Src tyrosine kinase mediates stimulation of Raf-1 and mitogen-activated protein kinase by the tumor promoter thapsigargin

Cancer Res. 1997 Aug 1;57(15):3168-73.

Abstract

Thapsigargin is a non-phorbol ester-type tumor promoter that elevates the intracellular Ca2+ (Ca(i)2+) levels by blocking the microsomal Ca2+ ATPase. At present, the consequence of this Ca(i)2+ increase and the nature of the tumorigenicity of thapsigargin still remain to be elucidated. Previously, we demonstrated that thapsigargin activates the mitogen-activated protein (MAP) kinase via Ca(i)2+ but independently of protein kinase C or Ca2+ influx. Here, we show that thapsigargin also rapidly stimulates the Src tyrosine kinase. Transfection of a v-Src gene into a hippocampal cell line (H19-7) renders a constitutive activation of MAP kinase, whereas transfection of a kinase-deficient Src mutant blocks the activation by thapsigargin, suggesting that Src is required for the thapsigargin-induced MAP kinase activation. Cotransfection of a dominant-inhibitory Raf-1 and the v-Src genes into H19-7 cells results in an inhibition of the otherwise constitutively elevated MAP kinase activity, suggesting that Raf-1 is required for the Src-dependent activation of MAP kinase. Similarly, in the LA-90 cells, expression of a temperature-sensitive allele of v-Src constitutively activates Raf-1 and MAP kinase, whereas expression of a dominant-inhibitory Raf-1 mutant abolishes the MAP kinase activation induced by either v-Src or thapsigargin treatment. Together, these results suggest that thapsigargin stimulates MAP kinase signaling via Src and Raf-1. The activation of this Src-MAP kinase pathway suggests a biochemical mechanism for the tumorigenic nature of thapsigargin.

Publication types

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

MeSH terms

  • Animals
  • Brain / drug effects
  • Brain / metabolism
  • Calcium-Calmodulin-Dependent Protein Kinases / metabolism*
  • Carcinogens / pharmacology
  • Cells, Cultured
  • Mutation
  • Protein-Serine-Threonine Kinases / genetics
  • Protein-Serine-Threonine Kinases / metabolism*
  • Proto-Oncogene Proteins / genetics
  • Proto-Oncogene Proteins / metabolism*
  • Proto-Oncogene Proteins c-raf
  • Proto-Oncogene Proteins pp60(c-src) / genetics
  • Proto-Oncogene Proteins pp60(c-src) / metabolism*
  • Rats
  • Thapsigargin / pharmacology*
  • Transfection

Substances

  • Carcinogens
  • Proto-Oncogene Proteins
  • Thapsigargin
  • Proto-Oncogene Proteins pp60(c-src)
  • Protein-Serine-Threonine Kinases
  • Proto-Oncogene Proteins c-raf
  • Calcium-Calmodulin-Dependent Protein Kinases