Fig. 4.

AMPK phosphorylates PGC-1α at threonine-177 and serine-538 in vitro and in cells. (A) PGC-1α interacts with AMPKα2 in cells. Expression vectors for FLAG-PGC-1α and Myc-AMPKα2 were transfected into BOSC cells, as indicated. Coimmunoprecipitation was performed as described in Materials and Methods. (B) Primary PGC-1α −/− myotubes stably expressing PGC-1α and PGC-1α T177A/S538A, respectively, were treated with vehicle or AICAR for 1 h in the presence of ³²P. (C) Purified recombinant GST-PGC-1α fragments (full-length 1–797, 1–190, 200–400, 395–565, 551–797) were incubated with purified AMPK, and phosphorylation was determined by incorporation of [γ-³²P]ATP. (D) Mass spectrometry identified threonine-177 and serine-538 as phosphorylated residues. The GST-PGC-1α fragment containing amino acid 1–190 T177A and the GST-PGC-1α fragment containing amino acids 395–565 S538A are not phosphorylated by AMPK. (E) The phosphorylation of PGC-1α protein by AMPK is required for elevated PGC-1α-dependent activity of the PGC-1α promoter. C2C12 muscle cells were transfected with a 2-kb PGC-1α promoter construct and expression plasmids for PGC-1α or PGC-1α T177A/S538A, respectively. After transfection, cells were differentiated for 1 day and treated with AICAR for 7.5 h before reporter gene levels were determined (∗, P < 0.01).