Genetic Analysis of FAK Activity Binding interactions between extracellular matrix proteins such as fibronectin and integrins play fundamental roles during development by controlling cell adhesion, motility, and survival. Signals generated by integrins at cell attachment sites termed focal adhesions are mediated by the recruitment of cytoskeletal and signaling proteins in a manner that remain under investigation. Focal adhesion kinase (FAK) is a cytoplasmic tyrosine kinase that is activated by integrins and hypothesized to regulate aspects of cell survival and motility during tumor progression. FAK and integrin function are essential during development as knockouts yield early embryonic lethal phenotypes. However, as FAK works as both a scaffolding protein and as a signaling kinase, knockout studies do not provide mechanistic insights in distinguishing these features of FAK action. Moreover, as FAK-null mouse embryo fibroblasts (MEFs) exhibit both proliferation and motility defects, it remains undetermined whether FAK activity is differentially involved in these events. We recently demonstrated that FAK promotes primary fibroblast proliferation through p53 inactivation in a kinase-independent manner via N-terminal FAK FERM (band 4.1, ezrin, radixin, moesin homology) domain- mediated nuclear translocation, p53 binding, and enhancement of p53 ubiquitination and turnover. Thus, we hypothesize that FAK FERM nuclear-association promotes cell survival by keeping p53 levels low. To support this model, we have generated a kinase-dead (KD) knock in point mutation (Lys-454 to Arg, R454) in exon 21 of mouse fak by homologous recombination. In the Preliminary Results, we find that homozygous KD FAK is embryonic lethal. However, unlike FAK-null MEFs that cannot grow (due to p53 activation), we find that homozygous KD FAK MEFs proliferate in culture, but show severe migration defects of enhanced focal adhesion formation and in directional motility. This shows that FAK catalytic activity is not essential for MEF proliferation-survival, but is required for cell movement in vitro and in vivo. To extend these findings, we propose 3 research aims. First, we will determine the role of FAK activity in vivo by analysis of KD FAK knock in embryos. This will involve comparisons to p53 activation in FAK-null embryos, pharmacological inhibition of FAK during development, and analysis of phospho-proteomic changes linked to FAK activity. Second, we will test whether FAK binding to and phosphorylation of talin are key events in both FAK activation and in focal adhesion turnover needed for motility. These studies will involve real-time imaging of WT, KD, and GFP-FAK reconstituted FAK-null MEFs as well as biochemical analysis of a linkage involving talin, FAK, and Src. Third, we will test the hypothesis that FAK activity promotes directionality motility-polarity via p190A RhoGAP complex formation, tyrosine phosphorylation, and selective leading-edge inhibition of RhoGTPase activity through a connection between FAK and p120RasGAP. Together, these studies will provide important insights into the molecular mechanism of cell movement underlying processes such as tumor invasion.

Public Health Relevance

Regulated and controlled cell migration is important in development and wound healing whereas uncontrolled motility promotes immune diseases and tumor spread. Our studies are focused on understanding the molecular mechanisms governing how FAK activity differentially promotes cell motility and survival. These studies will fill key gaps in our understanding of the basic signaling events regulating cell movement and underlying processes such as tumor spread.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM087400-02
Application #
7857976
Study Section
Intercellular Interactions (ICI)
Program Officer
Flicker, Paula F
Project Start
2009-06-01
Project End
2013-05-31
Budget Start
2010-06-01
Budget End
2011-05-31
Support Year
2
Fiscal Year
2010
Total Cost
$315,087
Indirect Cost
Name
University of California San Diego
Department
Obstetrics & Gynecology
Type
Schools of Medicine
DUNS #
804355790
City
La Jolla
State
CA
Country
United States
Zip Code
92093
Ward, Kristy K; Tancioni, Isabelle; Lawson, Christine et al. (2013) Inhibition of focal adhesion kinase (FAK) activity prevents anchorage-independent ovarian carcinoma cell growth and tumor progression. Clin Exp Metastasis 30:579-94
Lim, Ssang-Taek; Miller, Nichol L G; Chen, Xiao Lei et al. (2012) Nuclear-localized focal adhesion kinase regulates inflammatory VCAM-1 expression. J Cell Biol 197:907-19
Miller, Nichol L G; Lawson, Christine; Chen, Xiao Lei et al. (2012) Rgnef (p190RhoGEF) knockout inhibits RhoA activity, focal adhesion establishment, and cell motility downstream of integrins. PLoS One 7:e37830
Lawson, Christine; Schlaepfer, David D (2012) Integrin adhesions: who's on first? What's on second? Connections between FAK and talin. Cell Adh Migr 6:302-6
Lawson, Christine; Lim, Ssang-Taek; Uryu, Sean et al. (2012) FAK promotes recruitment of talin to nascent adhesions to control cell motility. J Cell Biol 196:223-32
Chen, Xiao Lei; Nam, Ju-Ock; Jean, Christine et al. (2012) VEGF-induced vascular permeability is mediated by FAK. Dev Cell 22:146-57
Tomar, Alok; Lawson, Christine; Ghassemian, Majid et al. (2012) Cortactin as a target for FAK in the regulation of focal adhesion dynamics. PLoS One 7:e44041
Li, Xiao-Yan; Zhou, Xiaoming; Rowe, R Grant et al. (2011) Snail1 controls epithelial-mesenchymal lineage commitment in focal adhesion kinase-null embryonic cells. J Cell Biol 195:729-38
Seong, Jihye; Ouyang, Mingxing; Kim, Taejin et al. (2011) Detection of focal adhesion kinase activation at membrane microdomains by fluorescence resonance energy transfer. Nat Commun 2:406
Yu, Hong-Gang; Nam, Ju-Ock; Miller, Nichol L G et al. (2011) p190RhoGEF (Rgnef) promotes colon carcinoma tumor progression via interaction with focal adhesion kinase. Cancer Res 71:360-70

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