Regulation of cPLA2 and Arachidonic Acid Release

85 kDa的胞质磷脂酶As （cPLA2）介导激动剂诱导的花生四烯酸释放，这是产生类二十烷和血小板活化因子的第一步。cPLA2受磷酸化和钙的调控，钙与C2结构域结合并诱导其向核膜的易位。然而，某些激动剂（PMA和冈田酸）诱导巨噬细胞释放cPLA2介导的花生四烯酸，而不增加细胞内钙，暗示cPLA2激活的新机制。假设细胞膜中特定的细胞质调节蛋白，新的磷酸化事件和磷脂酰肌醇-二磷酸（PIP2）水平的局部变化的作用。各种细胞模型，包括巨噬细胞，Sf9昆虫二磷酸（PIP2）在膜假设。包括巨噬细胞、Sf9昆虫细胞和小鼠肺成纤维细胞在内的各种细胞模型将用于阐明这些新机制。将测量促进花生四烯酸释放的激动剂诱导PIP2异构体合成的能力。通过过表达磷脂酰肌醇转移蛋白和I型磷脂酰肌醇-4-磷酸5激酶增加PIP2的合成对cpla2介导的花生四烯酸释放的影响将被确定。我们将开发一个渗透性细胞系统来重建激动剂诱导的、cpla2介导的花生四烯酸释放，以探索PIP2的作用并鉴定胞质调节蛋白。蛋白激酶C在cPLA2调控中的直接和间接作用也被提出。PKC直接磷酸化和调节cPLA2的能力将通过激活磷脂酶D，通过增加二酰基甘油或PIP2的产生进行测试。在冈田酸处理的巨噬细胞中，一个54 kDa激酶特异性地与cPLA2相关，这表明新的磷酸化事件的重要性。在冈田酸处理的巨噬细胞中，54 kDa激酶特异性地与cPLA2结合，这表明新的磷酸化事件的重要性。将鉴定54 kDa激酶并确定其在PLA2调节中的作用。最后，观察到环六铁和放线菌素D对激动剂诱导的巨噬细胞中花生四烯酸释放的抑制作用，提示其参与了一种快速翻转的蛋白质，其基础将被确定。这些研究将有助于更好地了解花生四烯酸释放和炎症脂质介质产生的调节。

The 85 kDa cytosolic phospholipase As (cPLA2) mediates agonist- induced arachidonic acid release, the first step in the production of eicosanoids and platelet-activating factor. cPLA2 is regulated by phosphorylation and by calcium, which binds to a C2 domain and induces its translocation to the nuclear envelope. However, certain agonists (PMA and okadaic acid) induce cPLA2-mediated arachidonic acid release in macrophages without increasing intracellular calcium implicating novel mechanisms for cPLA2 activation. A role for specific cytosolic regulatory proteins, novel phosphorylation events, and local changes in levels of phosphatidylinositol-bisphosphate (PIP2) in the membrane are hypothesized. Various cell models including macrophages, Sf9 insect bisphospate (PIP2) in the membrane are hypothesized. Various cell models including macrophages, Sf9 insect cells and mouse lung fibroblasts will be used to elucidate these novel mechanisms. The ability of agonists that promote arachidonic acid release to induce synthesis of PIP2 isomers will be measured. The effect of increasing synthesis of PIP2 by over-expressing phosphatidylinositol transfer protein and type I phosphatidylinositol-4- phosphate 5-kinase on cPLA2-mediated arachidonic acid release will be determined. A permeabilized cell system will be developed to reconstitute agonist-induced, cPLA2-mediated arachidonic acid release to explore the role of PIP2 and to identify cytosolic regulatory proteins. A role for protein kinase C in regulating cPLA2 by both direct and indirect mechanisms is also proposed. The ability of PKC to directly phosphorylate and regulate cPLA2 will through activation of phospholipase D by increasing production of diacylglycerol or PIP2 will be tested. A 54 kDa kinase specifically associates with cPLA2 in okadaic acid treated macrophages suggesting the importance of novel phosphorylation events. The 54 kDa kinase specifically associates with cPLA2 in okadaic acid treated macrophages suggesting the importance of novel phosphorylation events. The 54 kDa kinase will be identified and its role in regulating PLA2 determined. Finally, the observed inhibition of agonist-induced arachidonic acid release in macrophages by cyclohexamide and actinomycin D suggests the involvement of a rapidly turning-over protein, and the basis for this will be determined. These studies will lead to a better understanding of the regulation of arachidonic acid release and production of inflammatory lipid mediators.