Src tyrosine kinase mediates stimulation of Raf-1 and mitogen-activated protein kinase by the tumor promoter thapsigargin.
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.
Chao
T S
TS
The Ben May Institute for Cancer Research and Department of Pharmacological and Physiological Sciences, University of Chicago, Illinois 60637, USA.
Abe
M
M
Hershenson
M B
MB
Gomes
I
I
Rosner
M R
MR
D000818:Animals; D001921:Brain / Q000187:drug effects / Q000378:metabolism; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002273:Carcinogens / Q000494:pharmacology; D002478:Cells, Cultured; D009154:Mutation; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D019284:Thapsigargin / Q000494:pharmacology*; D014162:Transfection
D002273:Carcinogens; D011518:Proto-Oncogene Proteins; D019284:Thapsigargin; D016392:Proto-Oncogene Proteins pp60(c-src); D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D017871:Calcium-Calmodulin-Dependent Protein Kinases