Regulation of cPLA2 and Arachidonic Acid Release

cPLA2 和花生四烯酸释放的调节

• 批准号: 6611192
• 负责人: CHRISTINA Carroll LESLIE
• 金额: $ 22.05万
• 依托单位: NATIONAL JEWISH HEALTH
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-09-01 至 2003-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6611192
• 关键词: arachidonate; eicosanoid metabolism; enzyme activity; enzyme induction /repression; enzyme mechanism; laboratory mouse; macrophage; okadaic acid; phosphatidylinositols; phospholipase A2; phosphorylation; protein kinase C; protein protein interaction; tissue /cell culture

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.

85 kDa胞质磷脂酶As（cPLA 2）介导激动剂诱导的花生四烯酸释放，这是类花生酸和血小板活化因子产生的第一步。cPLA 2受磷酸化和钙调节，钙结合C2结构域并诱导其易位至核膜。然而，某些激动剂（PMA和冈田酸）诱导巨噬细胞中cPLA 2介导的花生四烯酸释放，而不增加细胞内钙，这涉及cPLA 2活化的新机制。特定的胞质调节蛋白，新的磷酸化事件，和磷脂酰肌醇二磷酸（PIP 2）在膜中的水平的局部变化的作用进行了假设。假设了各种细胞模型，包括巨噬细胞、膜中的Sf 9昆虫二磷酸盐（PIP 2）。各种细胞模型，包括巨噬细胞，Sf 9昆虫细胞和小鼠肺成纤维细胞将用于阐明这些新的机制。将测量促进花生四烯酸释放的激动剂诱导PIP 2异构体合成的能力。将确定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PIP 2合成对cPLA 2介导的花生四烯酸释放的影响。将开发透化细胞系统以重建激动剂诱导的cPLA 2介导的花生四烯酸释放，以探索PIP 2的作用并鉴定胞质调节蛋白。还提出了蛋白激酶C通过直接和间接机制调节cPLA 2的作用。将测试PKC通过增加二酰基甘油或PIP 2的产生来激活磷脂酶D而直接磷酸化和调节cPLA 2的能力。一个54 kDa的激酶特异性地与cPLA 2在冈田酸处理的巨噬细胞，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cPLA 2结合，表明新的磷酸化事件的重要性。将鉴定54 kDa激酶，并确定其在调节PLA 2中的作用。最后，所观察到的抑制激动剂诱导的花生四烯酸释放巨噬细胞环己酰胺和放线菌素D表明参与的快速翻转蛋白质，这将确定的基础。这些研究将导致更好地了解花生四烯酸的释放和炎症脂质介质的产生的调节。