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Engineering Approaches to Biomolecular Motors: From in vitro to in vivo Friday Speaker Abstracts
37
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