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.
Bibliographical noteFunding Information:
EU-H2020-ERC-POC, Grant/Award Number: 712730; ERC, Grant/Award Number: StG-335377; The Knell Family; Melanoma Research Alliance; The Hamburger Family; Israel Science Foundation, Grant/Award Number: 712811; Henry Chanoch Krenter Institute for Biomedical Imaging and Genomics; Estate of Alice Schwarz-Gardos; Estate of John Hunter; Peter and Patricia Gruber Award
Y.S. is supported by the Israel Science Foundation grant number 696/17. This project has received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (grant agreement No 754282), the ERC (StG-335377), the MRA, the Henry Chanoch Krenter Institute for Biomedical Imaging and Genomics, the Estate of Alice Schwarz-Gardos, the Estate of John Hunter, the Knell Family, the Peter and Patricia Gruber Award, and the Hamburger Family. M.K. is supported by an Israel Science Foundation grant number 1454/13.
© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
- endogenous epitope tagging
ASJC Scopus subject areas
- Biochemistry, Genetics and Molecular Biology (all)