Advertisement

AMP-activated Protein Kinase Up-regulates Mitogen-activated Protein (MAP) Kinase-interacting Serine/Threonine Kinase 1a-dependent Phosphorylation of Eukaryotic Translation Initiation Factor 4E*

  • Xiaoqing Zhu
    Affiliations
    Department of Molecular Genetics, CARIM School of Cardiovascular Diseases
    Search for articles by this author
  • Vivian Dahlmans
    Affiliations
    Department of Molecular Genetics, Maastricht University Medical Center, 6200 MD Maastricht, The Netherlands
    Search for articles by this author
  • Ramon Thali
    Affiliations
    Institute of Cell Biology, ETH Zurich, 8093 Zurich, Switzerland
    Search for articles by this author
  • Christian Preisinger
    Affiliations
    Proteomics Facility, Interdisciplinary Center for Clinical Research (IZKF), RWTH University Hospital Aachen, 52074 Aachen, Germany
    Search for articles by this author
  • Benoit Viollet
    Affiliations
    INSERM U1016, Institut Cochin, Department of Endocrinology, Metabolism and Diabetes, 75014 Paris, France

    CNRS UMR 8104, 75014 Paris, France

    Université Paris Descartes, Sorbonne Paris Cité, 75006 Paris, France
    Search for articles by this author
  • J. Willem Voncken
    Footnotes
    Affiliations
    Department of Molecular Genetics, Maastricht University Medical Center, 6200 MD Maastricht, The Netherlands
    Search for articles by this author
  • Dietbert Neumann
    Correspondence
    To whom correspondence should be addressed: Dept. of Molecular Genetics, CARIM School for Cardiovascular Diseases, Maastricht University, 6200 MD Maastricht, The Netherlands. Tel.: 31-43-388-1851; Fax: 31-43-388-4574
    Footnotes
    Affiliations
    Department of Molecular Genetics, CARIM School of Cardiovascular Diseases

    Institute of Cell Biology, ETH Zurich, 8093 Zurich, Switzerland
    Search for articles by this author
  • Author Footnotes
    * This work was supported by The Netherlands Organization for Scientific Research (NWO) (VIDI Grant 864.10.007) (to D. N.). This work was also supported by the Chinese Scholarship Council (to X. Z.). The authors declare that they have no conflicts of interest with the contents of this article.
    1 Both authors contributed equally to this work.
Open AccessPublished:July 13, 2016DOI:https://doi.org/10.1074/jbc.C116.740498
      AMP-activated protein kinase (AMPK) is a molecular energy sensor that acts to sustain cellular energy balance. Although AMPK is implicated in the regulation of a multitude of ATP-dependent cellular processes, exactly how these processes are controlled by AMPK as well as the identity of AMPK targets and pathways continues to evolve. Here we identify MAP kinase-interacting serine/threonine protein kinase 1a (MNK1a) as a novel AMPK target. Specifically, we show AMPK-dependent Ser353 phosphorylation of the human MNK1a isoform in cell-free and cellular systems. We show that AMPK and MNK1a physically interact and that in vivo MNK1a-Ser353 phosphorylation requires T-loop phosphorylation, in good agreement with a recently proposed structural regulatory model of MNK1a. Our data suggest a physiological role for MNK1a-Ser353 phosphorylation in regulation of the MNK1a kinase, which correlates with increased eIF4E phosphorylation in vitro and in vivo.

