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中负责脂肪酸的较高结构的一系列实验中，我们首先试图鉴定PKC和DGK中负责蛛网膜酸（AA）的域。 C1b而不是C1a，对于AA诱导的DGKγ靶向质膜是必需的。同样，C1b结构域参与了AA诱导的εpkc靶向Golgi复合物。另一方面，C1A或C1B足以用于蛛网膜酸介导的γPKC在质膜上的保留。要使用NMR，我们将DGKγC1A或C1B肽的融合蛋白融合到GST或MBP。最后，我们获得了约0.5 mg的MBP-DGKγC1A或MBP-DGKγC1B，但仅获得了少量的GST-DGKγC1A和GST-DGKγC1B。因此，用蛋白质Xa处理MBP-DGKγC1A和MBP-DGKγC1B，然后使用Superose 6hr应用于凝胶过滤。但是，我们无法获得足够数量的两个宠物。为了通过抗FLAG抗体监测少量的Petides，我们产生了MBP-DGKγC1A-FLAG和MBP-DGKγC1B-FLAG，然后我们发现DGKγC1A-FLAG和DGKγC1B-FLAG在Void中洗脱了，这表明了Petiess的聚集。在DGKγC1-FLAG，εpkcc1b-fag和εpkcc1-Flag的情况下可以看到聚集。另外，我们无法检测到MBP-εpkcc1b和Mbp-DGKγC1A与佛波尔酯的结合。这些结果表明，MBP融合蛋白的较高结构不是完整的。应考虑用于NMR的C1肽的另一种方法，即HA TAG或BACULO病毒系统。