SRC Phosphorylation Regulates the Human Kinesin-5, Eg5, and Disrupts the Binding of Eg5 Inhibitors

Abstract

The human kinesin-5 motor, Eg5, is required to establish and maintain the mitotic spindle. We show that Src kinase binds to a unique motif in the microtubule-binding interface of the Eg5 enzymatic head domain and phosphorylates three specific tyrosine residues in endogenous Eg5. These tyrosines are located near the nucleotide pocket and the functionally critical Loop 5 region within the Eg5 head. We have also found that phosphomimetic Eg5 motor proteins have altered motility characteristics relative to wild-type and non-phosphorylatable mutant proteins. Furthermore, cells expressing phosphomimetic Eg5 motors have increased spindle polarity defects. These results implicate Eg5 as a potential direct mitotic target of tyrosine kinases, most likely Src family kinases. Phosphomimetic motors also have greatly reduced affinity for the Eg5 inhibitor S-trityl-L-cysteine (STLC). In cells with high Src activity, including many types of cancers, the same mechanism may provide rapid resistance to therapy with Eg5 inhibitors.