Issandou M, Bayard F, Darbon J M
Institut National de la Santé et de la Recherche Médicale, U 168, Department of Endocrinology, CHU Rangueil, Université Paul Sabatier, Toulouse, France.
Cancer Res. 1988 Dec 1;48(23):6943-50.
We have investigated the effects of phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) and permeant diacylglycerol 1,2-dioctanoyl-sn-glycerol (DiC8) on MCF-7 cell proliferation and protein kinase C activity. DiC8 mimics the effects of TPA on both cell morphology and proliferation, with an ED50 value of 11 micrograms/ml for cell growth inhibition. As with TPA and phorbol 12,13-dibutyrate, DiC8 enhances the degree of phosphorylation of an endogenous Mr 28,000 protein in a time- and dose-dependent manner. The effect is measurable upon 5 min of cell treatment with each protein kinase C activator and reaches a maximum at 30 min. The ED50s observed are 5 ng/ml and 20 micrograms/ml, respectively, for phorbol esters and DiC8. The Mr 28,000 protein is found in the cytosolic fraction and is phosphorylated on serine residues by both TPA and DiC8. Further characterization of the phosphorylated proteins using a highly resolutive two-dimensional electrophoresis demonstrates that the two-protein kinase C activators lead to slightly distinct protein phosphorylation patterns with an extra set of proteins phosphorylated under TPA but not DiC8 stimulation. Contrary to TPA, DiC8 induces only a partial and transient translocation of protein kinase C activity from the cytosolic to the particulate compartment. Moreover, no down-regulation of protein kinase C is observed after prolonged treatment of MCF-7 cells with DiC8, while only 10% of the initial protein kinase C level remains present in cells treated with TPA for 48 h. However, this remainder enzymatic activity is sufficient to induce the phosphorylation of the Mr 28,000 protein at its maximal level. In conclusion, our results reinforce the hypothesis of a negative modulatory role of protein kinase C in MCF-7 cell proliferation but suggest that the two activators TPA and DiC8 could induce distinct molecular events with regard to the enzyme recruitment and activity as well as to its further processing.
我们研究了佛波酯12 - O - 十四烷酰佛波醇 - 13 - 乙酸酯（TPA）和渗透性二酰甘油1,2 - 二辛酰 - sn - 甘油（DiC8）对MCF - 7细胞增殖和蛋白激酶C活性的影响。DiC8模拟TPA对细胞形态和增殖的影响，细胞生长抑制的半数有效浓度（ED50）值为11微克/毫升。与TPA和佛波醇12,13 - 二丁酸酯一样，DiC8以时间和剂量依赖性方式增强内源性分子量28,000蛋白的磷酸化程度。用每种蛋白激酶C激活剂处理细胞5分钟后即可检测到这种效应，并在30分钟时达到最大值。观察到的佛波酯和DiC8的半数有效浓度分别为5纳克/毫升和20微克/毫升。分子量28,000的蛋白存在于胞质组分中，并且被TPA和DiC8磷酸化在丝氨酸残基上。使用高分辨率二维电泳对磷酸化蛋白进行进一步表征表明，这两种蛋白激酶C激活剂导致略有不同的蛋白磷酸化模式，在TPA刺激下有一组额外的蛋白被磷酸化，而DiC8刺激下则没有。与TPA相反，DiC8仅诱导蛋白激酶C活性从胞质到颗粒区室的部分和短暂转位。此外，用DiC8长时间处理MCF - 7细胞后未观察到蛋白激酶C的下调，而用TPA处理48小时的细胞中仅保留10%的初始蛋白激酶C水平。然而，这种剩余的酶活性足以诱导分子量28,000蛋白的最大程度磷酸化。总之，我们的结果强化了蛋白激酶C在MCF - 7细胞增殖中起负调节作用的假设，但表明两种激活剂TPA和DiC8在酶募集、活性及其进一步加工方面可能诱导不同的分子事件。
