Staddon J M, Bouzyk M M, Rozengurt E
Imperial Cancer Research Fund, London, United Kingdom.
J Biol Chem. 1992 Dec 15;267(35):25239-45.
Incubation of Swiss 3T3 cells with [2-3H]adenine, as in other cell types, reveals the ADP-ribosylation of GRP78 (the 78-kDa glucose-regulated protein, also known as BiP, the immunoglobulin heavy chain-binding protein), a resident endoplasmic reticulum protein that assists in the processing of proteins destined for secretion or cell surface expression. Here we show that Pasteurella multocida toxin, a potent growth factor for cultured fibroblasts, decreased the ADP-ribosylation of GRP78/BiP to 16 +/- 6% of the control value (n = 23). The action of the toxin occurred after a lag period, was blocked by lysosomotrophic agents, and potentiated by increased incubation time (ED50 4 ng/ml and 1 ng/ml in 4 and 8 h, respectively), thus indicating that the toxin enters the cells to act. Bombesin and platelet-derived growth factor (PDGF) similarly decreased the ADP-ribosylation of GRP78/BiP (ED50 0.5 nM and 2.5 ng/ml, respectively) but acted more rapidly than the toxin. Signaling pathways activated by the toxin, bombesin, and PDGF had effects on the ADP-ribosylation of GRP78/BiP. Thus, activation of protein kinase C alone by phorbol 12,13-dibutyrate was partially effective, and down-regulation of protein kinase C attenuated but did not block the action of the toxin, bombesin, and PDGF. Agents that mobilize Ca2+ from the endoplasmic reticulum (A23187, ionomycin, and thapsigargin) caused a decrease in the ADP-ribosylation of GRP78/BiP that was similar in magnitude to that achieved by the toxin, bombesin, and PDGF, implicating a role for inositol 1,4,5-trisphosphate-mediated Ca2+ mobilization in the action of the mitogenic agents. The growth factor-induced decrease in the ADP-ribosylation of GRP78/BiP may represent its conversion from an inactive to an active state.
与其他细胞类型一样，用[2-³H]腺嘌呤孵育瑞士3T3细胞，可显示GRP78（78 kDa的葡萄糖调节蛋白，也称为BiP，即免疫球蛋白重链结合蛋白）的ADP核糖基化，GRP78是一种内质网驻留蛋白，有助于处理 destined for secretion or cell surface expression的蛋白质。我们在此表明，多杀巴斯德氏菌毒素是一种对培养的成纤维细胞有效的生长因子，它将GRP78/BiP的ADP核糖基化降低至对照值的16±6%（n = 23）。毒素的作用在一段延迟期后发生，被溶酶体营养剂阻断，并因孵育时间延长而增强（4小时和8小时时的ED50分别为4 ng/ml和1 ng/ml），因此表明毒素进入细胞发挥作用。蛙皮素和血小板衍生生长因子（PDGF）同样降低了GRP78/BiP的ADP核糖基化（ED50分别为0.5 nM和2.5 ng/ml），但作用比毒素更快。毒素、蛙皮素和PDGF激活的信号通路对GRP78/BiP的ADP核糖基化有影响。因此，仅用佛波醇12,13-二丁酸激活蛋白激酶C有部分效果，而蛋白激酶C的下调减弱但并未阻断毒素、蛙皮素和PDGF的作用。从内质网动员Ca²⁺的试剂（A23187、离子霉素和毒胡萝卜素）导致GRP78/BiP的ADP核糖基化降低，其幅度与毒素、蛙皮素和PDGF所达到的相似，这表明肌醇1,4,5-三磷酸介导的Ca²⁺动员在促有丝分裂剂的作用中起作用。生长因子诱导的GRP78/BiP的ADP核糖基化降低可能代表其从无活性状态转变为活性状态。
