Mps1 regulates kinetochore-microtubule attachment stability via the Ska complex to ensure error-free chromosome segregation

The spindle assembly checkpoint kinase Mps1 not\ua0only inhibits anaphase but also corrects erroneous attachments that could lead to missegregation and aneuploidy. However, Mps1’s error correction-relevant substrates are unknown. Using a chemically\ua0tuned kinetochore-targeting assay, we show that Mps1 destabilizes microtubule attachments (K\ua0fibers) epistatically to Aurora B, the other major error-correcting kinase. Through quantitative proteomics, we identify multiple sites of Mps1-regulated phosphorylation at the outer kinetochore. Substrate modification was microtubule sensitive and opposed by PP2A-B56 phosphatases that stabilize chromosome-spindle attachment. Consistently, Mps1 inhibition rescued K-fiber stability after depleting PP2A-B56. We also identify the Ska complex as a key effector of Mps1 at the kinetochore-microtubule interface, as mutations that mimic constitutive phosphorylation destabilized K fibers in\ua0vivo and reduced the efficiency of the Ska complex's conversion from lattice diffusion to end-coupled microtubule binding in\ua0vitro. Our results reveal how Mps1 dynamically modifies kinetochores to correct improper attachments and ensure faithful chromosome segregation