Analysis of RAS protein interactions in living cells reveals a mechanism for pan-RAS depletion by membrane-targeted RAS binders

Proc Natl Acad Sci U S A. 2020 Jun 2;117(22):12121-12130. doi: 10.1073/pnas.2000848117. Epub 2020 May 18.


HRAS, NRAS, and KRAS4A/KRAS4B comprise the RAS family of small GTPases that regulate signaling pathways controlling cell proliferation, differentiation, and survival. RAS pathway abnormalities cause developmental disorders and cancers. We found that KRAS4B colocalizes on the cell membrane with other RAS isoforms and a subset of prenylated small GTPase family members using a live-cell quantitative split luciferase complementation assay. RAS protein coclustering is mainly mediated by membrane association-facilitated interactions (MAFIs). Using the RAS-RBD (CRAF RAS binding domain) interaction as a model system, we showed that MAFI alone is not sufficient to induce RBD-mediated RAS inhibition. Surprisingly, we discovered that high-affinity membrane-targeted RAS binding proteins inhibit RAS activity and deplete RAS proteins through an autophagosome-lysosome-mediated degradation pathway. Our results provide a mechanism for regulating RAS activity and protein levels, a more detailed understanding of which should lead to therapeutic strategies for inhibiting and depleting oncogenic RAS proteins.

Keywords: HRAS; KRAS; NRAS; protein–protein interaction; split-luciferase complementation.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Autophagosomes / metabolism*
  • Cell Membrane / metabolism*
  • Humans
  • Lysosomes / metabolism*
  • Models, Molecular
  • Protein Interaction Domains and Motifs
  • Protein Isoforms
  • Signal Transduction
  • ras Proteins / metabolism*


  • Protein Isoforms
  • ras Proteins