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Originally published In Press as doi:10.1074/jbc.M606676200 on October 5, 2006

J. Biol. Chem., Vol. 281, Issue 48, 36662-36672, December 1, 2006
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Regulation of AMP-activated Protein Kinase by Multisite Phosphorylation in Response to Agents That Elevate Cellular cAMP*Formula >

Rebecca L. Hurley{ddagger}, Laura K. Barré{ddagger}, Sumintra D. Wood{ddagger}, Kristin A. Anderson§, Bruce E. Kemp1, Anthony R. Means§, and Lee A. Witters{ddagger}2

From the {ddagger}Departments of Medicine and Biochemistry, Dartmouth Medical School, and the Department of Biological Sciences, Dartmouth College, Hanover, New Hampshire 03755, St. Vincent's Institute and Commonwealth Scientific and Industrial Research Organization Molecular and Health Technologies, Fitzroy, Victoria 3065, Australia, and the §Department of Pharmacology and Cancer Biology, Duke University, Durham, North Carolina 27710

Received for publication, July 13, 2006 , and in revised form, August 30, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The AMP-activated protein kinase (AMPK) and cAMP signaling systems are both key regulators of cellular metabolism. In this study, we show that AMPK activity is attenuated in response to cAMP-elevating agents through modulation of at least two of its {alpha} subunit phosphorylation sites, viz. {alpha}-Thr172 and {alpha}1-Ser485/{alpha}2-Ser491, in the clonal beta-cell line INS-1 as well as in mouse embryonic fibroblasts and COS cells. Forskolin, isobutylmethylxanthine, and the glucose-dependent insulinotropic peptide inhibited AMPK activity and reduced phosphorylation of the activation loop {alpha}-Thr172 via inhibition of calcium/calmodulin-dependent protein kinase kinase-{alpha} and -beta, but not LKB1. These agents also enhanced phosphorylation of {alpha}-Ser485/491 by the cAMP-dependent protein kinase. AMPK {alpha}-Ser485/491 phosphorylation was necessary but not sufficient for inhibition of AMPK activity in response to forskolin/isobutylmethylxanthine. We show that AMPK {alpha}-Ser485/491 can be a site for autophosphorylation, which may play a role in limiting AMPK activation in response to energy depletion or other regulators. Thus, our findings not only demonstrate cross-talk between the cAMP/cAMP-dependent protein kinase and AMPK signaling modules, but also describe a novel mechanism by which multisite phosphorylation of AMPK contributes to regulation of its enzyme activity.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The AMP-activated protein kinase (AMPK)3 is a member of a large metabolite-sensing protein kinase family found in eukaryotes. The mammalian enzyme is a heterotrimeric serine/threonine kinase consisting of a catalytic subunit, {alpha}, and two regulatory and targeting subunits, beta and {gamma}. Covalent activation of AMPK is mediated through its major regulatory phosphorylation site, {alpha}-Thr172, located within the "activation" loop on the {alpha} subunit. Phosphorylation of this site is essential for activity and can be catalyzed by four Thr172 AMPK kinases (AMPKKs), viz. LKB1 (1–3), calcium/calmodulin-dependent protein kinase kinase (CaMKK)-{alpha} and CaMKKbeta (4–7), and TAK1 (8). In addition to this phosphorylation site, other sites on both the {alpha} and beta subunits have been identified; however, their roles in regulation of AMPK have not been fully clarified (9–12). AMPK {alpha}1-Ser485 (equivalent to AMPK {alpha}2-Ser491) has been identified as both an autophosphorylation site (13) and a target site for protein kinase B/Akt (13, 14). Phosphorylation of {alpha}-Ser485/491 by Akt in response to insulin stimulation is likely to be involved in the insulin-mediated inhibition of AMPK activity (15–18).

The discovery that AMPK can be regulated not only by changes in the cellular AMP/ATP ratio, but also by the CaMKKs, which can be activated in response to several cell-surface ligands, has broadened the conceptual view of AMPK regulation (19, 20). Previous studies indicate that many extracellular ligands, including leptin, adiponectin, ghrelin, bradykinin, and catecholamines, can regulate AMPK activity (20). cAMP signaling systems are known to regulate many of the same metabolic pathways of carbohydrate, lipid, and protein metabolism that have more recently been shown to be regulated by the AMPK signaling system. Instances of cross-talk between cAMP signaling and AMPK pathways are well documented in muscle, liver, and adipose tissue. Physiologic stimuli such as exercise and fasting, as well as hormonal stimulation via beta-adrenergic receptors, may result in the simultaneous stimulation of both cAMP-dependent protein kinase (PKA) and AMPK (21–23). In these tissues, both kinases likely work in synergy to regulate key enzymes involved in glycogen metabolism, cholesterol synthesis, and fatty acid metabolism (23–27). However, AMPK and PKA can also antagonize one another, as is the case in their regulation of hormone-sensitive lipase, which regulates lipolysis in both adipose and muscle tissue (28).

In this study, we examined the relationship between the cAMP and AMPK signaling systems in an insulin-secreting cell line, INS-1. This cell line was chosen because it expresses many of the metabolic pathways regulated by both systems; responds to multiple ligands such as glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1, which work through Gs-coupled receptors that activate adenylyl cyclase; and has several characteristic phenotypic responses, including insulin secretion, that are influenced by activation of both pathways. Our results indicate an important intersection between these two signaling systems that involves regulation by cAMP signaling of AMPKKs active on both {alpha}-Thr172 and {alpha}-Ser485/491, underscoring the importance of multisite phosphorylation in the modulation of AMPK activity.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture, Incubation, and Extraction—INS-1 cells (a kind gift of Christopher Newgard, Duke University) were grown in RPMI 1640 medium supplemented with 10 mM HEPES, 1 mM sodium pyruvate, 50 µM beta-mercaptoethanol, 2 mM glutamine, 10% fetal bovine serum, 100 units/ml penicillin, and 100 mg/ml streptomycin. COS cells were purchased from American Type Culture Collection. Mouse embryonic fibroblasts (MEFs) from LKB1+/+ and LKB1–/– mice were kindly provided by Reuben Shaw (Harvard University). MEFs from CaMKKbeta+/+ and CaMKKbeta–/– mice4 were prepared as described (29). MEFs were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin. All cell lines were maintained at 37 °C in a humidified atmosphere containing 5% CO2. Cells were incubated in 6-well plates after various additions prior to extraction for immunoblotting and analysis of AMPK or AMPKK activity. Cell extracts were prepared in a 1% Triton X-100-containing buffer as described (30). Protein concentration was determined with a BCA assay (Pierce) according to the manufacturer's protocol.

Immunological Reagents and Immunoblotting—Cell extracts were examined by immunoblotting as described (30) employing a panel of different antibodies. These included anti-total AMPK{alpha} (reactive against both the {alpha}1 and {alpha}2 subunits), anti-AMPK {alpha}-phospho-Thr172, anti-AMPK {alpha}-phospho-Ser485/491, and anti-CaMKK{alpha}/beta (C terminus; BD Transduction Laboratories); anti-total Akt (pleckstrin homology domain; catalog no. 05-591, Upstate); and anti-Akt phospho-Thr308 (catalog no. 9275) and anti-Akt phospho-Ser473 (catalog no. 9271) (Cell Signaling Technology, Inc.). Where appropriate for quantitation, immunoblots were scanned into Adobe Photoshop using a UMAX Astra 6700 scanner with SilverFast software (Laser Soft Imaging AG, Kiel, Germany) at standardized settings. Band pixel intensity was quantified using IPLab Gel software.

