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

在一系列研究PKC和DGK中负责脂肪酸结构域的高级结构域的实验中，我们首先试图确定PKC和DGK中负责花生四烯酸（AA）的结构域。aa诱导的质膜靶向DGKγ需要C1B结构域，而不是C1A结构域。同样，C1B结构域也参与了aa诱导的εPKC靶向高尔基复合体。另一方面，C1A或C1B都足以使花生四烯酸介导的γPKC在质膜上的保留。利用核磁共振，我们将DGKγ C1A或C1B肽与GST或MBP融合。最后，我们得到了约0.5 mg的MBP-DGKγ C1A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Xa处理，然后用Superose 6HR进行凝胶过滤。然而，我们无法获得足够数量的这两种肽。为了用抗flag抗体检测少量多肽，我们制备了MBP-DGKγ C1A-FLAG和MBP-DGKγ C1B-FLAG，然后我们发现DGKγ C1A-FLAG和dgk - γC1B-FLAG在空隙体积中被洗脱，表明多肽聚集。DGKγC1-FLAG、εPKCC1B-FLAG和εPKCC1-FLAG均有聚集。此外，我们没有检测到MBP-εPKCC1B和MBP- dgkγ C1A与磷酯的结合。这些结果表明mbp融合蛋白的高层结构并不完整。C1肽核磁共振的另一种方法，即HA标签或Baculo病毒系统，应考虑。

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.