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.
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.
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