Figure 6.

AMPK phosphorylates human GR at serine 211 (232 of rat GR) through p38 MAPKα and modulates GR-induced transcriptional activity by changing the attraction of coregulators to DNA-bound GR. A, Replacement of human GR serine 211 by alanine abolishes AICAR-induced enhancement of GR transcriptional activity in CV-1 cells. CV-1 cells were transfected with the plasmid expressing wild-type (WT) GR or the GR mutant defective in serine 211, together with pMMTV-Luc (left panel) or pPEPCK-Luc (right panel), and pSV40-β-Gal. They were then incubated with 2 mm of AICAR in the presence or absence of 10⁻⁶ m of dexamethasone (Dex). Bars represent mean ± se values of the luciferase activity normalized for the β-galactosidase activity in the presence or absence of dexamethasone. *, P < 0.01, n.s., not significant, compared with the condition indicated. B, Replacement of human GR serine 211 by alanine blunts AICAR-induced regulation of GR transcriptional activity in HepG2 cells. HepG2 cells were transfected with the plasmid expressing wild-type GR or the GR mutant defective in serine 211, together with pMMTV-Luc (left panel) or pPEPCK-Luc (right panel), and pSV40-β-Gal. They were then incubated with 2 mm of AICAR in the presence or absence of 10⁻⁶ m of dexamethasone. Bars represent mean ± se values of the luciferase activity normalized for the β-galactosidase activity in the presence or absence of dexamethasone. The numbers above comparison lines indicate the percent increase of the transcriptional activity between the two conditions indicated. C, AMPK phosphorylates human GR at serine 211 through the phosphorylation/activation of p38 MAPKα in HepG2 cells. The HepG2 cells were treated with 2 mm of AICAR, 2 μm of SB202190 (p38 MAPK inhibitor), and/or 40 μm of compound C (AMPK inhibitor) in the presence or absence of 10⁻⁶ m of dexamethasone. Phosphorylated forms and entire proteins of the GR, p38 MAPKα, and the AMPKα catalytic subunit were examined in Western blots using their specific antibodies. D, AICAR phosphorylates rat GR at serine 232 by activating AMPK and p38 MAPKα in rat liver. Rats were injected with AICAR (0.7 g/kg per animal) and/or dexamethasone (1.5 mg/kg), and their livers were harvested after 2 h. Whole homogenates and nuclear extracts were prepared, and the phosphorylated forms and entire proteins of GR, p38 MAPKα, and the AMPK α-subunit were examined in Western blots using their specific antibodies. The kinase activity of the p38 MAPKα was also examined by using ATF-2 as a substrate (bottom gel). Samples from two rats in each treatment were run on the gels.