Inhibition of AMPK catabolic action by GSK3

Abstract

AMP-activated protein kinase (AMPK) regulates cellular energy homeostasis by inhibiting anabolic and activating catabolic processes. While AMPK activation has been extensively studied, mechanisms that inhibit AMPK remain elusive. Here we report that glycogen synthase kinase 3 (GSK3) inhibits AMPK function. GSK3 forms a stable complex with AMPK through interactions with the AMPK β regulatory subunit and phosphorylates the AMPK α catalytic subunit. This phosphorylation enhances the accessibility of the activation loop of the α subunit to phosphatases, thereby inhibiting AMPK kinase activity. Surprisingly, PI3K-Akt signaling, which is a major anabolic signaling and normally inhibits GSK3 activity, promotes GSK3 phosphorylation and inhibition of AMPK, thus revealing how AMPK senses anabolic environments in addition to cellular energy levels. Consistently, disrupting GSK3 function within the AMPK complex sustains higher AMPK activity and cellular catabolic processes even under anabolic conditions, indicating that GSK3 acts as a critical sensor for anabolic signaling to regulate AMPK.

Figure 1. GSK3 associates with the AMPK heterotrimeric kinase complex through the β regulatory subunit

(A) Endogenous AMPK α1 or α2 subunit co-IPs endogenous GSK3β in HEK293T cells. (B) All three AMPK subunits co-IP with GSK3β in HEK293T cells. (C) AMPK α1/α2 subunits are dispensable for the interaction between GSK3β and the β/γ heterodimer. HA- GSK3β sufficiently co-IPs with Flag-AMPK β1 subunit in AMPK α1/2 double knockout MEF cells. (D) AMPK α1 and γ1 subunits are dispensable for the interaction of GSK3β with the β/γ and α/β heterodimers, respectively. His-tagged AMPK β1 subunit and the indicated AMPK subunits were expressed with GSK3β in Sf9 insect cells. GSK3β co-purifies with the His-β1 AMPK subunit (Elution) in the absence of the α1 orγ 1 subunit expression. See also Figures S1.

Figure 2. AMPK α subunit is an atypical substrate of GSK3

(A) Location and sequence conservation of the ST-stretch of the αsubunit. (B) GSK3β-induced phosphorylation of the α1 subunit is largely abolished in T479A α1 mutant in HEK293T cells. The mobility shift was monitored by Phos-Tag^™ acrylamide gel electrophoresis. (C) GSK3β phosphorylates the α1 subunit in vitro. Myc-tagged wild type or kinase inactive GSK3β was purified from HEK293T cells. A GST-AMPK α1 subunit fragment (aa. 463–520) containing the ST-stretch purified from bacteria was used as α substrate. (D) T479A mutation of the α subunit abolishes GSK3β-induced AMPK phosphorylation in vitro. *p<0.05, **p<0.01 vs WT or 483A with GSK3, mean±SEM (n=3). (E) Stoichiometry analysis of GSK3β-induced ST stretch phosphorylation. The indicated polypeptides (10 μM) were subjected to in vitro kinase assay using GSK3β (0.23 μM) purified from Sf21 insect cells. Note that unprimed TSC2 peptide containing GSK3 phosphorylation sites was used in this assay. (F) GSK3 inhibitor-sensitive Thr479 phosphorylation of the α subunit. Levels of Thr479 phosphorylation of the IPed endogenous AMPK α1 subunit were detected with a phospho-specific Thr479 antibody. CHIR99021 (10 nM for 1 hr) treatment was performed. (G) Ablation of GSK3 expression abolishes Thr479 phosphorylation of the endogenous AMPK α subunit. GSK3α was knocked down in wild or GSK3β^−/− MEF cells. (H) GSK3β mutant (R96A) phosphorylates Thr479 of the α1 subunit in HEK293T cells. See also Figures S2.

