Intracellular signaling mechanisms leading to synergistic effects of endothelin-1 and stem cell factor on proliferation of cultured human melanocytes. Cross-talk via trans-activation of the tyrosine kinase c-kit receptor

J Biol Chem. 2000 Oct 27;275(43):33321-8. doi: 10.1074/jbc.M004346200.


We previously reported that activation of mitogen-activated protein kinase (MAPK) is involved in the mitogenic stimulation of normal human melanocytes (NHMC) by endothelin-1 (ET-1). In the present study, we determined signaling mechanisms upstream of MAPK activation that are involved in ET-1 stimulation and their synergism with stem cell factor (SCF). Pretreatment of cultured NHMC with ET(B) receptor antagonists, pertussis toxin, a specific phospholipase C inhibitor (), or a protein kinase C inhibitor (calphostine) blocked a transient tyrosine phosphorylation of MAPK induced by ET-1, whereas the addition of a calcium chelator (BAPTA) failed to inhibit that tyrosine phosphorylation of MAPK. Treatment with ET-1 and SCF together synergistically increased DNA synthesis, which was accompanied by synergism for MAPK phosphorylation. The time course of inositol 1,4,5-trisphosphate formation revealed that there is no difference in the level of inositol 1,4,5-trisphosphate stimulated by ET-1 + SCF or by ET-1 alone. Evaluations of the serine phosphorylation of MEK and Raf-1 activity showed a synergistic effect in SCF + ET-1-treated NHMC. Stimulation with SCF + ET-1 induced a more rapid and stronger tyrosyl phosphorylation of proteins corresponding to p52 and p66 Shc than did stimulation with SCF only, and this was accompanied by a stronger association of tyrosine-phosphorylated Shc with Grb2. Interestingly, a more rapid and marked tyrosine phosphorylation of c-kit was also detected in NHMC-treated with SCF + ET-1 than NHMC treated with SCF only. These data indicate that the synergistic cross-talk between SCF and ET-1 signaling is initiated through the pathway of tyrosine phosphorylation of c-kit, which results in the enhanced formation of the Shc-Grb(2) complex which leads in turn to the synergistic activation of the Ras/Raf-1/MEK/MAP kinase loop.

MeSH terms

  • Adaptor Proteins, Signal Transducing*
  • Adaptor Proteins, Vesicular Transport*
  • Cell Division / drug effects
  • Cells, Cultured
  • DNA / biosynthesis
  • Drug Synergism
  • Endothelin-1 / pharmacology*
  • GRB2 Adaptor Protein
  • Humans
  • Melanocytes / drug effects*
  • Melanocytes / physiology
  • Mitogen-Activated Protein Kinases / physiology
  • Protein Kinase C / physiology
  • Proteins / metabolism
  • Proto-Oncogene Proteins c-kit / physiology*
  • Proto-Oncogene Proteins c-raf / physiology
  • Shc Signaling Adaptor Proteins
  • Src Homology 2 Domain-Containing, Transforming Protein 1
  • Stem Cell Factor / pharmacology*
  • Transcriptional Activation*


  • Adaptor Proteins, Signal Transducing
  • Adaptor Proteins, Vesicular Transport
  • Endothelin-1
  • GRB2 Adaptor Protein
  • GRB2 protein, human
  • Proteins
  • SHC1 protein, human
  • Shc Signaling Adaptor Proteins
  • Src Homology 2 Domain-Containing, Transforming Protein 1
  • Stem Cell Factor
  • DNA
  • Proto-Oncogene Proteins c-kit
  • Proto-Oncogene Proteins c-raf
  • Protein Kinase C
  • Mitogen-Activated Protein Kinases