This proposal is based on a new finding that Src family kinases (SFKs) phosphorylate human Eg5, an essential mitotic kinesin family motor protein. SFKs are the original, canonical oncogenes and Eg5 is critical for spindle pole separation and stabilization of the mitotic spindle. Both proteins are anti-mitotic drug targets, with inhibitors in Phase I and II trials. Our preliminary data indicates that Src phosphorylates th enzymatic Eg5 heads at three tyrosines in vitro and in cells. These tyrosines are structurally very near the binding sites for Eg5 inhibitors. Our preliminary data also show that phosphomimetic mutations inhibit Eg5 activity and block the binding of an Eg5 inhibitor, STLC. We hypothesize that SFK phosphorylation of Eg5 heads alters Eg5 activity, localization, and action in bipolar spindle assembly and maintenance. We further hypothesize that SFK phosphorylation blocks the binding of several Eg5-targeted inhibitors. To begin our study, we will determine the mechanistic effects of SFK phosphorylation in vitro. We will develop functional phosphomimetic mutants and phospho-specific antibodies for the SFK sites in Eg5 heads. Next, we will examine the effects of SFK phosphoregulation of Eg5 on the progression of mitosis in fixed and live LLC-PK1 cells. We will develop new methods and cell lines for imaging the effects of SFK phosphorylation on mitotic targets, which will enable researchers to address the neglected issue of SFK activity in mitosis for any target of choice.
The final aim of this study isto test whether SFK phosphorylation directly affects Eg5 inhibitor binding and efficacy in vitro and in cells. These experiments will be a first step in evaluating a potential combination therapy regimen targeting SFKs and Eg5.

Public Health Relevance

This proposal is based on a new finding that Src family kinases (SFKs), which are canonical oncogenes, phosphorylate the kinesin family motor protein Eg5, which drives bipolar spindle assembly and maintenance during mitosis. Very few mitotic SFK targets have been identified, and this work will establish the direct effects of SFK phosphoregulation on Eg5 enzymatic activity and mitotic progression. Both SFKs and Eg5 are antimitotic drug targets. This work will demonstrate a direct functional link between them, which could lead to new combination therapy regimens targeting SFKs and Eg5.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM107209-01
Application #
8559178
Study Section
Macromolecular Structure and Function C Study Section (MSFC)
Program Officer
Gindhart, Joseph G
Project Start
2013-09-30
Project End
2017-05-31
Budget Start
2013-09-30
Budget End
2014-05-31
Support Year
1
Fiscal Year
2013
Total Cost
$368,496
Indirect Cost
$110,071
Name
Northwestern University at Chicago
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
005436803
City
Chicago
State
IL
Country
United States
Zip Code
60611
Smith, Kyle P; Gifford, Kathleen M; Waitzman, Joshua S et al. (2015) Survey of phosphorylation near drug binding sites in the Protein Data Bank (PDB) and their effects. Proteins 83:25-36
Waitzman, Joshua S; Rice, Sarah E (2014) Mechanism and regulation of kinesin-5, an essential motor for the mitotic spindle. Biol Cell 106:1-12