Anti-CaMKK isoform polyclonal antibodies employed in immunoprecipitation were raised by immunizing rabbits with peptide sequences specific for CaMKK{alpha} (CQDPRAELVERVAAIDCTH, corresponding to residues 9–27 of the human sequence) or CaMKKbeta (EALRGLSSLSIHLGMESF, corresponding to residues 35–52 of the human sequence). Antisera were then purified by immunoaffinity chromatography against each peptide sequence, and specificity for the isoforms was determined by immunoblotting of authentic recombinant proteins (data not shown).

AMPK Immunoprecipitation and Activity—AMPK was immunoprecipitated from experimental or control cell extracts prepared in triplicate, each representing a pooling of 2 wells treated under identical conditions. Protein-matched extracts (200–500 µg of total protein) were incubated with 40 µl of Protein A/G PLUS-agarose beads (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and 5 µl of serum containing antibodies immunoreactive against amino acids 2–20 of both AMPK{alpha}1 and AMPK{alpha}2 or with control preimmune serum in a total volume of 1 ml. Following incubation for 2 h at 4°C, extracts were spun down briefly for 20 s at 12,000 rpm to gently pellet the beads. The supernatant was carefully removed, and the beads were washed once with buffer containing 1% Triton X-100 and then subsequently washed two times with the same buffer containing no Triton. The beads were then resuspended in 50 µlof this buffer, and 10 µl of the resulting immune complexes was immediately assayed in duplicate for AMPK activity against the SAMS peptide at a saturating concentration of AMP as described (15).

CaMKK Immunoprecipitation and Activity—CaMKK{alpha} and CaMKKbeta were immunoprecipitated from experimental or control cell extracts prepared in triplicate, each representing a pooling of 2 wells treated under identical conditions. Protein-matched extracts were divided into three equal fractions and incubated with 40 µl of Protein A/G PLUS-agarose beads and purified rabbit polyclonal antibodies against either CaMKK{alpha} CaMKKbeta or rabbit normal IgG (Sigma) as a control. The concentration of each antibody used was 1 µg/100 µg of total extract protein. Following incubation for 2 h at 4°C, extracts were spun down briefly at 20 s for 12,000 rpm to gently pellet the beads. The supernatant was carefully removed, and the beads were washed once with buffer containing 1% Triton X-100 and then subsequently washed twice with reaction buffer (125 mM Tris-HCl (pH 7.6), 1.25 mM dithiothreitol, and 1.25 mg/ml bovine serum albumin). For determination of CaMKK{alpha} and CaMKKbeta enzyme activity, immune complexes were resuspended in a total volume of 80 µl of reaction buffer. 25 µl of each suspension (final concentration of 50 mM Tris-HCl (pH 7.6), 0.5 mM dithiothreitol, and 0.5 mg/ml bovine serum albumin) was then incubated in a final volume of 60 µl with recombinant AMPK{alpha}1-(1–312) (1 µg) (30) in the presence of 1 µM calmodulin, 200 µM ATP, 10 mM MgCl2, and either 1 mM EGTA or 1 mM CaCl2 for 20 min at 30 °C. Reactions were stopped by the addition of 4x Laemmli sample buffer, and samples were boiled for 5 min. Proteins were separated by SDS-PAGE and probed with antibody directed against AMPK {alpha}-phospho-Thr172 to detect phosphorylation of recombinant AMPK{alpha}1-(1–312).

AMPK {alpha}1-Ser485 Kinase Assays—AMPKK activity in cell extracts directed against the AMPK {alpha}-Ser485/491 site was determined by phosphorylation of recombinant AMPK{alpha}1-(250–548) as described (30), detected by immunoblotting with anti-{alpha}-phospho-Ser485/491 antibody following separation of reaction products by SDS-PAGE. {alpha}1-Ser485 activity of PKA against the same recombinant protein was determined by incubation of the recombinant PKA catalytic subunit (Calbiochem) with the AMPK{alpha}1-(250–548) fusion protein as a substrate. The catalytic subunit (22.4 units) was incubated with 1.6 µg of recombinant AMPK{alpha}1-(250–548) in the presence of either [{gamma}-32P]ATP or unlabeled ATP (200 µM) and Mg2+ (5 mM), and phosphate incorporation was monitored by either by trichloroacetic acid precipitation of radiolabeled proteins or by immunoblotting for {alpha}1-phospho-Ser485.

Preparation of AMPK Heterotrimer—COS cells were triply transfected with cDNAs expressing glutathione S-transferase-tagged full-length AMPK{alpha}1 (wild-type or S485A mutant) and AMPKbeta1 and hemagglutinin-tagged AMPK{gamma}1 (31) using FuGENE 6 (Roche Applied Science) according to the manufacturer's protocol. At 48 h post-transfection, cells were incubated in serum-free medium and then treated with forskolin (10 µM) and isobutylmethylxanthine (IBMX; 500 µM) or ethanol (1:50) for 30 min. Cells were lysed in buffer containing 1% Triton X-100 as described above. Cleared supernatants were absorbed to glutathione-agarose beads; active heterotrimer enzyme complexes were isolated as described (4); and AMPK activity was assayed as described (15). For immunoblot analysis, isolated heterotrimer (HT) samples were boiled in the presence of 4x Laemmli sample buffer and further diluted 1:1 with the sample buffer to reduce the effects of high salt on separation by SDS-PAGE. For in vitro kinase assays with recombinant CaMKKbeta, isolated complexes were first dephosphorylated and inactivated by incubation with 50 units of {lambda}-protein phosphatase (New England Biolabs, Beverly, MA) at 30 °C for 30 min according to manufacturer's protocol. Reactions were stopped with 10 µM sodium vanadate to inactivate phosphatase activity. Half of this reaction was then incubated with or without 10 µg of recombinant CaMKKbeta in the presence of 200 µM ATP and 5 mM Mg2+ as described (29) at 30 °C for 30 min, at which time reactions were stopped by the addition of 4x Laemmli sample buffer, and samples were boiled for 5 min. Proteins were separated by SDS-PAGE and probed with antibodies directed against AMPK {alpha}-phospho-Thr172, AMPK {alpha}-phospho-Ser485/491, and total AMPK{alpha}. The enzyme activities of the dephosphorylated and rephosphorylated heterotrimer complexes were measured against the SAMS peptide at a saturating concentration of AMP as described (15).

Miscellaneous Materials—Reagents, including forskolin, IBMX, and the PKA peptide inhibitor (PKI), were purchased from Sigma. GIP was purchased from Tocris Bioscience (Ellisville, MO).

