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Regulatory mechanisms and roles of phospholipase D (PLD) in cellular responses

磷脂酶 D (PLD) 在细胞反应中的调节机制和作用

基本信息

批准号：
08670143
负责人：
NAKASHIMA Shigeru
金额：
$ 1.47万
依托单位：
Gifu University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1997
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08670143/
关键词：
phospholipase D protein kinase C RhoA apoptosis differentiation protein tyrosine kinase growth factor transcription factor RhoA クロストリジウムトキシンB

项目摘要

We have found the alternatively spliced isoform of human phospholipase D1 (hPLD1). Moreover, the entire coding regions of rat PLD1 and PLD2 were isolated and sequenced. A shorter alternatively spliced form of rPLD1 (rPLD1b) was also identified. Chromosomal locations of these genes were determined in the rat and mouse by FISH.The PLD1 and PLD2 genes were localized to rat Chromosome 2q23.3-34 proximal end and Chromosome 10q23.3-24 proximal end, respectively. During cell differentiation or apoptotic processes, the expression of PLD1 and PLD2 mRNAs were drastically changed in rat phochromocytoma PC12 cells, human leukemic HL-60 cells and rat basophilic leukemia RBL-2H3 cells. In the presence of ethanol or butanol which is utilized for transphosphatidylation reaction of PLD to produce phosphatidylalcohol at the expense of phosphatidic acid, the expression of transcription factors, c-jun and c-fos in response to growth signal was inhibited, suggesting that PLD is closely involved in growth signal transduction. The regulatory mechanism of PLD was investigated in differentiated HL-60 cells and PC12 cells. Inhibitors of phosphatidylinositol 3-kinase (PI-3K), wortmannin and LY294002 revealed that PI-3K is located between receptor and PLD in differentiated HL-60 cells. Moreover, a kind of tyrosine kinase may work at the downstream of PI-3K.In PC12 cells, possible involvement of a calcium-sensitive tyrosine kinase in carbachol-induced PLD activation was suggested. Hydrogen peroxide (H_2O_2) greatly stimulated PLD activity in PC12 cells. An unidentified tyrosine kinase is also appeared to function to activate PLD in H_2O_2-stimulated PC12 cells. We are currently working on to identify the tyrosine kinase involved in these processes and also planning to use antisense oligonucleotides and cells transfected with PLD genes to further investigate the roles of PLD in cellular responses.

我们已经发现了人类磷脂酶D1（hPLD 1）的选择性剪接异构体。此外，分离并测序了大鼠PLD 1和PLD 2的整个编码区。还鉴定了一种较短的rPLD 1可变剪接形式（rPLD 1b）。用荧光原位杂交技术（FISH）对PLD 1和PLD 2基因在大鼠和小鼠的染色体定位进行了研究，PLD 1和PLD 2基因分别定位于大鼠染色体2q23.3-34近端和10q23.3-24近端。在细胞分化或凋亡过程中，PLD 1和PLD 2 mRNA在大鼠嗜铬细胞瘤PC 12细胞、人白血病HL-60细胞和大鼠嗜碱性白血病RBL-2 H3细胞中的表达发生显著变化。在乙醇或丁醇的存在下，PLD的转磷脂酰化反应，以产生磷脂酰醇的磷脂酸为代价，转录因子，c-jun和c-fos的表达响应于生长信号被抑制，这表明PLD是密切参与的生长信号转导。在分化的HL-60细胞和PC 12细胞中研究PLD的调控机制。磷脂酰肌醇3-激酶（PI-3 K）抑制剂wortmannin和LY 294002显示，PI-3 K在分化的HL-60细胞中位于受体和PLD之间。在PC 12细胞中，钙敏感性酪氨酸激酶可能参与了卡巴胆碱诱导的PLD激活过程。过氧化氢（H_2O_2）可显著刺激PC 12细胞PLD活性。在H_2O_2刺激的PC 12细胞中，一种未鉴定的酪氨酸激酶也具有激活PLD的功能。我们目前正致力于鉴定参与这些过程的酪氨酸激酶，并计划使用反义寡核苷酸和转染PLD基因的细胞来进一步研究PLD在细胞反应中的作用。