Study of Phospho-Regulation of a Mitotic Kinesin using a Directed Evolution Approach

Abstract

The S. cerevisiae Cin8 belongs to the kinsesin-5 sub-family of mitotic motor proteins. During mitosis, Cin8 orchestrates the mitotic spindle assembly and its elongation. Recent work from our laboratory indicated that phosphorylation of Cin8 by Cdk1 governs its localization to the mitotic spindle during mitosis. Here we tested the rigidity of phosphorylation sites in Cin8, and examined whether phosphorylation at newly created Cdk1 sites can mimic the known phospho-regulation or create new regulation. For this purpose, we generated phosphorylation-deficient mutant of Cin8 and introduced new Cdk1 sites by single amino acid replacement. This resulted in thirty-one novel Cdk1 phosphorylation sites. In part of the sites, partial and full Cdk1 consensus sites were created. Next we analyzed Cin8 localization to the spindle during anaphase. We found that only one novel Cdk1 phosphorylation site at position 276 is able to restore the original phospho-regulation of Cin8, and is located in high proximity to a native Cdk1 phosphorylation site (S277). Although several sites were created nearby, only this site exhibits localization pattern which is similar to WT-Cin8. This result suggests that phospho-regulation of Cin8 by Cdk1 at this region is rigid and highly dependent on the structural context. Several additional novel Cdk1 mutants exhibited new phenotypes, suggesting that there are regions in Cin8 where phsopho-regulation by Cdk1 is more flexible. These results imply that phospho-regulation of Cin8 is more elusive than previously anticipated and further study of its mechanism is required.