PKA phosphorylates and inactivates AMPKα to promote efficient lipolysis
2009; Springer Nature; Volume: 29; Issue: 2 Linguagem: Inglês
10.1038/emboj.2009.339
ISSN1460-2075
AutoresNabil Djouder, Roland Tuerk, Marianne Suter, Paolo Salvioni Chiabotti, Ramon F. Thali, Roland W. Scholz, Kari Vaahtomeri, Yolanda Auchli, Helene Rechsteiner, René Brunisholz, Benoı̂t Viollet, Tomi P. Mäkelä, Theo Wallimann, Dietbert Neumann, Wilhelm Krek,
Tópico(s)Lipid metabolism and biosynthesis
ResumoArticle26 November 2009free access PKA phosphorylates and inactivates AMPKα to promote efficient lipolysis Nabil Djouder Nabil Djouder Institute of Cell Biology, ETH Zurich, Zurich, Switzerland Competence Center for Systems Physiology and Metabolic Diseases, ETH Zurich, Zurich, Switzerland Search for more papers by this author Roland D Tuerk Roland D Tuerk Institute of Cell Biology, ETH Zurich, Zurich, Switzerland Search for more papers by this author Marianne Suter Marianne Suter Institute of Cell Biology, ETH Zurich, Zurich, Switzerland Search for more papers by this author Paolo Salvioni Paolo Salvioni Institute of Cell Biology, ETH Zurich, Zurich, Switzerland Search for more papers by this author Ramon F Thali Ramon F Thali Institute of Cell Biology, ETH Zurich, Zurich, Switzerland Search for more papers by this author Roland Scholz Roland Scholz Institute of Cell Biology, ETH Zurich, Zurich, Switzerland Search for more papers by this author Kari Vaahtomeri Kari Vaahtomeri Institute of Biotechnology and Genome-Scale Biology Program, University of Helsinki, Helsinki, Finland Search for more papers by this author Yolanda Auchli Yolanda Auchli Functional Genomics Center Zurich (FGCZ), University of Zurich, Zurich, Switzerland Search for more papers by this author Helene Rechsteiner Helene Rechsteiner Functional Genomics Center Zurich (FGCZ), University of Zurich, Zurich, Switzerland Search for more papers by this author René A Brunisholz René A Brunisholz Functional Genomics Center Zurich (FGCZ), University of Zurich, Zurich, Switzerland Search for more papers by this author Benoit Viollet Benoit Viollet Institut Cochin, Université Paris Descartes, CNRS (UMR 8104), Paris, France INSERM, U567, Paris, France Search for more papers by this author Tomi P Mäkelä Tomi P Mäkelä Institute of Biotechnology and Genome-Scale Biology Program, University of Helsinki, Helsinki, Finland Search for more papers by this author Theo Wallimann Theo Wallimann Institute of Cell Biology, ETH Zurich, Zurich, Switzerland Search for more papers by this author Dietbert Neumann Corresponding Author Dietbert Neumann Institute of Cell Biology, ETH Zurich, Zurich, Switzerland Search for more papers by this author Wilhelm Krek Corresponding Author Wilhelm Krek Institute of Cell Biology, ETH Zurich, Zurich, Switzerland Competence Center for Systems Physiology and Metabolic Diseases, ETH Zurich, Zurich, Switzerland Search for more papers by this author Nabil Djouder Nabil Djouder Institute of Cell Biology, ETH Zurich, Zurich, Switzerland Competence Center for Systems Physiology and Metabolic Diseases, ETH Zurich, Zurich, Switzerland Search for more papers by this author Roland D Tuerk Roland D Tuerk Institute of Cell Biology, ETH Zurich, Zurich, Switzerland Search for more papers by this author Marianne Suter Marianne Suter Institute of Cell Biology, ETH Zurich, Zurich, Switzerland Search for more papers by this author Paolo Salvioni Paolo Salvioni Institute of Cell Biology, ETH Zurich, Zurich, Switzerland Search for more papers by this author Ramon F Thali Ramon F Thali Institute of Cell Biology, ETH Zurich, Zurich, Switzerland Search for more papers by this author Roland Scholz Roland Scholz Institute of Cell Biology, ETH Zurich, Zurich, Switzerland Search for more