Activation of novel protein kinases C delta and C epsilon upon mitogenic stimulation of quiescent rat 3Y1 fibroblasts.
Rat fibroblast 3Y1 cells express at least three protein kinase C species, conventional PKC alpha (cPKC alpha), novel PKC delta (nPKC delta), and novel PKC epsilon (nPKC epsilon). The stimulation of quiescent 3Y1 cells by serum (or epidermal growth factor (EGF) but not 12-O-tetradecanoylphorbol-13-acetate (TPA) results in the induction of DNA synthesis. Upon stimulation by serum or EGF, endogenous PKC species showed no indication of activation such as translocation or down-regulation, whereas TPA or synthetic diacylglycerol caused activation of all these PKC species when judged by these criteria. The only indication of activation observed upon serum or EGF stimulation was an upward shift in the electrophoretic mobility of nPKC delta. The phosphorylation levels of endogenous PKC members determined by in vivo metabolic labeling experiments revealed increased phosphorylation of both nPKC delta and nPKC epsilon, but only a slight increase for cPKC alpha in response to serum or EGF. On the other hand, TPA caused increased phosphorylation of all three PKC species. Overexpression of these PKC members by introduction of the corresponding cDNA expression plasmids resulted in the enhancement of the cell response to TPA when monitored in terms of transcriptional activation through TPA- or serum-responsive elements. Such enhancement in transcriptional activation by overexpression of cPKC alpha, nPKC delta, or nPKC epsilon was also observed in response to diacylglycerol, indicating that all these PKC species are activated by diacylglycerol in cells. In contrast to these nonphysiological stimuli, serum (or EGF) stimulation of 3Y1 cells that overexpress the respective PKC members revealed a clear difference between cPKC and nPKC, in that overexpression of nPKC delta or nPKC epsilon resulted in a large increase in TPA- or serum-responsive element activation, whereas the overexpression of cPKC alpha increased activation only very slightly. These results indicate that the mitogenic stimulation of quiescent 3Y1 cells results in selective activation of endogenous nPKC members and that the modes of activation of cPKC and nPKC differ from each other.
Department of Molecular Biology, Yokohama City University School of Medicine, Japan.
S
S
Ohno
K
K
Mizuno
Y
Y
Adachi
A
A
Hata
Y
Y
Akita
K
K
Akimoto
S
S
Osada
S
S
Hirai
K
K
Suzuki
D000818:Animals; D001769:Blood; D002478:Cells, Cultured; D004789:Enzyme Activation; D004815:Epidermal Growth Factor / Q000494:pharmacology; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D008934:Mitogens / Q000494:pharmacology*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014158:Transcription, Genetic / Q000187:drug effects
D008934:Mitogens; D004815:Epidermal Growth Factor; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate