Ras recruits Raf-1 to the plasma membrane for activation by tyrosine phosphorylation

EMBO J. 1995 Jul 3;14(13):3136-45.

Abstract

A central feature of signal transduction downstream of both receptor and oncogenic tyrosine kinases is the Ras-dependent activation of a protein kinase cascade consisting of Raf-1, Mek (MAP kinase kinase) and ERKs (MAP kinases). To study the role of tyrosine kinase activity in the activation of Raf-1, we have examined the properties of p74Raf-1 and oncogenic Src that are necessary for activation of p74Raf-1. We show that in mammalian cells activation of p74Raf-1 by oncogenic Src requires pp60Src to be myristoylated and the ability of p74Raf-1 to interact with p21Ras-GTP. The Ras/Raf interaction is required for p21Ras-GTP to bring p74Raf-1 to the plasma membrane for phosphorylation at tyrosine 340 or 341, probably by membrane-bound pp60Src. When oncogenic Src is expressed with Raf-1, p74Raf-1 is activated 5-fold; however, when co-expressed with oncogenic Ras and Src, Raf-1 is activated 25-fold and this is associated with a further 3-fold increase in tyrosine phosphorylation. Thus, p21Ras-GTP is the limiting component in bringing p74Raf-1 to the plasma membrane for tyrosine phosphorylation. Using mutants of Raf-1 at Tyr340/341, we show that in addition to tyrosine phosphorylation at these sites, there is an additional activation step resulting from p21Ras-GTP recruiting p74Raf-1 to the plasma membrane. Thus, the role of Ras in Raf-1 activation is to bring p74Raf-1 to the plasma membrane for at least two different activation steps.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 3T3 Cells
  • Amino Acid Sequence
  • Animals
  • Binding Sites
  • Cell Membrane / metabolism
  • Cells, Cultured
  • Fibroblasts / metabolism
  • Fibroblasts / ultrastructure*
  • GTPase-Activating Proteins
  • Humans
  • Mice
  • Models, Biological
  • Molecular Sequence Data
  • Phosphorylation
  • Protein-Serine-Threonine Kinases / metabolism*
  • Protein-Tyrosine Kinases / metabolism
  • Proteins / metabolism*
  • Proto-Oncogene Proteins / metabolism*
  • Proto-Oncogene Proteins c-raf
  • Proto-Oncogene Proteins pp60(c-src) / metabolism
  • Signal Transduction*
  • Tyrosine / metabolism*
  • ras GTPase-Activating Proteins
  • ras Proteins / metabolism

Substances

  • GTPase-Activating Proteins
  • Proteins
  • Proto-Oncogene Proteins
  • ras GTPase-Activating Proteins
  • Tyrosine
  • Protein-Tyrosine Kinases
  • Proto-Oncogene Proteins pp60(c-src)
  • Protein-Serine-Threonine Kinases
  • Proto-Oncogene Proteins c-raf
  • ras Proteins