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 speci es, conventional PKC alpha (cPKC alpha), novel PKC delta (nPKC delta), and novel PKC epsilon (nPKC epsilon). The stimula tion of quiescent 3 Y1 cells by serum (or epidermal growth factor (EGF)) but not 12-O-tetr adecanoylphorbol-13-acetate (TPA) results in the induction of DNA synt hesis. 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 thes e PKC species when judged by these criteria. The only indication of ac tivation observed upon serum or EGF stimulation was an upward shift in the electrophoretic mobility of nPKC delta. The phosphorylation level s of endogenous PKC members determined by in vivo metabolic labeling e xperiments revealed increased phosphorylation of both nPKC delta and n PKC 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 intro duction of the corresponding cDNA expression plasmids resulted in the enhancement of the cell response to TPA when monitored in terms of tra nscriptional activation through TPA- or serum-responsive elements. Suc h enhancement in transcriptional activation by overexpression of cPKC alpha, nPKC delta, or nPKC epsilon was also observed in response to di acylglycerol, indicating that all these PKC species are activated by d iacylglycerol in cells. In contrast to these nonphysiological stimuli, serum (or EGF) stimulation of 3Y1 cells that overexpress the respecti ve PKC members revealed a clear difference between cPKC and nPKC, in t hat overexpression of nPKC delta or nPKC epsilon resulted in a large i ncrease in TPA- or serum-responsive element activation, whereas the ov erexpression of cPKC alpha increased activation only very slightly. Th ese results indicate that the mitogenic stimulation of quiescent 3Y1 c ells results in selective activation of endogenous nPKC members and th at the modes of activation of cPKC and nPKC differ from each other.