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Am J Physiol Endocrinol Metab 289: E474-E481, 2005. First published May 10, 2005; doi:10.1152/ajpendo.00003.2005
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Glycogen debranching enzyme association with {beta}-subunit regulates AMP-activated protein kinase activity

Hideyuki Sakoda,1 Midori Fujishiro,1 Junko Fujio,1 Nobuhiro Shojima,1 Takehide Ogihara,2 Akifumi Kushiyama,1 Yasushi Fukushima,1 Motonobu Anai,3 Hiraku Ono,3 Masatoshi Kikuchi,3 Nanao Horike,4 Amelia Y. I. Viana,4 Yasunobu Uchijima,4 Hiroki Kurihara,4 and Tomoichiro Asano4

2Division of Advanced Therapeutics for Metabolic Diseases, Center for Translational and Advanced Animal Research on Human Diseases, Tohoku University Graduate School of Medicine, Sendai; 1Department of Internal Medicine, Graduate School of Medicine; 3Institute for Adult Disease, Asahi Life Foundation; and 4Department of Physiological Chemistry and Metabolism, Graduate School of Medicine, University of Tokyo, Tokyo, Japan

Submitted 4 January 2005 ; accepted in final form 28 April 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
AMP-activated protein kinase (AMPK) regulates both glycogen and lipid metabolism functioning as an intracellular energy sensor. In this study, we identified a 160-kDa protein in mouse skeletal muscle lysate by using a glutathione-S-transferase (GST)-AMPK fusion protein pull-down assay. Mass spectrometry and a Mascot search revealed this protein to be a glycogen debranching enzyme (GDE). The association between AMPK and GDE was observed not only in the overexpression system but also endogenously. Next, we showed the {beta}1-subunit of AMPK to be responsible for the association with GDE. Furthermore, experiments using deletion mutants of the {beta}1-subunit of AMPK revealed amino acids 68–123 of the {beta}1-subunit to be sufficient for GDE binding. W100G and K128Q, both {beta}1-subunit mutants, are reportedly incapable of binding to glycogen, but both bound GDE, indicating that the association between AMPK and GDE does not involve glycogen. Rather, the AMPK-GDE association is likely to be direct. Overexpression of amino acids 68–123 of the {beta}1-subunit inhibited the association between endogenous AMPK and GDE. Although GDE activity was unaffected, basal phosphorylation and kinase activity of AMPK, as well as phosphorylation of acetyl-CoA carboxylase, were significantly increased. Thus it is likely that the AMPK-GDE association is a novel mechanism regulating AMPK activity and the resultant fatty acid oxidation and glucose uptake.

glutathione-S transferase; pull-down assay; mass spectrometry

AMP-ACTIVATED PROTEIN KINASE (AMPK) acts as an intracellular energy sensor and regulates both glycogen and lipid metabolism (1–3). An increased intracellular AMP-to-ATP ratio leads to a conformational change in AMPK; AMPK is then phosphorylated and activated by LKB1. Activated AMPK reportedly phosphorylates and inactivates acetyl-CoA carboxylase (ACC) and decreases in malonyl-CoA, which increases fatty acid oxidation. 3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase is also downregulated by AMPK, and cholesterol synthesis is thereby decreased. In skeletal muscle, contraction and 5-aminoimidazole-4-carboxamide-1-{beta}-D-ribofuranoside (AICAR) stimulation increase AMPK activity and thereby increase GLUT4 translocation and glucose uptake (4). In addition, expression of GLUT4 protein is increased by continuous AICAR stimulation (5, 6), although events downstream from AMPK involving translocation and gene transcription of GLUT4 have not yet been clarified.

Energy storage in cells depends mainly on glycogen and lipid. Recently, it was reported that a considerable amount of AMPK colocalizes with glycogen particles. This has been attributed to the {beta}-subunit of AMPK that contains a glycogen-binding domain (7–9). Although the physiological significance of this localization of AMPK with glycogen particles remains unclear, glycogen synthase was reported to be phosphorylated and deactivated by AMPK in vitro (10). In the skeletal muscle of AMPK{alpha}2 knockout mice, basal glycogen synthase activity is increased, and AICAR treatment fails to deactivate glycogen synthase (11). In addition, it was reported that the activation of AMPK{alpha}2 is increasingly suppressed as glycogen levels rise (12). Mutation of the AMPK {gamma}-subunit reportedly increase glycogen content in skeletal muscle and the heart (13, 14). Thus AMPK has a close relationship, not only locationally but also functionally, with glycogen.

