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Analysis of intracellular regulatory signaling for apoptosis induction

诱导细胞凋亡的细胞内调节信号分析

基本信息

批准号：
11480208
负责人：
YONEHARA Shin
金额：
$ 8.77万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B).
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-11480208/
关键词：
Fas cell death apoptosis Ras MAP kinase caspase FLASH Tax アデノウイルスE1B

项目摘要

We analyzed molecular mechanisms of intracellular signaling of Fas-mediated apoptosis and its regulation.1. We cloned a cDNA encoding a novel 220 kDa protein, designated FLASH, that interacts with the death effector domain (DED) of either caspase-8 or FADD through its DED-like region. Association of Fas and FLASH was observed following stimulation of Fas, indicating that FLASH is a component of DISC.Transient expression of FLASH promoted the activation of caspase-8.2. By an expression cloning method using Fas-transgenic Balb3T3 cells, we identified proto-oncogene c-k-ras to inhibit Fas-mediated apoptosis. Transient expression of K-Ras mutants revealed that oncogenic mutant K-Ras (RasV12) strongly inhibited, whereas dominant-inhibitory mutant K-Ras (RasN17) enhanced, Fas-mediated apoptosis by inhibitinz Fas-triggered activation of caspases without affecting an expression level of Fas. Among the target molecules of Ras, only constitutive active form of Raf could inhibit Fas-mediated apoptosis. Further analyses clearly indicate that the activation of MAPK and then CREB through Ras inhibits Fas-mediated apoptosis in Balb3T3 cells, which may play a role in oncogenesis.3. p40Tax, an oncogene product of HTLV-1, was shown to inhibit Fas-mediated apoptosis in Balb3T3 cells through activating CREB by analyses with various mutants of Tax and dominant-negative CREB.Surprisingly, in T lymphoma cell line, p40Tax inhibits Fas-mediated apoptosis through activating NF-kB but not CREB.Thus, p40Tax negatively regulates Fas-mediated apoptosis by different molecular mechanisms (activation of CREB or NF-kB) in different cells.

我们分析了fas介导的细胞内信号转导及其调控的分子机制。我们克隆了一个编码新的220 kDa蛋白的cDNA，该蛋白被命名为FLASH，通过其DED样区与caspase-8或FADD的死亡效应域（DED）相互作用。Fas刺激后观察到Fas与FLASH的关联，表明FLASH是DISC的一个组成部分。FLASH的瞬时表达促进了caspase-8.2的激活。通过fas转基因Balb3T3细胞的表达克隆方法，我们鉴定出原癌基因c-k-ras抑制fas介导的细胞凋亡。瞬时表达的K-Ras突变体表明，致癌性突变体K-Ras （RasV12）强烈抑制Fas介导的凋亡，而显性抑制突变体K-Ras （RasN17）通过抑制Fas触发的caspase的激活而增强Fas介导的凋亡，而不影响Fas的表达水平。在Ras的靶分子中，只有组成型活性Raf能够抑制fas介导的细胞凋亡。进一步分析清楚地表明，通过Ras激活MAPK再激活CREB可抑制fas介导的Balb3T3细胞凋亡，这可能在肿瘤发生中发挥作用。HTLV-1的致癌基因产物p40Tax通过激活CREB在Balb3T3细胞中抑制fas介导的凋亡，通过对各种突变的Tax和显性阴性CREB进行分析。令人惊讶的是，在T淋巴瘤细胞系中，p40Tax通过激活NF-kB抑制fas介导的细胞凋亡，而不是通过激活CREB。因此，p40Tax在不同细胞中通过不同的分子机制（激活CREB或NF-kB）负向调节fas介导的细胞凋亡。