Research ArticleCell Biology

IKK promotes cytokine-induced and cancer-associated AMPK activity and attenuates phenformin-induced cell death in LKB1-deficient cells

See allHide authors and affiliations

Science Signaling  10 Jul 2018:
Vol. 11, Issue 538, eaan5850
DOI: 10.1126/scisignal.aan5850
  • Fig. 1 AMPK Thr172 phosphorylation induced by inflammatory signals requires IKK.

    (A) HeLa cells were pretreated with either dimethyl sulfoxide (DMSO) or 5 μM Compound A (Cmpd A) and then treated with IL-1β (15 ng/ml) for the indicated time periods. Whole-cell lysates were then subjected to Western blot analysis using the indicated antibodies. Blots are representative of three independent experiments. (B) HeLa cells were transfected with either WT IKKβ or KD IKKβ and then treated with IL-1β (15 ng/ml) for 5 min. Whole-cell lysates were then subjected to Western blot analysis using the indicated antibodies. Blots are representative of two independent experiments. HA, hemagglutinin. (C) A549 cells were transfected with either noncoding siRNA (siNC) or siRNA targeting NEMO (siNEMO) and then stimulated with IL-1β (15 ng/ml) for 5 min and probed with the indicated antibodies. Blots are representative of three independent experiments. (D) WT or IKKβ−/− MEFs were treated with IL-1β for 5 min and immunoblotted with the indicated antibodies. Blots are representative of two independent experiments. (E) WT or AMPK−/− MEFs were treated with IL-1β for 30 min and then probed with antibodies recognizing ACC and ULK1 phosphorylated at their respective AMPK sites. Blots are representative of three independent biological experiments; the error bars represent the SEM of the normalized densitometry value measured for each experiment (n.d., not detected). (F) A549 cells were pretreated with either DMSO or Compound A for 15 min, then treated with IL-1β for 30 min, and then probed with antibodies recognizing ACC phosphorylated at the AMPK site. Blots are representative of three independent experiments. (G) A549 cells were treated with IL-1β (15 ng/ml) for the indicated time periods and probed for the autophagy marker LC3. The ratio of LC3-II/LC3-I and the normalized ratio of LC3-II to actin are displayed in the graph below (n = 3 independent biological replicates). A repeated measures linear mixed-effect model showed that there was a statistically significant trend of increasing LC3-II with time (P = 0.027). (H) A549 cells were pretreated with either with DMSO or 5 μM Compound A for 15 min and then with IL-1β for 20 min. Whole-cell extracts were then prepared and analyzed by Western blot for the indicated proteins. Blots are representative of three independent experiments. The error bars represent the SEM of the densitometry values obtained in each experiment. A two-way analysis of variance (ANOVA) was used to determine which groups were statistically different. *P < 0.05. (I) A549 cells were pretreated with DMSO, Compound A (5 μM), or Bafilomycin A1 (10 nM) for 15 min and then treated with IL-1β for 2 hours. The abundance of autophagosomes was assayed using a commercially available kit. Cells were then stained with a dye that is selective for acidified autophagosomes, and total fluorescence intensity (TFI) was measured. Error bars represent the SEM of the average total fluorescence measured in three independent experiments.

  • Fig. 2 IKK is downstream of TAK1 with respect to AMPK.

    (A) A549 cells were pretreated with either the IKK inhibitor Compound A (5 μM) or the TAK1 inhibitor 5z-7-oxozeanol (5z) (5 μM) for 15 min and then stimulated with IL-1β (15 ng/ml) or TNF-α (15 ng/ml) for 5 min. Blots are representative of three independent experiments. (B) HeLa cells were transfected with either an empty vector plasmid or plasmids encoding Flag-TAK1 and Flag-TAB1. Cells were then either treated with DMSO or Compound A (5 μM) for 30 min. Whole-cell lysates were then subjected to Western blot analysis with the indicated antibodies. Blots are representative of three independent experiments. A two-way ANOVA showed no difference between Compound A or DMSO treatment in TAK1/TAB1 overexpressing cells compared to cells transfected with empty vector (P = 0.82). (C) WT or TAK1−/− MEFs were treated with Compound A (5 μM) for 1 hour, and then, whole-cell lysates were analyzed by Western blot for AMPK Thr172 phosphorylation and total AMPK. Blots are representative of three independent experiments. The error bars represent the SEM of the normalized densitometry values measured in each experiment. A two-way ANOVA was used to determine which groups were statically different. ***P < 0.001. (D) TAK1−/− MEFs were transfected with siRNA targeting murine IKKβ and blotted for the indicated proteins. Blots are representative of three independent experiments. Error bars represent the SEM of the normalized densitometry values measured in each experiment. A ratio paired t test was performed to determine whether the control and siIKKβ groups were significantly different. *P < 0.05. (E) A549 cells were treated for 30 min with either 5z-7-oxozeanol or NG-25 to inhibit TAK1 and immunoblotted with the indicated antibodies. A549 cells were treated with Compound A at the indicated concentrations for 30 min and blotted for phospho-p38 (used as a marker of TAK1 activity) or phospho-p65 (used as a marker of IKKβ activity). Blots are representative of three independent experiments.

  • Fig. 3 IKK regulates basal AMPK Thr172 phosphorylation in cell lines.