papers by this author Kari Vaahtomeri Kari Vaahtomeri Institute of Biotechnology and Genome-Scale Biology Program, University of Helsinki, Helsinki, Finland Search for more papers by this author Yolanda Auchli Yolanda Auchli Functional Genomics Center Zurich (FGCZ), University of Zurich, Zurich, Switzerland Search for more papers by this author Helene Rechsteiner Helene Rechsteiner Functional Genomics Center Zurich (FGCZ), University of Zurich, Zurich, Switzerland Search for more papers by this author René A Brunisholz René A Brunisholz Functional Genomics Center Zurich (FGCZ), University of Zurich, Zurich, Switzerland Search for more papers by this author Benoit Viollet Benoit Viollet Institut Cochin, Université Paris Descartes, CNRS (UMR 8104), Paris, France INSERM, U567, Paris, France Search for more papers by this author Tomi P Mäkelä Tomi P Mäkelä Institute of Biotechnology and Genome-Scale Biology Program, University of Helsinki, Helsinki, Finland Search for more papers by this author Theo Wallimann Theo Wallimann Institute of Cell Biology, ETH Zurich, Zurich, Switzerland Search for more papers by this author Dietbert Neumann Corresponding Author Dietbert Neumann Institute of Cell Biology, ETH Zurich, Zurich, Switzerland Search for more papers by this author Wilhelm Krek Corresponding Author Wilhelm Krek Institute of Cell Biology, ETH Zurich, Zurich, Switzerland Competence Center for Systems Physiology and Metabolic Diseases, ETH Zurich, Zurich, Switzerland Search for more papers by this author Author Information Nabil Djouder1,2,‡, Roland D Tuerk1,‡, Marianne Suter1, Paolo Salvioni1, Ramon F Thali1, Roland Scholz1, Kari Vaahtomeri3, Yolanda Auchli4, Helene Rechsteiner4, René A Brunisholz4, Benoit Viollet5,6, Tomi P Mäkelä3, Theo Wallimann1, Dietbert Neumann 1 and Wilhelm Krek 1,2 1Institute of Cell Biology, ETH Zurich, Zurich, Switzerland 2Competence Center for Systems Physiology and Metabolic Diseases, ETH Zurich, Zurich, Switzerland 3Institute of Biotechnology and Genome-Scale Biology Program, University of Helsinki, Helsinki, Finland 4Functional Genomics Center Zurich (FGCZ), University of Zurich, Zurich, Switzerland 5Institut Cochin, Université Paris Descartes, CNRS (UMR 8104), Paris, France 6INSERM, U567, Paris, France ‡These authors contributed equally to this work *Corresponding authors. Institute of Cell Biology, ETH Zurich, Schafmattstrasse 18, Zurich 8093, Switzerland. Tel.: +41 44 633 3391; Fax: +41 44 633 11 74; E-mail: [email protected] or Tel.: +41 44 633 3447; Fax: +41 01 633 1357; E-mail: [email protected] The EMBO Journal (2010)29:469-481https://doi.org/10.1038/emboj.2009.339 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The mobilization of metabolic energy from adipocytes depends on a tightly regulated balance between hydrolysis and resynthesis of triacylglycerides (TAGs). Hydrolysis is stimulated by β-adrenergic signalling to PKA that mediates phosphorylation of lipolytic enzymes, including hormone-sensitive lipase (HSL). TAG resynthesis is associated with high-energy consumption, which when inordinate, leads to increased AMPK activity that acts to restrain hydrolysis of TAGs by inhibiting PKA-mediated activation of HSL. Here, we report that in primary mouse adipocytes, PKA associates with and phosphorylates AMPKα1 at Ser-173 to impede threonine (Thr-172) phosphorylation and thus activation of AMPKα1 by LKB1 in response to lipolytic signals. Activation of AMPKα1 by LKB1 is also blocked by PKA-mediated phosphorylation of AMPKα1 in vitro. Functional analysis of an AMPKα1 species carrying a non-phosphorylatable mutation at Ser-173 revealed a critical function of this phosphorylation for efficient release of free fatty acids and glycerol in response to PKA-activating signals. These