Role of CLASP2 phosphorylation in regulating kinetochore-microtubule interactions
Proper chromosome segregation requires dynamic regulation of kinetochore-microtubule attachments throughout mitosis. Multiple kinetochore proteins display microtubule-binding activity, yet how exactly these proteins are spatially and temporally regulated is unclear. Cytoplasmic linker-associating proteins (CLASPs) are present in the outer kinetochore, in close proximity to microtubule ends, and are required for mitosis. Here, we test whether phosphorylation of CLASP2 during mitosis serves as a mechanism to regulate kinetochore-microtubule interactions and the fidelity of chromosome segregation. We show that cyclin-dependent kinase 1 (Cdk1) and glycogen synthase kinase beta; (GSK3beta;)-dependent mitotic phosphorylation of CLASP2 within its microtubule end-binding domain inhibits its microtubule end-binding activity but does not affect CLASP2 kinetochore binding. Deregulation of CLASP2 phosphorylation does not affect initial chromosome congression, but weakens kinetochore-microtubule attachments. In the presence of endogenous CLASP2, phosphorylation-deficient CLASP2 increases average interkinetochore distance, while phosphomimetic CLASP2 decreases average interkinetochore distance measurements. Together, these results suggest that CLASP2 microtubule binding at the kinetochore is required for stable, tension-generating kinetochore-microtubule attachments. Furthermore, cells expressing CLASP2 phosphomutants have abnormal kinetochore dynamics and in some cells, kinetochore pairs flip about the metaphase plate, suggesting absent or imbalanced microtubule attachments. Expression of either nonphosphorylatable or phosphomimetic CLASP2 fails to rescue depletion of CLASP2 and ultimately leads to an increase in lagging chromosomes. Together, these results characterize specific phosphorylation sites in CLASP2 that negatively regulate microtubule binding and present a novel mechanism of Cdk1 and GSK3beta;-mediated control of kinetochore-microtubule interactions.