Function and regulation of the mitotic kinesin Kif18A

Abstract

During congression, vertebrate chromosomes align at the spindle equator and upon alignment these bi-oriented chromosomes continue to oscillate around the metaphase plate.Before sister chromatids are separated upon anaphase onset, these oscillatory movements are dampened resulting in the tight alignment of the chromosomes and the establishment of a thin metaphase plate. The formation of a thin metaphase plate is the key for accurate and faithful chromosome segregation. While it is well established that chromosome movements are coupled to the dynamic behaviour of kinetochore-microtubules and forces acting on the chromosomes, the molecular mechanism regulating dampening of chromosome oscillations and metaphase plate thinning remains largely unknown. RNA interference (RNAi)-mediated depletion of Kif18A, a member of the kinesin-8 family of motor proteins, increases the amplitude of chromosome oscillations, whereas over expression of Kif18A decreases it, suggesting that Kif18A dampens oscillations of bi-oriented chromosomes before anaphase. This function of Kif18A depends on its ability to accumulate at the plus-tips of kinetochore microtubules to act cooperatively in altering dynamics of kinetochore-microtubules.To dissect the regulation of Kif18A in its function to suppress chromosome oscillations, we analysed the post-translational modifications of Kif18A. By combining in-vitro assays with time-lapse microscopy imaging, we demonstrate that Kif18A is phosphorylated by cyclin-dependent kinase 1 (Cdk1) in complex with cyclin-B1 in cells and in-vitro and identified S674 and S684 as major Cdk1-phosphorylation sites on Kif18A. Cdk1 mediated inhibitory phosphorylation on Kif18A during early metaphase prevents Kif18A from accumulating at the plus-tips of kinetochore-microtubules. Consequently Kif18A is unable to suppress chromosome oscillations. Protein phosphatase 1 (PP1) removes these phosphorylations in metaphase in a reaction that depends on a conserved PP1-binding motif in the C-terminus of Kif18A. This dephosphorylation is required for correct accumulation of Kif18A at plus-tips of kinetochore-microtubules resulting in the suppression of oscillations.Based on our studies we propose a model in which a Cdk1/PP1 phosphorylation switch modulates Kif18A’s ability to accumulate at the plus-tips of kinetochore-microtubules to suppress chromosome oscillations, when kinetochore-microtubules are stably attached and under tension. This idea is in perfect agreement with the observation that metaphase plate thinning coincides with the global inactivation of Cdk1 and temporal localization of PP1 at kinetochores.