Differential Raf requirement for activation of mitogen-activated protein kinase by growth factors, phorbol esters, and calcium

J Biol Chem. 1994 Mar 11;269(10):7337-41.


Although a pathway that requires sequential activation of Ras, Raf, and MAP kinase kinase has been proposed as the major mechanism for stimulation of mitogen-activated protein kinase (MAP kinase), alternative pathways also exist. A wide variety of extracellular stimuli have been shown to activate MAP kinase; however, the precise mechanisms by which these stimuli mediate the signaling events have not been elucidated. Using a Balb/c-derived cell line expressing a dominant-negative mutant of Raf, we determined whether Raf is required for the activation of MAP kinase by growth factors, phorbol esters, and calcium. Insulin-like growth factor I (IGF-I), epidermal growth factor (EGF), and phorbol 12,13-dibutyrate activated Ras in both mutant and control cells. However, stimulation of MAP kinase by IGF-I was nearly abolished in the dominant-negative Raf mutant. Stimulation of MAP kinase by the Ca2+ mobilizer thapsigargin was also inhibited in the presence of the Raf mutant. In contrast, EGF and phorbol 12,13-dibutyrate remained potent stimulators of MAP kinase in the dominant-negative Raf cells. The activation of MAP kinase by these stimuli can be further distinguished by differential requirements for Ca2+ and protein kinase C. These results suggest that Raf is required for the activation of MAP kinase by IGF-I and calcium, whereas EGF and possibly phorbol esters may employ alternative Raf-independent pathways for MAP kinase activation.

Publication types

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

MeSH terms

  • 3T3 Cells
  • Animals
  • Calcium / pharmacology*
  • Down-Regulation
  • Enzyme Activation
  • Growth Substances / pharmacology*
  • Mice
  • Mice, Inbred BALB C
  • Mitogen-Activated Protein Kinase 1
  • Phorbol 12,13-Dibutyrate / pharmacology*
  • Protein Kinase C / metabolism
  • Protein Serine-Threonine Kinases / drug effects
  • Protein Serine-Threonine Kinases / metabolism*
  • Protein-Tyrosine Kinases / drug effects
  • Protein-Tyrosine Kinases / metabolism*
  • Proto-Oncogene Proteins / metabolism*
  • Proto-Oncogene Proteins c-raf
  • Terpenes / pharmacology
  • Thapsigargin


  • Growth Substances
  • Proto-Oncogene Proteins
  • Terpenes
  • Phorbol 12,13-Dibutyrate
  • Thapsigargin
  • Protein-Tyrosine Kinases
  • Protein Serine-Threonine Kinases
  • Proto-Oncogene Proteins c-raf
  • Protein Kinase C
  • Mitogen-Activated Protein Kinase 1
  • Calcium