Clustered, Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9-coupled Affinity Purification/Mass Spectrometry Analysis Revealed a Novel Role of Neurofibromin in mTOR Signaling.

Academic Article


  • 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.
  • Authors

    Published In


  • CRISPR-Cas Systems, Carrier Proteins, Cell Proliferation, Cell Survival, Gene Editing, HEK293 Cells, HeLa Cells, Humans, Intracellular Signaling Peptides and Proteins, Mass Spectrometry, Neoplasms, Neurofibromin 1, Protein Interaction Maps, Proteomics, Signal Transduction, TOR Serine-Threonine Kinases
  • Digital Object Identifier (doi)

    Pubmed Id

  • 16056214
  • Author List

  • Li X; Gao M; Choi JM; Kim B-J; Zhou M-T; Chen Z; Jain AN; Jung SY; Yuan J; Wang W
  • Start Page

  • 594
  • End Page

  • 607
  • Volume

  • 16
  • Issue

  • 4