In this study, we attempted to identify proteins that bind to AMPK by using a glutathione-S-transferase (GST)-AMPK fusion protein pull-down assay, and found GDE to be the AMPK-associated protein. Herein, we show that GDE associates with AMPK and thereby modulates its kinase activity.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Antibodies. The antibodies against the {alpha}- and {beta}-subunits of AMPK and GDE were prepared by immunization of rabbits with a GST{alpha}2 subunit, GST{beta}1 subunit, and GDE fusion protein, respectively. These antibodies were then affinity purified as previously described (15). Anti-Flag, anti-hyaluronic acid (HA), and anti-c-myc antibody were purchased from Sigma; anti-AMPK{beta}, anti-phospho-AMPK{alpha}-Thr172, and anti-phospho-ACC-Ser79 were purchased from Cell Signaling Technology; and anti-AMPK{alpha}1-specific antibody and anti-AMPK{alpha}2-specific antibody were purchased from Upstate Biotechnology.

Animals. Six-week-old male C57/BL6 mice and Sprague-Dawley rats were purchased from Tokyo Experimental Animals (Tokyo, Japan) and fed a standard rodent diet. Some mice were starved for 12 h; these mice were then anesthetized with pentobarbital sodium (60 mg/kg body wt inositol 1,4,5-trisphosphate), and lower limb skeletal muscles were dissected out. The study protocol was approved by the Institutional Review Board of the Institute for Adult Disease, Asahi Life Foundation. Animal care was in accordance with the policies of the University of Tokyo at all times.

Cell culture. COS-7 cells and HEK293 cells were maintained in DMEM supplemented with 5% fetal bovine serum under a 5% CO2 atmosphere at 37°C. Sf9 cells were maintained in Grace's medium supplemented with 5% fetal bovine serum at 27°C.

DNA constructs. cDNAs encoding human GDE and the {beta}1-subunit of human AMPK were produced by PCR with primers corresponding to sequences already reported, using the cDNA library from HEK293 cells. All GDE and AMPK{beta}1 constructs were designed to contain a Flag or HA tag at the COOH terminus. Human AMPK{alpha}1, -{alpha}2, -{beta}2, -{gamma}1, and -{gamma}2 cDNAs were kindly provided by Japan Tobacco, Central Pharmaceutical Research Institute (Osaka, Japan). W100G and K128Q, both {beta}1-subunit mutants, were produced by PCR as reported previously (8). Fragments of the NH2-terminal portion (amino acids 1–67), glycogen binding domain (GBD); (amino acids 68–163), and COOH- terminal portion (amino acids 164–270) of the {beta}1-subunit were amplified by PCR with sense primers attached to the BamHI site and a start codon: 5'-GGA TCC ATG GGC AAT ACC AGC AGT-3', 5'-GGA TCC ATG GAA GTG AAT GAT AAA GCT-3', or 5'-GGA TCC ATG GAT GCT TTA ATG GTG GA-3', respectively, and with antisense primers attached to the HA tag and a stop codon: 5'-TCA AGC GTA GTC TGG GAC GTC GTA TGG GTA CAG ATC ATG CTG CCA GGC-3', 5'-TCA AGC GTA GTC TGG GAC GTC GTA TGG GTA AAA TAC TTC AAA GTC-3', or 5'-TCA AGC GTA GTC TGG GAC GTC GTA TGG GTA TAT GGG CTT GTA TAA-3', respectively. The cDNAs encoding amino acids 68–113, 68–123, 68–133, 68–143, and 68–153 in the GBD of AMPK{beta}1 were amplified by PCR, with 5'-GGA TCC ATG GAA GTG AAT GAT AAA GCT-3' as the sense primer and 5'-TCA AAA GTT ATT GTG GCT TCT-3', 5'-TCA ATG CTC TCC TTC CGG-3', 5'-TCA CCA CTG ACC ATC CAC-3', 5'-TCA GGT TAC TAT GGG CTC-3', and 5'-TCA AAT GAT GTT GTT AAC-3' as antisense primers, respectively. The cDNA-encoding amino acids 113–163 of AMPK{beta}1 were amplified by PCR with 5'-GGA TCC ATG GTA GCC ATC CTG GAT CTG-3' as the sense primer and 5'-TCA GTA AAA TAC TTC AAA GTC-3' as the antisense primer. PCR products were cloned into a pCR2.1 plasmid vector (Invitrogen, San Diego, CA) and all sequences were confirmed using a CEQ2000 DNA Analysis System (Beckman Coulter).

