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Refinement of the endogenous epitope tagging technology allows the identification of a novel NRAS binding partner in melanoma
Author(s) -
Alon Michal,
Emmanuel Rafi,
Qutob Nouar,
Bakhman Anna,
Peshti Victoria,
Brodezki Alexandra,
Bassan David,
Kosloff Mickey,
Samuels Yardena
Publication year - 2018
Publication title -
pigment cell and melanoma research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.618
H-Index - 105
eISSN - 1755-148X
pISSN - 1755-1471
DOI - 10.1111/pcmr.12705
Subject(s) - neuroblastoma ras viral oncogene homolog , epitope , ubiquitin ligase , computational biology , biology , endogeny , mutant , effector , gene , mutation , ubiquitin , genetics , microbiology and biotechnology , kras , antibody , biochemistry
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.