Fig. 2

The E3 ubiquitin ligase MKRN1 ubiquitinates and degrades AMPKα subunits. a MKRN1 knockdown increases AMPKα1 protein levels. The HepG2 cells were transfected with MKRN1 siRNAs (#6 and #7). b MKRN1 knockout stabilises AMPKα. WT or MK1^−/− MEFs were treated with CHX (100 mg ml⁻¹) at the indicated time points. c, d MKRN1 expression promotes the proteasomal degradation of AMPKα subunits. The protein levels of ectopically expressed AMPK subunits were analysed using HEK293T cells. GFP was used as a transfection control (c). HepG2 cells were infected with retrovirus expressing MKRN1, followed by selection using puromycin. The cells were treated with 20 µM of MG132 for 6 h, and AMPKα1, α2, MKRN1 and actin were detected with the indicated antibodies (d). e MKRN1 induces both AMPKα1 and α2 ubiquitination. Constructs expressing FLAG/AMPKα1, α2, β1, γ1, 3.1/MKRN1 and HA/Ub were transfected into 293T cells. The ubiquitination assay was performed using cell lysates under denaturing conditions (in 1% SDS buffer). f, g MKRN1 directly ubiquitinates AMPKα subunits. In vitro ubiquitination of AMPKα1 (f) and α2 (g). h, i MKRN1 is required for the ubiquitination of AMPKα. Ubiquitinated endogenous AMPKα was determined under denaturing conditions using MG132-treated MEFs (h) and HepG2 cells (i). All the experiments with MEFs were conducted in cells within the first 3–6 passages. The data are representative of at least three independent experiments