Engineering Approaches to Biomolecular Motors

Engineering Approaches to Biomolecular Motors: From in vitro to in vivo Friday Speaker Abstracts

Src Phosphorylation Regulates the Human Kinesin-5, Eg5, and Disrupts the Binding of Eg5 Inhibitors Kathleen G. Bickel 1 , Joshua S. Waitzman 1 , Barbara Mann 2 , Melissa C. Gonzalez 1 , Patricia Wadsworth 2 , Sarah E. Rice 1 . 1 Northwestern University, Chicago, IL, USA, 2 University of Massachusetts-Amherst, Amherst, MA, USA. The human kinesin-5 motor, Eg5, is required to establish and maintain the mitotic spindle in organisms ranging from yeast to humans. Several phosphoregulatory mechanisms tightly control the localization and activity of Eg5 during mitosis. Phosphorylation of the Eg5 tail domain by Cdk1 is well known to regulate its localization to the mitotic spindle. Phosphoregulation of yeast and Drosophila Eg5 motor domains has also been reported, but the majority of the reported sites are not conserved in human or mammalian Eg5. Here, we examined human Eg5 motor domain phosphorylation. Using an anti-phospho-tyrosine antibody we show that Eg5 tyrosines are phosphorylated in human and mammalian-derived cells. Furthermore, using a chemical genetic approach we show that this phosphorylation is dependent on Src family kinase activity. Results from in vitro kinase assays and transfection experiments in mammalian-derived cells suggest that c-Src kinase binds to a SRC Homology 3 domain targeting motif (-PXXP-) within the Eg5 microtubule binding site and phosphorylates residues Y125, Y211, and Y231. These results implicate human Eg5 as a potential direct mitotic target of Src family tyrosine kinases. Additionally, we have evaluated the functional effects of this phosphoregulation. Phosphomimetic mutations within these three sites alter the in vitro motility characteristics of Eg5 motor domains relative to wild-type and non-phosphorylatable mutant proteins. Furthermore, mammalian cells expressing phosphomimetic Eg5 motors exhibit increased spindle polarity defects. Lastly, we report that phosphomimetic mutations reduce Eg5 affinity for the inhibitor S-trityl-L-cysteine (STLC). In cells with high Src activity, including many types of cancers, the same mechanism could provide rapid resistance to therapy using Eg5 inhibitors.

Light-mediated Motor Activities Control Cargo Distributions in Cells Bianxiao Cui Stanford University, Stanford, CA, USA No Abstract

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