VISUALIZING LOCALIZATION & TRANSLOCATION OF THREE TYPES OF PHOSPHOLIPASE A2

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。磷脂酶A2 （PLA2）通过生成溶血磷脂和花生四烯酸（AA）等脂肪酸，在包括基础代谢和信号转导在内的多种细胞反应中发挥重要作用，花生四烯酸是类二十烷酸的前体。迄今为止，哺乳动物pla25的许多亚型已被确定并分类为亚群。在本研究中，我们重点研究了它们的三个亚型；细胞质IV组PLA2 (cPLA2)，分泌组V （sPLA2）和细胞质Ca2+独立组VI PLA2 （iPLA2）。在前一年（2003年），我们研究了PLA2的分泌形式，并对慢性暴露于脂多糖后巨噬细胞中PLA2的激活进行了一系列研究。由于sPLA2是一种分泌酶，因此有人认为，在细胞受到刺激后，它必须被释放到细胞外介质中，并与外膜重新结合，从磷脂中释放花生四烯酸。使用共聚焦激光扫描显微镜和gfp标记的sPLA2版本，我们发现慢性暴露于脂多糖导致sPLA2与靠近核周区域的含小窝蛋白-2颗粒相关。这种关联被肝素（一种对sPLA2具有高亲和力的细胞不渗透化合物）阻断，表明颗粒是由先前与外细胞表面相关的sPLA2内化形成的。如果用IV组PLA2抑制剂甲基花生四烯酰基氟膦酸处理细胞，则未观察到核周定位，进一步表明cPLA2在激活过程中发挥重要作用。这些研究提供的证据表明，在细胞活化过程中，sPLA2被包裹成颗粒将酶带到核周包膜，在那里它可能更接近sPLA2和COX-2（环氧化酶-2），从而有效地合成前列腺素。该项目的研究结果发表在2003年的《生物化学杂志》上[Balboa et al.，在活化的P388D1巨噬细胞样细胞中，V组磷脂酶A2在小泡蛋白富集颗粒中的定位]（2003）。生物化学学报，2008(4),344 - 344。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Phospholipase A2 (PLA2) plays important roles in diverse cellular responses including fundamental metabolism and signal transduction by generating lysophospholipids and fatty acids such as arachidonic acid (AA), which is the precursor of eicosanoids. To date, many subtypes of mammalian PLA2s have been identified and classified into subgroups. In this study, we focus on three subtypes of them; the cytosolic group IV PLA2 (cPLA2), the secretory group V (sPLA2), and the cytosolic Ca2+independent group VI PLA2 (iPLA2). In the previous year (2003), we examined the secreted form of PLA2 and conducted a series of studies on its activation in macrophages following chronic exposure to lipopolysaccharide. Because sPLA2 is a secreted enzyme, it has been suggested that after cellular stimulation, it must be released to the extra-cellular medium and re-associates with the outer membrane to release arachidonic acid from phospholipids. Using confocal laser scanning microscopy and GFP-tagged versions of sPLA2, we found that chronic exposure to lipopolysaccharide results in sPLA2 being associated with caveolin-2- containing granules close to the perinuclear region. This association is blocked by heparin (a cell-impermeable compound with high affinity for sPLA2), suggesting that the granules are formed by the internalization of sPLA2 previously associated with the outer cell surface. Perinuclear localization is not observed if the cells are treated with the group IV PLA2 inhibitor methyl arachidonyl fluorophosphonate, further indicating the important role played by cPLA2 in the activation process. These studies provided evidence that the encapsulation of sPLA2 into granules brings the enzyme to the perinuclear envelope during cell activation where it may be closer to sPLA2 and COX-2 (cyclo-oxygenase-2) for efficient prostaglandin synthesis. Results from this project have been published on the Journal of Biological Chemistry in 2003 [Balboa et al., ¿Localization of group V phospholipase A2 in caveolin-enriched granules in activated P388D1 macrophage-like cells.¿(2003). Journal of Biological Chemistry, 278 (48), 48059-48065].