      Introduction

      Balancing catabolic and anabolic processes is fundamental to energy homeostasis and metabolic adaptation. The αβγ heterotrimeric AMP-activated protein kinase (AMPK)
      The abbreviations used are: AMPK
      AMP-activated protein kinase
      M/SAPK
      mitogen- and stress-activated protein kinase
      MNK
      MAP kinase-interacting serine/threonine protein kinase
      IVK
      in vitro kinase
      AICAR
      5-aminoimidazole-4-carboxamide ribonucleotide
      TPA
      12-O-tetradecanoylphorbol-13-acetate
      2PY
      double polyoma
      bACT
      β-actin
      IP
      immunoprecipitated
      p
      phosphorylated
      t
      total
      MEF
      mouse embryonic fibroblast
      CBB
      Coomassie Brilliant Blue
      aa
      amino acid(s)
      IB
      immunoblot.
      promotes ATP production and limits ATP consumption (
      • Hardie D.G.
      • Ross F.A.
      • Hawley S.A.
      AMPK: a nutrient and energy sensor that maintains energy homeostasis.
      ,
      • Steinberg G.R.
      • Kemp B.E.
      AMPK in health and disease.
      ). Shifts in the cellular AMP:ATP ratio are detected through the γ subunit, which cooperatively binds AMP. Consequential conformational changes within the heterotrimer stimulate AMPK kinase activity. Activation of AMPK further involves Thr172 phosphorylation in the activation loop of the α subunit kinase domain (
      • Stein S.C.
      • Woods A.
      • Jones N.A.
      • Davison M.D.
      • Carling D.
      The regulation of AMP-activated protein kinase by phosphorylation.
      ). Additional (auto-) phosphorylation events are known to regulate AMPK (
      • Woods A.
      • Vertommen D.
      • Neumann D.
      • Turk R.
      • Bayliss J.
      • Schlattner U.
      • Wallimann T.
      • Carling D.
      • Rider M.H.
      Identification of phosphorylation sites in AMP-activated protein kinase (AMPK) for upstream AMPK kinases and study of their roles by site-directed mutagenesis.
      ,
      • Oligschlaeger Y.
      • Miglianico M.
      • Chanda D.
      • Scholz R.
      • Thali R.F.
      • Tuerk R.
      • Stapleton D.I.
      • Gooley P.R.
      • Neumann D.
      The recruitment of AMP-activated protein kinase to glycogen is regulated by autophosphorylation.
      ,
      • Viollet B.
      • Foretz M.
      • Schlattner U.
      Bypassing AMPK phosphorylation.
      ). Through its established roles in lipid, glucose, and protein metabolism, AMPK functions at the crossroads of energy metabolism and basic cellular processes including cell proliferation, growth, and survival (
      • Dasgupta B.
      • Chhipa R.R.
      Evolving lessons on the complex role of AMPK in normal physiology and cancer.
      ,
      • Li W.
      • Saud S.M.
      • Young M.R.
      • Chen G.
      • Hua B.
      Targeting AMPK for cancer prevention and treatment.
      ,
      • Mihaylova M.M.
      • Shaw R.J.
      The AMPK signalling pathway coordinates cell growth, autophagy and metabolism.
      ). For example, AMPK-mediated regulation of acetyl CoA carboxylases ACC1 and ACC2 accelerates fatty acid synthesis and inhibits fatty acid oxidation, respectively (
      • Munday M.R.
      Regulation of mammalian acetyl-CoA carboxylase.
      ). AMPK is involved in control of cell growth via phosphorylation of regulatory-associated protein of mTOR (mechanistic target of rapamycin, Raptor) and tuberous sclerosis 2 (TSC2, tumor suppressor) (
      • Gwinn D.M.
      • Shackelford D.B.
      • Egan D.F.
      • Mihaylova M.M.
      • Mery A.
      • Vasquez D.S.
      • Turk B.E.
      • Shaw R.J.
      AMPK phosphorylation of raptor mediates a metabolic checkpoint.
      ,
      • Huang J.
      • Manning B.D.
      The TSC1-TSC2 complex: a molecular switchboard controlling cell growth.
      ).
      Despite its well known role as energy sensor, a lack of knowledge on the identity of direct AMPK targets and thus of pathways involved in these processes persists. A substantial number of functional screening approaches have been developed to identify AMPK downstream targets and interaction partners (
      • Ducommun S.
      • Deak M.
      • Sumpton D.
      • Ford R.J.
      • Núñez Galindo A.
      • Kussmann M.
      • Viollet B.
      • Steinberg G.R.
      • Foretz M.
      • Dayon L.
      • Morrice N.A.
      • Sakamoto K.
      Motif affinity and mass spectrometry proteomic approach for the discovery of cellular AMPK targets: identification of mitochondrial fission factor as a new AMPK substrate.
      ,
      • Thali R.F.
      • Tuerk R.D.