Statistical Analysis—Statistical analysis of experimental data was performed by a paired Student's t test with two-tailed distribution. In instances in which data sets could not be paired, analysis was done unpaired, but with equal variance between data sets. p < 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Forskolin and GIP Modulate AMPK Activity and Phosphorylation of AMPK {alpha}-Thr172 and {alpha}-Ser485/491
Treatment of INS-1 cells with forskolin, an activator of adenylyl cyclase, or with GIP, a gastrointestinal hormone whose Gs-coupled receptor can activate the cAMP pathway (32), leads to the significant inhibition of AMPK activity (Fig. 1A). IBMX, an inhibitor of cAMP phosphodiesterase, also had similar effects (data not shown). Coincident with this activity change, AMPK {alpha}-Thr172 phosphorylation was decreased (Fig. 1, B, middle panel; and C). Additionally, there was a notably slower electrophoretic migration of AMPK{alpha} in the presence of forskolin and GIP (Fig. 1B, left panel), raising the possibility of phosphorylation of AMPK{alpha} at additional sites. Further analysis demonstrated that there was increased phosphorylation of {alpha}-Ser485/491, most prominent on the slowest migrating {alpha} band, which was minimally phosphorylated at Thr172 (Fig. 1, B, right panel; and C). Quantitation of these changes in AMPK{alpha} site-specific phosphorylation by scanning densitometry revealed similar magnitude changes in response to forskolin and GIP (Fig. 1C).


Figure 1
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  		FIGURE 1.
Forskolin and GIP alter AMPK activity and phosphorylation in INS-1 cells. INS-1 cells were preincubated in serum-free INS-1 medium for 1 h, followed by treatment with forskolin (10 µM) or GIP (100 nM) for 30 min. Cells extracts were then prepared as described under "Experimental Procedures"; each extract (n) represents a pooling of 2 wells of a 6-well plate treated under identical conditions. A, AMPK was immunoprecipitated from protein-matched cell extracts in triplicate using pan-AMPK{alpha}-specific antibody, and the activity of the isolated enzyme was measured as described under "Experimental Procedures." Data (mean ± S.D., n = 9) are expressed as picomoles of 32Pi transferred to the SAMS peptide/min/mg of total protein subjected to immunoprecipitation. *, p < 0.0003 (compared with the control treatment by Student's t test); **, p < 0.022. B, shown is a representative immunoblot in which duplicate extracts from control or forskolin-treated (FSK) cells were matched for total protein and subjected to SDS-PAGE, followed by immunoblot analysis using antibodies directed against AMPK {alpha}-phospho-Thr172 ({alpha}T172p), AMPK {alpha}-phospho-Ser485/491 ({alpha}S485/491p), and total AMPK{alpha} (Total {alpha}). C, shown are the results from densitometric analysis of the immunoblots. White bars, control conditions; black bars, forskolin treatment; striped bars, GIP treatment. Data are expressed as the ratio of the phosphorylation state ({alpha}-phospho-Thr172 or {alpha}-phospho-Ser485/491) to total AMPK{alpha} (mean ± S.D., n = 3). The decreases in {alpha}-Thr172 phosphorylation and the increases in {alpha}-Ser485/491 phosphorylation compared with the control are all significant. *, p < 0.04; **, p < 0.004.

 


Figure 2
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  		FIGURE 2.
Effects of forskolin/IBMX on AMPK in MEFs. A and B, MEF lines lacking LKB1 (LKB1–/–) or CaMKKbeta (CaMKKbeta–/–), respectively, or WT MEFs (+/+) from each genetic strain were preincubated in serum-free Dulbecco's modified Eagle's medium for 1 h, followed by treatment with forskolin (10µM) and IBMX (500 µM) or ethanol (1:50) for 30 min in parallel. Cell extracts were prepared as described under "Experimental Procedures"; each extract (n) represents a pooling of 2 wells treated under identical conditions. Triplicate extracts were matched for protein and subjected to SDS-PAGE, followed by immunoblot analysis using antibodies directed against AMPK {alpha}-phospho-Thr172 ({alpha}T172p), AMPK {alpha}-phospho-Ser485/491 ({alpha}S485/491p), and total AMPK{alpha} (Total {alpha}). C, CaMKKbeta+/+ and CaMKKbeta–/– MEFs were treated as described above, and triplicate cell extracts were prepared by the digitonin lysis method for measurement of AMPK activity as described (4). Each extract (n) represents a pooling of 2 wells treated under identical conditions. Data (mean ± S.D., n = 3) are expressed as picomoles of 32Pi transferred to the SAMS peptide/min/mg of total protein.

 
Forskolin Regulation of AMPKKs Active on AMPK {alpha}-Thr172
Three upstream AMPKKs identified as LKB1, CaMKK{alpha}, and CaMKKbeta activate AMPK by phosphorylating {alpha}-Thr172 (1–7). To test whether any of these AMPKKs are required to mediate the cAMP-dependent inhibition of AMPK, we first utilized fibroblast cell lines derived from mouse embryos lacking either LKB1 (3, 4) or CaMKKbeta4; MEFs lacking CaMKK{alpha} are not presently available. To ensure a persistent elevation of cAMP levels, cells were treated with both IBMX and forskolin (Fig. 2). In wild-type MEFs, this treatment triggered an AMPK response similar to that observed in INS-1 cells, with a decrease in AMPK activity (Fig. 2C) concomitant with reduced {alpha}-Thr172 phosphorylation (Fig. 2, A and B, upper panels) and increased {alpha}-Ser485/491 phosphorylation (Fig. 2, A and B, middle panels). These effects were preserved in LKB1–/– MEFs (Fig. 2A), indicating that LKB1 is not required to mediate the effects of elevated cAMP. In contrast, loss of CaMKKbeta largely abrogated the cAMP-dependent reduction in both AMPK activity and {alpha}-Thr172 phosphorylation (Fig. 2, B, upper panel; and C), demonstrating that CaMKKbeta is an important intermediary in reducing AMPK {alpha}-Thr172 phosphorylation by agents that elevate cAMP. However, the absence of CaMKKbeta did not alter the ability of forskolin/IBMX to increase {alpha}-Ser485/491 phosphorylation (Fig. 2B, middle panel).

Reduced phosphorylation of AMPK {alpha}-Thr172 could be the result of cAMP-mediated inhibition of the activity of one or more of the three known AMPKKs. To test this question directly, we attempted to immunoprecipitate LKB1, CaMKK{alpha}, and CaMKKbeta from extracts of either control or forskolin-treated INS-1 cells and assay for AMPKK activity with a recombinant AMPK{alpha}1-(1–312) protein substrate. Although LKB1 was expressed in the INS-1 line (data not shown), we were unable to immunoprecipitate LKB1 activity with commercially available antibodies, leaving unanswered the question of whether this kinase can be directly regulated in response to elevated cAMP levels. However, both CaMKK{alpha} and CaMKKbeta were also expressed in the INS-1 cells, and their activities could be immunoprecipitated (Fig. 3, A and B). Consistent with other observations (33, 34), isolated CaMKK{alpha} and CaMKKbeta exhibited detectable kinase activity only in the presence of added Ca2+/calmodulin (data not shown). As shown in Fig. 3B, forskolin treatment led to a significant 40% decrease in both CaMKK{alpha} and CaMKKbeta activities, as measured in the presence of Ca2+/calmodulin, against the AMPK{alpha}1-(1–312) fusion protein (Fig. 3, B and C). Forskolin treatment did not affect the recovery of either CaMKK{alpha} or CaMKKbeta in the immunoprecipitates (data not shown). Taken together, the studies in CaMKKbeta–/– MEFs, coupled with the in vitro measurements of individual CaMKK activities in INS-1 cell extracts, show that CaMKK{alpha} and/or CaMKKbeta is required to mediate the forskolin-dependent inhibition of AMPK activity.