Generation of recombinant adenovirus and baculovirus. Recombinant adenovirus used to express human GDE was constructed by homologous recombination of the expression cosmid cassettes containing the corresponding cDNAs and the parental virus genome, as described previously (16). The full-length coding regions of AMPK{alpha}2, including the GST sequence at the NH2 terminus, AMPK{beta}1, AMPK{gamma}2, and GDE in the pBacPAK9 transfer vector (Clontech), and the baculoviruses were prepared according to the manufacturer's instructions. For protein production, Sf9 cells were infected with these baculoviruses and grown for 48 h.

Purification of GST-AMPK fusion protein. GST and the GST{alpha}2 subunit were overexpressed in Sf9 cells using baculoviruses and the cells were lysed with lysis buffer (PBS, 1% Triton X-100, 0.2 mmol/l PMSF). GST and the GST{alpha}2 subunit fusion protein were isolated and purified by affinity chromatography on glutathione-Sepharose 4B (Pharmacia Biotech). The purified GST and GST{alpha}2 subunit fusion protein were incubated with Sf-9 lysates of cells co-overexpressing the {beta}1- and {gamma}2-subunits of AMPK for 1 h and then washed six times with lysis buffer and used for the GST pull-down assay.

GST pull-down assay. Muscles were homogenized with 10 vol/wt homogenizing buffer [20 mmol/l Tris·HCl (pH 7.4), 1% Triton X-100, 0.25% sodium deoxycholate, 0.25 mol/l NaCl] containing 0.2 mmol/l PMSF and 5 µg/ml aprotinin and then centrifuged at 15,000 rpm for 30 min at 4°C. Next, the supernatants were recentrifuged at 100,000 g for 1 h. Then, 100 ml of supernatant (2 µg/ml protein concentration) were incubated with 1 ml of glutathione-Sepharose 4B for 1 h at 4°C to remove nonspecifically bound proteins, incubated with purified GST-AMPK fusion protein for 1 h, washed six times with homogenizing buffer, subjected to SDS-PAGE, and then silver stained. As a negative control, GST, preincubated only with Sf9 cells overexpressing the {beta}1- and {gamma}2-subunits, was incubated with tissue lysates from mouse skeletal muscle. GST-AMPK fusion protein not incubated with tissue lysates was also subjected to SDS-PAGE. The band specifically observed as an AMPK-associated protein was analyzed using mass spectrometry and Mascot search by Shimadzu Biotech (Ibaragi, Japan).

Purification of GST fusion protein and pull-down assay. cDNAs encoding full-length AMPK{alpha}2, AMPK{beta}1, and AMPK{gamma}1 and fragments of the {beta}1-subunit of AMPK were subcloned into a pGEX-4T-3 vector (Amersham Biosciences) that was used to transform Escherichia coli JM109. Transformed cells were grown to an A600 of 0.6 in LB medium supplemented with 0.1 mg/ml ampicillin and stimulated for 3 h with 1.0 mM isopropyl-{beta}-D-1-thiogalactopyranoside. GST fusion proteins were isolated and purified by affinity chromatography on glutathione-Sepharose 4B (17). GDE-overexpressing Sf9 cell lysates were immunoprecipitated with anti-Flag M2-agarose (Sigma), and GDE was purified and eluted from the agarose with 3x Flag peptide (Sigma). Purified GDE was incubated with GST, GST containing full-length AMPK{alpha}2, AMPK{beta}1, or AMPK{gamma}1, or various GST fragments of the {beta}1-subunit of AMPK for 1 h. After six washes with lysis buffer, glutathione-Sepharose 4B beads were boiled in Laemmli buffer, followed by SDS-PAGE and Western blotting.

