Chida K, Hashiba H, Sasaki K, Kuroki T
Cancer Res. 1986 Mar;46(3):1055-62.
The activation of protein kinase C and protein phosphorylation by tumor promoters were examined using quiescent cultures of BALB/3T3 and C3H/10T1/2 cells, because in these cells tumor promoters enhance chemically induced transformation and also induce DNA synthesis and ornithine decarboxylase. The cytosol and membrane fractions were partially purified, and the activity of protein kinase C was assayed. In quiescent cells, protein kinase C activity was found only in the cytosol fraction. Treatment with 100 ng of 12-O-tetradecanoylphorbol-13-acetate or teleocidin B per ml caused rapid translocation of protein kinase C from the cytosol to the membrane fraction. The activity in the cytosol disappeared almost completely after 15 min when the activity in the membrane reached a peak. The membrane activity gradually decreased to the control level after 6 h, while no activity reappeared in the cytosol within 6 h. Under these circumstances, a membrane protein with a molecular weight of 90,000 and pl of 4.0-4.4 (termed p90) was specifically phosphorylated, possibly by the activated protein kinase C, in both cell-free and intact-cell systems. On treatment of quiescent BALB/3T3 cells with 100 ng of 12-O-tetradecanoylphorbol-13-acetate, p90 phosphorylation increased 2-fold in 1 min, reaching a peak in 15 min of 3.4-fold the initial value. The phosphorylation of p90 increased with increase in the concentrations of 12-O-tetradecanoylphorbol-13-acetate between 0.1 and 10 ng/ml and reached a plateau at 10 ng/ml. p90 phosphorylation also occurred on exposure of the cells to non-phorbol ester tumor promoters (mezerein and teleocidin B) and growth factors, such as platelet-derived growth factor and fibroblast growth factor. p90 was not immunoprecipitated by antibody against the insulin receptor. Phosphorylation of p90 occurred at a serine residue. The present study suggests that activation of protein kinase C and phosphorylation of p90 by it are early events leading to tumor promotion.
利用BALB/3T3和C3H/10T1/2细胞的静止培养物，研究了肿瘤启动子对蛋白激酶C的激活作用以及蛋白磷酸化情况，因为在这些细胞中，肿瘤启动子可增强化学诱导的转化作用，还能诱导DNA合成和鸟氨酸脱羧酶的产生。对胞质溶胶和膜部分进行了部分纯化，并测定了蛋白激酶C的活性。在静止细胞中，蛋白激酶C活性仅存在于胞质溶胶部分。每毫升用100 ng的12 - O - 十四烷酰佛波醇 - 13 - 乙酸酯或替莱考丁B处理，可导致蛋白激酶C迅速从胞质溶胶转位至膜部分。15分钟后，胞质溶胶中的活性几乎完全消失，此时膜中的活性达到峰值。6小时后，膜中的活性逐渐降至对照水平，而6小时内胞质溶胶中未再次出现活性。在这些情况下，一种分子量为90,000、等电点为4.0 - 4.4的膜蛋白（称为p90）在无细胞和完整细胞系统中均被特异性磷酸化，可能是被激活的蛋白激酶C磷酸化。用100 ng的12 - O - 十四烷酰佛波醇 - 13 - 乙酸酯处理静止的BALB/3T3细胞，p90磷酸化在1分钟内增加2倍，15分钟时达到峰值，为初始值的3.4倍。p90的磷酸化随着12 - O - 十四烷酰佛波醇 - 13 - 乙酸酯浓度在0.1至10 ng/ml之间的增加而增加，并在10 ng/ml时达到平台期。细胞暴露于非佛波酯肿瘤启动子（美泽瑞因和替莱考丁B）以及生长因子（如血小板衍生生长因子和成纤维细胞生长因子）时，也会发生p90磷酸化。p90不能被抗胰岛素受体抗体免疫沉淀。p90的磷酸化发生在一个丝氨酸残基上。本研究表明，蛋白激酶C的激活及其对p90的磷酸化作用是导致肿瘤促进的早期事件。
