Proper assembly of kinetochores (KTs) during mitosis is required for bipolar attachment of spindle microtubules (MTs) and the accumulation of spindle assembly checkpoint (SAC) components. Here we show that testis-expressed protein 14 (Tex14), which has been implicated in midbody function, is recruited to KTs by Plk1 in a Cdk1-dependent manner during early mitosis. Exclusion of Tex14 from kinetochores results in an inability to efficiently localize outer KT components, impaired KT-MT attachment, chromosome congression defects, and whole-chromosome instability. In addition, we demonstrate that phosphorylation of Tex14 by Plk1 during metaphase promotes APC(Cdc20)-mediated Tex14 degradation. Inhibition of this phosphorylation event causes retention of Tex14 at KTs and results in delayed metaphase-to-anaphase transition and chromosome segregation defects. Our findings identify Tex14 as an important mediator of KT structure and function and the fidelity of chromosome separation.
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