The tumor suppressor LKB1 kinase directly activates AMP-activated kinase and regulates apoptosis in response to energy stress

Abstract

AMP-activated protein kinase (AMPK) is a highly conserved sensor of cellular energy status found in all eukaryotic cells. AMPK is activated by stimuli that increase the cellular AMP/ATP ratio. Essential to activation of AMPK is its phosphorylation at Thr-172 by an upstream kinase, AMPKK, whose identity in mammalian cells has remained elusive. Here we present biochemical and genetic evidence indicating that the LKB1 serine/threonine kinase, the gene inactivated in the Peutz-Jeghers familial cancer syndrome, is the dominant regulator of AMPK activation in several mammalian cell types. We show that LKB1 directly phosphorylates Thr-172 of AMPKalpha in vitro and activates its kinase activity. LKB1-deficient murine embryonic fibroblasts show nearly complete loss of Thr-172 phosphorylation and downstream AMPK signaling in response to a variety of stimuli that activate AMPK. Reintroduction of WT, but not kinase-dead, LKB1 into these cells restores AMPK activity. Furthermore, we show that LKB1 plays a biologically significant role in this pathway, because LKB1-deficient cells are hypersensitive to apoptosis induced by energy stress. On the basis of these results, we propose a model to explain the apparent paradox that LKB1 is a tumor suppressor, yet cells lacking LKB1 are resistant to cell transformation by conventional oncogenes and are sensitive to killing in response to agents that elevate AMP. The role of LKB1/AMPK in the survival of a subset of genetically defined tumor cells may provide opportunities for cancer therapeutics.

Fig. 1.

LKB1 phosphorylates Thr-172 of AMPKα in vitro and activates its kinase activity. (a) Lineup of known LKB1 in vitro phosphorylation sites with sites of phosphorylation in human AMPKα and its yeast homologue SNF1 protein (SNF1p). S.c., Saccharomyces cerevisiae; auto, autophosphorylation. (b) HT1080 cells were transfected with WT or kinase-dead (KD; K78I) LKB1 with or without its coactivating protein, STRAD. As indicated, LKB1 immunoprecipitates were tested for their ability to transphosphorylate bacterial MBP–AMPKα in an in vitro kinase assay. Parallel in vitro kinase assays were performed by using [γ-³²P]ATP followed by autoradiography or cold ATP followed by immunoblotting for phospho-Thr-172 AMPK (α-P-Thr172) and the indicated proteins. WT LKB1 immunoprecipitates were run in duplicate as shown. MBP–AMPK was also tested alone, as indicated. Results are typical of three separate experiments. (c) AMPKK assay. LKB1 phosphorylation of MBP–AMPK activates its kinase activity toward a peptide substrate (SAMS). LKB1 immunoprecipitates (as in a) were used to phosphorylate MBP–AMPK in vitro, and then MBP–AMPK was removed and tested for its ability to transphosphorylate the SAMS peptide in the presence of [γ-³²P]ATP. Results were obtained from two separate experiments in triplicate. LKB1 alone was incapable of detectably phosphorylating the SAMS peptide, and equivalent levels of LKB1 and MBP–AMPK were used in each reaction (data not shown). Samples without LKB1, without SAMS peptide, or without MBP–AMPK all gave similar levels of background (data not shown). KD, kinase-dead. (d) Coimmunoprecipitation of endogenous AMPKα with LKB1. HT1080 cells were transfected with FLAG-tagged vectors encoding WT or kinase-dead (KD) LKB1 (with or without STRAD), or FLAG-JNK or FLAG-PKC ζ. FLAG immunoprecipitates (IPs) were immunoblotted with anti-AMPKα pan antisera (Upstate Biotechnology); 5% of the total input is shown at right. A significant amount of endogenous AMPK coimmunoprecipitates with kinase-dead LKB1. TCEs, total cell extracts.

Fig. 2.