AMPK assay. After first serum-starving COS-7 cells for 2 h in serum-free DMEM, followed by preincubation for 1 h in Krebs-Ringer-HEPES buffer, the cells were stimulated with a 30-min incubation in Krebs-Ringer-HEPES buffer containing 2 mmol/l AICAR. Next, the COS-7 cells were lysed in buffer A and centrifuged. The supernatants were then immunoprecipitated with anti-AMPK{alpha} antibodies. AMPK activities in the immunoprecipitants were assayed using SAMS peptide, as described previously (15).

Glycogen debranching enzyme assay. After the serum starvation, preincubation, and AICAR stimulation previously described, COS-7 cells overexpressing GDE were collected with Tris buffer [20 mmol/l Tris·HCl (pH 7.5), 1 mmol/l sodium orthovanadate, 1 mmol/l {beta}-glycerophosphate, and 0.2 mmol/l PMSF] and then sonicated. The sonicated cells were centrifuged at 17,000 g for 20 min, and the supernatants were immunoprecipitated with anti-Flag antibody. The immunoprecipitants were washed twice with Tris buffer and twice more with GDE assay buffer [20 mmol/l Tris·HCl (pH 7.5), 50 mmol/l sodium acetate]. Next, the immunoprecipitants were incubated at 37°C for 30 min in the GDE assay buffer containing 15 mmol/l glucosyl-{beta}-cyclodextrin as the substrate (18–20), and GDE activities were determined by measuring the amount of glucose released from glucosyl-{beta}-cyclodextrin, using the glucose oxidase method (GLU CII, Wako).

Immunopreciptation and immunoblotting. The supernatants from skeletal muscle and COS-7 cells, prepared as previously described, were immunoprecipitated with 3 µg/ml antibodies for 2 h. The immunoprecipitants were boiled in Laemmli sample buffer containing 100 mmol/l DTT and then subjected to SDS-PAGE and Western blotting with each antibody, as previously described. (21).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
GST pull-down assay using GST-AMPK fusion protein-GST-AMPK{alpha}2 subunit fusion protein and the {beta}- and {gamma}-subunits were overexpressed in Sf9 cells using baculovirus. After the GST{alpha}2-subunit had been purified with glutathione-Sepharose 4B, Sf9 cell lysates co-overexpressing the {beta}1- and {gamma}2-subunits of AMPK were added to the GST-{alpha}2-subunit bound to glutathione-Sepharose 4B. Employing this procedure, a trimer consisting of {alpha}2-, {beta}1-, and {gamma}2-subunits was produced on glutathione-Sepharose 4B beads, although more than half of the {alpha}2-subunit was still not bound to the {beta}1- and {gamma}2-subunits. Next, purified GST-AMPK fusion proteins and GST alone were incubated with lysates from mouse skeletal muscle. GST-AMPK fusion and associated proteins were subjected to SDS-PAGE, followed by silver staining (Fig. 1A, lane 2). As a negative control, GST alone preincubated with {beta}1- and {gamma}2-subunits was incubated with tissue lysates from mouse skeletal muscle (Fig. 1A, lane 1). In lane 3 of Fig. 1A, corresponding to the GST-AMPK fusion protein, three subunits and several bands derived from Sf9 cell lysates are apparent. A comparison with lane 1, corresponding to the GST-alone protein, revealed the 160-kDa single band in lane 2 to be a protein binding specifically to AMPK. The 160-kDa band was trypsinized and analyzed using mass spectrometry and Mascot search. With the Mascot search method, protein scores exceeding 67 points are significant (P < 0.05). Five proteins had significant scores, and the highest, at 258 points, was rabbit GDE. The protein with the second highest score, 217 points, was rat GDE. Human GDE protein had a score of 174 points, the partial sequences of mouse GDE 94 and 75 points. All proteins with significant Mascot search results were GDEs, which consistently had very high scores. The identification of this 160-kDa protein as a GDE was confirmed with immunoblotting using anti-GDE antibody (data not shown). We thus concluded that the 160-kDa protein was, in fact, mouse GDE.



