Identification of FAK binding partners using immunoprecipitation coupled with nano-LC/MSMS technique

Abstract

Identification of FAK binding partners using immunoprecipitation coupled with nano-LC/MSMS technique Binh Thanh Nguyen1,2, Byeongsu Kim1,3, Min-Jung Kang1,2,3* 1Molecular Recognition Research Center, Korea Institute of Science and Technology, Hwarangno 14-gil 5, Seongbuk-gu,Seoul, 02792, Republic of Korea. 2Division of Bio-Medical Science & Technology, Korea University of Science and Technology, Daejeon 305-350,Korea. 3Division of Bio-Medical Science & Technology, KIST School, Korea University of Science and Technology, Daejeon 305-350, Korea Author’s E-mail: 614007@kist.re.kr Correspondence to: mjkang1@kist.re.kr Abstract: Focal Adhesion Kinase (FAK) is a non-tyrosine kinase that binds to itself and cellular partners. In integrin-mediated cell adhesion, FAK is activated via disruption of an auto-inhibitory molecular interaction between its amino terminal domain and the central kinase domain. Multiple downstream signaling pathways are identified to mediate FAK regulation in various cancer cells. Protein-protein interaction (PPIs) are essential to almost every process in a cell, so understanding PPIs is crucial for understanding cell physiology in disease states. In this work, we are interested in studying the binding network of FAK in human colon cancer cell. HCT-116 was employed as a model system. High expression of FAK was found in HCT-116 compared to the other cancer cells using Western blot analysis. FAK binding partners were pulled down by immunoprecipitation method and separated by SDS-PAGE gel. Trypsin digestic peptides were injected to nano-LC/MSMS system and protein identification was searched against Mascot search engine. Among candidates, zyxin, nesprin, beta-1,4-N-acetylgalactosaminyltransferase 3 and glycogen synthase kinase-3 alpha show high potential as new binding partners of FAK Key words: Proteomics, Focal Adhesion Kinase, Immunoprecipitation, HCT116