LKB1-deficient MEFs are defective in AMPK activation. (a) Littermate MEFs of the indicated LKB1 genotypes were left untreated (n.t.) or were treated with 0.1 mM H₂O₂ for 20 min or 2 mM AICAR for 2 h. Total cell extracts were immunoblotted for phospho-Thr-172 AMPK (P-AMPK) or phospho-Ser-79 ACC (P-ACC), as well as for total AMPK and LKB1. (b) An immortalized LKB1-deficient MEF cell line was reconstituted with human (hu) or mouse (ms) WT (wt) or kinase-dead (kd) LKB1-expressing retroviruses. v, Vector control cells; hu wt, human FLAG-tagged WT LKB1-expressing cells; hu kd, human FLAG-tagged kinase-dead LKB1-expressing cells; ms wt, untagged mouse WT LKB1-expressing cells; ms kd, untagged mouse kinase-dead LKB1-expressing cells. Cells were treated as in a. The asterisk indicates a background band that serves as a loading control.

Fig. 3.

LKB1 regulates activation of AMPK in response to the AMP analogue AICAR as well as to oxidative or osmotic stress in HT1080 cells. HT1080 cells stably expressing WT or kinase-dead (kd) LKB1 as in Fig. 2 were treated with 0.1 mM H₂O₂ for 20 min, 0.6 M sorbitol for 30 min, or 2 mM AICAR for 2 h. Total cell extracts were analyzed as in Fig. 2. v, Vector control cells; hu, human; ms, mouse; P-AMPK, phospho-Thr-172 AMPK; P-ACC, phospho-Ser-79 ACC.

Fig. 4.

LKB1 protects cells from apoptosis induced by agents that elevate intracellular AMP. (a) Two independent littermate-matched WT and LKB1-deficient primary MEF cell lines (plated in triplicate) were treated with 2 mM AICAR for 8 h. Cell viability was quantified by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and expressed as a percentage of untreated controls. Results represent three independent experiments. (Inset) Immunoblot demonstrating LKB1-deletion. The arrowhead indicates LKB1; the asterisk denotes the background band. (b) Phase-contrast images of HeLa cells stably expressing vector, WT LKB1, or kinase-dead (KD) LKB1 5 h after treatment with 2.5 mM AICAR. Results represent four independent experiments. (c) LKB1 expression protects HeLa cells from AICAR and peroxide, but not UV-induced cell death. Cell viability is expressed as a percentage of untreated controls and quantified by MTT assays run in triplicate on indicated HeLa stable cell lines treated with 2.5 mM AICAR, 100 μM H₂O₂, 50 J/cm² UV, or 10 mM metformin for 12 h. HeLa cells were stably infected with vector, WT LKB1-, or kinase-dead (KD) LKB1-expressing retroviruses as indicated.

Fig. 5.

Caspase-3, JNK, and p38 signaling are hyperactivated in LKB1-null MEFs in response to AICAR. Immunoblot analysis on LKB1 WT or -null MEFs without treatment (NT) or 5 h after treatment with 2 mM AICAR. Cleaved (activated) caspase-3, poly(ADP-ribose) polymerase (PARP), phospho-JNK (P-JNK), phospho-p38 (P-p38), phospho-AKT (P-AKT; Ser-473), and total Akt were detected by immunoblotting (Cell Signaling Technology).

Fig. 6.

Model for LKB1 as a sensor of low energy and negative regulator of tumorigenesis and apoptosis. Under basal conditions, LKB1 serves as a sensor of low energy, keeping ATP-consuming processes including protein synthesis in check via AMPK phosphorylation of TSC2. In response to stresses such as low glucose, hypoxia, nutrient deprivation, or mitochondrial poisons, LKB1 phosphorylates AMPK, which shuts off ATP-consuming processes and up-regulates ATP production to offset the elevated AMP/ATP ratio. This activity prevents the cells from going into apoptosis in response to elevated AMP. In LKB1-deficient cells, under some basal conditions, there may be increases in TOR signaling due to the lack of TSC2 phosphorylation by AMPK, resulting in increased growth or tumorigenic potential. In response to further increases in intracellular AMP, these cells have no mechanism to offset the elevated AMP and go straight into apoptosis.