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Identification of new binding proteins of focal adhesion kinase using immunoprecipitation and mass spectrometry

 Binh Thanh Nguyen  ;  Jae-Chul Pyun  ;  Sang-Guk Lee  ;  Min-Jung Kang 
 Scientific Reports, Vol.9(1) : 12908, 2019 
Journal Title
 Scientific Reports 
Issue Date
Focal adhesion kinase (FAK) is a 125 kDa protein recruited as a participant in focal adhesion dynamics and serves as a signaling scaffold for the assembly and subsequent maturation of focal contact. Identification of new FAK binding proteins could reveal potential signaling targets and contribute to further development of therapeutic drugs in the treatment of colon cancer. Here, we applied a functional proteomic strategy to identify proteins that interact with FAK in human colon cancer cell line HCT-116. Proteins were targeted by coimmunoprecipitation with an anti-FAK antibody and resolved on 1D-SDS-PAGE. The gel was excised, reduced, alkylated, and trypsin digested. Tryptic peptides were separated by nano-LC-MS/MS by an LTQ-Orbitrap-Velos spectrometer. We identified 101 proteins in the immunocomplex under epithelial growth factor (EGF) stimulation. Three proteins, zyxin, nesprin-1, and desmoplakin, were discovered and validated using reciprocal immunoprecipitation and Western blot analysis. Then, we sought to study the biological relevance of these proteins by siRNA transfection of HCT-116 cells. According to the results, zyxin might play a central role as an upstream regulator to mediate critical cancer-related signaling pathways. Zyxin and nesprin-1 depletion significantly impaired cell migration and invasion capabilities. Additionally, we performed ELISA assays on serum samples from patients with colon cancer instead of cell models to quantify the protein levels of zyxin and nesprin-1. Our results suggested that zyxin and nesprin-1 are not only promising therapeutic targets but also potential diagnostic biomarkers for colon cancer.
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1. College of Medicine (의과대학) > Dept. of Laboratory Medicine (진단검사의학교실) > 1. Journal Papers
Yonsei Authors
Lee, Sang-Guk(이상국) ORCID logo https://orcid.org/0000-0003-3862-3660
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