2 has multiple conformations, including the inactive open Mad2 (O-Mad2) and the active closed Mad2 (C-Mad2). The kinetochore-bound checkpoint protein complex Mad1–Mad2 promotes the conformational activation of O-Mad2 and serves as a catalytic engine of checkpoint signaling. The activated C-Mad2 binds to and inhibits Cdc20, an activator of APC/C, to prevent precocious anaphase onset. Deficient spindle checkpoint signaling leads to premature sister-chromatid separation and aneuploidy. Research in this thesis has provided several key insights into spindle checkpoint signaling at kinetochores. First, we show that the conformational transition of Mad2 is regulated by phosphorylation of S195 in its C-terminal region. The phospho-mimicking Mad2S195D mutant and the phospho-S195 Mad2 protein do not form C-Mad2 on their own. Mad2 phosphorylation inhibits its function through differentially regulating its binding to Mad1 and Cdc20. Our results establish for the first time that the conformational change of Mad2 is regulated by posttranslational mechanisms. Second, we have studied how Mad1 is targeted to kinetochores. We have determined the crystal structure of the conserved C-terminal domain (CTD) of human Mad1. The structure reveals unexpected fold similarity between Mad1 CTD and known kinetochore-binding modules. Functional studies then validate a role of Mad1 CTD in kinetochore targeting and implicate Bub1 as its receptor. Interestingly, deletion of the CTD does not abolish Mad1 kinetochore localization. Non-overlapping Mad1 fragments retain detectable kinetochore targeting. Our results indicate that the CTD–Bub1 connection is one of several mechanisms of targeting Mad1 to kinetochores. Finally, we show that the proper assembly of KMN is required for generating the spindle checkpoint signal at kinetochores. We have developed several strategies to inactivate KMN at kinetochores in human cells, and demonstrate its requirement for the spindle checkpoint in the absence of microtubules. We further show that two quasi-independent pathways mediate the mitosis-specific assembly of KMN at kinetochores. In one pathway, the centromeric kinase Aurora B phosphorylates the Mis12C component Dsn1, and strengthens Mis12C binding to the CCAN component CENP-C. In the second pathway, CENP-T anchors the CENP-H/I/K sub-complex at kinetochores, which in turn recruits Ndc80C. Inactivation of both pathways abolishes KMN at kinetochores and causes gross spindle checkpoint defects. In conclusion, combining cell biology and structural biology methods, our studies have defined a new posttranslational mechanism of Mad2 regulation, uncovered a critical way for targeting Mad1 to kinetochores, and dissected assembly pathways of the KMN checkpoint sensor at kinetochores.Item Spindle Checkpoint Silencing by TRIP13(2017-10-30) Brulotte, Melissa Lynn; DeBose-Boyd, Russell A.; Yu, Hongtao; Luo, Xuelian; Burma, Sandeep; Roth, Michael G.The spindle checkpoint is important for maintaining genomic stability and preventing aneuploidy, a hallmark of cancer. The checkpoint ensures that chromosome segregation does not occur until all sister chromatids are correctly attached to the mitotic spindle during metaphase. When this requirement is met, the checkpoint must be silenced for the cell to proceed to anaphase. Thyroid hormone receptor interacting protein 13 (TRIP13) is a hexameric AAA+ ATPase involved in spindle checkpoint silencing. TRIP13 functions by initiating a conformational change in mitotic arrest deficient 2 (Mad2), a key component of the mitotic checkpoint complex (MCC). This TRIP13-mediated conformational change of Mad2 causes MCC disassembly and relieves inhibition of the anaphase promoting complex/cyclosome (APC/C). The interaction between TRIP13 and Mad2 is dependent on the p31comet adaptor protein. In my first project, I show that TRIP13-p31comet disrupts the MCC by local unfolding of Mad2. I identify a binding surface on human TRIP13 for p31comet-Mad2 and key TRIP13 residues involved in its conformational dynamics. I propose that the flexibility of the hinge region of TRIP13 is important for coupling its ATPase activity to substrate unfolding. The hinge region is conserved in other eukaryotic AAA+ ATPases, and may also be important for energetic coupling in those systems. I have also reconstituted the process of spindle checkpoint silencing in vitro. Importantly, I show that TRIP13 can disrupt the free MCC complex, but not MCC bound to APC/C, providing an explanation for the coordination of the multiple mechanisms that work together to achieve spindle checkpoint silencing. In my second project, to provide a tool for future mechanistic studies and to examine the oncogenic activity of TRIP13, I attempted to identify chemical inhibitors for TRIP13 through high-throughput screening. I identified a series of lead compounds that indirectly inhibited TRIP13 as pan-assay interference compounds. These compounds are redox cyclers that generate hydrogen peroxide, which covalently modifies protein residues such as cysteines and tryptophans. No other potent lead compounds were discovered. This study revealed that TRIP13 may be a difficult protein to target, and that large compound libraries should be prescreened for redox cyclers before they are used in high-throughput inhibitor screening.