Figure 3. GSK3-induced Thr479 phosphorylation inhibits AL phosphorylation and kinase activity of the α subunit

(A) Reciprocal correlation between Thr479 and Thr172 (AL) phosphorylation of the α subunit. Levels of AL and Thr479 phosphorylation and the kinase activity of endogenous AMPK α1 subunit were monitored. Pretreatment with DMSO or GSK3 inhibitors, CHIR99021 (10 nM), BIO (5 μM), or LiCl (20 mM) treatment for 1 hr was performed before harvesting the HEK293T cells. **p<0.01 vs other groups; mean±SEM (n=3). (B) GSK3β inhibits AL phosphorylation through its kinase activity. Levels of AL and Thr479 phosphorylation were monitored in the presence or absence of GSK3 inhibitors in serum-starved HEK293T cells. (C) GSK3β inhibits AL phosphorylation via Thr479 phosphorylation. Levels of AL and Thr479 phosphorylation and the kinase activity were monitored in serum-starved HEK293T cells. **p<0.01 vs other groups; mean±SEM (n=3). (D) GSK3β inhibits AL phosphorylation via Thr479 but not Ser475 or Thr471 phosphorylation. GSK3β-induced reduction of AL phosphorylation in the indicated α1 mutant was monitored in serum-starved HEK293T cells. See also Figures S3.

Figure 4. GSK3-induced Thr479 phosphorylation enhances the sensitivity of phosphatase towards the AL site of theα subunit

(A) GSK3β inhibits AL phosphorylation in a manner independent of adenine nucleotide exchange on the γ regulatory subunit in serum-starved HEK293T cells. (B) The T479E α1 mutant is resistant to the induction of its AL phosphorylation upon glucose starvation. Transfected cells cultured in growth media containing 10% FBS were treated with glucose-free DMEM without serum for 2 hrs. The ratio of phospho-AMPK(T172): total AMPK was determined. Data were expressed as mean±SEM (n=3). (C and D) The T479A α1 mutant shows resistance to PP2Cα-induced AL de-phosphorylation in vitro. The indicated AMPK complex containing wild type or the T489A α1 subunit was purified from HEK293T cells and incubated with PP2Cα in vitro. 250 nM PP2Cα was used in the time course experiments (C). The PP2Cα titration experiments were incubated for 1 hr (D). Levels of AL phosphorylation were quantified. *p<0.05, **p<0.01 vs WT, mean±SEM (n=3). (E) T479A, but not T479E, α1 polypeptide interacts with the kinase domain of the α1 subunit. GST pull-down assays were performed using the indicated GST-ST-stretch polypeptides purified from E. coli and cell lysates expressing HA-AMPK α1 kinase domain (aa. 1–312, T172D). Intensity of the pulled down kinase domain was quantified. **p<0.01 vs. other groups, mean±SEM (n=3). See also Figures S4 and Supplemental Methods for additional details.

Figure 5. The PI3K-Akt pathway inhibits AMPK via GSK3-dependent Thr479 phosphorylation of the AMPK α subunit

