Integrin contacts with extracellular matrix play fundamental roles in vascular biology by promoting cell- adhesion, controlling cell motility, and regulating cell survival. As transmembrane integrin receptors do not possess intrinsic catalytic activity, signals generated by integrins must be mediated by associated proteins. Focal adhesion kinase (FAK) is a cytoplasmic tyrosine kinase that associates with and is activated by integrins. FAK also acts downstream of growth factor receptors such as VEGFr to promote endothelial cell (EC) motility and survival during the processes of angiogenesis. FAK function is essential for mouse EC cell biology as complete or conditional EC-specific FAK knockouts yield embryonic lethal phenotypes with defects in blood vessel morphogenesis. 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. Here, we will build upon a novel role for FAK in its ability to become nuclear-localized and function as a negative regulator of the p53 tumor suppressor under conditions of cellular stress. We found that FAK inactivates p53 in a kinase-independent manner through N-terminal FAK FERM (band 4.1, ezrin, radixin, moesin homology) domain-mediated nuclear translocation and enhancement of Mdm2-dependent p53 ubiquitination using primary mouse and human fibroblasts. We propose that FAK FERM nuclear-association promotes cell survival by keeping p53 levels low. As we find that FAK localizes to both integrin-associated signaling sites and to the nucleus of ECs, this proposal will test the overall hypothesis that FAK controls EC survival and motility- morphogenesis through differential kinase-independent (FERM-nuclear) and kinase-dependent (FAK-integrin) mechanisms, respectively.
Aim -1 will use lentiviral-mediated FAK shRNA knockdown and gain-of-function FAK re-expression studies to test whether nuclear FAK promotes EC survival in a kinase-independent manner and whether integrin-associated FAK activation is required to promote cell motility.
Aim -2 will determine the role of FAK signaling during developmental vasculogenesis-angiogenesis through the analysis of FAK knock-in mice and in vitro gain-of-function reconstitution studies using FAK-/- mouse embryonic stem cells stimulated to differentiate into ECs.
Aim -3 will use an inducible and conditional EC-specific FAK knockout in adult mice or pharmacological inhibition of FAK activity to determine the role of FAK in growth factor-stimulated angiogenesis. All three aims are also designed to differentiate the signaling differences between loss of FAK expression or inactivation of FAK catalytic activity in ECs. The results of these studies will provide important molecular insights for designing therapeutic agents for treatment of cardiovascular diseases.
In anti-cancer research, specific targeted anti-angiogenic therapeutic strategies are being adopted and used in combination with conventional anti-proliferative chemotherapeutic and radiotheraputic anti-tumor regimens. Our studies will provide important and novel insights into FAK function in controlling endothelial cell motility and survival. The results of these studies may serve as proof-of-principal for the future development of pharmacological inhibitors to FAK as anti-tumor and anti-angiogenic therapies.