results suggest a new mechanism of negative regulation of AMPK activity by PKA that is important for converting a lipolytic signal into an effective lipolytic response. Introduction White adipocytes have a central function in the control of whole-body energy homeostasis (Rosen and Spiegelman, 2006). These cells are able to accumulate and store dietary energy in the form of triacylglycerides (TAGs) through lipid synthesis and to mobilize the stored energy in times of caloric need by hydrolysing TAGs to generate non-esterified free fatty acids (NEFAs) and glycerol that are released into the circulation for use by other organs as energy substrates (Duncan et al, 2007). A breakdown in the regulation of adipocyte lipid storage and mobilization pathways can contribute to increased levels of NEFA in the circulation, which is an established risk factor for the development of insulin resistance in type II diabetes and related disorders (Gesta et al, 2006; Guilherme et al, 2008). In the basal state, the TAG pool in adipocytes is in a constant state of flux in that NEFAs are continuously released from TAG stores and reesterified again to produce TAG (Duncan et al, 2007). The simultaneously ongoing breakdown or lipolysis and resynthesis of TAG creates a ‘substrate cycle’ referred to as the TAG-NEFA cycle, which is characterized by energy expenditure in the absence of net conversion of substrate into product and allows adipocytes to respond rapidly to changes in peripheral requirements of NEFA (Kalderon et al, 2000; Large et al, 2004). Indeed, during periods of increased energy demands, the rate of lipolysis is enhanced through the action of lipolytic hormones that stimulate β-adrenergic signalling to activate the cAMP-PKA pathway (McKnight et al, 1998), which mediates phosphorylation and activation of lipolytic enzymes, including perilipin, adipose triglyceride lipase and hormone-sensitive lipase (HSL) (Khoo et al, 1972; Holm, 2003; Schweiger et al, 2006; Ducharme and Bickel, 2008; Granneman and Moore, 2008; Zimmermann et al, 2009). Phosphorylation of HSL occurs on multiple sites, including Ser-660, which stimulates catalytic activity and Ser-563, which is believed to be mutually exclusive with phosphorylation of HSL at the non-PKA site Ser-565 (Anthonsen et al, 1998; Watt et al, 2006). Thus, hormonal cues that signal systemic energy induce HSL phosphorylation at Ser-563 by PKA, which contributes to adipocyte lipolysis to maintain whole-body energy homeostasis. Existing evidence suggests that even during fasting as much as 30–40% of NEFAs released from TAG stores are reesterified (Reshef et al, 2003), providing a mechanism to limit NEFA release into the circulation. The reesterification step of the TAG-NEFA cycle is a highly energy-demanding process that consumes significant amounts of cellular ATP and also generates AMP, and when immoderate, can create a state of relative energy depletion (Gauthier et al, 2008). A key sensor of cellular energy level is AMPK (Hardie et al, 2006; Hue and Rider, 2007; Hardie, 2008), a heterotrimeric kinase complex, composed of the catalytic kinase α subunit and two associated regulatory subunits, β and γ, that maintains the balance between ATP production and consumption. AMPK is activated by phosphorylation of the critical activation loop threonine (Thr-172) in the α-subunit that is mediated by upstream kinases (Carling et al, 2008). Prominent among these is the tumour suppressor kinase LKB1 (Sakamoto et al, 2005; Shaw et al, 2005). Energy stress leads to an increase in the AMP:ATP ratio and AMP binds directly to the AMPKγ subunit, thereby stimulating the kinase activity allosterically and inducing a conformational change that inhibits the deactivation by phosphatases (Suter et al, 