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  		Fig. 1. Pull-down assays using glutathione-S-transferase-AMP-activated protein kinase (GST-AMPK) fusion proteins. A: GST and GST-AMPK{alpha}2 fusion proteins were overexpressed in Sf9 cells by using a baculovirus and then purified with glutathione-Sepharose 4B. Purified GST alone and GST-AMPK{alpha}2 fusion proteins were incubated with Sf9 cell lysates overexpressing AMPK{beta}1 and AMPK{gamma}2 and then with a tissue lysate from mouse skeletal muscle for 1 h. Beads were washed 6 times with homogenizing buffer followed by SDS-PAGE and silver staining. Lane 1: GST alone was incubated with AMPK{beta}1 and AMPK{gamma}2 and then with lysates from mouse skeletal muscle. Lane 2: GST-AMPK{alpha}2 was incubated with AMPK{beta}1 and AMPK{gamma}2 and then with lysates from mouse skeletal muscle. Lane 3: GST-AMPK{alpha}2 was incubated with AMPK{beta}1 and AMPK{gamma}2 but not with the mouse skeletal muscle lysate. A 160-kDa band (arrow, lane 2) was identified as glycogen debranching enzyme (GDE) by mass spectrometry and Mascot search technique. B: lysates from mouse skeletal muscle were immunoprecipitated (IP) with control IgG or anti-AMPK{beta} antibodies, followed by SDS-PAGE and Western blotting with anti-AMPK{beta} (top) or anti-GDE (bottom) antibodies. C: Flag-tagged GDE was overexpressed in COS-7 cells with adenoviruses. After being serum starved or not for 12 h, cells were lysed and immunoprecipitated with anti-Flag antibody. Immunoprecipitants were subjected to SDS-PAGE and Western blotting using anti-AMPK{beta} (top), and anti-GDE (bottom) antibodies.

 
Subsequently, we examined the in vivo association between AMPK{alpha} and GDE. Mouse skeletal muscle lysates were immunoprecipitated with anti-AMPK{beta} antibody (Cell Signaling) or control IgG, and the immunoprecipitants were subjected to SDS-PAGE and immunoblotting using anti-GDE antibody. As shown in Fig. 1B, GDE was detected in the AMPK{beta} immunoprecipitant but not in the control IgG precipitant, which indicates a physiological association between AMPK and GDE. In addition, this association did not differ significantly, depending on whether the mice were fasted or fed.

GDE bound to the AMPK{beta}1-subunit directly. AMPK consists of a catalytic {alpha}-subunit and regulatory {beta}- and {gamma}-subunits. To examine which subunit binds GDE, each subunit and GDE were co-overexpressed in COS-7 cells and immunoprecipitated with each of the anti-tag antibodies. The {alpha}1-, {alpha}2-, {beta}1-, and {beta}2-subunits of AMPK were coimmunoprecipitated with GDE, whereas the {gamma}1- and {gamma}2-subunits were not (Fig. 2, A, B, and C). The 312 NH2-terminal amino acid portion of the {alpha}-subunit does not bind to the {beta}- and {gamma}-subunits, since the association with the {beta}-subunit is via the COOH-terminal portion of the {alpha}-subunit. The 312 NH2-terminal amino acids of the {alpha}-subunit were found to not coimmunoprecipitate with GDE (Fig. 2A, lower middle). This raised the possibility that the coimmunoprecipitation of the {alpha}-subunit with GDE did not represent a direct association but rather binding with the {beta}- or {gamma}-subunit of AMPK.



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  		Fig. 2. {beta}-Subunit of AMPK binds to GDE. A: GDE (Flag-tagged) and AMPK{alpha}2, 312 NH2-terminal amino acids of AMPK{alpha}2, and -{alpha}1 (myc-tagged) were co-overexpressed in COS-7 cells. Cell lysates were immunoprecipitated with anti-myc or anti-Flag antibodies followed by SDS-PAGE and Western blotting (IB) with anti-myc or anti-Flag antibodies. B: GDE (Flag-tagged) and AMPK{beta}1, -{beta}2 [(HA)-tagged], and AMPK{gamma}1 (HA-tagged) were co-overexpressed in COS-7 cells. Cell lysates were immunoprecipitated with anti-HA or anti-Flag antibodies followed by SDS-PAGE and Western blotting with anti-HA or anti-Flag antibodies. C: GDE (Flag-tagged) and AMPK{alpha}2, and -{gamma}2 (myc-tagged) were co-overexpressed in COS-7 cells. Cell lysates were immunoprecipitated with anti-myc or anti-Flag antibodies followed by SDS-PAGE and Western blotting with anti-myc or anti-Flag antibodies, respectively. D: GDE (Flag-tagged) and wild-type, W100G, and K128Q AMPK{beta}1 (HA-tagged) were overexpressed in Sf9 cells by baculoviruses. Cell lysates were immunoprecipitated with anti-HA or anti-Flag antibodies followed by SDS-PAGE and Western blotting with anti-HA or anti-Flag antibodies. E: GST fusion proteins containing full-length AMPK{alpha}2, -{beta}1, or -{gamma}1 were purified using glutathione-Sepharose 4B, as described in MATERIALS AND METHODS. GDE was purified using anti-FLAG M2-agarose (Sigma) and 3x Flag peptide (Sigma). Purified GDEs were incubated with GST, GST-AMPK{alpha}2, GST-AMPK{beta}1, or GST-AMPK{gamma}1 for 1 h. After 6 washes with lysis buffer, glutathione-Sepharose 4B beads were boiled in Laemmli buffer followed by SDS-PAGE and Western blotting using anti-Flag antibody.

