title: Mapping the interactome of overexpressed RAF kinase inhibitor protein in a gastric cancer cell line.
abstract: BACKGROUND
  Gastric cancer (GC) is a threat to human health with increasing incidence and mortality worldwide. Down-regulation or absence of RAF kinase inhibitor protein (RKIP) was associated with the occurrence, differentiation, invasion, and metastasis of GC. This study aims to investigate the molecular mechanisms and biological functions of RKIP in the GC biology.
  
  
  METHODS
  The fusion expression plasmid pcDNA3.1-RKIP-3xFLAG was transfected into SGC7901 cells, the RKIP fusion proteins were purified with anti-flag M2 magnetic beads, and the RKIP-interacting proteins were identified with tandem mass spectrometry (MS/MS), and were analyzed with bioinformatics tools. Western blot and co-immunoprecipitation were used to confirm the interaction complex.
  
  
  RESULTS
  A total of 72 RKIP-interacting proteins were identified by MS/MS. Those proteins play roles in enzyme metabolism, molecular chaperoning, biological oxidation, cytoskeleton organization, signal transduction, and enzymolysis. Three RKIP-interaction protein network diagrams were constructed with Michigan Molecular Interactions, functional linage network, and Predictome analysis to address the molecular pathways of the functional activity of RKIP. The MS/MS-characterized components of the existing interaction complex (RKIP, HSP90, 14-3-3ε, and keratin 8) were confirmed by Western blot analysis and co-immunoprecipitation.
  
  
  CONCLUSIONS
  This study is the first discovery of the interaction of RKIP with HSP90, 14-3-3, and keratin. The present data would provide insight into the molecular mechanisms of how RKIP inhibits the occurrence and development of GC.
authors.forename: Huan
authors.initials: H
authors.lastname: Gu
authors.affiliation: Department of Gastroenterology, Xiangya Hospital, Central South University, Changsha, Hunan 410008, China. yjzhan2011@gmail.com.
authors.forename: Xianquan
authors.initials: X
authors.lastname: Zhan
authors.forename: Guiying
authors.initials: G
authors.lastname: Zhang
authors.forename: Lu
authors.initials: L
authors.lastname: Yan
authors.forename: William Cs
authors.initials: WC
authors.lastname: Cho
authors.forename: Maoyu
authors.initials: M
authors.lastname: Li
authors.forename: Ting
authors.initials: T
authors.lastname: Liu
authors.forename: Zhuchu
authors.initials: Z
authors.lastname: Chen
mesh_terms: D002352:Carrier Proteins / Q000378:metabolism; D045744:Cell Line, Tumor; D015870:Gene Expression; D006801:Humans; D049488:Phosphatidylethanolamine Binding Protein / Q000235:genetics* / Q000378:metabolism*; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods; D060066:Protein Interaction Maps; D015203:Reproducibility of Results; D013274:Stomach Neoplasms / Q000235:genetics* / Q000378:metabolism*; D014162:Transfection
chemical_list: D002352:Carrier Proteins; C511482:PEBP1 protein, human; D049488:Phosphatidylethanolamine Binding Protein