Regulating Kinetochore Function

Chromosome segregation must occur with high fidelity, with even minor defects leading to cell inviability or disease. We have defined specific phosphorylation events in kinetochore proteins that are critical for ensuring accurate chromosome segregation. First, in the presence of inappropriate microtubule attachments, such as both sister kinetochores attaching to the same spindle pole, incorrect kinetochore-microtubule attachments must be eliminated to allow new, proper attachments to be formed. We demonstrated Aurora B kinase phosphorylation inhibits the microtubule binding activities of multiple kinetochore components thereby inactivating the kinetochore-microtubule interface. Importantly, these substrates are differentially phosphorylated in response to changes in kinetochore tension and attachment status, providing a flexible mechanism to correct improper kinetochore-microtubule interactions. Second, during the course of mitosis, kinetochore function must be altered to facilitate the changing requirements to capture microtubules, stabilize attachments, and segregate chromosomes. We found that phosphorylation plays a key role in controlling the kinetochore assembly state during mitosis, changing kinetochore function by altering its components rather than regulating their activities. Third, most kinetochore proteins localize to centromeres only during mitosis, but the mechanisms that control mitotic kinetochore assembly and disassembly were unknown. We demonstrated that cyclin-dependent kinase (CDK) controls kinetochore assembly by directly phosphorylating CENP-T and other kinetochore proteins to promote interactions between the constitutively localized inner kinetochore proteins and mitosis-specific outer kinetochore components. We are currently analyzing kinetochore phospho-regulation downstream of additional mitotic kinases to define the paradigms that control kinetochore assembly and activity.