 
To confirm that the {beta}- but not the {gamma}-subunit binds to GDE, GDE alone or GDE plus the {beta}1-, {beta}2-, {gamma}1-, or {gamma}2-subunit was overexpressed in COS-7 cells. In the GDE immunoprecipitant, the {beta}1- and {beta}2- but not the {gamma}1- and {gamma}2-subunits were detected (Fig. 2, B and C, lower middle). Similarly, GDE was detected in the {beta}1- and {beta}2- but not the {gamma}1- and {gamma}2-subunit immunoprecipitants (Fig. 2, B and C, bottom).

In addition, pull-down assays were performed using GST-AMPK fusion protein with purified GDE. As shown in Fig. 2E, GDEs were pulled down by purified GST-AMPK{beta}1 fusion protein.

Thus we concluded that the {beta}-subunit is responsible for the association of AMPK with GDE.

Recently, AMPK{beta}1 was reported to possess a glycogen binding domain (8, 9). GDE also binds to glycogen, and thus we speculate that AMPK{beta}1 and GDE coimmunoprecipitate through their glycogen binding domains rather than associating directly. To examine this possibility, {beta}1-subunit mutants W100G and K128Q, both of which are incapable of binding glycogen (8), were overexpressed with or without GDE, as well as the wild-type {beta}1-subunit (Fig. 2D). It was clearly demonstrated that these glycogen-binding, defective {beta}1-subunit mutants still coimmunoprecipitate with GDE, just like the wild-type {beta}1-subunit. This indicates that the association between the {beta}-subunit and GDE does not involve glycogen but rather is direct.

GDE binding to an AMPK site. To identify the GDE binding site in the {beta}1-subunit, we prepared various deletion mutants of the {beta}1-subunit as GST fusion proteins (Fig. 3A). By use of these GST-AMPK{beta}1 fragment fusion proteins, pull-down assays were performed.



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  		Fig. 3. Amino acids 68–123 of AMPK{beta}1 include the GDE binding site. A: schematic presentation of GST fragment of AMPK{beta}1 fusion protein. B: GST-AMPK{beta}1 fragment fusion proteins were overexpressed in JM109 and purified with glutathione-Sepharose 4B. Purified GST-AMPK fusion proteins were incubated with GDE (Flag-tagged) overexpressing Sf9 cell lysates for 1 h and then washed 6 times with lysis buffer followed by SDS-PAGE and Western blotting with anti-Flag antibody. The amino acids 68–123 sequence of the {beta}1-subunit bound GDE most strongly. C and D: GDE and amino acids 1–67, 68–123, or 164–270 of the {beta}1-subunit were overexpressed in COS-7 cells with adenoviruses. C: cells were lysed and immunoprecipitated with anti-Flag antibody. Immunoprecipitants were subjected to SDS-PAGE and Western blotting using anti-AMPK{beta} and anti-GDE antibodies. D: overexpression of amino acids 68–123 of the {beta}1-subunit inhibited association of AMPK{beta} with GDE. Total cell lysates were subjected to SDS-PAGE and Western blotting using anti-AMPK{alpha}1- or -{alpha}2-specific antibody.

