Regulation of cPLA2 and Arachidonic Acid Release

85 kDa胞浆磷脂酶AS(CPLA2)介导激动剂诱导的花生四烯酸释放，这是产生二十烷类化合物和血小板激活因子的第一步。CPLA2受磷酸化和钙的调节，钙结合C2结构域并诱导其移位到核膜上。然而，某些激动剂(PMA和冈田酸)在不增加细胞内钙离子的情况下诱导cPLA2介导的花生四烯酸在巨噬细胞中的释放，这意味着cPLA2激活的新机制。特定的胞浆调节蛋白的作用，新的磷酸化事件，以及膜上磷脂酰肌醇-二磷酸(PIP2)水平的局部变化被假设。假设了多种细胞模型，包括巨噬细胞、膜上的Sf9昆虫双磷酸盐(PIP2)。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被用来阐明这些新的机制。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。过表达磷脂酰肌醇转运蛋白和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。一个通透性的细胞系统将被开发来重建激动剂诱导的、cPLA2介导的花生四烯酸释放，以探索PIP2的作用并鉴定胞浆调节蛋白。还提出了蛋白激酶C通过直接和间接机制调节cPLA2的作用。PKC直接磷酸化和调节cPLA2的能力将通过增加二酰甘油或PIP2的产生而激活磷脂酶D来进行测试。在冈田酸处理的巨噬细胞中，54 kDa的激酶与cPLA2特异性结合，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的激酶与cPLA2特异性结合，提示新的磷酸化事件的重要性。54 kDa的激酶将被鉴定，并确定其在调节PLA2中的作用。最后，观察到环己胺和放线菌素D抑制激动剂诱导的花生四烯酸在巨噬细胞中的释放，这表明一种快速翻转蛋白参与其中，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性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.