We have shown previously that overexpression of the e isoform of protein kinase C (PKCe) in rat colonic epithelial cells causes malignant transformation, possibly by interacting with the ras signal transduction pathway (Oncogene 12: 847, 1996). We have now performed experiments to examine certain early steps in the ras signaling pathway. A marked increase of Raf-1 phosphorylation was detected in tumorigenic ras-transformed D/ras as well as in D/e cells (overexpressing PKCe), compared to the nontumorigenic D/WT parental line. Moreover, in the PKCe-transformed D/e cell line, stable transfection with a dominant-negative raf-1 (DNraf) sequence caused complete regression of the neoplastic phenotype. These results suggested that PKCe-induced transformation was associated with increased Raf-1 activation, and that DNraf could block the oncogenic e ect of PKCe. Furthermore, transfection of D/WT cells with dominant-negative ras induced arrest of cell growth, and subsequent transfection with PKCe cDNA enhanced cell proliferation and induced neoplastic transformation. These results suggest that ras acts upstream of PKCe, and that overexpression of PKCe circumvents the block in cell proliferation caused by dominant-negative ras. We conclude that PKCe exerts its oncogenic activity in rat colonic cells by a ecting the ras signaling cascade at the level of Raf-1 activation.Keywords: PKCe; Raf-1; colon cancer; oncogeneWe have demonstrated that the epsilon isoform of protein kinase C (PKCe) is oncogenic when overexpressed in rat colonic epithelial cells . In the previous report, we analysed the expression of several PKC isoforms in a nontumorigenic rat colonic epithelial cell line FRC/TEX CLD (D/WT), and in a derivative tumorigenic Ha-rastransformed FRC/TEX CLD/H-ras line (D/ras) (Pories et al., 1993). PKCe was markedly increased in D/ras cells when compared to the non-neoplastic D/ WT line. To assess whether overexpression of PKCe was involved in the transformed phenotype, we stably transfected D/WT cells with a PKCe cDNA construct. Overexpression of PKCe induced complete in vitro and in vivo neoplastic transformation.Because PKCe expression was found to be altered in D/ras cells, our ®ndings suggested that PKCe might exert its oncogenic activity by interacting with the ras signal transduction pathway. In particular, although it has been shown that p21ras can recruit Raf-1 kinase to the plasma membrane, this interaction is not su cient for complete Raf-1 activation, which occurs via phosphorylation of speci®c serine residues by a Raf kinase-kinase (Daum et al., 1994). Several reports have suggested that PKCe and PKCa are likely candidates for this kinase in mammalian cells (reviewed in: Perletti and Monti, 1996;Daum et al., 1994;Malarkey et al., 1995). We, therefore, focused new experiments on the role of PKCe in ras signal transduction, at the level of Raf-1 activation.Based on the ®ndings described above, we hypothesize that PKCe interacts with the ras signaling pathway, likely by activating Raf-1.
Raf-1 phosphorylation is in...
