Clustered, Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9-coupled Affinity Purification/Mass Spectrometry Analysis Revealed a Novel Role of Neurofibromin in mTOR Signaling.
Mol Cell Proteomics
; 16(4): 594-607, 2017 04.
Article
em En
| MEDLINE
| ID: mdl-28174230
ABSTRACT
Neurofibromin (NF1) is a well known tumor suppressor that is commonly mutated in cancer patients. It physically interacts with RAS and negatively regulates RAS GTPase activity. Despite the importance of NF1 in cancer, a high quality endogenous NF1 interactome has yet to be established. In this study, we combined clustered, regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated gene knock-out technology with affinity purification using antibodies against endogenous proteins, followed by mass spectrometry analysis, to sensitively and accurately detect NF1 protein-protein interactions in unaltered in vivo settings. Using this system, we analyzed endogenous NF1-associated protein complexes and identified 49 high-confidence candidate interaction proteins, including RAS and other functionally relevant proteins. Through functional validation, we found that NF1 negatively regulates mechanistic target of rapamycin signaling (mTOR) in a LAMTOR1-dependent manner. In addition, the cell growth and survival of NF1-deficient cells have become dependent on hyperactivation of the mTOR pathway, and the tumorigenic properties of these cells have become dependent on LAMTOR1. Taken together, our findings may provide novel insights into therapeutic approaches targeting NF1-deficient tumors.
Texto completo:
1
Bases de dados:
MEDLINE
Assunto principal:
Proteínas de Transporte
/
Neurofibromina 1
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Proteômica
/
Serina-Treonina Quinases TOR
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Edição de Genes
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Neoplasias
Tipo de estudo:
Prognostic_studies
Limite:
Humans
Idioma:
En
Revista:
Mol Cell Proteomics
Assunto da revista:
BIOLOGIA MOLECULAR
/
BIOQUIMICA
Ano de publicação:
2017
Tipo de documento:
Article