Refinement of the endogenous epitope tagging technology allows the identification of a novel NRAS binding partner in melanoma

• Published in: Pigment cell and melanoma research 2018-09, Vol.31 (5), p.641-648
• Main Authors: Alon, Michal, Emmanuel, Rafi, Qutob, Nouar, Bakhman, Anna, Peshti, Victoria, Brodezki, Alexandra, Bassan, David, Kosloff, Mickey, Samuels, Yardena
• Format: Article
• Language: English
• Subjects: Binding; Drug development; endogenous epitope tagging; Epitope Mapping - methods; Epitopes; Epitopes - genetics; Epitopes - metabolism; GTP Phosphohydrolases - genetics; GTP Phosphohydrolases - metabolism; Humans; Marking; Melanoma; Melanoma - genetics; Melanoma - metabolism; Melanoma - pathology; Membrane Proteins - genetics; Membrane Proteins - metabolism; mRNA; Mutation; NRAS; Polyadenylation; Protein Interaction Domains and Motifs; Proteins; Proteomics; Proteomics - methods; Proto-Oncogene Proteins c-cbl - genetics; Proto-Oncogene Proteins c-cbl - metabolism; Tumor Cells, Cultured; Ubiquitin; Ubiquitin-protein ligase
• ISSN: 1755-1471; 1755-148X
• DOI: 10.1111/pcmr.12705

Summary The NRAS oncoprotein is highly mutated in melanoma. However, to date, no comprehensive proteomic study has been reported for NRAS. Here, we utilized the endogenous epitope tagging (EET) approach for the identification of novel NRAS binding partners. Using EET, an epitope tag is added to the endogenously expressed protein, via modification of its genomic coding sequence. Existing EET systems are not robust, suffer from high background, and are labor‐intensive. To this end, we present a polyadenylation signal‐trap construct for N’‐tagging that generates a polycistronic mRNA with the gene of interest. This system requires the integration of the tagging cassette in frame with the target gene to be expressed. Using this design, we demonstrate, for the first time, endogenous tagging of NRAS in melanoma cells allowing the identification of the E3 ubiquitin ligase c‐CBL as a novel NRAS binding partner. Thus, our developed EET technology allows the characterization of new RAS effectors, which could be beneficial for the design of future drugs that inhibit constitutive signaling of RAS oncogenic mutants.