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

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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.