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

Michal Alon, Rafi Emmanuel, Nouar Qutob, Anna Bakhman, Victoria Peshti, Alexandra Brodezki, David Bassan, Mickey Kosloff, Yardena Samuels

Research output: Contribution to journalArticlepeer-review

Abstract

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.

Original languageEnglish
Pages (from-to)641-648
Number of pages8
JournalPigment Cell and Melanoma Research
Volume31
Issue number5
DOIs
StatePublished - Sep 2018

Bibliographical note

Publisher Copyright:
© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

Keywords

  • NRAS
  • endogenous epitope tagging
  • melanoma

ASJC Scopus subject areas

  • General Biochemistry, Genetics and Molecular Biology
  • Dermatology
  • Oncology

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