Ohno S, Mizuno K, Adachi Y, Hata A, Akita Y, Akimoto K, Osada S, Hirai S, Suzuki K
Department of Molecular Biology, Yokohama City University School of Medicine, Japan.
J Biol Chem. 1994 Jul 1;269(26):17495-501.
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.
大鼠成纤维细胞3Y1至少表达三种蛋白激酶C亚型，即传统的蛋白激酶Cα（cPKCα）、新型蛋白激酶Cδ（nPKCδ）和新型蛋白激酶Cε（nPKCε）。血清（或表皮生长因子（EGF），而非12 - O - 十四烷酰佛波醇 - 13 - 乙酸酯（TPA））刺激静止的3Y1细胞会诱导DNA合成。在血清或EGF刺激下，内源性蛋白激酶C亚型未显示出如转位或下调等激活迹象，而根据这些标准判断，TPA或合成二酰甘油会导致所有这些蛋白激酶C亚型的激活。在血清或EGF刺激下观察到的唯一激活迹象是nPKCδ的电泳迁移率向上移动。通过体内代谢标记实验测定的内源性蛋白激酶C成员的磷酸化水平显示，nPKCδ和nPKCε的磷酸化均增加，但cPKCα对血清或EGF的反应仅略有增加。另一方面，TPA导致所有三种蛋白激酶C亚型的磷酸化增加。通过引入相应的cDNA表达质粒过表达这些蛋白激酶C成员，当通过TPA或血清反应元件监测转录激活时，会导致细胞对TPA的反应增强。在对二酰甘油的反应中也观察到，cPKCα、nPKCδ或nPKCε的过表达导致转录激活增强，表明所有这些蛋白激酶C亚型在细胞中都被二酰甘油激活。与这些非生理性刺激相反，血清（或EGF）对过表达各自蛋白激酶C成员的3Y1细胞的刺激揭示了cPKC和nPKC之间的明显差异，即nPKCδ或nPKCε的过表达导致TPA或血清反应元件激活大幅增加，而cPKCα的过表达仅使激活略有增加。这些结果表明，静止3Y1细胞的促有丝分裂刺激导致内源性nPKC成员的选择性激活，并且cPKC和nPKC的激活方式彼此不同。
