Complexes between the LKB1 tumor suppressor, STRAD alpha/beta and MO25 alpha/beta are upstream kinases in the AMP-activated protein kinase cascade

Abstract

Background: The AMP-activated protein kinase (AMPK) cascade is a sensor of cellular energy charge that acts as a 'metabolic master switch' and inhibits cell proliferation. Activation requires phosphorylation of Thr172 of AMPK within the activation loop by upstream kinases (AMPKKs) that have not been identified. Recently, we identified three related protein kinases acting upstream of the yeast homolog of AMPK. Although they do not have obvious mammalian homologs, they are related to LKB1, a tumor suppressor that is mutated in the human Peutz-Jeghers cancer syndrome. We recently showed that LKB1 exists as a complex with two accessory subunits, STRAD alpha/beta and MO25 alpha/beta.

Results: We report the following observations. First, two AMPKK activities purified from rat liver contain LKB1, STRAD alpha and MO25 alpha, and can be immunoprecipitated using anti-LKB1 antibodies. Second, both endogenous and recombinant complexes of LKB1, STRAD alpha/beta and MO25 alpha/beta activate AMPK via phosphorylation of Thr172. Third, catalytically active LKB1, STRAD alpha or STRAD beta and MO25 alpha or MO25 beta are required for full activity. Fourth, the AMPK-activating drugs AICA riboside and phenformin do not activate AMPK in HeLa cells (which lack LKB1), but activation can be restored by stably expressing wild-type, but not catalytically inactive, LKB1. Fifth, AICA riboside and phenformin fail to activate AMPK in immortalized fibroblasts from LKB1-knockout mouse embryos.

Conclusions: These results provide the first description of a physiological substrate for the LKB1 tumor suppressor and suggest that it functions as an upstream regulator of AMPK. Our findings indicate that the tumors in Peutz-Jeghers syndrome could result from deficient activation of AMPK as a consequence of LKB1 inactivation.

Figure 1

Two AMPKKs can be resolved from rat liver extracts and both contain LKB1, STRADα and MO25α. (a) Separation of two activities that activate the GST-AMPKα1 catalytic domain by Q-Sepharose chromatography. The graph shows AMPKK activity in 4.5 ml fractions (red circles and red line), absorbance at 280 nm (continuous black line) and conductivity in the eluate (dashed black line) plotted against fraction number. (b) Probing of blots of column fractions after SDS gel electrophoresis (1 μl per lane) using anti-LKB1, anti-STRADα or anti-MO25α antibodies. In the three bottom panels, fractions 26–30 were concentrated from 4.5 ml to 250 μl using Amicon Ultra-4 30,000 MWCO centrifugal concentrators, and reanalyzed by western blotting using 2 μl per lane. (c) The effect of protein phosphatase treatment on the mobility of LKB1. The peak fractions of AMPKK1 (0.2 units) or AMPKK2 (0.8 units) were incubated in a final volume of 20 μl with or without 5 mM MgCl2 and 200 μM ATP for 15 min at 30°C. Protein phosphatases (PP1γ, 8 mU; or PP2A₁, 1 mU) or buffer were added and incubation continued for a further 15 min before stopping the reactions in SDS sample buffer and analyzing by SDS gel electrophoresis and western blotting using anti-LKB1 antibody.

Figure 2

AMPKK activity (that is, the ability to activate AMPKα1 catalytic domain), and LKB1, STRADα and MO25α polypeptides, can be immunoprecipitated from rat liver AMPKK1 and AMPKK2 using anti-LKB1 antibody. (a) Depletion of AMPKK activity from supernatant. Sheep anti-human LKB1 or pre-immune control immunoglobulin (IgG) was prebound to Protein G-Sepharose beads and cross-linked with dimethylpimelimidate as described [47], except that a final wash of the beads with 100 mM glycine, pH 2.5, was performed. Bead-bound antibodies (40 μl) were incubated with the peak fraction of AMPKK1 (0.04 units), AMPKK2 (0.03 units) or recombinant GST-LKB1:STRADα:MO25α complex (0.06 units) for 120 minutes and the beads removed in a microcentrifuge (14,000 × g for 2 min). AMPKK activity was determined in the supernatants and is expressed as a percentage of the value obtained using the control IgG. (b) The pellets from the experiment in (a) were resuspended in the original volume and samples of the supernatants and pellets analysed by western blotting with anti-LKB1 antibody. The recombinant LKB1 migrates at a higher molecular mass because of the GST tag. (c) As in (a), except that the amounts of AMPKK1, AMPKK2 and recombinant GST-LKB1:STRADα:MO25α complex were increased to 0.44, 0.70 and 1.4 units, respectively, and the activities were determined in the resuspended pellets. In this experiment the amount of antibody was limiting, so only a fraction of the activity was precipitated. (d) The pellets from the experiment in (c) were resuspended and samples analyzed by western blotting with anti-LKB1, anti-STRADα and anti-MO25α antibodies.