 
First, GST protein containing three fragments corresponding to the NH2 terminus, GBD, and COOH terminus of the {beta}1-subunit were purified with glutathione-Sepharose 4B, and the association with GDE was assessed by incubation with the lysate from GDE overexpressing Sf9 cells. Figure 3B, panel 1, shows clearly that the sequence responsible for the association with GDE is included in the GBD of the {beta}1-subunit (Fig. 3B, panel 1). Next, GBD was separated into two fragments: amino acids 68–113 and amino acids 113–163 of the {beta}1-subunit. However, we found that these fragments did not bind GDE (Fig. 3B, panel 2). Thus we prepared four additional {beta}1-subunit deletion mutants of GBD: amino acids 68–123, 68–133, 68–143, and 68–153. Amino acids 68–123, but not 68–113, were demonstrated to be sufficient for binding with GDE (Fig. 3B, panel 3). Amino acid sequences 68–133 and 68–143 exhibited weaker binding with GDE than amino acids 68–123, although the reason for this is unclear. The binding of amino acids 68–123 with GDE was not abolished by washing with high-salt buffer (containing 500 mM NaCl), whereas those with full-length GBD and amino acid sequences 68–133, 68–143, or 68–153 were apparently reduced (Fig. 3B, panel 4). Thus it is likely that amino acids 68–123 of the {beta}1-subunit possess a high affinity for GDE.

Inhibition of the association between GDE and AMPK increases AMPK activity. Amino acids 68–123 were used to inhibit the association between AMPK and GDE. Full-length GDE and amino acids 68–123 of the {beta}1-subunit were overexpressed, and GDE was immunoprecipitated with anti-Flag antibody. As shown in Fig. 3C, overexpression of amino acids 68–123 of the {beta}1-subunit inhibited the association between GDE and AMPK{beta}1, whereas the AMPK{alpha}1 and {alpha}2 protein expression levels were unchanged (Fig. 3D).

Under these conditions, we examined GDE or AMPK activities in COS-7 cells. Overexpressing amino acids 68–123 of the {beta}1-subunit did not change GDE activities (Fig. 4A). On the other hand, basal AMPK activity increased significantly, by ~30%, compared with control cells (Fig. 4B). Basal phosphorylation of AMPK{alpha}-Thr172 was also increased by 35% (Fig. 4C). Phosphorylation of ACC-Ser79, which is reportedly phosphorylated by AMPK, was also approximately doubled when amino acids 68–123 of the {beta}1-subunit were overexpressed without AICAR stimulation (Fig. 4D). Thus it is very likely that inhibition of the association between GDE and AMPK increases AMPK activity.



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  		Fig. 4. Inhibition of association between AMPK and GDE increases basal phosphorylation of AMPK{alpha} and acetyl-CoA carboxylase (ACC) and also increases basal AMPK activity. A: after serum starvation, preincubation, and 5-aminoimidazole-4-carboxamide-1-{beta}-d-ribofuranoside (AICAR) stimulation, as described in RESULTS, GDE-overexpressing COS-7 cells were collected with Tris buffer and then sonicated. Sonicated cells were centrifuged at 17,000 g for 20 min, and supernatants were immunoprecipitated with anti-Flag antibody. GDE activities in immunoprecipitants were assayed using glucosyl-{beta}-cyclodextrin as substrate, as described in MATERIALS AND METHODS. Bars depict means ± SE of 3 independent experiments. B: COS-7 cells overexpressing amino acids 1–67, 68–123, or 164–270 of the {beta}1-subunit were treated with or without 2 mmol/l AICAR for 30 min. Cells were then lysed and immunoprecipitated with anti-AMPK{alpha} antibody. AMPK activities in immunoprecipitants were assayed using SAMS peptide. Bars depict means ± SE of 3 independent experiments. C and D: COS-7 cells overexpressing amino acids 1–67, 68–123, or 164–270 of the {beta}1-subunit were incubated with or without 2 mmol/l AICAR for 30 min. Cells were then lysed followed by SDS-PAGE and Western blotting using anti-phospho-AMPK{alpha}-Thr172 (C), andanti-phospho-ACC-Ser79 (D). *P < 0.05 vs. control cells (basal).

