Schievella A R, Regier M K, Smith W L, Lin L L
Small Molecule Drug Discovery Group, Genetics Institute, Inc., Cambridge, Massachusetts 02140, USA.
J Biol Chem. 1995 Dec 22;270(51):30749-54. doi: 10.1074/jbc.270.51.30749.
Cytosolic phospholipase A2 (cPLA2) is activated by a wide variety of stimuli to release arachidonic acid, the precursor of the potent inflammatory mediators prostaglandin and leukotriene. Specifically, cPLA2 releases arachidonic acid in response to agents that increase intracellular Ca2+. In vitro data have suggested that these agents induce a translocation of cPLA2 from the cytosol to the cell membrane, where its substrate is localized. Here, we use immunofluorescence to visualize the translocation of cPLA2 to distinct cellular membranes. In Chinese hamster ovary cells that stably overexpress cPLA2, this enzyme translocates to the nuclear envelope upon stimulation with the calcium ionophore A23187. The pattern of staining observed in the cytoplasm suggests that cPLA2 also translocates to the endoplasmic reticulum. We find no evidence for cPLA2 localization to the plasma membrane. Translocation of cPLA2 is dependent on the calcium-dependent phospholipid binding domain, as a calcium-dependent phospholipid binding deletion mutant of cPLA2 (delta CII) fails to translocate in response to Ca2+. In contrast, cPLA2 mutated at Ser-505, the site of mitogen-activated protein kinase phosphorylation, translocates normally. This observation, combined with the observed phosphorylation of delta CII, establishes that translocation and phosphorylation function independently to regulate cPLA2. The effect of these mutations on cPLA2 translocation was confirmed by subcellular fractionation. Each of these mutations abolished the ability of cPLA2 to release arachidonic acid, establishing that cPLA2-mediated arachidonic acid release is strongly dependent on both phosphorylation and translocation. These data help to clarify the mechanisms by which cPLA2 is regulated in intact cells and establish the nuclear envelope and endoplasmic reticulum as primary sites for the liberation of arachidonic acid in the cell.
胞质型磷脂酶A2（cPLA2）可被多种刺激激活，以释放花生四烯酸，而花生四烯酸是强效炎症介质前列腺素和白三烯的前体。具体而言，cPLA2会响应增加细胞内Ca2+的因子而释放花生四烯酸。体外数据表明，这些因子会诱导cPLA2从细胞质转移至细胞膜，而其底物就位于细胞膜上。在此，我们利用免疫荧光来观察cPLA2向不同细胞膜的转移。在稳定过表达cPLA2的中国仓鼠卵巢细胞中，在用钙离子载体A23187刺激后，这种酶会转移至核膜。在细胞质中观察到的染色模式表明，cPLA2也会转移至内质网。我们没有发现cPLA2定位于质膜的证据。cPLA2的转移依赖于钙依赖性磷脂结合结构域，因为cPLA2的钙依赖性磷脂结合缺失突变体（delta CII）在受到Ca2+刺激时无法转移。相比之下，在丝裂原活化蛋白激酶磷酸化位点Ser-505处发生突变的cPLA2能正常转移。这一观察结果，结合所观察到的delta CII的磷酸化，证实转移和磷酸化在调节cPLA2时发挥独立作用。这些突变对cPLA2转移的影响通过亚细胞分级分离得到了证实。这些突变中的每一个都消除了cPLA2释放花生四烯酸的能力，这表明cPLA2介导的花生四烯酸释放强烈依赖于磷酸化和转移。这些数据有助于阐明在完整细胞中cPLA2的调节机制，并确定核膜和内质网是细胞内花生四烯酸释放的主要部位。