Figure 3

Recombinant LKB1:STRAD:MO25 complexes can efficiently activate the AMPKα1 catalytic domain via phosphorylation at Thr172. (a) The indicated combinations of GST-tagged wild-type LKB1 (WT, lanes 1–9), or kinase-dead (D194A; KD, lanes 10–13) LKB1 mutant, or GST-alone (lane 14), FLAG-tagged STRADα or STRADβ, and Myc-tagged MO25α or MO25β were coexpressed in HEK-293T cells, purified on glutathione-Sepharose and tested for their ability to activate GST-AMPKα1 catalytic domain (top panel). The results are expressed as the increase in the units of AMPK activity generated per mg full-length GST-AMPKα1 catalytic domain. Samples from each incubation were also analyzed by western blotting and probed using the indicated antibodies (from top to bottom): anti-pT172; anti-AMPKα1 catalytic domain (GST-AMPKα1); anti-GST to detect GST-LKB1; anti-FLAG to detect STRADα and STRADβ, and anti-Myc to detect MO25α and MO25β. All proteins migrated with the expected mobility, taking into account the epitope tags. The bottom three blots were conducted on blank reactions lacking GST-AMPKα1 catalytic domain, as the latter appeared to cause some interference with detection. (b) Recombinant GST-LKB1:STRADα:MO25α complex was used to phosphorylate wild-type GST-AMPKα1 catalytic domain (GST-α1-WT) or a T172A mutant (GST-α1-T172A) using [γ-³²P]ATP as described in Materials and methods. The reaction was analyzed by SDS gel electrophoresis and autoradiography. Arrows show the migration of GST-LKB1 (which autophosphorylates) and GST-AMPKα1 catalytic domain.

Figure 4

Activation and phosphorylation of heterotrimeric AMPK complexes by AMPKK1, AMPKK2 and recombinant GST-LKB1:STRADα:MO25α complexes, and the effect of AMP. (a) Activation of α1β1γ1 and α2β1γ1 complexes separated from purified rat liver AMPK. The AMPKα1- or AMPKα2-containing complexes were purified by immunoprecipitation and activation of the resuspended immunoprecipitates by the three AMPKK preparations examined. The results are expressed as activation relative to the control without added AMPKK. (b) Quantification by western blotting of the relative amounts of LKB1, STRADα and MO25α polypeptides in the three AMPKK preparations used in (a). A small amount of degradation is detectable due in part to the heavy loadings of the GST-LKB1 and FLAG-STRADα. The identity of the polypeptide labeled '?' in the anti-LKB1 blot is not known. (c) Effect of AMP on the activation of α1β1γ1 and α2β1γ1 heterotrimers of AMPK, and of GST-AMPKα1 catalytic domain, by AMPKK1, AMPKK2 and the recombinant GST-LKB1:STRADα:MO25α complex. AMPKK activity was measured as in Figure 3 with or without 200 μM AMP. The results are expressed as ratios of the activities obtained with and without AMP.

Figure 5

Endogenous AMPKK activity (that is, ability to activate AMPKα1 catalytic domain) can be immunoprecipitated from 293 cells using anti-LKB1 antibody, but activity can only be immunoprecipitated from HeLa cells if they stably express wild-type LKB1, but not a catalytically-inactive mutant. (a) LKB1 was immunoprecipitated from 0.5 mg cell extract derived from untransfected HEK-293T cells (lanes 1,2), untransfected HeLa cells (control; lanes 3,4), or HeLa cells stably expressing wild-type LKB1 (WT; lanes 5,6) or a kinase-dead LKB1 mutant (D194A; KD, lanes 7,8). Immunoprecipitation used anti-LKB1 (lanes 1, 3, 5, 7) or a pre-immune control immunoglobulin (IgG; lanes 2, 4, 6, 8). Samples of each immunoprecipitate were used to assay activation of GST-AMPKα1 catalytic domain, to analyze phosphorylation of GST-AMPKα1 catalytic domain on Thr172 (middle panel), and to determine by western blotting the recovery of LKB1 and its accessory subunits (bottom panels). In lanes 5 and 7 some immunoglobulin heavy chain (IgG-H) had eluted from the protein G-Sepharose despite the fact that it had been cross-linked: this explains why LKB1 may not appear to comigrate in lanes 1, 5 and 7. Also shown at left in the top panel is the basal activity obtained when the GST-AMPKα1-catalytic domain was incubated with MgATP on its own (no addition). (b) Whole cell lysates from the same cells were analyzed by SDS gel electrophoresis and blots probed using anti-LKB1, anti-STRADα, and anti-MO25α antibodies. They were also probed with anti-ERK1/2 antibodies as loading controls.

Figure 6

Restoration of the ability of AMPK to be activated, and AMPK and acetyl-CoA carboxylase to be phosphorylated, by AICA riboside and phenformin in HeLa cells following expression of LKB1. Control HeLa cells (lanes 1,2,3), HeLa cells expressing wild-type LKB1 (WT; lanes 4,5,6) or kinase-inactive mutant LKB1 (D194A; KD, lanes 7,8,9) were incubated for 60 min with no further addition, with 2 mM AICA riboside or 10 mM phenformin, and lysed. (a) Endogenous AMPK was immunoprecipitated from the cell extracts and assayed. (b) The cell lysates was immunoblotted with antibodies recognizing AMPKα1 phosphorylated at Thr172 or total AMPKα1; the results were analyzed using the LI-COR Odyssey™ IR imager as described in the Materials and methods section, and are expressed as a ratio of the two signals. (c) The cell lysates were analyzed by western blotting and the membranes probed with antibodies recognizing ACC phosphorylated at Ser79, or streptavidin to determine total AMPKα1. The results were analyzed using the LI-COR imager as for (b).

Figure 7

AMPK is activated and phosphorylated in response to AICA riboside and phenformin in LKB1^+/+ but not in LKB1^-/- MEF cells. Cells immortalized from control embryos LKB1^+/+ and LKB1^-/- knockout embryos [14], were incubated with AICA riboside (2 mM) or phenformin (10 mM) for 1 hour. Lysates were prepared and AMPK activity (expressed as units per mg total lysate protein) determined in immunoprecipitates made using a mixture of anti-AMPKα1 and anti-AMPKα2 antibodies. Lysates were also analyzed by SDS gel electrophoresis and blots probed using anti-LKB1, anti-AMPKα1/β2, and anti-pT172 antibodies.