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Discovery and analysis of dovel lipid signal transduction complexes

Dovel脂质信号转导复合物的发现和分析

基本信息

批准号：
18590274
负责人：
KANOH Hideo
金额：
$ 2.49万
依托单位：
Sapporo Medical University
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-18590274/
关键词：
Diacylglycerol kinase Phosphatidic acid phosphatase Melanoma Apoptosis NF-κB Racl β2-Chimaerin Lipid phosphate phosphatase ラフト

项目摘要

Diacylglycerol (DAG) kinase (DGK) modulates the balance between the two signaling lipids, DAG and phosphatidic acid (PA), by phosphorylating DAG to yield PA. To date, ten mammalian DGK isozymes have been identified. Beyond our expectations, recent studies have revealed that DGK isozymes play pivotal roles in a wide variety of signal transduction pathways.Recently, we found that DGKa is expressed in several human melanoma cell lines but not in noncancerous melanocytes. Intriguingly, the overexpression of wild-type DGKα, but not of its kinase-dead mutant, markedly suppressed tumor necrosis factor-a-induced apoptosis of AKI human melanoma cells. In the reverse experiment, small interfering RNA-mediated knockdown of DGKα significantly enhanced the apoptosis, thus convincingly indicating that this isoform possesses a cytoprotective function. Moreover, we obtained several lines of evidence suggesting that DGKα suppresses tumor necrosis factor-a-induced melanoma cell apoptosis through activat … More ion of nuclear factor κB. Thus, this transcription factor is likely to be one of the key factors downstream of DGKα during cell proliferation and survival.We had already demonstrated that DGKγ functions through its catalytic action as an upstream suppressor of Racl, and consequently, lamellipodium/membrane ruffle formation. Recently, we further found that DGKγ specifically interacts with and activates β2-chimaerin, a Rac-specific GTPase-activating protein, in response to cell stimulation with epidermal growth factor and hydrogen peroxide/phorbol 12-myristate 13-acetate (PMA). PMA was found to be mainly required for a conversion of 82-chimaerin to an active form. On the other hand, H_2O_2 was suggested to induce a release of Zn^<2+> from the Cl domain of β2-chimaerin. By stepwise deletion analysis, we demonstrated that the Src homology 2 (SH2) and Cl domains of β2-chimaerin interacted with the N-terminal half of catalytic region of DGKγ. Taken together, these results suggest that the functional link between DGKγ and β2-chimaerin (and Racl) has a broad significance in response to a wide range of cell stimuli. Our work offers a novel mechanism of protein-protein interaction, that is, the phosphotyrosine-independent interaction of the SH2 domain acting in cooperation with the Cl domain.Lipid phosphate phosphatases (LPPs, type 2 phosphatidic acid phosphatases), integral membrane proteins with six transmembrane domains, dephosphorylate a variety of extracellular lipid phosphates. Although LPP3 is already known to bind to Triton X-100-insoluble rafts, we found that LPP1 is also associated with lipid rafts (Triton X-100-soluble but CHAPS-insoluble) distinct from those harboring LPP3. LPP1 and LPP3 are distributed in distinct lipid rafts that may provide unique microenvironments defining their non-redundant physiological functions. Less

二酰基甘油(DAG)激酶(DGK)通过使DAG磷酸化而生成PA，从而调节DAG和PA两种信号脂之间的平衡。到目前为止，已鉴定出10种哺乳动物DGK同工酶。最近的研究表明，DGK同工酶在多种信号转导途径中发挥着关键作用。最近，我们发现DGKA在几个人黑色素瘤细胞系中都有表达，但在非癌性黑素细胞中没有表达。有趣的是，野生型DGKα的过表达，而不是其激酶死亡突变体的过表达，显著抑制了肿瘤坏死因子-a诱导的人黑色素瘤细胞的凋亡。在反向实验中，小干扰RNA介导的DGKα的敲除显著促进了细胞的凋亡，从而有力地表明该异构体具有细胞保护功能。此外，我们还获得了一系列证据，表明dgkα通过激活…抑制肿瘤坏死因子-a诱导的黑色素瘤细胞的凋亡。因此，该转录因子可能是Dgkα下游在细胞增殖和存活过程中的关键因子之一。我们已经证明了dgkγ的功能是通过其上游抑制因子Racl的作用而发挥作用的，从而导致片层/膜褶皱的形成。最近，我们进一步发现，DGKγ在表皮生长因子和过氧化氢/佛波醇12-肉豆蔻酸13-醋酸酯刺激的细胞刺激下，与β2-嵌合体特异地相互作用并激活GTP酶激活蛋白。研究发现，PMA主要是82-嵌合体转化为活性形式所必需的。另一方面，H_2O_2可诱导β_2-嵌合体Cl域中的锌离子释放。通过逐步缺失分析，我们证明了DGK2-嵌合体的Src同源2(SH2)和Cl域与βγ催化区的N-末端部分相互作用。综上所述，这些结果表明，DGKγ和β2-嵌合体(和Racl)之间的功能联系在响应广泛的细胞刺激方面具有广泛的意义。我们的工作提供了一种新的蛋白质-蛋白质相互作用机制，即SH2结构域与Cl域协同作用的不依赖磷酸酪氨酸的相互作用。脂磷酸盐磷酸酶(LPPs)是具有六个跨膜结构域的完整膜蛋白，它能使多种胞外脂磷酸盐去磷酸化。虽然已知LPP3与Triton X-100不溶的脂筏结合，但我们发现LPP1也与脂筏(Triton X-100可溶，但CHAPS不溶)有关，与含有LPP3的脂筏不同。LPP1和LPP3分布在不同的脂筏中，可能提供了独特的微环境来定义它们的非冗余生理功能。较少