Src Kinase Regulates The Human Kinesin-5, Eg5, By Phosphorylating Tyrosines In The Eg5 Motor Domain

The human kinesin-5, Eg5, is required to establish and maintain the mitotic spindle. using in silico, in vitro and cell culture methods, we show that Src kinase phosphorylates specific tyrosine residues in Eg5. These residues are located near the nucleotide pocket and the functionally critical L5 loop. Phosphomimetic and non-phosphorylatable Eg5 mutant proteins have diminished ATPase activity and microtubule sliding relative to wild-type Eg5. We also report that phosphomimetic proteins have greatly reduced affinity for the Eg5 inhibitor S-trityl-L-cysteine. This finding suggests that Src phosphorylation of Eg5 may provide cells a non-mutagenesis-dependent strategy to evolve resistance to anti-mitotic Eg5 inhibitors. In this case, combination treatment targeting both SFKs and Eg5 may inhibit mitosis more effectively than anti-Eg5 treatment alone. Ultimately, Src phosphorylation of Eg5 represents a novel regulatory mechanism for a human kinesin, and links the chemical and physical processes that cause mitosis.