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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MAPK活化和神经元分化。我们发现，同时加入溶血磷脂、溶血磷脂酰胆碱(LPC)和鞘氨醇磷脂酰胆碱(SPC)，显著增强了NGF诱导的MAP激酶的激活(通过其磷酸化)和突起生长。一致认为，LPC和SPC增强了NGF诱导的即刻早期基因表达。对胞外Ca~(2+)的去除没有表现出任何效果。不同抑制剂的研究表明，神经生长因子和LPC/SPC的协同作用需要激活特定的细胞信号通路(S)。AoPlaA-EGFP融合蛋白的定位分析表明，它定位于线粒体，生化分离实验支持这一结果。为了检测N-末端是否含有线粒体靶向信号，表达了EGFP融合蛋白，并对其定位进行了分析。经Ni^&lt；2+&gt；柱层析纯化的AoPlaA-HA-His_6的N-末端氨基酸序列分析表明存在可切割的线粒体靶向序列。在一项独立的实验中，突变菌株OFA。产生了对AopaA破坏的lyzae，并对其进行了表型分析。