Figure 1.
GST isoforms and GST-tag interact with full-length AMPK in pull-down assays.
Pull-down of recombinant AMPK 221WT with GST-Sj (S. japonicum), GSTM1 or GSTP1 (R. norvegicus). In all assays, AMPK (0.075 mg/ml) was incubated with or without (negative control) GST proteins (0.075 mg/ml). Pull-down with Glutathione Sepharose 4B was subjected to immunoblot analysis using anti-AMPKα antibody. Left: representative data; right: quantification (mean ± SD, n = 3; * p<0,01 and # p<0,05 versus no GST).
Figure 2.
Y2H analysis identifies GST isoforms as AMPK interaction partners and the AMPK interaction domain.
Two different cytosolic Y2H systems were applied to analyze interaction of AMPK with Schistosoma japonicum GST (GST-Sj) and mammalian (rat) GSTM1 and GSTP1. (A) Cyto-Y2H: interacting proteins lead to reconstitution of ubiquitin and a transcriptional readout allowing growth on medium lacking adenine and histidine (SD-AHWL). Spots represent yeast grown for 72 h at 30°C. (B) Split-Trp-Y2H: interacting proteins lead to reconstitution of Trp1p, an enzyme in tryptophan biosynthesis, and allow growth on medium lacking tryptophan (SD-UWL). Yeast was grown for 8–9 days at 27°C. Δβ, N-terminal domain of AMPK β-subunit. Controls: LT, Large T Antigen of Simian Virus (amino acids 84–704; negative control); GST, GST-Sj (positive control). A representative data set out of three independent experiments is shown. For more details see Materials and Methods and Supporting Information.
Figure 3.
AMPK interacts with endogenous GST isoforms in rat liver.
GST immunoprecipitation (A) or pull-downs (B, C) were performed with rat liver extract. AMPK in immunoprecipitates or pull-down fractions was detected by immunoblot analysis with anti-αAMPK antibody. The main liver GST isoforms in pull-down fractions were detected by Ponceau staining and mass spectroscopy. (A) Immunoprecipitation of endogenous AMPK by anti-GSTM1/2 or anti-GSTP1 antibodies. (B) GST pull-down of endogenous liver AMPK by liver GST isoforms in absence or presence of glutathione. Note: Addition of glutathione reduces pull-down of GSTA isoforms without affecting pull-down of AMPK. (C) GST pull-down of added AMPK 221WT or constitutively active 221TD. Left: representative data sets; right: quantification (mean ± SD, n = 3; * p<0,01 and # p<0,05 versus no GST (A), GSTM1 (B) or no extract (C)). Extr, liver extract.
Figure 4.
Surface plasmon resonance identifies high affinity interactions between GSTs and AMPK.
Freshly diluted, recombinant full-length AMPK was injected onto immobilized GST. (A) GST-Sj binding of 10 nM AMPK 221WT (black full line), constitutive active AMPK 221TD (grey full line) or BSA (grey dotted line) at a flow rate of 20 µl/min (surface: self-assembled monolayer). (B) Equilibrium response from (A), mean ± SD, 12 (221TD), 6 (221WT) or 3 (BSA) independent experiments (* p<0,01 versus control; § p<0,01 versus AMPK-TD). (C) Comparison of GSTM1 (black) or GSTP1 (grey) association and dissociation kinetics of 10 nM AMPK 221WT (full lines) or 100 nM of BSA (dotted lines) and a flow rate of 30 µl/min (surface: CM5). (D) GSTM1 association and dissociation kinetics of AMPK 221WT at different concentrations (dashed black lines) and a flow rate of 30 µl/min (surface: CM5), single exponential fit of experimental data (grey lines) and corresponding residuals (to assess the quality of the fit, lower panel). Representative sensorgrams of at least two repetitions are shown. Bars on the top of sensorgrams indicate protein injection (association, black) or injection of running buffer (white).
Figure 5.
AMPK 221WT (4 pmol) pre-activated by CamKKβ (1 pmol) does not phosphorylate GST-Sj and phosphorylates GSTM1 and -P1 only at low levels as compared to ACC (all at 200 pmol). In vitro phosphorylation assays were run for 3 min (GST-ACC), 30 min (GSTP1, GSTM1) or 60 min (GST-Sj) and analyzed by SDS-PAGE and Typhoon phosphoimager. Note the autophosphorylation of the AMPK β-subunit. Representative data with quantification are shown. Detailed phosphorylation kinetics is shown in Fig. S2.
Table 1.
Enzyme kinetic parameters of GSTP1 in presence or absence of AMPK or BSA.
Figure 6.
Glutathionylation of AMPK is facilitated by GST.
Glutathionylation assays were performed (A) with AMPK 221WT (1 μM) in absence or presence of GSTM1 or -P1 (0,5 μM) and 10 mM glutathione or (B) with AMPK 221WT (1 μM, additionally pre-reduced with ß-mercaptoethanol) in absence or presence of GSTM1 or -P1 (10 μM) and 0,1 mM glutathione. AMPK modification was detected either as a molecular mass shift of GST protein in SDS-PAGE (see arrows in Ponceau protein stain, “protein”) or by direct detection of glutathione by immunoblotting (“glutathionylation”). Note: As soon as glutathione is present, AMPK is almost quantitatively glutathionylated in (A), while additional presence of GST is needed for glutathionylation in (B). Left: representative data; right: quantification (mean, n = 2).
Figure 7.
AMPK glutathionylation does not affect its phosphorylation by CamKKβ, but increases phosphorylation of downstream substrate.
(A) GSTM1 or -P1 (62,5 pmol) were pre-incubated with AMPK 221WT (12.5 pmol) with or without 10 mM glutathione, in presence of ATP prior to addition of CamKKβ (0.63 pmol). Phosphorylation assays were subjected to immunoblot analysis using anti-P-T172-α AMPK antibody. (B) AMPK 221WT pre-activated with CamKKβ in kinase buffer with cold ATP and glutathionylated with 0,1 mM glutathione in presence or absence of GSTM1 or -P1, both as described above and in Fig. 6, were incubated with ACC (200 pmol) and [γ-32P]ATP. In vitro phosphorylation assays were analyzed by SDS-PAGE, Ponceau protein staining (lower panel) and Typhoon phosphoimager (upper panel) are shown. Left: representative data; right: quantification of lanes in presence of glutathione (mean ± SD, n = 4; * = p<0,01 versus no GST). A control experiment lacking CamKKβ is shown in Fig. S4. Note: AMPK autophosphorylation of α- and β-subunits.