A phosphorylation switch governs KIF11’s mechanical output during mitosis

The kinesin-5 motor protein KIF11 is crucial for mitotic spindle assembly, driving the separation of spindle poles through microtubule sliding. Src-family kinases phosphorylate KIF11 at multiple tyrosine residues within its motor domain, but the mechanistic consequences of these modifications remain unclear. Here, we dissect the role of phosphorylation at Y211 using phospho-mimetic (Y211E) and nonphosphorylatable mutants (Y211F) in biochemical, biophysical, and cellular assays. Optical trapping and Förster resonance energy transfer analyses reveal that Y211 phosphorylation slows neck-linker docking, reducing motor velocity and force generation under load. In human cells, Y211E expression impairs bipolar spindle formation and decreases spindle pole separation velocity, while Y211F shortens steady-state spindle length. Fluorescence recovery after photobleaching analyses show that Y211E accelerates motor turnover on spindle microtubules, consistent with the mutant motor’s heightened load sensitivity. Together, these findings support a model in which Src-mediated phosphorylation at Y211 acts as a rheostat to tune KIF11 mechanochemistry and spindle assembly dynamics, linking cancer-relevant kinase signaling to mitotic force generation.

Accurate chromosome segregation during mitosis depends on the formation of a bipolar spindle, a dynamic structure composed of microtubules (MTs) and numerous associated proteins. Spindle assembly requires the coordinated nucleation, organization, and sliding of MTs to establish a bipolar configuration that captures, aligns, and segregates paired sister chromatids accurately. This process depends on a balance of forces generated by molecular motor proteins and MT dynamics to maintain spindle length and tension while preventing collapse into a monopolar configuration. Among these motors, kinesin-5 family members play a central role by crosslinking and sliding antiparallel MTs apart to drive pole separation and stabilize spindle bipolarity. While kinesin-5 motors have been the subject of intense investigation, how their force production is regulated during mitosis to optimize spindle function remains incompletely understood.