      • Scholz R.
      • Yoho-Auchli Y.
      • Brunisholz R.A.
      • Neumann D.
      Novel candidate substrates of AMP-activated protein kinase identified in red blood cell lysates.
      ,
      • Tuerk R.D.
      • Thali R.F.
      • Auchli Y.
      • Rechsteiner H.
      • Brunisholz R.A.
      • Schlattner U.
      • Wallimann T.
      • Neumann D.
      New candidate targets of AMP-activated protein kinase in murine brain revealed by a novel multidimensional substrate-screen for protein kinases.
      ,
      • Banko M.R.
      • Allen J.J.
      • Schaffer B.E.
      • Wilker E.W.
      • Tsou P.
      • White J.L.
      • Villén J.
      • Wang B.
      • Kim S.R.
      • Sakamoto K.
      • Gygi S.P.
      • Cantley L.C.
      • Yaffe M.B.
      • Shokat K.M.
      • Brunet A.
      Chemical genetic screen for AMPKα2 substrates uncovers a network of proteins involved in mitosis.
      ,
      • Moreno D.
      • Viana R.
      • Sanz P.
      Two-hybrid analysis identifies PSMD11, a non-ATPase subunit of the proteasome, as a novel interaction partner of AMP-activated protein kinase.
      ). High-density protein microarrays enable the rapid identification of potentially novel human kinase substrates at a proteomic scale (
      • Mok J.
      • Im H.
      • Snyder M.
      Global identification of protein kinase substrates by protein microarray analysis.
      ). Using this strategy, we identified MAP kinase-interacting serine/threonine protein kinase 1 (MNK1) as a putative novel AMPK target (
      • Thali R.F.
      ). The human MKNK1 gene encodes two splice variants, MNK1a and MNK1b, which differ in their C-terminal sequences. In contrast to other MNKs, MNK1a has low basal activity and is highly induced upon activation (
      • Hou J.
      • Lam F.
      • Proud C.
      • Wang S.
      Targeting Mnks for cancer therapy.
      ). MNK1a is activated by mitogen- and stress-activated protein kinases (M/SAPK), ERK (extracellular signaling-regulated kinase), and P38, via phosphorylation of two threonine residues (Thr209 and Thr214) within the activation/T-loop (
      • Goto S.
      • Yao Z.
      • Proud C.G.
      The C-terminal domain of Mnk1a plays a dual role in tightly regulating its activity.
      ,
      • Waskiewicz A.J.
      • Flynn A.
      • Proud C.G.
      • Cooper J.A.
      Mitogen-activated protein kinases activate the serine/threonine kinases Mnk1 and Mnk2.
      ). MNK1 and MNK2 isoforms phosphorylate the eukaryotic translation initiation factor and mRNA cap-binding protein 4E (eIF4E) on serine 209 (
      • Waskiewicz A.J.
      • Johnson J.C.
      • Penn B.
      • Mahalingam M.
      • Kimball S.R.
      • Cooper J.A.
      Phosphorylation of the cap-binding protein eukaryotic translation initiation factor 4E by protein kinase Mnk1 in vivo.
      ,
      • Wang X.
      • Flynn A.
      • Waskiewicz A.J.
      • Webb B.L.
      • Vries R.G.
      • Baines I.A.
      • Cooper J.A.
      • Proud C.G.
      The phosphorylation of eukaryotic initiation factor eIF4E in response to phorbol esters, cell stresses, and cytokines is mediated by distinct MAP kinase pathways.
      ,
      • Pyronnet S.
      • Imataka H.
      • Gingras A.C.
      • Fukunaga R.
      • Hunter T.
      • Sonenberg N.
      Human eukaryotic translation initiation factor 4G (eIF4G) recruits Mnk1 to phosphorylate eIF4E.
      ). Although the exact biological relevance of this phosphorylation event is still under debate, cellular eIF4E plays an important role in the regulation of mRNA translation, in which interaction with the 5′-cap structure of mRNA appears pivotal (
      • Bhat M.
      • Robichaud N.
      • Hulea L.
      • Sonenberg N.
      • Pelletier J.
      • Topisirovic I.
      Targeting the translation machinery in cancer.
      ). Deregulation of eIF4E has been linked to tumorigenesis (
      • Bhat M.
      • Robichaud N.
      • Hulea L.
      • Sonenberg N.
      • Pelletier J.
      • Topisirovic I.
      Targeting the translation machinery in cancer.
      ,
      • Siddiqui N.
      • Sonenberg N.
      Signalling to eIF4E in cancer.
      ); inhibition of protein translation, e.g. via MNK, is being considered for cancer treatment (
      • Hou J.
      • Lam F.
      • Proud C.
      • Wang S.
      Targeting Mnks for cancer therapy.
      ).
      Here we show that MNK1a is a genuine AMPK target in vitro and in vivo. MNK1a phosphorylation at Ser353 by AMPK increases its kinase activity toward eIF4E phosphorylation. The relevance of our findings for human metabolic and neoplastic conditions is discussed.

