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 Ca 2+ ATPase, At present, the consequence of this Ca-i(2+) increase and t he nature of the tumorigenicity of thapsigargin still remain to be elu cidated. Previously, we demonstrated that thapsigargin activates the m itogen-activated protein (MAP) kinase via Ca-i(2+) but independently o f protein kinase C or Ca2+ influx, Here, we show that thapsigargin als o rapidly stimulates the Src tyrosine kinase, Transfection of a v-Src gene into a hippocampal cell line (H19-7) renders a constitutive activ ation of MAP kinase, whereas transfection of a kinase-deficient Src mu tant blocks the activation by thapsigargin, suggesting that Src is req uired for the thapsigargin-induced MAP kinase activation, Cotransfecti on 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-depend ent activation of MAP kinase, Similarly, in the LA-90 cells, expressio n 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 o r thapsigargin treatment. Together, these results suggest that thapsig argin stimulates MAP kinase signaling via Src and Raf-1. The activatio n of this Src-MAP kinase pathway suggests a biochemical mechanism for the tumorigenic nature of thapsigargin.