Molecular mechanism of cellular response induced by dynamic metabolism of membrane phospholipid

最近的技术发展使我们能够使用针对特定脂质的探针来监测活细胞中脂质信使的产生、移动和信号传递。在这个项目中，我们使用不同的脂质信使探针进行了时空分析，以阐明在生理细胞反应中，脂质信号发生在细胞内的时间和地点。结果，我们获得了以下结果：1)吞噬过程中信号分子(蛋白激酶C亚型、Rac亚型、p47；lt；Phox；gt；、p40；lt；Phox&gt；)的实时成像。每个具有不同脂结合能力的分子在不同的时间段内转移到特定的细胞内。2)二酰甘油(DG)-PKC-DG激酶(DGK)通路的功能和分子相互作用。用实时成像技术证明了PKC和DGK之间的直接结合以及结合对每个酶活性的调节。PKC与质膜上的DGK结合，并在S776和S779处磷酸化DGK。然后，磷酸化的DGK被激活，并通过将DG转化为质膜上的PA来终止PKC途径。3)DGKGamma的核转位需要它的Cl域，而不是激酶的活性。4)突变型PKCγ在细胞内的聚集可能导致脊髓小脑型共济失调14型(SCA14)。

Recent technical development has enabled us to monitor the production, movement and signaling of lipid messengers in living cells using probes for specific lipids. In this project, we performed the spatio-temporal analysis using various probes for lipid messengers to elucidate when and where the lipid signaling occurs within the cells in physiological cellular responses. As a result, we obtained the results below.1) Live imaging of signaling molecules (Protein kinase C (PKC) subtypes, Rac subtypes, p47^<Phox>, p40^<Phox>) in phagocytosis. Each molecule having different lipid binding ability translocated to the specific intracellular compartment in different time course. The finely tuned control of targeting mechanism of signaling molecules to lipid regulate the superoxide production in phagocytosis.2) Functional and molecular interaction of Diacylglycerol (DG)-PKC-DG kinase (DGK) pathway. Direct binding between PKC and DGK and the regulation of each enzymatic activity by the binding were demonstrated using live-imaging techniques. PKC binds to DGK on the plasma membrane and phosphorylates DGK at S776and S779. The phosphorylated DGK is then activated and terminates PKC pathway by convert DG to PA on the plasma membrane.3) Nuclear translocation of DGKgamma requires its Cl domain but not the kinase activity. Nuclear DGKgamma controls cell cycle.4) Aggregation of mutant PKCgamma within cells may cause Spinocerebellar ataxia type 14 (SCA14).