Christopher Ritchie1, Andrew Mack1, Logan Harper1, Ayna Alfadhli1, Philip J S Stork2, Xiaolin Nan3, Eric Barklis4. 1. Department of Molecular Microbiology and Immunology, Oregon Health & Science University, Portland, OR, U.S.A. 2. Department of Vollum Institute, Oregon Health & Science University, Portland, OR, U.S.A. 3. Department of Biomedical Engineering, Oregon Health & Science University, Portland, OR, U.S.A. 4. Department of Molecular Microbiology and Immunology, Oregon Health & Science University, Portland, OR, U.S.A. barklis@ohsu.edu.
Abstract
BACKGROUND: Mutations of the human K-Ras 4B (K-Ras) G protein are associated with a significant proportion of all human cancers. Despite this fact, a comprehensive analysis of K-Ras interactions is lacking. Our investigations focus on characterization of the K-Ras interaction network. MATERIALS AND METHODS: We employed a biotin ligase-tagging approach, in which tagged K-Ras proteins biotinylate neighbor proteins in a proximity-dependent fashion, and proteins are identified via mass spectrometry (MS) sequencing. RESULTS: In transfected cells, a total of 748 biotinylated proteins were identified from cells expressing biotin ligase-tagged K-Ras variants. Significant differences were observed between membrane-associated variants and a farnesylation-defective mutant. In pancreatic cancer cells, 56 K-Ras interaction partners were identified. Most of these were cytoskeletal or plasma membrane proteins, and many have been identified previously as potential cancer biomarkers. CONCLUSION: Biotin ligase tagging offers a rapid and convenient approach to the characterization of K-Ras interaction networks. Copyright
BACKGROUND: Mutations of the humanK-Ras 4B (K-Ras) G protein are associated with a significant proportion of all humancancers. Despite this fact, a comprehensive analysis of K-Ras interactions is lacking. Our investigations focus on characterization of the K-Ras interaction network. MATERIALS AND METHODS: We employed a biotin ligase-tagging approach, in which tagged K-Ras proteins biotinylate neighbor proteins in a proximity-dependent fashion, and proteins are identified via mass spectrometry (MS) sequencing. RESULTS: In transfected cells, a total of 748 biotinylated proteins were identified from cells expressing biotin ligase-tagged K-Ras variants. Significant differences were observed between membrane-associated variants and a farnesylation-defective mutant. In pancreatic cancer cells, 56 K-Ras interaction partners were identified. Most of these were cytoskeletal or plasma membrane proteins, and many have been identified previously as potential cancer biomarkers. CONCLUSION:Biotin ligase tagging offers a rapid and convenient approach to the characterization of K-Ras interaction networks. Copyright
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