Immunocytochemistry of phospholipase D1 and D2 in cultured cells
Immunocytochemistry of phospholipase D1 and D2 in cultured cells
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培养细胞中磷脂酶 D1 和 D2 的免疫细胞化学
DOI：
10.1016/j.bbrc.2022.07.118
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发表时间：
2022
影响因子：
3.1
通讯作者：
Nakano Tomoyuki
中科院分区：
文献类型：
--
作者：
Hozumi Yasukazu;Yamazaki Masakazu;Nakano Tomoyuki
Gq蛋白偶联受体的激活触发了磷脂酶C(PLC)途径，从而产生一对第二信使：二酰甘油(DG)和1，4，5-三磷酸肌醇(IP3)。DG激酶(DGK)使DG磷酸化，产生另一种第二信使磷脂酸(PA)。与PLC-DGK途径一样，PA是由磷脂酶D(PLD)直接作用于膜上的主要磷脂：磷脂酰胆碱(PC)而产生的。PA在磷脂酸性磷酸酶的作用下转化为DG，提示PLD和DGK是调节DG和PA的关键酶。PLD参与了广泛的细胞过程。然而，由于缺乏针对PLD的特异性抗体，PLD的细胞表达和亚细胞定位仍然难以捉摸。在这项研究中，我们提出了针对哺乳动物主要PLD亚型的特异性抗体：PLD1和PLD2。用特异性抗体进行免疫细胞化学分析表明，在培养的细胞中，天然的PLD1和PLD2以点状结构定位于不同的亚细胞区域。PLD1主要定位于细胞膜，而PLD2主要定位于细胞质。这些发现表明，PLD1和PLD2在不同亚细胞区域的肌醇磷脂信号通路中具有不同的作用。
Activation of Gq protein-coupled receptors triggers the phospholipase C (PLC) pathway, which yields a pair of second messengers: diacylglycerol (DG) and inositol 1,4,5-trisphosphate (IP3). DG kinase (DGK) phosphorylates DG to produce phosphatidic acid (PA), which serves as another second messenger. Along with PLC-DGK pathway, PA is produced directly by the action of phospholipase D (PLD), which hydrolyzes the major membrane phospholipid: phosphatidylcholine (PC). PA is converted to DG by phosphatidic acid phosphatase, suggesting that PLD, together with DGK, is a key enzyme regulating DG and PA. PLD has been implicated in a broad range of cellular processes. However, cellular expression and subcellular localization of PLD remain elusive because of a lack of specific antibodies against PLDs. For this study, we raised specific antibodies against major mammalian PLD isoforms: PLD1 and PLD2. Immunocytochemical analysis using specific antibodies showed clearly that native PLD1 and PLD2 localize to distinct subcellular regions as dot-like structures in cultured cells. PLD1 predominantly localizes to the plasma membrane, whereas PLD2 mostly localizes within the cytoplasm. These findings suggest that PLD1 and PLD2 have different roles in the phosphoinositide signaling pathway in distinct subcellular regions.