We have analysed the expression of three calciumindependent isoforms of protein kinase C (PKC), PKCd, PKCe and PKCz, in

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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...

Malonic acid as well as its model substrate methylmalonic acid are covalently bound to two chemically different sites of the fatty acid synthetase complex of yeast. When radioactively labeled malonyl CoA or methylmalonyl CoA are incubated with the multienzyme complex, radioactive malonyl and methylmalonyl enzymes are formed and may be precipitated with trichloroacetic acid. Treatment with performic acid splits only a minor proportion of the (methyl) malonyl enzyme bonds. Most of them are resistant to the oxidant and, hence, prove to be non-thioester linkages.Methy1-[3-W]malonyl enzyme was digested with pepsin. By DEAE-Sephadex chromatography of the peptic hydrolysate the radioactive (methy1)malonyl peptides were separated into three Werent fractions A, B and C. I n peptides B and C (methy1)malonic acid is bound by performic acid stable linkages, whereas (methy1)malonyl peptide A apparently is a thioester and can be split by oxidation. Peptide C is always a minor satellite of peptide B, the quantitative relation of both varying somewhat in different preparations. It is concluded that both are derived from a common original peptide sequence in the enzyme.Methyl-[3-14C]malonyI peptide B was further purified by the following procedures : Dowex-BOX2 chromatography, subtilisin S digestion, DEAE-Sephadex chromatography, Dowex-50 X 2 chromatography, high voltage paper electrophoresis and paper chromatography. Two methylmalonyl peptides, B, end B,, resulted from subtilisin S treatment. They h l l y were obtained in analytically pure form and proved to be a pentapeptide and a heptapeptide, respectively, with the following amino acid composition: Bl = Leu,Ala,Gly,Ser,His and B, = Leu, Ah,Gly(S x),Glu,Ser,Hk. Leucine was the C-terminal amino acid in peptide Bl. No cysteine was found in either peptide.A preparation of [3-14C]malonyl peptide B was found to be analytically pure after DEAESephadex, Dowex-50 X2 and paper chromatography. Its amino acid composition was identical to that of methylmalonyl peptide B,.The acyl peptides B and C were highly sensitive against alkaline hydrolysis. It is concluded that the (methy1)malonic acid in peptides B and C is bound to serine by an 0-ester linkage which is unusually activated by some surrounding amino acid, possibly histidine.[3-14c]Malonal peptide A was shown to contain stoichiometric amounts of cysteamine, 8-alanine, and organic phosphate, corresponding to the quantity of malonate bound. These data suggest a thioester linkage between malonate and the SH-group of enzyme-bound 4'-phosphopantetheine. 4'-Phospho-pantetheine, therefore, represents the so called "central" SH-group in yeast fatty acid synthetase. The other malonyl binding site is thought to be the active oenter of the malonyl transferase in the complex.After alkali treatment of the purified fatty acid synthetase under defined conditions 4'-phosphopantetheine was isolated from the hydrolysate and characterized as S-benzoyl-4'-phosphopantetheine. One molecule of the multienzyme complex contained between 3.5 and 6 molecules ...

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