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

在研究PKC和DGK中负责脂肪酸的结构域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。C1 B结构域是AA诱导DGKγ靶向细胞膜所必需的，而C1 A结构域则不是必需的。同样，C1 B结构域参与AA诱导εPKC靶向高尔基复合体。另一方面，C1 A或C1 B足以使花生四烯酸介导的γPKC滞留在质膜上。利用核磁共振技术，将DGKγ C1 A或C1 B肽分别与GST或MBP融合。最终得到约0.5mg的MBP-DGKγ C1 A或MBP-DGKγ C1 B，而仅得到少量的GST-DGKγ C1 A和GST-DGKγ C1 B。因此，用蛋白酶因子Xa处理MBP-DGKγ C1 A和MBP-DGK γ C1 B，然后使用Superose 6 HR进行凝胶过滤。然而，我们不能获得足够量的两种肽。为了用抗FLAG抗体监测小肽的含量，我们制备了MBP-DGKγ C1 A-FLAG和MBP-DGKγ C1 B-FLAG，发现DGKγ C1 A-FLAG和DGKγ C1 B-FLAG都在空体积中被洗脱，表明小肽聚集。DGKγC1-FLAG、ε PKCC 1 B-FLAG和ε PKCC 1-FLAG均可见聚集。此外，未检测到MBP-ε PKCC 1B和MBP-DGKγ C1 A与佛波酯的结合。这些结果表明，MBP融合蛋白的高级结构不是完整的。用于NMR的C1肽的另一种方法，即，HA标签或Baculo病毒系统。

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.