Extended Data Fig. 3: LCA triggers the lysosomal AMPK pathway through deacetylation of v-ATPase.
From: Lithocholic acid binds TULP3 to activate sirtuins and AMPK to slow down ageing
a, Deacetylation is required for LCA-induced activation of AMPK. MEFs were pre-treated with 2 μM TSA and 8 mM NAM for 12 h, followed by incubating with 1 μM LCA for 4 h. Cells were then lysed, and the AMPK activities were determined by immunoblotting. b-e, V1E1-3KR renders the lysosomal AMPK pathway constitutively active. HEK293T with ectopic expression of V1E1, V1E1-3KR (d; triple mutation of K52, K99 and K191 of V1E1 to arginine; mimicking the deacetylated state of V1E1), or other single K to R mutations (b) of V1E1, or MEFs with stable expression of single, double, or triple mutation of K52, K99 and K191 residues (c) were lysed, followed by determination of the activity of AMPK (b, in which the statistical analysis results are shown as mean ± s.e.m.; n = 4 replicates for each treatment, and P value by two-sided Student’s t-test; and d) or the expression levels of V1E1 (c, e). f, V1E1-3KQ impairs the LCA-induced lysosomal translocation of AXIN and v-ATPase inhibition. MEFs with stable expression of V1E1-3KQ, mimicking the constitutively acetylated states of V1E1, were treated with 1 μM LCA for 4 h, followed by determination of the activity of v-ATPase (upper panel), and the lysosomal localization of AXIN (lower panel). Statistical analysis data are shown as mean ± s.e.m.; n = 23 (right panel, LCA group) or 22 (others) cells for each treatment, and P value by two-sided Student’s t-test. g-j, V1E1-3KR activates AMPK through the lysosomal pathway. LAMTOR1−/− (g-i) and AXIN−/− (j) MEFs, and control MEFs, were infected with lentivirus carrying HA-tagged V1E1-3KR, followed by determination for AMPK activation (g, j), activity of v-ATPase (h), and the lysosomal localization of AXIN (i). Statistical analysis data in h, i are shown as mean ± s.e.m.; n = 20 (h), 22 (V1E1 of i) or 23 (V1E1-3KR of i) cells, and P value by two-sided Student’s t-test. k, Acetylation does not affect ubiquitination of V1E1. HEK293T cells were transfected with HA-tagged V1E1 or V1E1-3KR, along with FLAG-tagged Ub. At 12 h post-transfection, cells were treated with 20 nM MG-132 for another 12 h, and the ubiquitination levels of V1E1 were determined by immunoprecipitation of HA-tag, followed by immunoblotting (left panel). See also the right panel for the AMPK activation in MEFs after treatment of 20 nM MG-132 for 12 h and 1 μM LCA for 4 h. l, Validation of the antibody against K99-acetylated V1E1. HEK293T cells were transfected with V1E1-K99R, or V1E1-K52R, V1E1-K191R and the wildtype V1E1 as controls, followed by determination of the reaction specificity of the Ac-K99-V1E1 antibody by immunoblotting. Experiments in this figure were performed three times, except b four times.