      Results

      MNK1a Is a Novel AMPK Target in Vitro

      The online tool Scansite predicted the presence of a putative AMPK phosphorylation motif LQRNSSTMDL in MNK1a; this domain is absent in MNK1b. The putative AMPK target site in this motif, Ser353, is evolutionarily highly conserved among mammals and lower vertebrates (Fig. 1A). To validate the initial phospho-protein microarray finding, cell-free in vitro kinase (IVK) assays were performed using recombinant AMPK and MNK1a. Mass spectrometric analysis of in vitro phosphorylated recombinant MNK1aWT indicated Ser353 as the primary phospho-site in the MNK1a-specific tryptic peptide sequence NSSTMDLTLFAAEAIALNR (Ser353 underlined; localization probability: 74%; Fig. 1A). To further probe the specificity of this phospho-event in vitro, the GST tag was also proteolytically removed from recombinant GST-MNK1a to exclude interference by GST tag phosphorylation (
      • Klaus A.
      • Zorman S.
      • Berthier A.
      • Polge C.
      • Ramirez S.
      • Michelland S.
      • Sève M.
      • Vertommen D.
      • Rider M.
      • Lentze N.
      • Auerbach D.
      • Schlattner U.
      Glutathione S-transferases interact with AMP-activated protein kinase: evidence for S-glutathionylation and activation in vitro.
      ). Both GST-MNK1aWT and MNK1aWT protein were phosphorylated specifically and only in the presence of AMPK (Fig. 1B). The absence of radiolabeled MNK1WT protein in AMPK-free control reactions suggested that MNK1aWT phosphorylation in vitro was AMPK-dependent and that MNK1aWT protein did not show auto-phosphorylation under these conditions. A commercially available AMPK substrate antiserum, which detects a common AMPK-phosphorylated LXRXXpS/pT motif, recognized in vitro phosphorylated MNK1aWT, indicating that MNK1a phosphorylation occurred at an AMPK substrate-like motif (Fig. 1C). The absence of detectable MNK1aWT phosphorylation using a kinase-dead AMPK further corroborated the notion that MNK1 phosphorylation is dependent on the kinase activity of AMPK in vitro (Fig. 1D) To definitively identify the phosphorylated MNK1a residue, a recombinant MNK1a Ser353 to alanine mutant (MNK1aS353A) was tested in cell-free assays. To exclude the possibility that the neighboring Ser352 residue was phosphorylated by AMPK under these conditions, we also generated a MNK1aS352A mutant. Although Mnk1aWT and MNK1aS352A were clearly detected by autoradiography, the MNK1aS353A mutant protein was not radiolabeled under these conditions (Fig. 1E). Analogously, comparative analysis of MNK1aWT and MNK1aS353A showed that MNK1aS353A, in contrast to MNK1aWT, was not detected by the AMPK substrate antiserum (Fig. 1F). These combined data strongly support the idea that AMPK phosphorylates MNK1aWT at Ser353 in vitro.
      Figure thumbnail gr1
      FIGURE 1.MNK1a-Ser353 is a novel AMPK target. A, upper panel, evolutionary conservation of putative MNK1a phosphorylation site. Sequence alignments of MNK1a proteins of human (Q9BUB5), mouse (O08605), rat (Q4G050), bovine (Q58D94), pig (B8XSK1), and Western clawed frog (Q66JF3) (www.uniprot.org) are shown. Lower panel, identification of MNK1a phospho-peptide (aa 351–369; Swiss-Prot: Q9BUB5-2). Intensities of phospho-peptide MS spectra in the presence or absence of ATP (±ATP) are shown. Probabilities for each phosphorylatable residue are indicated between brackets in the aa sequence. B, IVK assays. Purified GST-MNK1a protein was incubated with or without active AMPK in the presence of [γ-32P]ATP. Protease-mediated removal of the GST tag was done as indicated. Upper panel, pMNK1a autoradiograph (autorad). Lower panel, CBB staining image (used as loading control). C, IVK assays as in B, using “cold” ATP instead. Protease-mediated removal of the GST tag was performed as indicated in the figure. Immunoblot (IB) detection of phosphorylated MNK1a using phosphorylated AMPK substrate (AMPK-sub) antiserum or antiserum against total Mnk1 protein (tMNK1) is shown. D, as in C. IVK analyses include: active (WT), kinase-dead (KD), or no AMPK. IB detection was done using the indicated antisera. E, IVK. Purified recombinant GST-MNK1aWT and mutant GST-MNK1aS353A or GST-MNK1aS352A proteins were incubated with active AMPK and [γ-32P]ATP. Upper panel, pMNK1a autoradiograph. Lower panel, CBB image (used as loading control). F, as in C. IVK analyses were performed with cold ATP. IB detection was done using the indicated antisera. G, MNK1a and AMPK protein levels in HeLa cervical adenocarcinoma, U2OS, MEF, TIG3 human primary fibroblasts, HEK293T human embryonic kidney, and HL-1 immortal murine cardiomyocytes. IB analyses of total cell extracts using the indicated antisera were performed. H, U2OS cells expressing 2PY-MNK1aWT or MNK1aS353A (control: empty vector (ev)) were treated as indicated (c: control conditions; ss: serum-starved; AI: ss+AICAR). MNK1a was IP using PY antiserum; IB analysis of immunoprecipitation and input material was performed using the indicated antisera. I, as in C. IVK analyses using purified recombinant GST-MNK1aWT protein, active AMPK, and cold ATP were performed. Protease-mediated removal of the GST tag was performed as indicated in the figure. IB of phosphorylated MNK1a were performed using the indicated antisera. J, as in H. IVK analyses using purified GST-MNK1aWT or GST-MNK1aS353A with active AMPK and cold ATP were performed. IB was done using the indicated antisera. K, U2OS cells expressing 2PY-MNK1aWT, 2PY-MNK1aS353A, or transduced with empty vector (ev) were treated as indicated (serum-starved (ss) or ss+AICAR (AI)).

