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

Michal AlonRafi EmmanuelNouar QutobAnna BakhmanVictoria PeshtiAlexandra BrodezkiDavid BassanMickey KosloffYardena Sameuls
Published in: Pigment cell & melanoma research (2018)
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.
Keyphrases
  • wild type
  • copy number
  • binding protein
  • genome wide
  • monoclonal antibody
  • dna binding
  • human immunodeficiency virus
  • dna methylation
  • transcription factor
  • basal cell carcinoma
  • hiv infected