BubR1 in the Coordination of Kinetochore-Microtubule Attachment and Spindle Assembly Checkpoint Signaling

Abstract

BubR1 is a key protein constituting the mitotic checkpoint complex (MCC). During mitosis, the spindle assembly checkpoint (SAC) acts to delay anaphase onset until all chromosomes are attached to mitotic spindles at kinetochores. The SAC works through generation of MCC, which inhibits the anaphase-promoting complex/cyclosome E3 ligase (APC/C) in the cytoplasm.

This study addresses on first, how BubR1 coordinated kinetochore-microtubule (KT-MT) attachments and SAC signaling. Secondly, it also addresses on how a regulator in mitosis, tumor suppressor BRCA2 serves as a signaling platform during SAC.

In order to understand the physiological role of BubR1 acetylation, acetylation deficient knock-in mice (K243R/+) were generated. After 60 weeks, 38% of mice spontaneously developed tumors at various tissues. K243R/+ Mouse Embryonic Fibroblasts (MEFs) were highly aneuploid and had weakened SAC. At kinetochores, unstable KT-MT attachments were observed due to reduced recruitment of PP2AB56a. Insufficient amount of PP2AB56a could not counterbalance the excessive Aurora B activity at kinetochores. All together, unstable KT-MT attachment and failure in maintaining MCC formed in mitosis led to accumulation of chromosome instability (CIN) in K243R/+. These CIN manifested in various forms in the acetylation deficient mice generated a mutation-prone cellular environment favoring tumorigenesis.

Previous works have shown that in prometaphase, BubR1 acetylation occurs at kinetochores, only if BRCA2 is present to support the BubR1-P300/CBP-associated factor (PCAF) interaction (Choi et al., 2012). My research showed that BubR1 was deacetylated when SAC was silenced. Deacetylation of BubR1 was a cue to SAC silencing, as cells expressing acetylation mimetic form of BubR1 could not exit mitosis after the metaphase delay. Also, acetylation of BubR1 diminished when SAC silencing was mimicked in mitotic cells. Deacetylation of BubR1 was mediated by class I HDACs, and BRCA2 was needed for HDACs to interact with BubR1 at prometaphase. Hence, one can conclude that BRCA2 not only regulates BubR1 acetylation, but it presents the acetylated BubR1 to HDACs at prometaphase for deacetylation. Analysis on the BRCA2 complex in mitosis suggested that BRCA2 acts as a signaling platform within the SAC signaling network by specifying the interaction and localization of essential proteins at kinetochores.

In essence, my study provides further insight into the following key questions in mitosis. First, the question of how KT-MT attachment is sensed and relayed to SAC was further explained by elucidating the role of BubR1 in coordinating KT-MT attachment and SAC signaling. Second, the question of how complex SAC signaling is punctually regulated by multiple proteins during metaphase to anaphase transition was addressed through elucidating the role of BRCA2 as a scaffold for BubR1 acetylation/deacetylation at kinetochores.