Multisite phosphorylation determines the formation of Ska-Ndc80 macro-complexes that are essential for chromosome segregation during mitosis.

The human Ska complex (Ska) localizing to both spindle microtubules and kinetochores is essential for proper chromosome segregation during mitosis. Although several mechanisms have been proposed to explain how Ska is recruited to kinetochores, it is still not fully understood. By analyzing Ska3 phosphorylation, we identified six critical Cdk1 sites, including the previously identified Thr358 and Thr360. Mutations of these sites to phospho-deficient alanine (6A) in cells completely abolished Ska3 localization to kinetochores and Ska functions in chromosome segregation. In vitro, Cdk1 phosphorylation on Ska enhanced WT, not phospho-deficient 6A, binding to Ndc80C. Strikingly, the phosphomimetic Ska 6D complex formed a stable macro-complex with Ndc80C, but Ska WT failed to do so. These results suggest that multisite Cdk1 phosphorylation-enabled Ska-Ndc80 binding is decisive for Ska localization to kinetochores and its functions. Moreover, we found that Ska decrease at kinetochores triggered by the microtubule-depolymerizing drug nocodazole is independent of Aurora B but can be overridden by Ska3 overexpression, suggestive of a role of spindle microtubules in promoting Ska kinetochore recruitment. Thus, based on the current and previous results, we propose that multisite Cdk1 phosphorylation is critical for the formation of Ska-Ndc80 macro-complexes that are essential for chromosome segregation.