Analysis of K-Ras interactions by biotin ligase tagging

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 proximitydependent 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.