PLASTICITY VS. RIGIDITY OF PHOSPHO-REGULATION OF A MITOTIC NANO-MOTOR

Alina Goldstein ^1,2 Darya Goldman ^1,2 Ervin Valk ³ Mart Loog ³ Liam Holt ⁴ Leah Gheber ^1,2

¹Department of Chemistry, Ben-Gurion University of the Negev
²Ilse Katz Institute for Nanoscale Science and Technology, Ben-Gurion University of the Negev
³Institute of Technology University, University of Tartu
⁴Department of Molecular and Cell Biology, UC Berkeley

The S. cerevisiae Cin8 is a member of the kinesin-5 subfamily and plays key roles during cell division. Previous work from our laboratory showed that during mitosis Cin8 undergoes phospho-regulation by Cyclin dependent kinase-1 (Cdk1). We showed that phosphorylation in the motor domain of Cin8 is required for its detachment from the spindle during late anaphase. In this study, we examined the rigidity of this phospho-regulation and the ways it affects the function of Cin8. For this purpose we generated a phospho-deficient variant of Cin8 and systematically introduced novel Cdk1 sites by mutations. We tested the mutant’s functionality and observed their localization during cell division. We found that only one mutant bearing a novel Cdk1 site was able to mimic the native phospho-regulation of Cin8 and is located in close proximity to a native Cdk1 site. Two other positions resulted in novel regulation and function of Cin8. These results indicate that, in contrast to many flexible phospho-regulation examples, the phospho-regulation of Cin8 by Cdk1 is a non-flexible, tight and precise process that is likely based on the specific mechanism of kinesin activity.