Spindle formation in mammalian cells requires precise spatial and temporal regulation of the kinesin-5 motor proteins which generate outward force to establish spindle bipolarity. A growing body of work suggests that phosphorylation of kinesin-5 motor domains is an important regulator of their motor characteristics and function in cells. My thesis work demonstrates that the human kinesin-5, Eg5, is phosphorylated on tyrosine residues in its motor domain in a Src family kinase (SFK)-dependent manner. Additionally, c-Src kinase phosphorylates Eg5 motor domains at three sites in vitro: Y125, Y211, and Y231. Mutation of these sites to the phosphomimetic glutamate diminishes motor activity in vitro, and replacement of endogenous Eg5 with phosphomimetic Eg5-Y211E Em in LLC-Pk1 cells results in monopolar spindles, consistent with loss of Eg5 activity. Cells treated with SFK inhibitors show defects in spindle formation, similar to those in cells expressing the non-phosphorylatable Y211 mutant, and distinct from inhibition of other mitotic kinases. This phosphoregulatory mechanism may tune Eg5 enzymatic activity for optimal spindle morphology. Phosphomimetic Eg5 mutants also have decreased binding affinity for small molecule inhibitors of Eg5. These results suggest that phosphoregulation of Eg5 motor domains may also have implications for Eg5 as a cancer therapy target.

Last modified

• 10/12/2018

Creator

• Kathleen Gifford Bickel

DOI

• https://doi.org/10.21985/N2GX7T

Subject

• Driskill Graduate Training Program in Life Sciences

Keyword

• mitosis,
• kinesin-5
• cytoskeleton
• phosphorylation
• Src family kinases
• drug resistance

Date created

• 2017-01-01

Resource type

• Dissertation

Rights statement

• In Copyright