2006; Sanders et al, 2007). In the context of adipocytes, evidence suggests that AMPK is activated as a consequence of constitutively ongoing reesterification that consumes energy (Gauthier et al, 2008). An immediate consequence of enhanced AMPK activity is the phosphorylation of HSL at Ser-565, which precludes activation of HSL by PKA (Garton et al, 1989; Daval et al, 2005). Thus, stimulation of adipocyte lipolysis through PKA activation triggers, in turn, a negative feedback mechanism involving AMPK to match the rate of lipolysis to energy supply. The mechanism(s) underlying coordination of these apparently opposing activities under conditions of acute systemic metabolic needs remain elusive. Hence, we explored whether PKA and AMPK kinase pathways crosstalk in adipocytes as part of their lipolysis regulatory functions. Results PKA and AMPK associate in vivo Primary mouse adipocytes were treated with isoproterenol, an epinephrine analogue and β-adrenergic agonist that simulates the physiological stimulus for lipolysis during fasting, to activate the cAMP-PKA pathway. The activation state of AMPK was monitored using an antibody specific for AMPKα that is phosphorylated on Thr-172, the critical residue in the activation loop of AMPKα phosphorylated by LKB1 (Hawley et al, 1996). As shown in Figure 1A, 30 min of 200 nM isoproterenol treatment suppressed basal AMPKα phosphorylation at Thr-172 (compare lanes 2 and 1). Simultaneous addition of 1 μM H89, a pharmacological inhibitor of PKA, prevented this (lane 3). The effect of PKA signalling on Thr-172 phosphorylation of AMPKα under AMPK-activating conditions, such as in response to glucose deprivation or addition of 1 mM of the AMP-mimicking agent aminoimidazole carboxamide ribonucleotide (AICAR), was also evaluated. As expected, these treatments induced AMPKα Thr-172 and Ser-79 AMPK substrate acetyl-CoA-carboxylase (ACC) phosphorylation (compare lanes 4, 7 and 1). Induction of AMPKα phosphorylation was, however, blocked by isoproterenol (lanes 5 and 8) and recovered in the presence of H89 inhibitor (lanes 6 and 9). Thus, signals that activate PKA reduce AMPKα phosphorylation at Thr-172. In line with these data, isoproterenol inhibits AMPK activity as assessed by the ability of AMPKα1 immunoprecipitates to phosphorylate SAMS peptide (Supplementary Figure S1A). Consistent with this finding, the phosphorylation state of an established common effector of PKA and AMPK in the regulation of lipolysis, HSL, changed accordingly. Conditions that activated PKA-induced phosphorylation of HSL at Ser-660 and Ser-563 and suppressed phosphorylation of HSL at the AMPK site Ser-565 (Figure 1A). Conversely, inhibition of PKA signalling reversed these effects, resulting in increased phosphorylation of HSL at the AMPK site Ser-565 (Figure 1A). Similar changes in the phosphorylation states of AMPKα, ACC and HSL were observed when PKA signalling was induced by 20 μM forskolin, a potent inhibitor of phosphodiesterases that leads to cAMP increase, or suppressed by 100 μM of a specific myristoylated protein kinase A inhibitor (PKi) (Figure 1B). Activation of Ca2+ signalling by ionomycin did not interfere with Thr-172 phosphorylation of AMPKα in adipocytes (Supplementary Figure S1B). The negative effect of forskolin on AMPK Thr-172 phosphorylation was first detectable around 20 min after treatment (Figure 1C) and correlated with increased phosphorylation of HSL at Ser-660 and the cAMP response element-binding protein (CREB) at Ser-133 (both PKA mediated) and decreased phosphorylation of ACC at Ser-79 and HSL at Ser-565 (both AMPK mediated) (Figure 1C). In vitro kinase assays using purified PKA and AMPK further support the notion that phosphorylation of HSL at Ser-563 and Ser-565 is mutually