Dynamic phosphorylation of CENP-N by CDK1 guides accurate chromosome segregation in mitosis.

In mitosis, accurate chromosome segregation depends on super-molecular machinery kinetochore that couples dynamic spindle microtubules to centromeric chromatin. However, the structure-activity relationship of the constitutive centromere-associated network (CCAN), during mitosis remains uncharacterized. Building on our recent cryo-electron microscopy structure of human CCAN, here we reveal the molecular basis of how dynamic phosphorylation of human CENP-N regulates accurate chromosome segregation. Our mass spectrometric analyses revealed mitotic phosphorylation of CENP-N by CDK1 kinase, which modulates the CENP-L-CENP-N interaction for accurate chromosome segregation and CCAN organization. Perturbation of CENP-N phosphorylation is shown to prevent proper chromosome alignment and activate the spindle assembly checkpoint. These analyses provide mechanistic insight into a previously undefined link between the centromere-kinetochore network and accurate chromosome segregation.