SRC Kinase Phospho-Regulation of the Human Mitotic Kinesin Eg5

Eg5 is a human kinesin-5 that drives spindle pole separation during the early phases of mitosis. While a substantial body of work has revealed the role that Eg5 plays during mitosis, relatively little is known about how Eg5 activity is regulated throughout the cell cycle. Our data shows that endogenous Eg5 in HEK cells is phosphorylated on tyrosine residues. In silico predictors suggest three tyrosines in the Eg5 motor domain as targets of Src kinase phosphorylation. We show that these residues are phosphorylated specifically by Src kinase, but not by Wee1 kinase in vitro. Furthermore, cells expressing Eg5 constructs with a phosphomimetic mutation of Y211 to glutamate (Y211E) show significantly higher percentages of monopolar spindles than cells expressing wild-type Eg5. The Y211E mutation also significantly decreases Eg5 ATPase rates and motility in vitro. The proximity of the potential phosphorylation sites to the binding sites of small molecule inhibitors targeting Eg5 suggested that phosphorylation may interfere with drug binding. Isothermal calorimetry experiments show that phosphomimetic Eg5 constructs have significantly decreased affinity for S-trityl-L-cysteine (STLC). Together, these data suggest a role for the mitotic kinase Src in regulating Eg5 throughout the cell cycle by directly altering its motor characteristics. Such a role for post-translational modifications has not yet been investigated in Eg5. Additionally, the effect of phosphomimetic mutations on STLC binding suggests that phosphorylation may decrease the efficacy of Eg5 inhibitors that are currently in clinical trials. Future work will seek to confirm Src kinase as a regulator of Eg5 activity in cells and investigate phosphorylation as a potential cancer resistance mechanism.