      Cellular MNK1a Is Phosphorylated at Ser353 upon AMPK Activation

      To establish that MNK1a phosphorylation at Ser353 occurs in living cells, we first compared MNK1a expression levels in different normal diploid and cancer cells. The osteosarcoma cell line U2OS was found to express endogenous MNK1a at a very low level, whereas its AMPK level was relatively high (Fig. 1G). The functional interaction between AMPK and MNK1a was studied in these cells using retrovirally expressed 2PY-MNK1a. Cells were treated with the AMPK-activating compound 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR); AMPK activity was assessed by pACC1 measurement. Although relatively low under control conditions (i.e. non-stimulated or serum-starved), AICAR dramatically increased 2PY-MNK1aWT phosphorylation at an AMPK substrate-like motif (Fig. 1H). Importantly, in contrast to immunoprecipitated (IP) 2PY-MNK1aWT, IP 2PY-MNK1aS353Amutant protein did not show any detectable phosphorylation (Fig. 1H). These data are congruent with cellular AMPK-mediated phosphorylation of MNK1a at Ser353.
      To be able to study the biological context and relevance of MNK1a phosphorylation at Ser353, we generated a polyclonal antiserum specifically recognizing Ser353-phosphorylated MNK1a. Both phosphorylated recombinant GST-MNK1aWT and proteolytically released MNK1aWT were detected by the MNK1a-Ser(P)353 antiserum (Fig. 1I). The finding that recombinant GST-MNK1aS353A was no longer detectable by the MNK1a-Ser(P)353 antiserum under similar assay conditions corroborated the specificity of the newly generated MNK1a-Ser(P)353 antiserum (Fig. 1J). To obtain direct evidence that MNK1a-Ser353 phosphorylation occurs in living cells, U2OS cells expressing MNK1aWT or MNK1aS353A were treated with AICAR. As expected, AICAR increased AMPK-Thr(P)172 (pAMPK) and ACC1-pS79 (pACC1) in both cell types (Fig. 1K). Importantly, AMPK activation correlated well with increased MNK1a-Ser(P)353 signal, and the MNK1aS353A mutant was not recognized by the MNK1a-Ser(P)353 antiserum (Fig. 1K).