exclusive. Specifically, phosphorylation of purified glutathione-S-transferase (GST)-HSL fusion protein at Ser-563 by PKA renders the substrate resistant to subsequent phosphorylation at Ser-565 by AMPK complexes in which the Thr-172 residue of the AMPKα1 subunit has been mutated to an aspartic acid, referred to as AMPK(T172D) to yield constitutive active kinase complexes (Figure 1D). In contrast, when GST-HSL was first phosphorylated by AMPK(T172D), PKA failed to phosphorylate HSL at Ser-563 (Figure 1D). In keeping with these observed changes in the phosphorylation state of HSL, isoproterenol-stimulated NEFA release from adipocytes, whereas addition of H89 inhibitor blocked both basal and isoproterenol-induced NEFA release (Figure 1E), whereas glucose deprivation or treatment of cells with AICAR that activates AMPK caused reduced basal and isoproterenol-induced NEFAs release (Figure 1E). As phosphorylation of AMPK at Thr-172 is required for kinase activation, these findings imply that activation of PKA in adipocytes suppresses, directly or indirectly, AMPK activity and thus the extend to which the downstream effector of PKA, HSL, is phosphorylated. Finally, we detected PKAα in endogenous AMPKα1 immunoprecipitates derived from primary adipocytes whole-cell extracts (Figure 1F, lane 3), suggesting that PKAα and AMPKα1 physically interact in vivo. Stable complex formation of these kinases was, however, not affected by treatment of cells with either isoproterenol or ionomycin (Supplementary Figure S1C). Figure 1.PKA signalling inhibits Thr-172 phosphorylation of AMPKα. (A) Primary adipocytes were cultivated for 30 min either in the presence of 25 mM glucose (+Glc) (lanes 1–3 and 7–9) or the absence of glucose (−Glc) (lanes 4–6) and treated with 1 mM AICAR (lanes 7–9), 200 nM isoproterenol (Iso) (lanes 2, 5 and 8) or 200 nM isoproterenol in combination with 1 μM H89 (lanes 3, 6 and 9). Protein lysates were prepared and probed with the indicated antibodies. (B) Similar experiment as in (A) except that 20 μM Forskolin (FSK) and 100 μM myristoylated PKi were added instead of isoproterenol and H89, respectively. (C) Primary adipocytes were glucose starved for 60 min (−Glc) and incubated with 20 μM Forskolin (FSK) for indicated times. FSK was either present ab initio (lane 8), added during the last 5, 10, 20, 30 or 45 min of starvation (lanes 3–8) or omitted (lane 2). As control, cells were grown in glucose-rich medium (+Glc) (lane 1). Equal amounts of protein lysates were subjected to immunoblotting with the indicated antibodies. (D) Phosphorylation of HSL by PKA and AMPK at Ser-563 and Ser-565, respectively, is mutually exclusive. In vitro kinase assay of GST-HSL in the presence of PKA (lanes 2, 4 and 5) and constitutively active AMPK(T172D) (lanes 3, 4 and 5). PKA was either added before (lane 4) or after incubation of HSL with AMPK(T172D) (lane 5). Proteins were subjected to immunoblotting with the indicated antibodies. (E) NEFA release in response to PKA signalling. Primary adipocytes were incubated for 30 min with 200 nM isoproterenol alone (Iso) or in combination with 1 μM H89. Furthermore, this treatment was done in the presence (+Glc) or absence (−Glc) of glucose or in the presence of glucose and 1 mM AICAR. NEFA were measured in the incubation medium. Bars represent the mean NEFA release from three independent experiments. (F) Whole-cell extracts of primary mouse adipocytes (WCE, lane 1) were prepared and aliquots were subjected to immunoprecipitation with control IgG (lane 2) or anti-AMPKα1 antibodies (lane 3) and immunoblotted for AMPKα1 and PKAα. Immunoblots are representative of at least three independent experiments. Download figure Download PowerPoint PKA phosphorylates AMPKα1 at Ser-173, Ser-485, Ser-497 