BubR1 blocks substrate recruitment to the APC/C in a KEN-box-dependent manner

Abstract

SummaryThe spindle assembly checkpoint (SAC) is a signalling network that delays anaphase onset until all the chromosomes are attached to the mitotic spindle through their kinetochores. The downstream target of the spindle checkpoint is the anaphase-promoting complex/ cyclosome (APC/C), an E3 ubiquitin ligase that targets several anaphase inhibitors for proteolysis, including securin and cyclin B1. In the presence of unattached kinetochores, the APC/C is inhibited by the mitotic checkpoint complex (MCC), a tetrameric complex composed of three SAC components, namely BubR1, Bub3 and Mad2, and the APC/C co-activator Cdc20. The molecular mechanisms underlying exactly how unattached kinetochores catalyse MCC formation and how the MCC then inhibits the APC/C remain obscure. Here, using RNAi complementation and in vitro ubiquitylation assays, we investigate the domains in BubR1 required for APC/C inhibition. We observe that kinetochore localisation of BubR1 is required for efficient MCC assembly and SAC response. Furthermore, in contrast to previous studies, we show that the N-terminal domain of BubR1 is the only domain involved in binding to Cdc20-Mad2 and the APC/C. Within this region, an N-terminal KEN box (KEN1) is essential for these interactions. By contrast, mutation of the second KEN box (KEN2) of BubR1 does not interfere with MCC assembly or APC/C binding. However, both in cells and in vitro, the KEN2 box is required for inhibition of APC/C when activated by Cdc20 (APC/C Cdc20 ). Indeed, we show that this second KEN box promotes SAC function by blocking the recruitment of substrates to the APC/C. Thus, we propose a model in which the BubR1 KEN boxes play two very different roles, the first to promote MCC assembly and the second to block substrate recruitment to APC/C Cdc20 .

The Cdc20-binding Phe Box of the Spindle Checkpoint Protein BubR1 Maintains the Mitotic Checkpoint Complex During Mitosis

“…4A). These results were consistent with published reports showing that the KEN1 box, but not the KEN2 box, of BubR1 was required for MCC formation in human cells (23). Strikingly, despite retaining proper Bub3 binding, Myc-BubR1 ⌬PheD bound to Cdc20 and Mad2 much less efficiently, at 70 and 30% of the WT levels (Fig.…”

“…Using a seat belt-like structural element, Mad2 in its active, closed conformation traps the Mad2-interacting motif of Cdc20 in a topological embrace (26 -28). The N-terminal region of BubR1 (BubR1N) contains two KEN boxes and interacts directly with Cdc20 through the first KEN box (KEN1) (16,23). Cdc20-bound Mad2 also makes direct contact with Mad3 (and quite possibly BubR1N) (17,29), buttressing the weak Cdc20-KEN1 interaction.…”

Bimodal activation of BubR1 by Bub3 sustains mitotic checkpoint signaling

“…BubR1 contains two Cdc20 binding sites, a conserved site in the N-terminal Mad3-homology domain and an internal site that is present only in higher eukaryotes. The two Cdc20 binding sites of BubR1 have been shown to be able, respectively, to mediate inhibition of Cdc20 activation of APC/C in Mad2-dependent (13,41) and Mad2-independent manners (42). We tested the contributions of the individual Cdc20 binding sites of BubR1 in Bub3-stimulated inhibition of APC/C Cdc20 ubiquitination of cyclin B in vitro.…”

“…Our evidence in the current study has established that Bub3 promotes this BubR1-Cdc20 interaction, thereby uncovering a previously unknown role for Bub3 in production of the mitotic checkpoint inhibitor through action at the most downstream step of the signaling pathway. Two previous studies proposed that the first KEN box in the N-terminal Cdc20 binding site of BubR1 is responsible for BubR1 binding to Cdc20 (14,41). Based on this, it is plausible that Bub3 binding may alter the conformation of the initially rod-shaped BubR1 N terminus (34,50) in a way to either promote the initial assembly or stabilization of a BubR1-Cdc20 complex for generating the characteristic, selective inhibition of APC/C Cdc20 .…”

“…Failure of Bub3 binding to BubR1 has been shown to weaken the mitotic checkpoint (39)(40)(41). However, it has remained unclear how Bub3 stimulates mitotic checkpoint signaling through binding to BubR1.…”