Synthetic-Evolution Approach Reveals Context-Specific Regulation of a Mitotic Kinesin-5 Nano-Motor

Kinesin-5 mitotic nano-motors play essential roles in mitotic spindle dynamics, by cross-linking and sliding apart antiparallel microtubules of the spindle. Recently, we found that the S. cerevisiae kinsesin-5 Cin8 is phospho-regulated by Cdk1 at three sites in its catalytic motor domain, which governs its localization to the mitotic spindle during mitosis. We also determined that each of these Cdk1 sites plays a different role in regulation of Cin8. Here we tested the rigidity of phospho-regulation of Cin8, and examined whether novel Cdk1 sites created by a single amino acid replacement can mimic the known phospho-regulation or create new phenotypes. For this purpose, we systematically generated Cin8 mutants carrying a novel Cdk1 site as a sole source for Cdk1 phospho-regulation. We found that out of 29 novel sites that we have generated, only one site in the motor domain, in high proximity to a native Cdk1 site, recapitulated the phospho-regulation of the adjacent native site, although several sites were created nearby. This result suggests that phospho-regulation of Cin8 by Cdk1 at this site is rigid and highly dependent on the structural context. Two other sites resulted in novel phospho-regulation of Cin8; however they resulted in a less optimized phospho-regulation. Interestingly multiple-sequence-alignment revealed that one of these sites is present in other organisms, suggesting that phospho-regulation at this site is flexible throughout evolution and may occur according to the needs of the organism.