Translocation of cytosolic phospholipase A2 to the nuclear envelope elicits topographically localized phospholipid hydrolysis.
Translocation of cytosolic phospholipase A2 to the nuclear envelope elicits topographically localized phospholipid hydrolysis.
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胞浆磷脂酶 A2 易位至核膜引起局部磷脂水解。
DOI：
10.1042/bj3180797
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发表时间：
1996
期刊：
影响因子：
--
通讯作者：
Brock,T
中科院分区：
文献类型：
--
作者：
Peters-Golden,M;Song,K;Marshall,T;Brock,T
胞浆磷脂酶 A2 (cPLA2) 是介导激动剂刺激的膜磷脂释放花生四烯酸 (AA) 的良好候选者。在多种细胞类型中，这种酶经历 Ca2+ 依赖性从胞质溶胶到膜位点的易位，该位点最近被确定为白细胞中的核膜。这一发现的功能相关性尚未确定。因此，本研究旨在确定 cPLA2 向核膜的易位是否与该位点的局部磷脂水解有关。先前显示含有 cPLA2 的大鼠肺泡上皮细胞用 [3H]AA 预先标记，并用模型激动剂离子载体 A23187 刺激。离子载体诱导的 AA 释放表现出 cPLA2 介导反应的典型特征，即 Ca2+ 依赖性、sn-2 AA 选择性，并被花生四烯基三氟甲基酮抑制。通过间接免疫荧光显微镜分析以及免疫印迹的亚细胞分级确定，离子载体处理导致 cPLA2 蛋白从细胞质易位至核膜。为了确定核膜是否确实是释放的 AA 的来源，在存在或不存在 A23187 的情况下孵育预标记的细胞，然后对核膜和非核膜部分中的磷脂放射性进行定量。 [3H]AA分布在核膜磷脂和非核膜磷脂中。 A23187刺激后，核膜磷脂中[3H]AA的损失占磷脂总损失的88.1±5.8%，占释放到培养基中的[3H]AA总量的92.9±2.3%。这些结果首次证明激动剂刺激的 cPLA2 易位至核膜与优先定位于该位点的磷脂水解相关。
Cytosolic phospholipase A2(cPLA2) is a good candidate for mediating the agonist-stimulated release of arachidonic acid (AA) from membrane phospholipids. This enzyme undergoes a Ca2+-dependent translocation from the cytosol to a membrane site in a variety of cell types, and this site has recently been identified as the nuclear envelope in leucocytes. The functional correlate of this finding has not yet been established. The present study was therefore undertaken to determine whether translocation of cPLA2to the nuclear envelope was associated with localized phospholipid hydrolysis at this site. Rat alveolar epithelial cells, previously shown to contain cPLA2, were prelabelled with [3H]AA and stimulated with the model agonist, ionophore A23187. Ionophore-induced AA release exhibited characteristics typical of a cPLA2-mediated response, in that it was Ca2+-dependent,sn-2 AA-selective, and inhibited by arachidonyl trifluoromethyl ketone. As determined by indirect immunofluorescence microscopic analysis as well as subcellular fractionation with immunoblotting, ionophore treatment resulted in a translocation of cPLA2protein from the cytoplasm to the nuclear envelope. To determine whether the nuclear membrane was indeed the source of released AA, prelabelled cells were incubated in the presence or absence of A23187, after which the phospholipid radioactivity was quantified in nuclear and non-nuclear membrane fractions. [3H]AA was distributed in both nuclear and non-nuclear membrane phospholipids. Following A23187 stimulation, the loss of [3H]AA from nuclear membrane phospholipids accounted for 88.1±5.8% of the total loss from phospholipids and for 92.9±2.3% of the total [3H]AA released into the medium. These results demonstrate for the first time that agonist-stimulated translocation of cPLA2to the nuclear envelope is associated with phospholipid hydrolysis which is preferentially localized to that site.