    (A) A549 (LKB1-deficient) and MDA-MB-231 (expressing LKB1) cells were transfected with siRNA targeting NEMO or noncoding siRNA (N.C.) for 48 hours and immunoblotted with the indicated antibodies. Blots are representative of three independent experiments. Error bars represent the SEM of the normalized densitometry values measured for each experiment. A two-way ANOVA was used to determine which groups were statistically different. **P < 0.01, *P < 0.05. (B) A549 cells were treated with siRNA targeting either IKKα or IKKβ for 48 hours and immunoblotted as indicated. Blots are representative of three independent experiments. Error bars represent the SEM of the normalized densitometry values measured in each experiment. A two-way ANOVA was used to determine which groups were statically different. *P < 0.05. (C) Whole-cell extracts from WT, IKKα−/−, and IKKβ−/− MEFs were analyzed by Western blot with antibodies for either AMPK phospho-Thr172 or total AMPK. Blots are representative of three independent experiments. Error bars represent the SEM of the normalized densitometry values measured in each experiment. A two-way ANOVA was used to determine which groups were statically different. *P < 0.05. (D) A549 and MDA-MB-231 cells were grown in full medium [Dulbecco’s modified Eagle’s medium (DMEM), 10% fetal bovine serum (FBS)], treated with the indicated concentrations of Compound A for 1 hour and immunoblotted as indicated. Blots are representative of three independent experiments. Error bars represent the SEM of the normalized densitometry values measured in each experiment. (E) A549 and MDA-MB-231 cells were treated with Compound A (5 μM) for the indicated time periods and then analyzed by Western blot for AMPK Thr172 phosphorylation. Blots are representative of three independent experiments. Error bars represent the SEM of the normalized densitometry values measured in each experiment.

  • Fig. 4 IKK phosphorylates AMPK Thr172 in cell-free kinase assays and induces its activity.

    (A) Increasing amounts of GST-IKKβ were incubated with recombinant AMPKα1/β1/γ1 trimers in cell-free kinase assays and immunoblotted as indicated. Blots are representative of three independent experiments. (B) Cell-free kinase assays were performed as in (A) but using SAMS peptide as substrate. AMPK activity was calculated after subtraction of the blank reaction and the reaction with only IKKβ. This experiment was performed on three separate occasions. The graph represents the mean activity calculated from the three experiments, and the error bars represent the SEM. (C) A GST-tagged KD mutant of the AMPKα1 kinase domain (AMPK D157A) was purified from human embryonic kidney (HEK) 293T cells and then used as a substrate for cell-free kinase assays with active IKKβ. Blots are representative of three independent experiments. (D) Recombinant IKKα and IKKβ were incubated with AMPKα1/β1/γ1 trimers as in (A). The amount of AMPK Thr172 phosphorylation by IKKβ was normalized to the amount of AMPK Thr172 phosphorylation by IKKα and is displayed in the graph. The blot is representative of three independent experiments. The error bars represent the SEM of the normalized densitometry values measured in each experiment. (E) A GST-tagged construct of the AMPK kinase domain (amino acids 1 to 312) or with an empty vector (E.V.) was transfected into HEK293T cells and used to coprecipitate IKKα or IKKβ. Blots are representative of two independent experiments. (F) Effect of Compound A on the phosphorylation of AMPK Thr172 by recombinant IKKβ, TAK1/TAB1, or CAMKK2. Blots are representative of five independent experiments for each kinase, the ratio of phosphorylated to total AMPK is plotted in the graphs below, and the error bars represent the SEM of the normalized densitometry values measured in each experiment.

  • Fig. 5 IKK/AMPK provide resistance to phenformin in LKB1−/− cells.

    (A) A549 and MDA-MB-231 cells were transfected with either noncoding siRNA (siNC) or siRNA targeting NEMO (siNEMO), treated with phenformin (Phen.; 1 mM) for 18 hours, and immunoblotted as indicated. Blots are representative of three independent experiments. (B) A549 or MDA-MB-231 cells were pretreated with Compound A (5 μM) or DMSO for 15 min and then with phenformin (1 mM) for 45 min and immunoblotted as indicated. Blots are representative of three independent experiments. (C) A549 and MDA-MB-231 cells were transfected with either noncoding siRNA (siNC) or siRNA targeting NEMO (siNEMO) and treated with increasing concentrations of phenformin for 18 hours. Caspase 3/7 activity was measured (n = 3 biological replicates; the error bars represent the SEM; RLU is relative luminescence units). (D) A panel of LKB1-deficient (A549, HeLa, NCI-H460, and NCI-H23) and LKB1 WT cell lines (IMR90, NCI-H441, MDA-MB-231, and MiaPaca-2) were treated with either DMSO, phenformin (1 mM), Compound A (5 μM), or both (Combo) for 18 hours. Caspase 3/7 activity of each cell line was measured. The average of three independent experiments is shown in the graph (a two-way ANOVA was performed to determine which groups were statistically different). **P < 0.01.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/11/538/eaan5850/DC1

    Fig. S1. Further characterization of the effect of IL-1 and IKK on AMPK signaling.

    Fig. S2. Validation of Compound A and siRNA specificity.

    Fig. S3. IKK regulates AMPK independently of changes in energy status.

    Fig. S4. Identification of AMPK Thr172 phosphorylation by MS.

  • This PDF file includes:

    • Fig. S1. Further characterization of the effect of IL-1 and IKK on AMPK signaling.
    • Fig. S2. Validation of Compound A and siRNA specificity.
    • Fig. S3. IKK regulates AMPK independently of changes in energy status.
    • Fig. S4. Identification of AMPK Thr172 phosphorylation by MS.

    [Download PDF]

Stay Connected to Science Signaling

Navigate This Article