      MNK1a-Ser353 Phosphorylation Is AMPK-dependent and Involves Direct Physical Interaction

      To obtain genetic evidence for the AMPK dependence of MNK1a-Ser353 phosphorylation, 2PY-MNK1a-transduced mouse embryonic fibroblasts (MEFs) derived from wild type (AMPKWT) or AMPKα1/α2 double null mutant mice (AMPKdKO) were treated with either AICAR or A769662, the latter being an AMPK activator with a distinct mode of action (
      • Göransson O.
      • McBride A.
      • Hawley S.A.
      • Ross F.A.
      • Shpiro N.
      • Foretz M.
      • Viollet B.
      • Hardie D.G.
      • Sakamoto K.
      Mechanism of action of A-769662, a valuable tool for activation of AMP-activated protein kinase.
      ,
      • Guigas B.
      • Sakamoto K.
      • Taleux N.
      • Reyna S.M.
      • Musi N.
      • Viollet B.
      • Hue L.
      Beyond AICA riboside: in search of new specific AMP-activated protein kinase activators.
      ). Both AICAR and A769662 enhanced pAMPK and pACC1 phosphorylation, which correlated well with enhanced MNK1a-Ser353 phosphorylation in AMPKWT, but not in AMPKdKO MEFs (Fig. 2A). These data convincingly support the notion that MNK1a is phosphorylated at Ser353 in an AMPK-dependent fashion in vivo. We then asked whether AMPK physically interacts with MNK1a in living cells. Endogenous AMPK was IP from 2PY-MNK1aWT U2OS cells. The interaction between MNK1a was evident under basal conditions and increased dramatically upon AMPK activation by AICAR or glucose deprivation (Fig. 2B). Accordingly, AMPK-MNK1a complex formation is induced in response to AMPK-activating stimuli.
      Figure thumbnail gr2
      FIGURE 2.Metabolic stress-induced MNK1a-Ser353 phosphorylation enhances MNK1a kinase activity toward eIF4E. A, AMPKWT and AMPKdKO MEFs expressing 2PY-MNK1aWT were treated with AICAR or A769662 as indicated. IB analyses of total cell extracts using the indicated antisera were performed. B, U2OS cells expressing 2PY-MNK1aWT (control: empty vector, ev), were treated as indicated: c, control conditions, serum-starved (ss), ss+AICAR (AI) (cf. G), or glucose-deprived (GD)). IB analyses of immunoprecipitation and input material using the indicated antisera were performed; bACT, loading control. C, U2OS cells expressing 2PY-MNK1aWT were glucose-deprived (GD, indicated in hours). #, cells were glucose-deprived, and glucose was re-administered for 1 h. D, lower panel, U2OS cells expressing 2PY-MNK1aWT, 2PY-MNK1aS353A, or 2PY-MNK1aS353D (control: empty vector, ev). IB analyses were performed using the indicated antisera. bACT, loading control. Upper panel, quantitation of peIF4E (FC: -fold change) in U2OS cells expressing 2PY-MNK1aWT or 2PY-MNK1aS353D (n = 5, *, p < 0.005). Error bars indicate means ± S.E. E, upper panels, U2OS cells expressing 2PY-tagged MNK1aWT, MNK1aS353A, MNK1aS353D, MNK1aT2→A2 (a double T209A/T214A mutant), or MNK1aK→M (a kinase-dead K78M mutant) were treated as indicated: AICAR, TPA, or combination treatment (all preceded by serum starvation (ss)). AI, ss+AICAR. 2PY-MNK1a was IP using PY antiserum (middle panels). IB analyses of IP and input material were performed using the indicated antisera. Immunoprecipitations were used in IVKs with recombinant GST-eIF4E and [γ-32P]ATP. White bars between sections indicate separate blots. Lower panels, peIF4E autoradiograph (autorad); CBB image (used as loading control). F, a functional role for Ser(P)353 in MNK1a activation based on a previously suggested conformational change model (
      • Goto S.
      • Yao Z.
      • Proud C.G.
      The C-terminal domain of Mnk1a plays a dual role in tightly regulating its activity.
      ): inactive MNK1a is maintained in a “closed” conformation (upper schematic). ERK and P38 (M/SAPK) binding of the MNK1a C-terminal binding domain (C-term) and subsequent M/SAPK-mediated phosphorylation (encircled P) at MNK1a-Thr209/Thr214 (bold TT) renders MNK1a receptive to AMPK-dependent phosphorylation at Ser353 (bold S). In the context of metabolic stress, AMPK-mediated MNK1a-Ser353 phosphorylation stabilizes an active “open” conformation. Replacement of Thr209/Thr214 by two non-phosphorylatable alanine residues (bold AA) desensitizes MNK1a to M/SAPK signaling (“locked” state).

