Focal adhesion kinase (FAK) is a 125 kDa protein that colocalizes with integrins at focal adhesions upon cell adhesion to the extracellular matrix. FAK provides catalytic and scaffolding functions in integrin-mediated signaling events that control cell motility and survival. Multiple lines of evidence suggest that FAK may function in the pathology of human cancer and vascular disease and is therefore considered to be a potential target for drug development. The C-terminal focal adhesion targeting (FAT) domain of FAK mediates localization of FAK to discrete regions in the cell called focal adhesions and is important for FAK signaling since disruption of localization prevents the activation of FAK and phosphorylation of downstream substrates in response to integrin-dependent cell adhesion. Paxillin, a focal adhesion-associated adaptor protein that has been implicated in regulating cell motility, binds to the FAT domain and promotes FAK localization to focal adhesions. Moreover, phosphorylation of a strictly conserved tyrosine in the FAT domain modulates FAK localization, FAK signaling and focal adhesion turnover. We have previously solved NMR solution structures of the FAT domain in the presence and absence of a paxillin-derived peptide. We have also developed a novel methodology that integrates hydrogen exchange (HX) data into discrete molecular dynamics (DMD) simulations. The DMD/HX methodology was applied to the FAT domain of FAK and revealed the presence of a FAT intermediate state. The presence of this intermediate state is fully supported by experimental data leading us to propose that conformational dynamics of the FAT domain modulates paxillin binding and phosphorylation and therefore FAK function. The primary goal of this proposal is to investigate structural and dynamic features of the FAT domain that facilitate `switching'between phosphorylated and paxillin bound states of FAK using nuclear magnetic resonance (NMR) experiments and mutation studies combined with biochemical and biophysical approaches. Results from these studies are likely to shed light on how conformational dynamics of the FAT domain regulates FAK function and may provide information helpful for inhibition of FAK function by altering FAT domain ligand-binding and phosphorylation. Focal adhesion kinase (FAK) functions as both a scaffold protein and kinase that regulates a plethora of cellular processes such as cell proliferation, cell death and motility. Aberrant regulation of FAK can result in cancer and vascular disease. The focal adhesion targeting domain (FAT) is located at the C-terminus of the protein. Since the FAT domain regulates FAK function, the proposed investigation should provide information helpful for inhibiting FAK function by altering FAT domain ligand binding- binding and phosphorylation.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM080568-04
Application #
8015604
Study Section
Special Emphasis Panel (ZRG1-BCMB-B (02))
Program Officer
Flicker, Paula F
Project Start
2008-02-15
Project End
2014-01-31
Budget Start
2011-02-01
Budget End
2014-01-31
Support Year
4
Fiscal Year
2011
Total Cost
$263,290
Indirect Cost
Name
University of North Carolina Chapel Hill
Department
Biochemistry
Type
Schools of Medicine
DUNS #
608195277
City
Chapel Hill
State
NC
Country
United States
Zip Code
27599
Case, Lindsay B; Baird, Michelle A; Shtengel, Gleb et al. (2015) Molecular mechanism of vinculin activation and nanoscale spatial organization in focal adhesions. Nat Cell Biol 17:880-92
Tolbert, Caitlin E; Thompson, Peter M; Superfine, Richard et al. (2014) Phosphorylation at Y1065 in vinculin mediates actin bundling, cell spreading, and mechanical responses to force. Biochemistry 53:5526-36
Thompson, Peter M; Tolbert, Caitlin E; Shen, Kai et al. (2014) Identification of an actin binding surface on vinculin that mediates mechanical cell and focal adhesion properties. Structure 22:697-706
Tolbert, Caitlin E; Burridge, Keith; Campbell, Sharon L (2013) Vinculin regulation of F-actin bundle formation: what does it mean for the cell? Cell Adh Migr 7:219-25
Thievessen, Ingo; Thompson, Peter M; Berlemont, Sylvain et al. (2013) Vinculin-actin interaction couples actin retrograde flow to focal adhesions, but is dispensable for focal adhesion growth. J Cell Biol 202:163-77
Beck, Moriah R; Dixon, Richard D S; Goicoechea, Silvia M et al. (2013) Structure and function of palladin's actin binding domain. J Mol Biol 425:3325-37
Thompson, Peter M; Tolbert, Caitlin E; Campbell, Sharon L (2013) Vinculin and metavinculin: oligomerization and interactions with F-actin. FEBS Lett 587:1220-9
Cable, Jennifer; Prutzman, Kirk; Gunawardena, Harsha P et al. (2012) In vitro phosphorylation of the focal adhesion targeting domain of focal adhesion kinase by Src kinase. Biochemistry 51:2213-23
Shen, Kai; Tolbert, Caitlin E; Guilluy, Christophe et al. (2011) The vinculin C-terminal hairpin mediates F-actin bundle formation, focal adhesion, and cell mechanical properties. J Biol Chem 286:45103-15
Beck, Moriah R; Otey, Carol A; Campbell, Sharon L (2011) Structural characterization of the interactions between palladin and α-actinin. J Mol Biol 413:712-25