Structural and functional analyses of a germline KRAS T50I mutation provide insights into Raf activation

JCI Insight. 2023 Sep 8;8(17):e168445. doi: 10.1172/jci.insight.168445.

Abstract

A T50I substitution in the K-Ras interswitch domain causes Noonan syndrome and emerged as a third-site mutation that restored the in vivo transforming activity and constitutive MAPK pathway activation by an attenuated KrasG12D,E37G oncogene in a mouse leukemia model. Biochemical and crystallographic data suggested that K-RasT50I increases MAPK signal output through a non-GTPase mechanism, potentially by promoting asymmetric Ras:Ras interactions between T50 and E162. We generated a "switchable" system in which K-Ras mutant proteins expressed at physiologic levels supplant the fms like tyrosine kinase 3 (FLT3) dependency of MOLM-13 leukemia cells lacking endogenous KRAS and used this system to interrogate single or compound G12D, T50I, D154Q, and E162L mutations. These studies support a key role for the asymmetric lateral assembly of K-Ras in a plasma membrane-distal orientation that promotes the formation of active Ras:Raf complexes in a membrane-proximal conformation. Disease-causing mutations such as T50I are a valuable starting point for illuminating normal Ras function, elucidating mechanisms of disease, and identifying potential therapeutic opportunities for Rasopathy disorders and cancer.

Keywords: Leukemias; Oncogenes; Oncology.

Publication types

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

MeSH terms

  • Animals
  • Disease Models, Animal
  • Germ Cells
  • Germ-Line Mutation
  • Leukemia*
  • Mice
  • Mutation
  • Proto-Oncogene Proteins p21(ras)* / genetics
  • ras Proteins

Substances

  • Proto-Oncogene Proteins p21(ras)
  • ras Proteins
  • Hras protein, mouse