Date of Graduation

2017

Document Type

Dissertation

Degree Type

PhD

College

School of Medicine

Committee Chair

Michael D Schaller

Committee Co-Chair

Steven M Frisch

Committee Member

Visvanathan Ramamurthy

Committee Member

David P Siderovski

Committee Member

David M Smith

Abstract

Focal adhesion kinase is an essential nonreceptor tyrosine kinase that plays an important role in development, in homeostasis and in the progression of human disease. Multiple stimuli activate FAK, which requires a change in structure from an autoinhibited to activated conformation. In the autoinhibited conformation the FERM domain associates with the catalytic domain of FAK and PI(4,5)P2 binding to the FERM domain plays a role in the release of autoinhibition, activating the enzyme. This work aims to better characterize the residues involved in the release of the autoinhibitory conformation. The first aim was to characterize the effects of point mutations found in the COSMIC database. None of these mutations had an impact on the FAK autoinhibited conformation, thus classifying FAK as a kinase in which overexpression, and not mutation, is the mechanism by which its activity is increased in cancer. The second aim was to assess the role of FERM domain basic residues at the interface of the FERM and kinase domains in the autoinhibited conformation, R184, K190, and K191. While mutation of these residues does not cause a significant shift in FAK conformation, the results do suggest a minor role in binding to the membrane through PI(4,5)P2 and interacting with the kinase domain to maintain the autoinhibited conformation. The third aim was to characterize residues on the catalytic domain that were modeled binding with the membrane in silico. Constructs with these residues mutated to alanine exhibited defects in phosphorylation and failed to completely rescue the phenotype associated with fak -/- phenotype fibroblasts demonstrating the importance of these residues in FAK function. The catalytic domain of FAK exhibited PI(4,5)P2 binding in vitro and binding activity was lost upon mutation of putative PI(4,5)P2 binding site basic residues. Collectively, these studies further characterize the structural elements that aid in maintenance of both the open and autoinhibited conformations of FAK.

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