The Motile Properties of Kinesin-5 Mitotic Motor Cin8 are Regulated by Three Cdk1 Phosphorylation Sites in its Catalytic Domain

Cin8 is S. cerevisiae bipolar kinesin-5 biological nano-motor. These motors use ATP-driven motility along microtubules (MTs) to perform their essential functions in mitotic spindle dynamics such as crosslinking and sliding apart antiparallel MTs of the spindle. It was previously demonstrated in our lab, that Cin8 is differentially phosphorylated during anaphase in at least one of the three cyclin-dependent kinase 1 (Cdk1) sites located in its catalytic domain: S277, T285 and S493. It was also shown that this phosphorylation affects Cin8 localization to the mitotic spindle, and mitotic spindle elongation. Moreover, it was found that although the three Cdk1 sites undergo similar phosphorylation in vitro, in vivo they differ in the timing of their phosphorylation, and the strength of their effect. Thus, they exhibit differential regulation of Cin8 activity during anaphase in cells, with the S277 site being most important in regulating Cin8 localization to the mitotic spindle, while the S493 site affecting the rate of spindle elongation.

In vitro study of the motile properties of phospho-mimic variant in which all three sites were replaced with the phospho-mimetic aspartic acid exhibited decreased attachment to MTs and increased minus-end directed motility, in comparison to the wild type Cin8 and phospho-deficient variant in which the three sites were replaced with alanine. This indicates that phosphorylation of at least one of these sites is required for Cin8 detachment from the mitotic spindle and for controlling its directionality. To understand the role of the individual sites in controlling the motor functions of Cin8, in this present study, we examine the motile properties of phospho-variants of Cin8 containing combinations of phospho-mimic and phospho-deficient mutations of the Cdk1 sites. We examine these variants of Cin8, overexpressed and purified from the S. cerevisiae cells, using single-molecule fluorescence motility assay. Results will be presented and discussed.