In vitro Reconstitution Unveils the Phospho-regulated Ska-Ndc80 Macro-complex

Abstract

ABSTRACTThe human Ska (spindle and kinetochore associated) complex is essential for proper kinetochore-microtubule interactions. Its loading onto the kinetochore complex Ndc80 (Ndc80C) plays a critical role in such function; however, the key determinant that docks Ska onto Ndc80C still remain elusive. Here, we address this question using biochemical reconstitution followed by functional analysis. We identified six Cdk1 sites in Ska3 distributed in three conserved regions. In vitro, Cdk1 phosphorylation on the Ska complex enhanced WT, not phospho-deficient 6A, binding to Ndc80C. Strikingly, the phospho-mimetic Ska 6D complex formed a stable macro-complex with Ndc80C, but Ska WT failed to do so, suggesting that Cdk1 phosphorylation is necessary and sufficient for the formation of the Ska-Ndc80 macro-complex. In cells, Ska3 6A completely lost its localization to kinetochores and its functions in chromosome segregation; whereas Ska3 6D partially restored them. Altogether, our findings not only for the first time reveal the key phospho-regulated Ska-Ndc80 macro-complex essential for faithful chromosome segregation, but also pave the way for mechanistic analyses of kinetochore-microtubule interactions.