FIG 6.

Metformin or salicylate activates p53 mainly through AMPK-mediated MDMX phosphorylation and binding to 14-3-3 in vitro and in vivo. (A) Both metformin and salicylate activate AMPK and p53 in a dose-dependent manner. HCT116 colon cancer cells were treated with 10 mM metformin or 10 mM salicylate and harvested at the indicated time points. Forty micrograms of protein was loaded into each lane to measure the expression level and activation of AMPK, p53, and their targets. (B) Metformin or salicylate remarkably enhanced the binding of endogenous MDMX to 14-3-3γ in HCT116 cells treated with metformin or salicylate for 12 h. Seven hundred micrograms of total protein per sample was incubated with 2 μg of anti-14-3-3γ (Ab-2) for 4 h at 4°C. (C) Metformin increases p53 level and activity by activating AMPK and enhancing the binding of MDMX to 14-3-3γ in WT but not MDMX-3SA MEFs. Both cell lines were treated with 10 mM metformin for 12 h. Forty micrograms of total protein per sample was used for Western blot analysis and 1.2 mg of total protein for co-IP experiments. For IP analysis, lysates were incubated with 6 μg of anti-MDMX (D-19) for 4 h at 4°C. (D) Salicylate activates p53 mainly through AMPK-mediated MDMX phosphorylation and binding to 14-3-3γ in mouse livers. WT or MDMX-3SA mice were treated with salicylate for 1.5 h, and liver tissues were harvested for WB and co-IP analysis, as described in Materials and Methods. The conditions for WB and co-IP analysis were similar to those described for panel C. (E) A model demonstrates that some metabolic stresses activate p53 by AMPK phosphorylating MDMX at S342, further inducing the binding of 14-3-3 to phosphorylated MDMX, which, in turn, inhibits the activity of MDMX.