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
AMPK is a regulator of the key enzymes involved in glucose and lipid metabolism, functioning as a fuel gauge sensor. ACC and HMG-CoA reductase are well known to be substrates of AMPK; however, downstream from AMPK, their roles in glucose uptake and glycogen metabolism remain unclear. In this study, we identified GDE as an AMPK-binding protein by using a GST-AMPK pull-down assay. Coimmunoprecipitation of AMPK with GDE was observed not only in overexpression experiments but also endogenously (Fig. 1B). In addition, the {beta}-subunit was demonstrated to be responsible for the association with GDE. Interestingly, the portion of the {beta}-subunit that binds with GDE (amino acids 68–123 of AMPK{beta}1) is included in the sequence that reportedly associates with glycogen (amino acids 68–163 of AMPK{beta}1). Thus we suspected that the association between the {beta}-subunit and GDE occurs via the binding of glycogen to both the {beta}-subunit and GDE. However, W100G and K128Q, glycogen nonbinding mutants of the {beta}1-subunit, fully retained the ability to bind with GDE, suggesting that the association between the {beta}-subunit and GDE does not involve glycogen binding. Furthermore, we found that adding glycogen to the buffer did not alter the in vitro association between the {beta}-subunit and GDE (data not shown). Taking these two observations into consideration, it is reasonable to assume that the association of the {beta}-subunit of AMPK with GDE is direct.

GDE, a 160-kDa monomeric protein, is widely distributed in bacteria, yeasts, plants, and animals. GDE contains two independent catalytic activities, transferase and glucosidase (22, 23), that are responsible for glycogen degradation. Although glycogen phosphorylase degrades glycogen from its nonreducing ends, leaving dextrin with shortened chains, GDE transfers maltosyl units from the shortened chain to another chain employing its transferase activity. GDE then removes the glycosyl stub using its glucosidase activity. Genetic deficiency of GDE in humans is known to cause a type III glycogen storage disorder called Cori's disease (24), which is characterized by hepatomegaly, hypoglycemia, short stature, and muscle weakness (25–27).

A considerable portion of AMPK reportedly colocalizes with glycogen particles, although the physiological significance of this subcellular localization remains unclear. Our results suggest that this subcellular localization of AMPK may be due to binding to GDE but not glycogen or to binding both GDE and glycogen. In addition, we demonstrated herein that amino acids 68–123 of the {beta}1-subunit contain the GDE binding domain. We also showed that overexpression of amino acids 68–123 of the {beta}1-subunit effectively interrupts the binding of endogenous AMPK with GDE and significantly enhances AMPK activity. Thus it is very likely that the association with GDE suppresses the basal kinase activity of AMPK, although how release of the {beta}-subunit from GDE affects the kinase activity of the {alpha}-subunit of AMPK remains unknown. We speculate that a decrease in glycogen content may induce dissociation of AMPK from GDE, thereby increasing AMPK activity, which leads to various metabolic actions, including increased glucose uptake and fatty acid oxidation. However, we did not examine the relationship between cellular glycogen contents and the association between AMPK and GDE herein. It is reasonable that upregulating AMPK activity would increase glucose uptake and fatty acid oxidation when cellular glycogen is in short supply. This hypothesis appears to be supported by a previous study showing AMPK activity to be higher in skeletal muscle containing less glycogen (12).

We also speculate that the localization of AMPK is important for its regulation of kinase activity. Many proteins, including protein phosphatase, bind to glycogen, and this binding determines their cellular localizations (8, 9). Furthermore, dissociation from glycogen may change certain protein interactions. AMPK is reportedly activated via phosphorylation of its {alpha}-subunit in response to ATP depletion, which is caused by muscle contraction, metabolic poisoning, oxidatative stress, hypoxia, and nutrient deprivation. Increased cellular AMP changes the conformation of the AMPK{alpha}{beta}{gamma} complex, revealing the Thr172 of the {alpha}-subunit to be phosphorylated by LKB-1 (28–30). Thus it is reasonable to speculate that the conformational change in AMPK induced by GDE binding affects the association of AMPK with LKB-1 or some other phosphatase(s). Unfortunately, however, we detected no significant change in the association between AMPK and GDE in response to either fasting or feeding of the mice or with serum starvation of COS-7 cells. Thus although this is the first study to demonstrate a direct association between AMPK and GDE, further studies are needed to clarify how this association is regulated. In addition, it is also necessary to elucidate the resultant physiological significance of this association, which may occur via the regulation of AMPK activity or subcellular localization.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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REFERENCES
 
This work was supported by a Grant-in-Aid for Suzuken Memorial foundation (04-042).


   FOOTNOTES
 

Address for reprint requests and other correspondence: Department of Physiological Chemistry and Metabolism, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo Bunkyo-ku, Tokyo, Japan (e-mail: asano-tky{at}umin.ac.jp)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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