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@2') levels by blocking the microsomal Ca2@ ATPase. At present, the consequence of this Ca@2@ increase and the nature of the tumorigenicity of thapsigargin still remain to be elucidated. Previ ously, we demonstrated that thapsigargin activates the mitogen-activated protein (MAP) kinase via Ca@2' but independently of protein kinase C or Ca2― Influx. Here, we show that thapsIgar@n 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 doednant-inifibitory 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. Sinai lady, 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 activa tion 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.