Analysis on the physiological roles of phospholipases A_2

磷脂酶A_2的生理作用分析

基本信息

批准号：
18580067
负责人：
ARIOKA Manabu
金额：
$ 2.43万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2006
资助国家：
日本
起止时间：
2006 至 2007
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-18580067/
关键词：
phospholipase A_2 lysophospholipid MAP kinase PC12 Aspergillus oryzae ホスホリパーゼA2 nerve growth factor MAP kinase lysophosphatidylcholine sphingosylphosphorylcholine neurite outgrowth secretory phospholipase A_2

项目摘要

Nerve growth factor (NGF) induces MAP kinase activation and neuronal differentiation in PC12cells. We found that simultaneous addition of lysophospholipids, lysophosphatidylcholine (LPC) and sphingosylphosphorylcholine (SPC), markedly enhances NGF-induced MAP kinase activation, as examined by its phosphorylation, and neurite outgrowth. In agreement, the induction of immediate early gene expression by NGF was potentiated by LPC and SPC. Removal of extracellular Ca^<2+> did not show any effect. Studies using various inhibitors of suggested that the synergistic effect of NGF and LPC/SPC requires the activation of specific cellular signaling pathway(s).Putative cytosolic phospholipase A_2 gene, AoplaA, uniquely found in Aspergillus oryzae genome was studied. Localization analysis using AoPlaA-EGFP fusion protein demonstrated that it localizes to mitochondria, which was supported by the biochemical fractionation experiment. To examine whether the N-terminal region contains the mitochondrial targeting signal, EGFP-fusion proteins were expressed and their localization was analyzed. The presence of cleavable mitochondrial targeting sequence was suggested by the N-terminal amino acid sequence analysis of AoPlaA-HA-His_6 purified on a Ni^<2+> column chromatography. In an independent experiment, a mutant strain ofA. olyzae disrupted for AoplaA was generated and tested for the phenotypic analyses.

神经生长因子 (NGF) 诱导 PC12 细胞中 MAP 激酶激活和神经元分化。我们发现，同时添加溶血磷脂、溶血磷脂酰胆碱 (LPC) 和鞘氨醇磷酸胆碱 (SPC)，可显着增强 NGF 诱导的 MAP 激酶激活，通过其磷酸化和神经突生长进行检查。一致认为，LPC 和 SPC 增强了 NGF 对立即早期基因表达的诱导。细胞外Ca ^ 2+ 的去除没有显示出任何效果。使用各种抑制剂的研究表明，NGF 和 LPC/SPC 的协同作用需要激活特定的细胞信号通路。对米曲霉基因组中独特发现的假定的胞质磷脂酶 A_2 基因 AoplaA 进行了研究。使用 AoPlaA-EGFP 融合蛋白进行的定位分析表明，它定位于线粒体，这得到了生化分级分离实验的支持。为了检查 N 末端区域是否包含线粒体靶向信号，表达了 EGFP 融合蛋白并分析了它们的定位。通过在Ni 2+ 柱层析上纯化的AoPlaA-HA-His_6的N-末端氨基酸序列分析表明可切割的线粒体靶向序列的存在。在一项独立实验中，A 的突变株。生成 AoplaA 破坏的奥莱菌并进行表型分析测试。