AMPK {alpha}-Ser485/491 Phosphorylation
Autophosphorylation—Although the cAMP-dependent inhibition of AMPK activity might be accounted for by the attenuation of phosphorylation of AMPK {alpha}-Thr172, the unexpected finding that another site in AMPK, {alpha}-Ser485/491, shows simultaneously a significant increase in phosphorylation raises several important questions regarding not only the nature of this phosphorylation event, but also how phosphorylation of the {alpha}-Ser485/491 site might contribute to overall enzyme activity. Regulation of phosphorylation of the {alpha}-Ser485/491 site has not been well characterized to date. There are reports suggesting that it is likely to be both an autophosphorylation site (13) as well as a target for protein kinase B/Akt (13, 14). To directly test the ability of AMPK to phosphorylate AMPK {alpha}-Ser485/491, we utilized an in vitro kinase kinase assay in which bacterially expressed truncated AMPK{alpha}1-(1–312) was first activated by recombinant CaMKKbeta as described (30) and then incubated with a recombinant AMPK{alpha}1 protein substrate containing an unoccupied {alpha}1-Ser485 site. As shown in Fig. 4A, phosphorylation of {alpha}1-Ser485 occurred only in the presence of active AMPK{alpha}1-(1–312), whereas both inactive AMPK{alpha} and CaMKKbeta were unable to phosphorylate the site. In intact wild-type MEFs, treatment with known AMPK activators (ionomycin and 2-deoxyglucose) led to stimulation of AMPK activity (4) concomitant with increased {alpha}-Thr172 phosphorylation (Fig. 4B, lower panel). This enhanced activity was also accompanied by a significant increase in {alpha}-Ser485/491 phosphorylation (Fig. 4B, upper panel). Although these results are not inconsistent with an autophosphorylation event, the extent of {alpha}-Ser485/491 phosphorylation does not parallel {alpha}-Thr172 phosphorylation with these two AMPK activators, leaving open the possibility that other {alpha}-Ser485/491-directed AMPKKs may play a role in {alpha}-Ser485/491 phosphorylation during AMPK activation.


Figure 3
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  		FIGURE 3.
Forskolin inhibits CaMKK activity in INS-1 cells. INS-1 cells were preincubated in serum-free medium for 1 h, followed by treatment with forskolin (10 µM) for 30 min. Cells extracts were prepared as described under "Experimental Procedures"; each extract (n) represents a pooling of 2 wells treated under identical conditions. Extracts were protein-matched, and each one was divided into three equal fractions for immunoprecipitation (IP) with anti-CaMKK{alpha} or anti-CaMKKbeta antibody or with control normal rabbit IgG as indicated. A, shown is an immunoblot of the immunoprecipitated enzyme complexes from INS-1 cells probed with a mouse monoclonal antibody that recognizes C-terminal sequences common to both CaMKK{alpha} and CaMKKbeta. B, isolated CaMKK complexes were incubated with recombinant AMPK{alpha}1-(1–312) in the presence of Ca2+, calmodulin, and ATP/Mg2+ for 20 min at 30 °C as described under "Experimental Procedures." Reactions were stopped by the addition of 4x Laemmli sample buffer, and samples were boiled for 5 min. Proteins were separated by SDS-PAGE and probed with anti-AMPK {alpha}-phospho-Thr172 antibody to detect phosphorylation of recombinant AMPK{alpha}1, C, shown are the results from densitometric analysis of CaMKK activity at AMPK {alpha}-Thr172. White bars, control conditions; black bars, forskolin treatment. Data (mean ± S.D., n = 6 for control and n = 5 for forskolin-treated) are expressed as the measured pixel density of {alpha}-Thr172 phosphorylation in the recombinant AMPK{alpha} substrate. The decrease in AMPKK activity of both CaMKK{alpha} and CaMKKbeta following forskolin treatment was significant versus the control. *, p < 0.0005.

 
Evidence for an {alpha}-Ser485/491 AMPKK—Because AMPK activity is substantially inhibited under conditions of elevated cAMP levels, the enhanced phosphorylation of {alpha}-Ser485/491 in response to forskolin and GIP is unlikely to be due to an autophosphorylation event, suggesting the existence of a separate {alpha}-Ser485/491 AMPKK. Such an activity was demonstrated using an in vitro kinase kinase assay in which INS-1 cell extracts made from control or forskolin- or IBMX-treated cells were incubated with a recombinant AMPK{alpha}1 protein substrate containing an unoccupied {alpha}1-Ser485 site. As shown in Fig. 4C, forskolin- and IBMX-treated cells extracts showed significantly enhanced AMPKK activity against {alpha}1-Ser485, with little activity detectable in unstimulated cells.


Figure 4
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  		FIGURE 4.
AMPK {alpha}-Ser485/491 phosphorylation. A, shown is a representative immunoblot of an in vitro kinase kinase assay in which bacterially expressed truncated AMPK{alpha}1-(1–312) was first activated by recombinant CaMKKbeta as described (29) and then incubated in the presence or absence of recombinant AMPK{alpha}1-(250–548) along with ATP/Mg2+ for 20 min at 30 °C. As a control, CaMKKbeta or inactive AMPK{alpha}1-(1–312) was also incubated with the substrate in parallel. Reactions were stopped by the addition of 4x Laemmli sample buffer, and samples were boiled for 5 min. Proteins were separated by SDS-PAGE and probed with anti-AMPK {alpha}-phospho-Ser485/491 antibody. B, WT MEFs were stimulated with either ionomycin (1 µM) for 5 min or 2-deoxyglucose (2-DG; 50 mM) for 15 min. Cell extracts were prepared by the digitonin lysis method as described (4). Each extract (n) represents a pooling of 2 wells treated under identical conditions. Duplicate extracts were matched for protein and subjected to SDS-PAGE, followed by immunoblot analysis using anti-AMPK {alpha}-phospho-Ser485/491 ({alpha}S485/491p) and anti-AMPK {alpha}-phospho-Thr172 ({alpha}T1729) antibodies. C, INS-1 cells were preincubated in serum-free medium for 1 h, followed by treatment with either forskolin (10 µM), IBMX (500 µM), or ethanol (control; 1:100) for 30 min. Extracts were prepared in triplicate, each representing 1 well of a 6-well plate, and then matched for total protein. Each extract was incubated in the presence of recombinant AMPK{alpha}1-(250–548) along with ATP/Mg2+ for 20 min at 30 °C. Reactions were stopped by the addition of 4x Laemmli sample buffer, and samples were boiled for 5 min. Proteins were separated by SDS-PAGE and probed with anti-AMPK {alpha}-phospho-Ser485/491 antibody.

