Synthetic-evolution reveals narrow paths to regulation of the saccharomyces cerevisiae mitotic kinesin-5 cin8

Cdk1 has been found to phosphorylate the majority of its substrates in disordered regions, but some substrates maintain precise phosphosite positions over billions of years. Here, we examined the phosphoregulation of the kinesin-5, Cin8, using synthetic Cdk1-sites. We first analyzed the three native Cdk1 sites within the catalytic motor domain. Any single site conferred regulation, but to different extents. Synthetic sites were then systematically generated by single amino-acid substitutions, starting from a phosphodeficient variant of Cin8. Out of 29 synthetic Cdk1 sites, 8 disrupted function; 19 were neutral, similar to the phospho-deficient variant; and only two gave rise to phosphorylation-dependent spindle phenotypes. Of these two, one was immediately adjacent to a native Cdk1 site. Only one novel site position resulted in phospho-regulation. This site was sampled elsewhere in evolution, but the synthetic version was inefficient in S. cerevisiae. This study shows that a single phosphorylation site can modulate complex spindle dynamics, but likely requires further evolution to optimally regulate the precise reaction cycle of a mitotic motor.