Site Directed Mutagenesis of PPARalpha Phosphorylation Sites S6, S12 and S21

Peroxisome Proliferator Activated Receptor alpha regulates transcription of genes involved in fat metabolism. In addition to ligand activation by fatty acids, eicosanoids and anti‐lipidemic agents, phosphorylation of PPARα can affect its activity. Phosphorylation at serines 6, 12 and 21 in the N‐terminal A/B domain in vivo is insulin‐dependent. Previous work in our laboratory demonstrated that treatment with the phosphatase inhibitor okadaic acid decreased the ability of transfected PPARα to activate a PPRE‐containing luciferase reporter in HepG2 cells, and treatment with DHEA, an activator of PPARα, resulted in decreased phosphorylation at S12 and S21. To further investigate this, we generated S6A, S12A or S21A mutants either alone or in combination. The negative effect of okadaic acid on luciferase activity was not completely eliminated by any of the modifications. However, the S21A mutant had transcriptional activity equal to or greater than that of wt PPARα, while the S12A mutant had decreased ability to stimulate promoter‐luciferase activity. We also generated S12D and S21D mutants of PPARαeither alone or in combination. These mutants presumably mimic permanent phosphorylation at their respective sites. The S21D mutant either alone or in combination with S12D had lower activity than wt PPARα. The S12D mutant alone had an activity intermediate between the wild type and S21D PPARα. These results suggest the potential importance of S21 phosphorylation in the transcriptional activity of PPARα. Supported in part by USPHS grant 1 R01 DK 54774.