Chromosome segregation fidelity is controlled by small changes in kinetochore phospho-occupancy

Kinetochore protein phosphorylation promotes the correction of erroneous microtubule attachments to ensure faithful chromosome segregation during cell division. Determining how phosphorylation executes error correction requires an understanding of whether kinetochore substrates are completely (i.e. all-or-none) or only fractionally phosphorylated. Using quantitative mass spectrometry (MS), we measured phospho-occupancy on the conserved kinetochore protein Hec1 (NDC80) that directly binds microtubules. None of the positions measured exceeded [~]50% phospho-occupancy, and the cumulative phospho-occupancy changed by only [~]20% in response to changes in microtubule attachment status. The narrow dynamic range of phospho-occupancy is maintained by ongoing phosphatase activity. Further, both Cdk1-Cyclin B1 and Aurora kinases phosphorylate Hec1 to enhance error correction in response to different types of microtubule attachment errors. Thus, networks of kinases and phosphatases maintain low inherent phospho-occupancy to promote microtubule attachment to kinetochores while providing for high sensitivity of kinetochore-microtubule attachments to very small changes in phospho-occupancy to ensure high mitotic fidelity.