and AMPKβ1 at Ser-24 Given the observation that PKA signalling inhibits AMPK activation and that these two kinases associate in vivo, we asked next whether PKA phosphorylates AMPK in vitro. For these assays, we produced heterotrimeric AMPK complexes with a catalytically inactive AMPKα1 subunit in which Asp-157 has been mutated to alanine in bacteria. We refer to this complex as AMPK(D157A). It follows that incorporation of radioactive phosphate into any of the AMPK subunits would then be solely due to the action of PKA and not due to autophosphorylation activity of AMPK. Indeed, the constitutively active catalytic subunit of PKA (PKA[cat]) or a partially purified PKA holoenzyme (PKA[holo]) that is dependent on cAMP for full activity, both phosphorylated the catalytic α1- and regulatory β1-subunits of AMPK(D157A) (Figure 2A). Phosphorylation of the AMPKγ1 subunit by PKA was not observed under these conditions. Conversely, PKAα did not serve as a substrate for active AMPK in vitro (data not shown). Phosphorylation site mapping by mass spectrometry and solid-phase sequencing revealed that PKA phosphorylated the AMPKα1 subunit at Ser-173, Ser-485 and Ser-497 and the β1-subunit at Ser-24 (Figure 2B and C, respectively). All these sites are highly conserved between human, mouse and rat AMPK subunits (Supplementary Figure S2). In addition, the amino-acid sequence encompassing the Ser-173 site in AMPKα1 is also conserved in rat AMPKα2 (Supplementary Figure S2A). It has been reported that α1-containing AMPK is the predominant complex in adipocytes accounting for >90% of total AMPK activity detectable in these cells (Lihn et al, 2004; Daval et al, 2005). Therefore, we focused our further analysis on the regulation of AMPKα1 by PKA. Mutation of each of the above-noted serine residues to alanine as well as combined mutations of these critical serines were introduced as secondary mutations in AMPKα1(D157A)- or β1-subunits and the corresponding AMPK complexes exposed to PKA. Only complexes harbouring the triple mutant of AMPKα1(S173A/S485/S497A) (Figure 2D) or the single mutant AMPKβ1(S24A) (Figure 2E) proved to be resistant to PKA phosphorylation, suggesting that each of these sites can be phosphorylated by PKA in vitro. Figure 2.PKA phosphorylates catalytic α- and regulatory β-subunits of AMPK at multiple sites. (A) PKA phosphorylates AMPK in vitro. Autoradiograph of α1β1γ1 catalytically inactive AMPK(D157A) complexes incubated in the presence of [γ-32P]ATP with either the cAMP-dependent PKA holoenzyme, PKA[holo], or the constitutively active, cAMP independent, catalytic domain of PKA, PKA[cat]. Arrows indicate phosphorylated AMPK(D157) at the α1- and β1-subunits. (B) Mass fingerprinting of isolated phosphopeptides derived from HPLC was performed by MALDI-ToF MS. Peptides differing by +80 Da (HPO3) from computed masses were then selected for MS/MS and phosphorylation was confirmed by a neutral loss of −98 Da (H3PO4) during fragmentation. Further verification of the phosphosites was attempted by solid-phase sequencing of the radiolabelled peptides. After each cycle of N-terminal Edman degradation, liberated single amino acids were collected and spotted onto DEAE cellulose. The respective phosphorylated residues were then detected by autoradiography. The annotated mass spectra indicates the phosphorylated residues of AMPKα1 pSer-173, pSer-485 and pSer-497. (C) As in (B) but related to AMPKβ1 pSer-24. Dha, dehydroala. (D) Analysis of AMPKα1 phosphorylation site mutants. Purified AMPK(D157A) (control), or the indicated serine to alanine mutants thereof, were incubated with PKA in the presence of [γ-32P]ATP and processed for SDS–PAGE and autoradiography. (E) Verification of in vitro AMPK β1-phosphorylation sites by mutational