Bubristatins inhibit BubR1-dependent signaling by interrogating BubR1-CENP-E interaction

The mitotic checkpoint prevents premature anaphase onset through generation of an inhibition to the E3 ubiquitin ligase APC/C, whose ubiquitination of cyclin B and securin targets them for degradation. BUBR1 is a core component of the mitotic checkpoint and is required for formation of stable kinetochore-microtubule attachments. BubR1 interacts with mitotic kinesin CENP-E but its mechanism of action has remained elusive. Here we show our identification and characterization of BubR1 chemical inhibitors Bubristatins. Interestingly, the compounds bubristatin-1 and bubristatin-3 inhibit BubR1 kinase activity and the binding of BubR1 to its effector CENP-E. The binding of the bubristatin-1 to BubR1 was confirmed by enzymatic assay and site-specific mutagenesis. Bubristatin compounds show selectivity for BubR1 relative to other mitotic kinases such as Bub1. A combination of in vitro reconstitution and cell-based assays has enabled us to identify Ser2639 of CENP-E as a cognate substrate of BubR1. Our real-time i...