Fig. 7: Dephosphorylation of β1-pS182 and β2-pS184 is not a prerequisite for nuclear AMPK transport but for β2 promotes cell proliferation under conditions of amino acid stress.

Cytosolic and nuclear fractions were prepared from α1β2γ1-, α2β1γ1- or α2β2γ1-transfected HEK293T cells incubated in complete media. A Stoichiometries of β2-pS184 on FLAG-α1β2γ1 in cytosolic and nuclear fractions determined by LC-MS using peptide and phosphopeptide area under the curve. Data presented as mean % stoichiometry ± SEM, n = 3. Statistical analyses were performed by unpaired t test. GST-tagged (GST-α) (B) α2β1γ1- and (C) α2β2γ1-expressing HEK293T cells were incubated with torin1 (250 nM) for up to 24 h, cytosolic and nuclear fractions were immunoblotted as indicated. D Data from (B) and (C) are presented as relative AMPK β content (nucleus:cytosol ratio) ± SEM, n = 4. Statistical analyses were performed by unpaired t tests vs. basal (β1 vs. β2, **P < 0.01; 0 h vs. 24 h, ns not significant). Real-time proliferation analysis of HEK293 cells expressing FLAG-fusions of either (E) α2β2 (WT or β2-S184A/E mutants) or (F) α2β1 (WT or β1-S182A/E mutants), after switching to arginine- and lysine-free (−Arg/Lys) medium from complete growth medium. Data presented as mean % confluence ± SEM, n = 6. Statistical analyses at each time point were performed by 2-way ANOVA vs. respective WT. *P < 0.05, **P < 0.01. Validation of HEK293T β1-S182A and β2-S184A KI cell lines by (G) DNA sequencing, and (H) immunoblot. I Nuclear and cytosolic fractions were prepared from WT, β1-S182A KI and β2-S184A KI HEK293T cells and immunoblotted as indicated. Data presented as mean β1 or β2 signal (a.u. arbitrary units) ± SEM, n = 3. Statistical analyses were performed by one-way ANOVA with Dunnett’s multiple-comparisons test. *P < 0.05, ****P < 0.0001. J Adenine nucleotides were PCA-extracted from WT and β2-S184A KI HEK293T cells incubated in complete growth media and quantified by LC-MS/MS. AMP/ATP and ADP/ATP ratios are depicted here, ± SEM, n = 3. Statistical analyses were performed by one-way ANOVA with Dunnett’s multiple-comparisons test. n.s. not significant. K Data from (I) are presented as mean α-pT172/α (a.u. arbitrary units) ± SEM, n = 2. Statistical analyses were performed by one-way ANOVA with Dunnett’s multiple-comparisons test. n.s. not significant. L Cytosolic and nuclear fractions were prepared from β1/2-dKO immortalised MEFs expressing β2 WT or S184A mutant, and activities of FLAG-immunoprecipitated AMPK measured by radiolabelling of SAMS synthetic peptide. Data presented as fold change in mean AMPK specific activity vs. cytosolic WT ± SEM, n = 4. Statistical analyses were performed by 2-way ANOVA vs. WT nuclear activity. ****P < 0.0001. Representative immunoblots from three independent experiments are shown.