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

Research output: Contribution to journalArticle

Abstract

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.

Original languageEnglish
Article number12908
JournalScientific reports
Volume9
Issue number1
DOIs
Publication statusPublished - 2019 Dec 1

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Zyxin
Focal Adhesion Protein-Tyrosine Kinases
Immunoprecipitation
Mass Spectrometry
Carrier Proteins
Colonic Neoplasms
Focal Adhesions
Proteins
Desmoplakins
HCT116 Cells
Proteomics
Trypsin
Small Interfering RNA
Cell Movement
Transfection
Polyacrylamide Gel Electrophoresis
Intercellular Signaling Peptides and Proteins
Therapeutics
Biomarkers
Western Blotting

All Science Journal Classification (ASJC) codes

  • General

Cite this

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title = "Identification of new binding proteins of focal adhesion kinase using immunoprecipitation and mass spectrometry",
abstract = "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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Identification of new binding proteins of focal adhesion kinase using immunoprecipitation and mass spectrometry. / Nguyen, Binh Thanh; Pyun, Jae Chul; Lee, Sang Guk; Kang, Min Jung.

In: Scientific reports, Vol. 9, No. 1, 12908, 01.12.2019.

Research output: Contribution to journalArticle

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AU - Nguyen, Binh Thanh

AU - Pyun, Jae Chul

AU - Lee, Sang Guk

AU - Kang, Min Jung

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