Analysis on the physiological roles of phospholipases A_2

神经生长因子（NGF）诱导pc12细胞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）和鞘酰基磷酸胆碱（SPC），通过其磷酸化和神经突生长来检测，显著增强ngf诱导的MAP激酶激活。与此一致的是，LPC和SPC增强了NGF对即时早期基因表达的诱导。去除细胞外Ca^<2+>没有任何效果。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。对米曲霉基因组中可能存在的胞质磷脂酶A_2基因（AoplaA）进行了研究。利用AoPlaA-EGFP融合蛋白进行定位分析，证实其定位于线粒体，生化分离实验也证实了这一点。为了检验n端区域是否含有线粒体靶向信号，我们表达了egfp融合蛋白并分析了其定位。用Ni^<2+>柱层析纯化的AoPlaA-HA-His_6的n端氨基酸序列分析表明存在可切割的线粒体靶向序列。在一个独立的实验中，a的突变株。产生了AoplaA的裂解酶，并进行了表型分析。

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.