Browsing by Subject "Cdc20 Proteins"
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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 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.