 
Identity of the Forskolin-stimulated {alpha}-Ser485/491 AMPKK—Based on the studies done in the intact MEFs, forskolin-induced enhancement of {alpha}-Ser485/491 phosphorylation was maintained in the absence of both LKB1 and CaMKKbeta (Fig. 2, A and B). By direct measurement, both CaMKK{alpha} and CaMKKbeta activities were inhibited by forskolin and exhibited little activity in the absence of added Ca2+/calmodulin, as in the conditions of our lysate kinase kinase assay (Fig. 3C). We were also unable to demonstrate in vitro that either of the CaMKKs can phosphorylate the {alpha}-Ser485/491 site (Fig. 4A and data not shown). Taken together, these observations make it unlikely that any of the three known {alpha}-Thr172 AMPKKs double as the cAMP-stimulated {alpha}-Ser485/491 AMPKK.


Figure 5
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  		FIGURE 5.
Wortmannin does not block the effects of forskolin in INS-1 cells. INS-1 cells preincubated in serum-free medium for 30 min were incubated with either wortmannin (200 nM) or Me2SO (DMSO; control; 1:1000, v/v) for 45 min, followed by treatment with forskolin (10 µM) or ethanol (control; 1:100, v/v) for 30 min. Cells extracts were prepared as described under "Experimental Procedures"; each extract (n) represents a pooling of 2 wells treated under identical conditions. A, AMPK was immunoprecipitated from proteinmatched cell extracts in triplicate using a pan-AMPK{alpha}-specific antibody, and the activity of the isolated enzyme was measured as described "Experimental Procedures." White bars, control incubations; black bars, treatment with forskolin. Data (mean ± S.D., n = 3) are expressed as picomoles of 32Pi transferred to the SAMS peptide/min/mg of total protein subjected to immunoprecipitation. Wortmannin treatment did not significantly block the basal level of activity (*, p < 0.7) or forskolin-mediated inhibition of AMPK activity (**, p < 0.3). B and C, the same protein-matched extracts as in A were subjected to SDS-PAGE, followed by immunoblot analysis using anti-AMPK {alpha}-phospho-Thr172 ({alpha}T172p) and anti-AMPK {alpha}-phospho-Ser485/491 ({alpha}S485/491p) antibodies (triplicate extracts) (B) or anti-Akt phospho-Thr308 (T308p), anti-Akt phospho-Ser473 (S473p), and anti-total Akt antibodies (duplicate extracts) (C).

 
Recent studies have identified protein kinase B/Akt as an {alpha}-Ser485/491 AMPKK that is, in part, responsible for inhibition of AMPK activity during insulin signaling (13, 14). This information, coupled with reports that Akt can be activated in some cell lines by forskolin, GIP, and glucagon-like peptide-1 (35–38), led us to investigate whether Akt might be the {alpha}-Ser485/491 AMPKK involved in this cAMP-mediated AMPK response in INS-1 cells. INS-1 cells were treated with or without forskolin in the presence of wortmannin, a specific inhibitor of phosphatidylinositol 3-kinase, the upstream activator of Akt. Enhanced {alpha}-Ser485/491 phosphorylation was maintained in the presence of wortmannin (Fig. 5B), even though Akt was effectively inhibited, as determined by immunoblot analysis of its two main regulatory phosphorylation sites, Thr308 and Ser473 (Fig. 5C). Wortmannin was also unable to block the inhibitory effects of forskolin on AMPK activity (Fig. 5A) or on {alpha}-Thr172 phosphorylation (Fig. 5B, upper panel). Treatment with wortmannin also failed to alter the effects of GIP on {alpha}-Thr172 and {alpha}-Ser485/491 phosphorylation (supplemental Fig. 1). Additionally, INS-1 extracts in which >95% of endogenous Akt was removed by immunoprecipitation still retained unaltered forskolin-stimulated AMPKK activity at {alpha}-Ser485/491 as determined by an in vitro kinase kinase assay (data not shown). Taken together, these data effectively exclude Akt as the {alpha}-Ser485/491 AMPKK stimulated under conditions of forskolin or GIP stimulation in this cell line.

PKA Is the {alpha}-Ser485/491 AMPKK—In INS-1 cells, as in many other cells types, cAMP-mediated signal transduction is mediated through PKA. Both PKA and Akt have overlapping substrate specificities and have been shown to phosphorylate the same residues on several shared target proteins (39–42). The sequence surrounding the AMPK {alpha}-Ser485/491 site (RSGSIS) loosely conforms to the consensus target sites for both PKA and Akt, kinetically favoring an arginine at position –3 and a hydrophobic residue at position +1 (43, 44). Thus, PKA becomes a possible candidate for the {alpha}-Ser485/491 AMPKK. In assays of INS-1 cell extracts, the forskolin-stimulated {alpha}1-Ser485 AMPKK activity was totally inhibited by PKI, the specific peptide inhibitor of the catalytic subunit of PKA (Fig. 6, A and B). To directly assess whether PKA can phosphorylate {alpha}1-Ser485, the recombinant catalytic subunit of PKA was incubated with recombinant AMPK{alpha}1-(250–548) in the presence of either radiolabeled (Fig. 6C) or unlabeled (Fig. 6D) ATP. These results show that PKA catalyzed the incorporation of nearly 1 mol/mol phosphate into the fusion protein (Fig. 6C), whereas immunoblot analysis confirmed that phosphate was incorporated into the {alpha}1-Ser485 site (Fig. 6D). Analysis of AMPK{alpha} phosphorylation sites by electrospray mass spectrometry confirmed that AMPK {alpha}-Ser485/491 is a bona fide target site for PKA (data not shown). Taken together, these data strongly suggest that PKA mediates AMPK {alpha}-Ser485/491 phosphorylation under conditions of forskolin or GIP stimulation of INS-1 cells.

{alpha}-Ser485/491 Phosphorylation and AMPK Activity—The identification of AMPKK-mediated phosphorylation of {alpha}-Ser485/491 suggests the possibility that this phosphorylation might contribute to overall AMPK activity. In the CaMKKbeta-deficient MEFs, forskolin/IBMX treatment enhanced {alpha}-Ser485/491 phosphorylation, yet overall enzyme activity was not inhibited (Fig. 2, B and C), indicating that phosphorylation of this site may not directly regulate AMPK activity. This interpretation is complicated, however, by the fact that phosphorylation of other sites in AMPK {alpha} and beta subunits, which we are unable to monitor, may act in conjunction to provide cooperative regulation of the enzyme. To test more directly whether the phosphorylation of {alpha}-Ser485/491 contributes to cAMP-dependent inhibition of AMPK, we utilized a reconstituted AMPK HT in which the {alpha}1-Ser485 site had been abolished by mutation to alanine ({alpha}1-S485A). COS cells were triply transfected with subunits coding for either a wild-type (WT) heterotrimer ({alpha}1beta1{gamma}1) or a mutant heterotrimer ({alpha}1(S485A)beta1{gamma}1). Cells were then treated with or without forskolin and IBMX prior to heterotrimer isolation by glutathione bead absorption. The activity of individual enzyme complexes was measured in a peptide-based assay. The WT heterotrimer isolated from forskolin/IBMX-treated cells showed a decrease in both enzyme activity (Fig. 7A) and {alpha}1-Thr172 phosphorylation (Fig. 7B, upper panel) accompanied by a significant increase in {alpha}1-Ser485 phosphorylation (middle panel) compared with the WT heterotrimer isolated from control cells. Interestingly, the {alpha}1-S485A mutant HT, which did not exhibit a significant difference in basal activity compared with its WT counterpart, was unresponsive to the forskolin/IBMX treatment. AMPK activity was no longer suppressed (Fig. 7A), with little change in {alpha}1-Thr172 phosphorylation (Fig. 7B, upper panel). These results provide evidence that, at least in the COS cell line, phosphorylation of {alpha}1-Ser485 is required for the cAMP-mediated inhibition of AMPK and suggest that phosphorylation of {alpha}-Ser485/491 may influence phosphorylation of {alpha}-Thr172.


