Figure 8.

Threonine-129 (T129) and serine-163 (S163) of PPARα are phosphorylated by PKA. (A) Two synthetic peptides containing residues 123 through 135 and 157 through 169 of PPARα were phosphorylated by using 1 U of PKA-Cα in the presence of [γ-³²P]-ATP. Some phosphorylated proteins were dephosphorylated by calf alkaline phosphatase to indicate the covalent binding of ³²P to threonine or serine. (B) The mutated short peptides with their PKA phosphorylation sites replaced by alanine were incubated with PKA and [γ-³²P]-ATP in phosphorylation buffer. (C) Western blot of immunoprecipitated nuclear PPARα from wild or mutated PPARα expression plasmid transfected HEK293 cells. (D) EMSA using extracts from wild-type or mutated PPARα expression plasmid transfected HEK293 cells. The PPRE bindings were observed in wild-type, Ser⁹³A and Thr¹²⁹A, but not in Ser¹⁶³A and Thr¹²⁹A/Ser¹⁶³A cells. (E) Luciferase activity in HEK293 cells transfected with reporter plasmid (0.5 µg pGL3-SV40-3PPRE with 0.01 µg pRL-CMV as control) and either wild-type or mutated PPARα expression plasmid (0.1 µg) and 0.1 µg pSG5-RXRα plasmids. Cells were treated with forskolin + bilobetin (both 1 µmol·L⁻¹) or fenofibrate acid (1 µmol·L⁻¹) for 4 h. Data are means ± SEM. n= 3. ^aP < 0.01 versus pSG5-Ser⁹³APPARα transfected cells, ^bP < 0.01 versus pSG5-Thr¹²⁹APPARα-transfected cells (anova).