The BUBR1 pseudokinase domain promotes efficient kinetochore PP2A-B56 recruitment to regulate spindle checkpoint silencing and chromosome alignment

Abstract

The balance of phospho-signalling at outer-kinetochores during mitosis is critical for the accurate attachments between kinetochores and the mitotic spindle and timely exit from mitosis. In humans, a major player in determining this balance is the PP2A-B56 phosphatase which is recruited to the Kinase Attachment Regulatory Domain (KARD) of the Spindle Assembly Checkpoint protein Budding Uninhibited by Benzimidazole 1-related 1 (BUBR1) in a phospho-dependent manner. This event unleashes a rapid, switch-like phosphatase relay that reverses phosphorylation at the kinetochore, extinguishing the checkpoint and promoting anaphase entry. Here, we conclusively demonstrate that the pseudokinase domain of human BUBR1 lacks phosphotransfer activity and that it was maintained in vertebrates because it allosterically promotes KARD phosphorylation. Mutation or removal of this domain results in decreased PP2A-B56 recruitment to the outer kinetochore, attenuated checkpoint silencing and errors in chromosome alignment as a result of imbalance in Aurora B activity. We demonstrate that the functions of the BUBR1 pseudokinase and the BUB1 kinase domains are intertwined, providing an explanation for retention of the pseudokinase domain in certain eukaryotes.

A bifunctional kinase-phosphatase module integrates mitotic checkpoint and error-correction signalling to ensure mitotic fidelity

“…To create the phosphatase-dominant situation, B56γ was tethered to the Cterminus of BUBR1 in place of the KARD and pseudokinase domain (BUBR1 B56γ ). Note, that the only known function of the pseudokinase is to regulate KARD phosphorylation and therefore PP2A-B56 recruitment (Gama Braga et al, 2020). To create the analogous kinase-dominant situation, that KARD was deleted together with the pseudokinase domain by removing the entire Cterminus (BUBR1 ΔPP2A(ΔC) ).…”