Figure 6
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  		FIGURE 6.
PKA is an AMPKK active on AMPK {alpha}1-Ser485. A, INS-1 cells were preincubated in serum free medium, followed by treatment with forskolin (10 µM) or ethanol (control) for 30 min. Extracts were prepared in triplicate, each representing 1 well of a 6-well plate. Extracts were then matched for total protein and incubated in the presence of recombinant AMPK{alpha}1-(250–548) along with ATP/Mg2+ for 20 min at 30 °C following a 10-min preincubation in the presence or absence of 1 µM PKI. Reactions were stopped by the addition of 4x Laemmli sample buffer, and samples were boiled for 5 min. Proteins were separated by SDS-PAGE and probed with anti-AMPK {alpha}-phospho-Ser485/491 antibody. B, shown are the results from densitometric analysis of the immunoblots. White bars, control incubation; black bars, incubation with PKI. Data (mean ± S.D., n = 6 for control and n = 5 for forskolin-treated) are expressed as the measured pixel density of {alpha}1-Ser485 phosphorylation ({alpha}1S485p). PKI significantly inhibited AMPKK activity that was stimulated by treatment with forskolin. *, p < 0.0004. C, the recombinant PKA catalytic subunit (22.4 units) was incubated with recombinant AMPK{alpha}1-(250–548) along with [{gamma}-32P]ATP (200 µM) and Mg2+ (5 mM) for 10, 30, and 60 min in triplicate at 30 °C, and 32Pi incorporation was determined as described "Experimental Procedures." Data (mean ± S.D.) are plotted as moles of 32Pi incorporated per mol of total substrate versus time (minutes). D, reactions (as in C) containing unlabeled ATP were carried out in parallel. These reactions were stopped by the addition of 4x Laemmli sample buffer, and samples were boiled for 5 min. Proteins were separated by SDS-PAGE and probed with anti-AMPK {alpha}-phospho-Ser485/491 antibody.

 
The ability of recombinant CaMKKbeta to phosphorylate {alpha}-Thr172 in the reconstituted AMPK heterotrimer in vitro was unimpaired in the {alpha}1-S485A mutant versus the wild type (Fig. 7C). Isolated WT and {alpha}1-S485A mutant HT complexes were treated with {lambda}-phosphatase to dephosphorylate AMPK{alpha} (Fig. 7C), resulting in nearly complete inactivation of complex activity (>95% reduction) (data not shown). Rephosphorylation of these HT complexes with CaMKKbeta led to a significant increase in {alpha}-Thr172 phosphorylation in both heterotrimer complexes (Fig. 7C, third and sixth lanes). Indeed, the {alpha}1-S485A mutant appeared to be phosphorylated to a greater extent and then exhibited higher AMPK activity (0.09 pmol of 32Pi transferred to the SAMS peptide/min) compared with its WT counterpart (0.05 pmol/min), suggesting that the absence of {alpha}1-Ser485 phosphorylation may render the {alpha}-Thr172 site more accessible to phosphorylation by its regulatory AMPKKs. These data also indicate that {alpha}1-Ser485 phosphorylation is not required for {alpha}-Thr172 phosphorylation and activation of the enzyme. The increase in {alpha}1-Ser485 phosphorylation seen in the WT heterotrimer (Fig. 7C, middle panel) in these incubations is likely the result of an autophosphorylation event, as CaMKKbeta is not a Ser485 AMPKK (Fig. 4A).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
This study has demonstrated an important intersection between the cAMP and AMPK signaling pathways. AMPK activity can be attenuated in response to cAMP-elevating agents through changes in at least two of its phosphorylation sites, viz. {alpha}-Thr172 and {alpha}-Ser485/491. Forskolin, IBMX, and/or the insulinotropic hormone GIP inhibited AMPK activity by reducing phosphorylation of {alpha}-Thr172 and by enhancing phosphorylation of {alpha}-Ser485/491 in several cell lines, including a clonal beta-cell line (INS-1), as well as MEFs and COS cells. Moreover, these findings elucidate a novel mechanism in which multisite phosphorylation of AMPK contributes to the regulation of overall enzyme activity.

The reduction in phosphorylation of AMPK {alpha}-Thr172 in response to cAMP-elevating agents appears to be due, in part, to cAMP-dependent inhibition of one or both CaMKKs active on the site. In MEFs lacking CaMKKbeta, but not in LKB1–/– cells, both cAMP-mediated inhibition of AMPK activity and attenuation of {alpha}-Thr172 phosphorylation were abolished. (CaMKK{alpha}–/– cells are not presently available for study.) By direct measurement in INS-1 cell extracts, the activities of both CaMKKbeta and CaMKK{alpha} showed a 40% inhibition following forskolin/IBMX treatment. Although cAMP-mediated inhibition of CaMKKbeta activity has not been reported, Wayman et al. (45) have shown that forskolin treatment of PC12 cells, cultured hippocampal neurons, and Jurkat cells leads to inhibition of CaMKK{alpha} activity by ~40–60%. The inhibition is attributed to PKA-mediated phosphorylation of several sites within the catalytic domain (Thr108) and the calmodulin-binding domain (Ser458) of CaMKK{alpha} (45, 46). Additionally, phosphorylation of another site (Ser74) by PKA allows for an interaction with the adapter protein 14-3-3{gamma}, which significantly inhibits kinase activity by maintaining phosphorylation of the Thr108 site (47). It seems likely that the forskolin-induced inhibition of CaMKKbeta that we observed is mediated in a similar way, as both CaMKK isoforms contain the same regulatory sequences. PKA has been reported to phosphorylate LKB1 as well, although it is not clear how/if this phosphorylation affects its activity (48, 49). Based on our results in MEFs, LKB1 does not appear to be necessary for cAMP-mediated inhibition of AMPK.


