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.

我们发现人磷脂酶D1（HPLD1）的剪接同工型。此外，分离并测序了大鼠PLD1和PLD2的整个编码区域。还确定了较短的RPLD1（RPLD1B）的剪接形式。这些基因的染色体位置是在大鼠和小鼠中通过鱼类确定的。PLD1和PLD2基因分别定位于大鼠染色体2Q223.3-34近端末端和染色体10q23.3-24近端近端。在细胞分化或凋亡过程中，在大鼠嗜铬细胞瘤PC12细胞，人类白血病HL-60细胞和大鼠嗜碱性白血病RBL-2H3细胞中，PLD1和PLD2 mRNA的表达发生了巨大变化。在存在乙醇或丁醇的存在下，该乙醇用于PLD的磷脂酰化反应以产生磷脂酰醇，以磷脂酸为代价，转录因子的表达抑制了转录因子，C-JUN和C-FOS对生长信号的响应，表明PLD是PLD是与生长信号转导密切相关。在分化的HL-60细胞和PC12细胞中研究了PLD的调节机制。磷脂酰肌醇3-激酶（PI-3K），Wortmannin和Ly294002的抑制剂显示，PI-3K位于分化的HL-60细胞中受体和PLD之间。此外，某种酪氨酸激酶可以在PC12细胞的下游，可能参与钙敏感的酪氨酸激酶参与卡尔巴乔诱导的PLD激活。过氧化氢（H_2O_2）在PC12细胞中极大地刺激了PLD活性。似乎还可以在H_2O_2刺激的PC12细胞中激活PLD，从而激活PLD。我们目前正在努力鉴定酪氨酸激酶在这些过程中涉及的酪氨酸激酶，并计划使用使用PLD基因转染的反义寡核苷酸和细胞进一步研究PLD在细胞反应中的作用。