The recruitment of Raf-1 to membranes is mediated by direct interaction with phosphatidic acid and is independent of association with Ras

J Biol Chem. 2000 Aug 4;275(31):23911-8. doi: 10.1074/jbc.M001553200.


The serine/threonine kinase Raf-1 is an essential component of the MAPK cascade. Activation of Raf-1 by extracellular signals is initiated by association with intracellular membranes. Recruitment of Raf-1 to membranes has been reported to be mediated by direct association with Ras and by the phospholipase D product phosphatidic acid (PA). Here we report that insulin stimulation of HIRcB fibroblasts leads to accumulation of Ras, Raf-1, phosphorylated MEK, phosphorylated MAPK, and PA on endosomal membranes. Mutations that disrupt Raf-PA interactions prevented recruitment of Raf-1 to membranes, whereas disruption of Ras-Raf interactions did not affect agonist-dependent translocation. Expression of a dominant-negative Ras mutant did not prevent insulin-dependent Raf-1 translocation, but inhibited phosphorylation of MAPK. Finally, the PA-binding region of Raf-1 was sufficient to target green fluorescent protein to membranes, and its overexpression blocked recruitment of Raf-1 to membranes and disrupted insulin-dependent MAPK phosphorylation. These results indicate that agonist-dependent Raf-1 translocation is primarily mediated by a direct interaction with PA and is independent of association with Ras.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Biological Transport
  • Endosomes / metabolism
  • Humans
  • Insulin / pharmacology
  • Intracellular Membranes / metabolism*
  • Mitogen-Activated Protein Kinases / metabolism
  • Models, Biological
  • Phosphatidic Acids / metabolism*
  • Phosphorylation
  • Protein Binding
  • Proto-Oncogene Proteins c-raf / metabolism*
  • Rats
  • Receptor, Insulin / agonists
  • Receptor, Insulin / genetics
  • Recombinant Fusion Proteins / metabolism
  • Signal Transduction
  • ras Proteins / genetics
  • ras Proteins / metabolism


  • Insulin
  • Phosphatidic Acids
  • Recombinant Fusion Proteins
  • Receptor, Insulin
  • Proto-Oncogene Proteins c-raf
  • Mitogen-Activated Protein Kinases
  • ras Proteins