Figure 7
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  		FIGURE 7.
Role of AMPK {alpha}1-Ser485 in the regulation of AMPK. COS cells were triply transfected with cDNAs expressing the full-length {alpha}1 (WT or S485A mutant), beta1, and {gamma}1 subunits of AMPK, followed by incubation for 48 h. Cells were then incubated for 1 h in serum-free Dulbecco's modified Eagle's medium, followed by treatment with either forskolin (10 µM) and IBMX (500 µM) or ethanol (control; 1:50) for 30 min. A, AMPK HT complexes were isolated by glutathione bead absorption and assayed for AMPK activity as described under "Experimental Procedures." White bars, HT isolated from control incubations; black bars, HT isolated from cells incubated with forskolin/IBMX. Data (mean ± S.D., n = 4 for wild-type isolates and n = 5 for mutant isolates) are expressed as picomoles of 32P incorporated into the SAMS peptide/min normalized to total AMPK{alpha} content for each isolated heterotrimer, which was determined by pixel density quantitation of the immunoblots. Forskolin/IBMX treatment significantly inhibited the activity of the WT heterotrimer (*, p < 0.0001 versus the WT control), but not that of the {alpha}1-S485A mutant heterotrimer (p < 0.664 versus the mutant control). There was no significant difference between the WT and {alpha}1-S485A mutant heterotrimers at basal levels (p < 0.48). B, shown is a representative blot of eight independently isolated HT complexes that were subjected to SDS-PAGE, followed by immunoblot analysis using anti-AMPK {alpha}-phospho-Thr172 ({alpha}T172p), anti-AMPK {alpha}-phospho-Ser485/491 ({alpha}1S485p), and anti-total AMPK{alpha} (Total {alpha}) antibodies. C, shown is the in vitro phosphorylation of the isolated WT or {alpha}1-S485A mutant heterotrimer with recombinant CaMKKbeta. COS cells were triply transfected with cDNAs expressing the full-length {alpha}1 (wild-type or S485A mutant), beta1, and {gamma}1 subunits of AMPK, followed by incubation for 48 h. AMPK HT complexes were isolated by glutathione bead absorption as described under "Experimental Procedures." Isolated HT complexes were dephosphorylated with 50 units of {lambda}-protein phosphatase as described under "Experimental Procedures" and rephosphorylated with 10 µg of recombinant CaMKKbeta or buffer. Reactions were stopped by the addition of 4x Laemmli sample buffer, and samples were boiled for 5 min. Proteins were separated by SDS-PAGE and probed with anti-AMPK {alpha}-phospho-Thr172, anti-AMPK {alpha}-phospho-Ser485/491, and anti-total AMPK{alpha} antibodies.

 
The critical involvement of the CaMKKs, but not LKB1, in mediating the AMPK response to increased levels of cellular cAMP highlights an important facet of AMPK regulation. In the MEF cell lines, the basal level of {alpha}-Thr172 phosphorylation remained unaltered in the absence of either LKB1 (Fig. 2A) or CaMKKbeta (Fig. 2B), suggesting that different AMPKKs may contribute to the basal phosphorylation of AMPK, perhaps varying as a function of the overall cell/tissue phenotype. This finding is not surprising given that, in yeast, the upstream kinases active on the AMPK homolog Snf1 exhibit functional redundancy (50). However, there is mounting evidence, including that presented here, suggesting that, in response to a variety of regulatory stimuli, the known mammalian {alpha}-Thr172 AMPKKs are not always functionally redundant, but likely have different roles, perhaps in a tissue-specific context (19).

The phosphorylation state of the major regulatory site, AMPK {alpha}-Thr172, is reflective of the overall activity level of the enzyme. Thus, the cAMP-dependent attenuation of AMPK activity can be correlated with the reduction in phosphorylation of this site. The enhancement of phosphorylation of {alpha}-Ser485/491 was an unexpected finding, raising several important questions regarding not only the nature of this phosphorylation event, but also whether phosphorylation of the {alpha}-Ser485/491 site could contribute to the inhibition of AMPK activity. The {alpha}-Ser485/491 site is both an autophosphorylation site and a target for upstream kinases such as protein kinase B/Akt (13, 14). As forskolin, IBMX, and GIP treatment led to the inhibition of AMPK activity, it is unlikely that the increased phosphorylation of this site reflects an autophosphorylation event. In support of this, in vitro kinase assays provided clear evidence that, under conditions of elevated levels of cAMP, stimulated AMPKK activity is directed to {alpha}-Ser485/491.

It is unlikely that any of the three {alpha}-Thr172 AMPKKs double as the stimulated {alpha}-Ser485/491 AMPKK under these conditions. Neither LKB1 nor CaMKKbeta is required to establish or maintain enhanced phosphorylation of {alpha}-Ser485/491. In addition, both CaMKK{alpha} and CaMKKbeta are partially inhibited under elevated cAMP conditions.

Recent findings have demonstrated that Akt can phosphorylate AMPK at {alpha}-Ser485/491 in response to insulin stimulation (13, 14), which has long been known to inhibit AMPK via an unknown mechanism (15–18). Because agonists acting through cAMP, such as glucagon-like peptide-1 and GIP, have been shown to stimulate Akt in INS-1 cells (35, 37, 38), Akt became a likely candidate for the {alpha}-Ser485/491 AMPKK. However, treatment of INS-1 cells with wortmannin, a classical inhibitor of phosphatidylinositol 3-kinase and thus of Akt, did not alter the effects of forskolin or GIP on either {alpha}-Ser485/491 phosphorylation or AMPK activity, indicating that Akt does not serve as the {alpha}-Ser485/491 AMPKK in this response.

As the site surrounding {alpha}-Ser485/491 conforms, in part, to the consensus target site for PKA, this kinase became another candidate. In vitro, the stimulated activity at {alpha}1-Ser485 seen in INS-1 cells treated with forskolin or IBMX was abolished in the presence of PKI, a specific peptide inhibitor of PKA. Additionally, the recombinant catalytic subunit of PKA phosphorylated {alpha}1-Ser485 in vitro stoichiometrically to nearly 1 mol/mol phosphate. To address whether PKA is the {alpha}-Ser485/491 AMPKK in intact cells involved in this response, we noted that H-89, a cell-permeant PKA inhibitor (51), led to a significant decrease in forskolin-stimulated {alpha}-Ser485/491 phosphorylation (data not shown). However, interpretation of this observation is complicated because H-89 can also inhibit AMPK activity (51), thus making it difficult to distinguish the potential effects of the inhibitor on AMPK from a PKA-mediated change. Nonetheless, taken together, our data support the conclusion that PKA is the {alpha}-Ser485/491 AMPKK that mediates the cAMP-dependent inhibition of AMPK.


Figure 8
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  		FIGURE 8.
Regulation of AMPK by multisite phosphorylation. Phosphorylation of AMPK {alpha}-Thr172 ({alpha}T172p) which is required for the activity of AMPK, is mediated by LKB1, the CaMKKs, and TAK1. LKB1-mediated phosphorylation is influenced by the binding of AMP to the AMPK {gamma} subunit during energy depletion, whereas CaMKK-mediated phosphorylation may be initiated by several cell-surface ligands that increase intracellular Ca2+. AMPK {alpha}-Thr172 can be attenuated by phosphorylation of AMPK {alpha}-Ser485/491 (S485/491p) in response to insulin (acting through protein kinase B (PKB)/Akt stimulation) or in response to cAMP-elevating agents that activate PKA. AMPK {alpha}-Ser485/491 phosphorylation may decrease the accessibility of the {alpha}-Thr172 site to its AMPKKs. Additionally, PKA can directly phosphorylate and inactivate the CaMKKs. Finally, coincident with AMPK activation, autophosphorylation of AMPK {alpha}-Ser485/491 could serve to prevent excessive AMPK activation.

