A bifunctional kinase–phosphatase module balances mitotic checkpoint strength and kinetochore–microtubule attachment stability.

Two major mechanisms safeguard genome stability during mitosis: the mitotic checkpoint delays mitosis until all chromosomes have attached to microtubules, and the kinetochore–microtubule error‐correction pathway keeps this attachment process free from errors. We demonstrate here that the optimal strength and dynamics of these processes are set by a kinase–phosphatase pair (PLK1‐PP2A) that engage in negative feedback from adjacent phospho‐binding motifs on the BUB complex. Uncoupling this feedback to skew the balance towards PLK1 produces a strong checkpoint, hypostable microtubule attachments and mitotic delays. Conversely, skewing the balance towards PP2A causes a weak checkpoint, hyperstable microtubule attachments and chromosome segregation errors. These phenotypes are associated with altered BUB complex recruitment to KNL1‐MELT motifs, implicating PLK1‐PP2A in controlling auto‐amplification of MELT phosphorylation. In support, KNL1‐BUB disassembly becomes contingent on PLK1 inhibition when KNL1 is engineered to contain excess MELT motifs. This elevates BUB‐PLK1/PP2A complex levels on metaphase kinetochores, stabilises kinetochore–microtubule attachments, induces chromosome segregation defects and prevents KNL1‐BUB disassembly at anaphase. Together, these data demonstrate how a bifunctional PLK1/PP2A module has evolved together with the MELT motifs to optimise BUB complex dynamics and ensure accurate chromosome segregation. Synopsis: The mitotic checkpoint and kinetochore–microtubule error‐correction pathways safeguard chromosome segregation during mitosis. This work shows that a kinase–phosphatase recruitment module in BUBR1 integrates both processes by balancing PLK1 and PP2A levels at kinetochore scaffold KNL1. The kinase PLK1 and phosphatase PP2A‐B56 are engaged in an intramolecular negative feedback loop on BUBR1, which binds to kinetochores via phosphorylated MELT motifs in KNL1.This feedback balances PLK1 and PP2A levels at KNL1 to optimise strength and dynamics of the mitotic checkpoint (PLK1) and kinetochore–microtubule attachments (PP2A).The number of MELT motifs in KNL1 is also crucial for setting the right PLK1/PP2A levels because elevating MELT number causes defects that are associated with high PLK1/PP2A activity. [ABSTRACT FROM AUTHOR]