Analysis of higher structure of fatty aid -responsible domains in PKC and DGK

PKC 和 DGK 中脂肪辅助责任域的高级结构分析

基本信息

批准号：
15570115
负责人：
SHIRAI Yasuhito
金额：
$ 2.37万
依托单位：
Kobe University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2004
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15570115/
关键词：
Protein kinase C Diacylglycerol kinase C1 domain NMR Fatty acid Lipid messenger Ceramide PKC DGK

项目摘要

In the series of the experiments to study higher structure of domains responsible for fatty acids in PKC and DGK we first tried to identify the domains responsible for arachidonic acid (AA) in PKC and DGK. The C1B, but not C1A, domain was necessary for AA-induced plasma membrane targeting of DGKγ. Similarly, the C1B domain was involved in the AA-induced targeting of εPKC to the Golgi complex. On the other hand, either C1A or C1B was enough for arachidonic acid-mediated retention of γPKC on the plasma membrane. To use NMR, we made fusion protein of DGKγ C1A or C1B peptide to GST or MBP. Finally, we obtained about 0.5 mg of MBP-DGKγ C1A or MBP-DGKγ C1B but got only small amount of GST-DGKγ C1A and GST-DGKγ C1B. Therefore, MBP-DGKγ C1A and MBP- DGKγ C1B were treated with a protease, Factor Xa, and then applied to gel filtration using Superose 6HR. However, we could not obtain enough amount of both peptides. To monitor small amount of the peptides by anti-FLAG antibody, we produced MBP-DGKγ C1A-FLAG and MBP-DGKγ C1B-FLAG, and then we found both DGKγ C1A-FLAG and DGKγC1B-FLAG were eluted in the void volume, suggesting the peptides aggregated. The aggregation was seen in the cases of DGKγC1-FLAG, εPKCC1B-FLAG and εPKCC1-FLAG. In addition, we could not detect the binding of MBP-εPKCC1B and MBP-DGKγ C1A to phorbol ester. These results suggest that higher structure of the MBP-fusion proteins are not intact. Another approaches to the C1 peptides for NMR, i.e., HA tag or Baculo virus system, should be considered.

在研究 PKC 和 DGK 中负责脂肪酸的高级结构的一系列实验中，我们首先尝试鉴定 PKC 和 DGK 中负责花生四烯酸 (AA) 的结构域，C1B 结构域是必需的，但 C1A 结构域不是必需的。 AA 诱导的质膜靶向 DGKγ 类似，C1B 结构域参与 AA 诱导的 εPKC 靶向高尔基复合体。足以使花生四烯酸介导的γPKC保留在质膜上。为了使用NMR，我们制备了DGKγC1A或C1B肽与GST或MBP的融合蛋白，最后，我们获得了约0.5mg的MBP-DGKγC1A或MBP-DGKγ。 C1B，但只得到少量的GST-DGKγ C1A和GST-DGKγ C1B，因此，MBP-DGKγ。 C1A 和 MBP-DGKγ C1B 用蛋白酶、因子 Xa 处理，然后使用 Superose 6HR 进行凝胶过滤，但是，我们无法获得足够量的两种肽，以通过抗 FLAG 抗体监测少量肽。我们制作了MBP-DGKγ C1A-FLAG和MBP-DGKγ C1B-FLAG，然后我们发现DGKγ C1A-FLAG和DGKγC1B-FLAG 在空隙体积中洗脱，这表明在 DGKγC1-FLAG、εPKCC1B-FLAG 和 εPKCC1-FLAG 的情况下观察到肽聚集。此外，我们无法检测到 MBP-εPKCC1B 和 MBP 的结合。 -DGKγ C1A 至佛波酯这些结果表明 MBP 融合蛋白的高级结构并非如此。应考虑用于 NMR 的 C1 肽的另一种方法，即 HA 标签或杆状病毒系统。