analysis. Catalytically inactive AMPK(D157A) complexes containing a regulatory β1-subunit mutated at Ser-24 and/or Ser-25 were incubated with [γ-32P]ATP in the presence or absence of PKA. Phosphorylation was visualized by SDS–PAGE and autoradiography. PKA was unable to phosphorylate AMPKβ1 mutated in Ser-24 (lane 4) but not Ser-25 (lane 6), showing that only Ser-24 is targeted by PKA. Control, AMPK(D157A) containing no additional mutations. Immunoblots are representative of at least three independent experiments. Download figure Download PowerPoint Phosphorylation of AMPKα1 at Ser-173 by PKA precludes phosphorylation of AMPKα1 at Thr-172 by LKB1 Next, we assessed whether phosphorylation of AMPKα1 by either LKB1 or PKA interferes with subsequent phosphorylation by PKA and LKB1, respectively. As shown above, PKA phosphorylates AMPK(D157A/S485A/S497A) complexes at Ser-173 as AMPK(D157A/S485A/S497A/S173A) complexes that harbour an additional mutation at Ser-173 are resistant to phosphorylation by this kinase (see Figure 2D, compare lanes 7 and 8). Consistent with these data, PKA phosphorylated AMPKα1(D157A/S485A/S497A) at Ser-173 as evidenced by 32P-incorporation and recognition by anti-phospho Ser-173 antibodies (Figure 3A, lane 3). When AMPK(D157A/S485A/S497A) complexes were incubated with LKB1 before the addition of PKA, phosphorylation of AMPKα1(D157A/S485A/S497A) by PKA still occurred to a similar extend as indicated by 32P-incorporation (Figure 3A, lane 4). However, the anti-phospho Ser-173 antibodies failed now to recognize the phospho-Ser173 epitope (Figure 3A, lane 4). These results indicate that Thr-172 phosphorylation of AMPKα1 by LKB1 does not preclude subsequent PKA phosphorylation of AMPKα1 at Ser-173 in vitro. Moreover, they further suggest that the anti-phospho Ser-173 antibodies recognize the Ser-173 epitope only when AMPKα1 is not phosphorylated at Thr-172. Figure 3.PKA-mediated phosphorylation of AMPKα1 prevents its phosphorylation by LKB1 at Thr-172 in vitro and phosphorylation of AMPK at Thr172 and Ser-173 is not mutually exclusive. (A) LKB1 was allowed to phosphorylate AMPKα1(D157S485A/S497A) in the presence of ATP before addition of PKA and [γ-32P]ATP. Signals obtained by autoradiography indicate phosphorylation at Ser-173 (lanes 3 and 4). Phosphorylation of Thr-172 and Ser-173 is coexistent, if PKA is allowed to phosphorylate AMPK after preincubation with LKB1 (lane 4). In this double-phosphorylated form, phosphorylation of Thr-172 but not Ser-173 was recognized by the corresponding antibodies. PKi, PKA inhibitor. Samples were processed for immunoblotting with the specified antibodies. (B) PKA phosphorylation of AMPKα1 prevents LKB1-mediated Thr-172 phosphorylation in vitro. AMPKα1(D157A) was preincubated with/without PKA and non-radioactive ATP as indicated, followed by LKB1 assays in the presence of [γ-32P]ATP. LKB1 was unable to phosphorylate AMPK after PKA phosphorylation. Samples were processed for immunoblotting with the specified antibodies. (C) PKA-mediated AMPKα1 phosphorylation inhibits AMPK activity. AMPK was preincubated with different amounts of PKA before in vitro kinase reactions with LKB1. AMPK activity assessed as the amount of phosphate incorporated into the synthetic peptide substrate SAMS. (D) Immunoblot corresponding to (B) probed with the indicated antibodies. (E) PKA-phosphorylated AMPK was either left untreated or dephosphorylated with λ-PPase using the indicated concentrations, then processed for in vitro kinase assays with or without LKB1. AMPK activity quantified using the SAMS assay. (F) Immunoblot corresponding to (E) probed with the indicated antibodies. All activity measurements were done in triplicates. Error bars represent±s.e.m. Immunoblots are