Figure 3.

p160^MBP's interaction with PGC-1α is modulated by p38 MAPK phosphorylation. (A) GST-PGC1(200-400) was phosphorylated in vitro by activated p38 MAPK in the presence of ATP [GST-PGC1(200-400)-P] or mock phosphorylated in the absence of ATP [GST-PGC1(200-400)]. The GST proteins were then incubated with C2C12 nuclear extracts and interacting proteins were visualized by Western blot by using antibodies against p160^MBP or against cdc16 (Santa Cruz Biotechnology). (B) Flag-tagged PGC-1α (f:PGC1α) was cotransfected with myc-tagged p160^MBP (myc-p160^MBP) in BOSC cells and after 24 h treated with the cytokines IL-1α, IL-1β, and TNFα overnight in DMEM + 0.5% BSA. Whole-cell extracts were immunoprecipitated with M2 anti-Flag antibodies (Sigma) and Western blots were conducted with antibodies against myc or PGC-1α (Santa Cruz Biotechnology). The left panel is a Western blot of 5% of the whole-cell extract before immunoprecipitation and the right panel is a Western blot of the immunoprecipitated proteins. (C) Gal4-PGC1α was cotransfected in C2C12 cells with a UAS-luciferase reporter and a vector expressing MKK6, the upstream activator of p38 MAPK. Medium was changed to DMEM + 0.5% BSA after 24 h, and luciferase activity was measured after 48 h. (Asterisks) P < 0.05, paired t-test. (D) Myc-p160^MBP was cotransfected with f:PGC1α, f:PGC1α with the phosphorylation sites mutated to aspartic acid (f:PGC1-3D), or f:PGC1α with selected leucines at L2 and L3 mutated to alanine (f:PGC1-L2/3A). Extracts were immunoprecipitated with anti-Flag antibodies as described earlier. (E) PGC-1α or PGC1-3D were cotransfected with PPARγ, RXR, and DR-1 luciferase reporter in HIB1B cells. Cells were harvested after 24 h and fold activation over PPARγ was graphed. (Asterisks) P < 0.05, paired t-test. (F) Gal4-PGC1 or Gal4-PGC1-L2/3A were cotransfected with a UAS-TATA-luciferase reporter in C2C12 cells, and luciferase activity was measured after 24 h. (Asterisks) P < 0.05, paired t-test.