 
Based on our findings, cAMP-mediated alteration of AMPK activity is the result of at least two limbs of regulation that coordinate inhibition via modulation of multiple phosphorylation sites (Fig. 8). In one limb, inhibition of the CaMKKs, mediated by PKA, contributes to the reduced phosphorylation of {alpha}-Thr172. In the second limb, PKA directly phosphorylates {alpha}-Ser485/491. Phosphorylation of this {alpha}-Ser485/491 site appears to be required but not sufficient to inhibit AMPK under conditions of elevated cAMP. In the reconstituted heterotrimer in COS cells, forskolin/IBMX treatment did not significantly reduce phosphorylation of {alpha}1-Thr172 when the {alpha}1-Ser485 site was mutated, suggesting that there may be some interplay between the two sites. Indeed, prior phosphorylation of {alpha}1-Ser485 in vitro with Akt attenuates LKB1-mediated phosphorylation of {alpha}1-Thr172, concomitant with a 30% decrease in AMPK activity (13). In addition, an AMPK heterotrimer lacking the {alpha}1-Ser485 site could be phosphorylated to a greater extent at {alpha}-Thr172 by CaMKKbeta than its WT counterpart (Fig. 7C). Taken together, these data raise the possibility that PKA-mediated phosphorylation of {alpha}-Ser485/491 may alter the accessibility of the {alpha}-Thr172 site to its regulatory AMPKKs. Coupled with the fact that the CaMKKs are themselves inhibited, this provides one model in which regulation of multiple phosphorylation sites contributes to overall enzyme inhibition.

Nevertheless, our results do not exclude contributions from other phosphorylation sites that may be modulated under these conditions. In addition to confirming AMPK {alpha}-Ser485/491 as a target regulatory site for PKA, preliminary phosphopeptide analysis revealed that PKA phosphorylated at least one other site in vitro in AMPK{alpha}, viz. Ser497/503 (data not shown). However, more work will be required to determine whether this phosphorylation event also contributes to cAMP-mediated inhibition of AMPK in vivo, although the data shown in Fig. 7A suggest that Ser485/491 phosphorylation by itself may be sufficient for enzyme regulation.

Enhanced phosphorylation of AMPK {alpha}-Ser485/491 has now been shown to correlate with inhibition of AMPK activity during both insulin signaling and cAMP-mediated signaling, yet phosphorylation of this same site is also enhanced under conditions of AMPK activation. Phosphorylation events at this site, mediated by autophosphorylation, other Ser485-directed AMPKKs, or conceivably altered enzyme dephosphorylation, could represent a regulatory mechanism by which AMPK activity can be tempered following stimulation. This "overshoot protection" model would likely limit excessive activation of AMPK, which could otherwise have deleterious effects in the cell.

Potential cross-talk between the cAMP/PKA pathway and AMPK has been well documented. In muscle, liver, and adipose tissue, physiologic stimuli such as exercise and fasting result in the simultaneous stimulation of both PKA and AMPK (21–23). Given that PKA and AMPK share a number of downstream targets, it is not hard to imagine that these two kinases might act in concert with one another to regulate a number of biosynthetic pathways in these tissues. In support of this, both kinases have been reported to regulate, either directly or indirectly, key enzymes involved in glycogen metabolism (inhibition of glycogen synthase) (23, 24), cholesterol synthesis (inhibition of hydroxymethylglutaryl-CoA reductase) (23), and lipid metabolism (inhibition of acetyl-CoA carboxylase) (23). In the case of the latter, although PKA can phosphorylate this enzyme in vitro, its regulatory role in vivo is less certain, as AMPK is considered to be the predominant regulator of inhibition (25–27). Additionally, endothelial nitric-oxide synthase can be activated by both PKA and AMPK via phosphorylation at a common site, Ser1179 (Ser1177 in humans) (40, 52). The relationship between AMPK and PKA is not always synergistic, however, as is the case with hormone-sensitive lipase, an enzyme found in both the adipocyte and muscle that catalyzes the hydrolysis of triglycerides to fatty acids. Regulation of hormone-sensitive lipase illustrates a relationship between PKA and AMPK in which the AMPK-mediated inactivation of hormone-sensitive lipase antagonizes its activation by PKA, resulting in attenuated lipolysis (28).

In both 3T3-L1 adipocytes and perfused liver, beta-adrenergic or glucagon stimulation leads to activation of both PKA and AMPK (53–56). This is in contrast to our findings in the clonal beta-cell line INS-1, in which cAMP-mediated signaling results in significant inhibition of AMPK. Thus, further investigation will be required to identify the potential tissue-specific differences in AMPK regulation under these conditions.

In cells of islet origin such as INS-1 cells, there appears to be an antagonistic relationship between cAMP-mediated and AMPK signaling. Insulinotropic hormone peptides such as GIP and glucagon-like peptide-1, which can act through G-protein-coupled receptors to elevate beta-cell cAMP, stimulate insulin secretion (32). Stimulation of AMPK in islet cells has been shown, however, to coincide with inhibition of insulin secretion, although the mechanism underlying this is not known (57–59). Additional studies will be necessary to establish whether cAMP-mediated inhibition of AMPK is necessary for cAMP-mediated stimulation of insulin secretion. In pancreatic beta-cells, fatty acids are necessary to mediate normal insulin secretion (60). Optimal glucose-stimulated insulin secretion, as well as insulin secretion potentiated by forskolin (61, 62), results from the rapid increase in cellular stores of fatty acids either through hydrolysis of stored triglycerides (lipolysis) or by increasing uptake into the cell (60). Whether an active AMPK attenuates insulin secretion by inhibiting lipolysis and promoting fatty acid oxidation is not clear. Nevertheless, other factors that potentiate insulin secretion in beta-cells independently of elevated cAMP levels, such as high glucose levels (58, 59) and amino acids (63), have also been shown to inhibit AMPK activity, suggesting that AMPK inhibition may be a necessary step for optimal insulin secretion.

The recognition that hormones and other agents working through cell-surface receptors can regulate AMPK through classical signaling pathways (e.g. cAMP-dependent, Ca2+-regulated) has significantly expanded the roles for AMPK signaling in the overall regulation of metabolism and other events in vivo, belied by its name "AMP-activated," which suggests that it is important only during states of energy depletion. We favor AMPK being renamed protein kinase E ("energy" or "economy"), recognizing its central roles in the energy economy of both single cells and a complex multicellular organism (19).


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grant DK35712 (to L. A. W.) and Grants DK074701 and GM33976 (to A. R. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. 1. Back

1 Australian Research Council Federation Fellow supported by grants from the Australian Research Council, the National Health and Medical Research Council, and the National Heart Foundation. Back

2 To whom correspondence should be addressed: Dartmouth Medical School, Remsen 322, N. College St., Hanover, NH 03755-3833. Tel.: 603-650-1909; Fax: 603-650-1727; E-mail: lee.a.witters{at}dartmouth.edu.

3 The abbreviations used are: AMPK, AMP-activated protein kinase; AMPKKs, AMP-activated protein kinase kinases; CaMKK, calcium/calmodulin-dependent protein kinase kinase; PKA, cAMP-dependent protein kinase; GIP, glucose-dependent insulinotropic polypeptide; MEFs, mouse embryonic fibroblasts; IBMX, isobutylmethylxanthine; HT, heterotrimer; PKI, protein kinase A peptide inhibitor; WT, wild-type. Back

4 K. A. Anderson and A. R. Means, manuscript in preparation. Back



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 ABSTRACT
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 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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