- The homoterameric bipolar kinesin-5 motors perform essential functions in mitotic spindle dynamics by crosslinking and sliding apart antiparallel microtubules. S. cerevisiae cells express two kinesin-5s Cin8 and Kip1, which overlap in function. We have recently demonstrated that Cin8 and Kip1 are minus-end directed on the single-molecule level and can switch directionality under a number of conditions (Duselder et al., 2015; Fridman et al., 2013; Gerson-Gurwitz et al., 2011). The mechanism of this directionality switch and its physiological significance remain unclear. We have also demonstrated that Cin8 is differentially phosphorylated during late anaphase at three cyclin-dependent kinase 1 (Cdk1) sites located in its motor domain. This phosphorylation regulates Cin8 activity during anaphase (Avunie-Masala et al., 2011), but its mechanism remains unclear. Here we examined the hierarchical regulation of the activity of Cin8 by in vitro kinase assay, live-cell imaging and in vitro motor motility assay. We also developed quantitative analyses to compare the contributions of the three Cdk1 sites in the catalytic domain of Cin8 to regulation of its intracellular activity. We found that the three Cdk1 sites undergo phosphorylation in vitro by Cdk1 kinase and that this phosphorylation affects the motile properties of Cin8. Our analysis also reveals that each one of the Cdk1-specific sites in the catalytic domain of Cin8 control its localization to the anaphase spindles. Phospho-deficient mutation at the S277 site exerts the strongest effect, indicating that phosphorylation of this site primarily by Cdk1, regulates the dynamics of Cin8 attachment to- and detachment from the anaphase spindles and contributes to the regulation of its function. 1 Fridman, V. et al. J Cell Sci 126, 4147-4159, doi:10.1242/jcs.125153 (2013). 2 Gerson-Gurwitz, A. et al. Embo J 30, 4942-4954 (2011). 3 Duselder, A. et al. J Biol Chem 19, 620799 (2015). 4 Avunie-Masala, R. et al. J Cell Sci 124, 873-878 (2011).