representative of at least three independent experiments. Download figure Download PowerPoint In reciprocal experiments, LKB1 phosphorylated AMPK (D157A) complexes on Thr-172 as evidenced by 32P-incorporation and recognition of AMPKα1(D157A) by anti-phospho Thr-172 antibodies (Figure 3B, lane 2). Importantly, when AMPK(D157A) complexes were incubated with PKA before the addition of LKB1, Ser-173 phosphorylation occurred but autoradiography of in vitro kinase assays show that subsequent phosphorylation of AMPKα1(D157A) at Thr-172 by LKB1 was blocked (Figure 3B, lane 4). Phosphorylation of AMPKα1(D157A) by LKB1 at Thr-172 was, however, observed when PKA was inhibited by PKA inhibitory peptide PKi (Figure 3B, lane 3). Addition of PKi alone did not negatively affect LKB1-mediated AMPKα1(D157A) phosphorylation at Thr-172 in the absence of PKA (Figure 3B, lane 1). These results suggest that PKA-mediated phosphorylation of AMPKα1 at Ser-173 interferes with subsequent LKB1 phosphorylation of AMPKα1 at Thr-172. Accordingly, LKB1-mediated activation of AMPK, as measured by the ability of AMPK to phosphorylate the synthetic peptide substrate SAMS, decreased, as increasing amounts of PKA were added to the reaction mix (Figure 3C). The observed decline in AMPK activity correlated with a decrease in Thr-172 phosphorylation and an increase in Ser-173 phosphorylation of AMPKα1 (Figure 3D). Moreover, when PKA-phosphorylated AMPK was treated with λ phosphatase before the addition of LKB1, activation of AMPK was recovered (Figure 3E). Immunoblotting revealed that λ phosphatase caused dephosphorylation of AMPKα1 at Ser-173 and that LKB1 phosphorylated AMPKα1 at the adjacent Thr-172 residue (Figure 3F). These results suggest that PKA phosphorylation of AMPKα1 prohibits subsequent activation of AMPK by LKB1 at Thr-172. Ser-173 phosphorylation of AMPKα1 antagonizes phosphorylation of AMPKα1 at Thr-172 by LKB1 To elucidate the molecular mechanism underlying PKA-mediated inhibition of AMPK activation by LKB1, we generated a non-phosphorylatable mutant of AMPKα1 in which Ser-173 was mutated to an alanine residue. The AMPKα1(S173A) mutant protein proved to be resistant to Thr-172 phosphorylation by LKB1, but was still phosphorylated at Ser-485 and Ser-497 by PKA (Figure 4A). One possible explanation of a failure of LKB1 to phosphorylate Thr-172 in AMPKα1 in the context of a Ser-173 to alanine mutation may relate to the fact that these two sites are located within the recognition sequence of LKB1. Alternatively, the mutation may disrupt the epitope of the phospho-Thr-172 antibody. However, we also failed to observe any 32P-incorporation into the AMPKα1(S173A) mutant protein on exposure to LKB1 (Figure 4A). Therefore, it is likely that the introduction of an alanine residue at Ser-173 affects the ability of LKB1 to phosphorylate Thr-172. We note that equivalent alanine mutations of AMPKα1 at Ser-485 and/or Ser-497 (Figure 4B) or AMPKβ1 at Ser-24 (Supplementary Figure S3) did not negatively affect Thr-172 phosphorylation of AMPKα1 by LKB1. Furthermore, the ability of PKA to inhibit subsequent activation by LKB1 was preserved in AMPKα1(S485A), AMPKα1(S497A) and AMPKβ1(S24A) mutant complexes, thus suggesting that these sites are not responsible for PKA-mediated inhibition. Figure 4.Phosphorylation of AMPKα1 at Ser-173 mediates the inhibitory effect by PKA. (A) Mutagenesis of Ser-173 to alanine blocks phosphorylation of AMPK at Thr-172 by LKB1. AMPK(D157A/S173A) was phosphorylated by PKA with non-radioactive ATP and subsequently by LKB1 in the presence of [γ-32P]ATP in the absence or presence of PKi as indicated. Note that LKB1 is unable to phosphorylate this mutant AMPK protein, suggesting th
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