Investigation of phosphorylation and ligand binding of the focal adhesion targeting domain of focal adhesion kinase Public Deposited

Downloadable Content

Download PDF
Last Modified
  • March 20, 2019
  • Cable, Jennifer L.
    • Affiliation: School of Medicine, Department of Biochemistry and Biophysics
  • Focal adhesion kinase (FAK) is a nonreceptor tyrosine kinase that localizes to focal adhesions upon integrin activation. FAK plays a key role in cell migration, cell cycle progression, and apoptosis. Because of its role in these processes, it is not surprising that FAK is also involved in tumor progression and metastasis. Overexpression of FAK often correlates with increased metastasis. The C-terminal focal adhesion targeting (FAT) domain of FAK is required for proper localization and subsequent activation of FAK. Phosphorylation of the FAT domain at Y926 by Src kinase is believed to delocalize FAK from focal adhesions and promote cell migration, angiogenesis, and tumor metastasis. Because of its role in such important processes, phosphorylation at Y926 is likely to be tightly regulated. Because the inherent conformation of Y926 is not favorable for Src recognition, phosphorylation of Y926 is thought to be regulated by changes in the conformation or dynamics of the region surrounding Y926. However, what regulates these conformational changes is unknown. In this study, we provide evidence that Src-mediated phosphorylation of Y926 is sensitive to pH in vitro and reveal a second site of Src-mediated phosphorylation in the FAT domain: Y1008. NMR studies of the FAT domain reveal pH-dependent changes in backbone dynamics in regions shown to be important for phosphorylation. In addition to understanding how phosphorylation is regulated, we also were interested in investigating how phosphorylation itself affects the characteristics of the FAT domain. To address this issue, we characterized several Y926 mutants of the FAT domain and determined that perturbation of Y926 affects paxillin binding. Therefore, it is likely that phosphorylation is incompatible with paxillin binding, which supports the hypothesis that phosphorylation delocalizes FAK from focal adhesions. Finally, we have investigated a possible interaction between the FAT domain and the protein talin. This interaction has been proposed as a secondary mechanism by which the FAT domain localizes FAK to focal adhesions. However, we were unable to detect an interaction under our conditions.
Date of publication
Resource type
Rights statement
  • In Copyright
  • "... in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Biochemistry and Biophysics."
  • Campbell, Sharon
Place of publication
  • Chapel Hill, NC
  • Open access

This work has no parents.