A mitotic motor protein undergoes complex phospho-regulation pathways as revealed by single molecule motility and live imaging microscopy analysis

Kinesin-5 motor proteins perform pivotal roles in mitotic spindle formation and elongation. S. cerevisiae cells express two kinesin-5 homologues, Cin8 and Kip1, which partially overlap in function. Recently it was found that Cin8 and Kip1 are phospho-regulated by Cdk1, which governs their localization to the mitotic spindle during mitosis. Cin8 contains five putative Cdk1 phosphorylation sites, three in the motor domain and two in the stalk and tail. Although it was demonstrated that phospho-regulation of Cin8 at its motor domain by Cdk1 regulates its localization, the contribution of each of the three Cdk1 sites was never explored. We first established the role of each of the three Cdk1 sites in the motor domain by employing an analysis method specifically developed for quantifying Cin8 distribution in live cells. We then combined these results to analysis by in vitro single molecule assay conducted on purified phosphor-variants of Cin8. We found that one of the sites, S277, located in a large insert in loop 8, is the major regulator of Cin8 phospho-regulation in vivo, and responsible for most of Cin8 phospho-regulation of localization and functionality in vivo, however the mostly conserved site at position S493 is responsible for regulating Cin8 motility and association to MTs. Furthermore, we tested the rigidity of phospho-regulation of Cin8, and examined whether phosphorylation at newly created Cdk1 sites can mimic the known phospho-regulation or create new phenotypes. We found that phospho-regulation of Cin8 by Cdk1 is rigid and highly dependent on position and structural context.