(A) Serum stimulation inhibits AL phosphorylation of the endogenous α subunit in a PI3K- and GSK3-dependent manner. Serum- and glucose-starved MEF cells were stimulated with 10% dialyzed serum for the indicated times in the presence or absence of PI3K inhibitor (LY294002: 20 μM; preincubation for 2 hrs) or GSK3 inhibitors (LiCl: 20 mM, BIO: 5 μM; preincubation for 2 hrs). Levels of AL phosphorylation of endogenous AMPK α subunit were quantified. *p<0.05, **p<0.01 vs time 0, mean±SEM (n=3). (B) Serum stimulation induces GSK3-dependent Thr479 phosphorylation of the α subunit. MEF cells were treated with the indicated concentrations of serum for 30 min in the presence or absence of GSK3 inhibitors. (C) Insulin enhances GSK3-dependent Thr479 AMPK phosphorylation. The effect of insulin (100 nM for 60 min) on Thr479 or AL phosphorylation, or the kinase activity of the α1 subunit was monitored in the presence or absence of the GSK3 inhibitors in serum-starved HEK293E cells. **p<0.01 vs other groups, mean±SEM (n=3). (D) Ablation of GSK3 attenuates insulin-induced Thr479 phosphorylation and AL de-phosphorylation. GSK3α was knocked down in GSK3β^−/− MEF cells. The effects of insulin (100 nM) on AL and Thr479 phosphorylation of the endogenous α subunit were monitored. (E) Insulin-dependent Ser485 phosphorylation is required for GSK3-induced Thr479 phosphorylation. HEK293E cells were transfected with the indicated AMPK α1 subunits. The cells were treated with insulin (100 nM) for 60 min. (F) Active Akt induces AL dephosphorylation through GSK3-dependent Thr479 phosphorylation in HEK293E cells. (G) Insulin induces AL dephosphorylation in a GSK3-dependent manner in vivo. C57BL/6J male mice starved for 16 hours were treated with insulin (1 mU/g, ip) for 60 min. LiCl (200 μg/g, ip) was injected 2 hrs before obtaining heart tissues. **p<0.01 vs other groups, mean±SEM (n=5). (H) PI3K activity is required for GSK3-dependent AMPK inhibition in vivo. Wild type and muscle-specific TSC1 knockout male mice were treated as Figure 5G. **p<0.01 vs other groups, NS indicates “not significant”, mean±SEM (n=3). See also Figures S5.

Figure 6. Loss of GSK3-dependent AMPK inhibition causes metabolic inflexibility

(A) Insulin fails to suppress ACC1/2 and Raptor phosphorylation in T479A MEF cells. The indicated MEF cells were serum-starved for 16 hours and then stimulated with insulin (100 nM) for 60 min. See also Figures S6. (B) Lower levels of protein synthesis are observed in T479A MEF cells. The indicated MEF cells were serum-starved for 16 hours and then stimulated with serum (10%) for 4 hours. Nascent protein synthesis was determined (upper panel). The rate of nascent protein synthesis was quantified by the expression of β-actin. *p<0.05, mean±SEM (n=3). (C) Higher autophagic activity is observed in T479A MEF cells. Autophagosome formation was monitored in the indicated MEF cells in the presence of serum stimulation (10% for 60 min). The number of LC3-puncta per cell was measured and quantified in the indicated MEF cells. **p<0.01, mean±SEM (n=15). (D) Higher autophagic flux is observed in T479A MEF cells. MEF cells were stimulated with serum (10% for 60 min) in the presence of NH₄Cl. The ratio of LC3-II/LC3-I was quantified. *p<0.05, vs WT 30 min, mean±SEM (n=3). (E) Schematic model of AMPK inhibition by GSK3. Under catabolic conditions (low energy and low PI3K-Akt activity), the binding of AMP or ADP to the γ subunit promotes the association of the α-hook with the γ subunit and the subsequent interaction of the kinase domain (KD) with the β subunit. Simultaneously, the non-phosphorylated ST-stretch and its adjacent region of the α subunit (blue bar) may sterically hinder phosphatase accessibility for the activation loop (left panel). In response to anabolic stimuli (high PI3K-Akt activity), successive phosphorylations on the ST-stretch of the α subunit induced by Akt and GSK3 may promote dissociation of the ST-stretch from the KD, thereby allowing the phosphatase to dephosophorylate AL loop (middle panel). Under anabolic conditions (high energy), the KD of the α subunit may be further exposed to the phosphatase through additional conformational changes in the α-hook and linker region (right panel). A previous structure study proposed that the dissociation of the α-hook from the γ subunit precedes nucleotide exchange (AMP/ADP to ATP) on the γ subunit (Xiao et al., 2011). Whether this steric change of the ST-stretch triggers dissociation of the α hook from the γ subunit remains to be resolved.