Figure 4.

Physical interaction between PGC-1α and GABP requires phosphorylation and HCF. (A) BOSC cells were transfected with expression plasmids for Flag-tagged PGC-1α, myc-tagged GABPA and GABPB1, constitutively active MEKK (MEKK*), and HCF as indicated. Cells were harvested 48 h after transfection, 1 mg of total protein from the cell lysates was incubated with anti-myc beads, and the bound proteins as well as input (10 μg protein) were blotted with anti-Flag and anti-myc antibodies. (B) BOSC cells were transfected with expression plasmids for Flag-tagged PGC-1α, myc-tagged GABPA and GABPB1, the phosphorylation-deficient alleles Flag-PGC-1α 3A, myc-GABPA P-mut, and myc-GABPB1 P-mut, respectively, constitutively active MEKK (MEKK*), and HCF as indicated. Cells were harvested 48 h after transfection, 1 mg of total protein from the cell lysates was incubated with anti-myc beads, and the bound proteins as well as input (10 μg protein) were blotted with anti-Flag and anti-myc antibodies. (C) C2C12 myotubes were transfected with expression plasmids for Flag-tagged PGC-1α, myc-tagged GABPA and GABPB1, constitutively active MEKK (MEKK*), and HCF as indicated. The cells were differentiated for 2 d and simultaneously treated with 5 nM recombinant NRG-1. Cells were harvested, 1 mg of total protein from the cell lysates was incubated with anti-myc beads, and the bound proteins as well as input (10 μg protein) were blotted with anti-Flag and anti-myc antibodies. (D) GABPA and GABPB1 bind as heterotetramers and upon phosphorylation of GABPB1 recruit phosphorylated PGC-1α and HCF. These phosphorylation events are induced by NRG-1 signaling or constitutive MAPK activation.