      Metabolic Stress-induced MNK1a-Ser353 Phosphorylation Controls Its Kinase Activity toward eIF4E

      To chart the consequences of AMPK-mediated MNK1a-Ser353 phosphorylation status as a function of time, 2PY-MNK1aWT U2OS cells were glucose-deprived and AMPK and MNK1 phosphorylation was monitored. MNK1a-Ser(P)353 and pACC1 were both induced upon glucose deprivation; both phosphorylation events correlated well with AMPK activation (Fig. 2C). Relevantly, 1 h after replenishment of glucose, cells down-regulated MNK1-Ser(P)353 and pACC1, in parallel with pAMPK (Fig. 2C). These results are strongly suggestive of direct dynamic control of reversible MNK1a phosphorylation at Ser353 by AMPK.
      AMPK activation aims to conserve cellular ATP by tuning energy-consuming processes such as mRNA translation to metabolic conditions. MNK1 is known to phosphorylate the eukaryotic initiation factor 4E (eIF4E) at Ser209, a rate-limiting component of the translation apparatus (
      • Lu C.
      • Makala L.
      • Wu D.
      • Cai Y.
      Targeting translation: eIF4E as an emerging anticancer drug target.
      ). To explore the potential functional relevance of AMPK-mediated MNK1a-Ser353 phosphorylation in regard to eIF4E-Ser209 phosphorylation (peIF4E), we compared eIF4E phosphorylation status in the U2OS model transduced with MNK1aS353A or MNK1aS3535D mutants. Cells expressing MNK1aS353D showed almost 2-fold increased eIF4E phosphorylation at basal levels when compared with cells expressing MNK1aWT or MNK1aS353A (Fig. 2D). Combined, these data suggest AMPK-dependent stimulation of MNK1a toward its downstream target eIF4E, via MNK1a-Ser353 phosphorylation.
      MNK1-Thr209/Thr214 are phosphorylated within the activation T-loop by the M/SAPKs ERK and P38 (
      • Goto S.
      • Yao Z.
      • Proud C.G.
      The C-terminal domain of Mnk1a plays a dual role in tightly regulating its activity.
      ,
      • Waskiewicz A.J.
      • Flynn A.
      • Proud C.G.
      • Cooper J.A.
      Mitogen-activated protein kinases activate the serine/threonine kinases Mnk1 and Mnk2.
      ). The C terminus of MNK1a, harboring Ser353, is thought to play a dual role in the regulation of MNK1a kinase activity: the C terminus acts repressive in the basal state while allowing full stimulation upon ERK/P38-dependent phosphorylation of Thr209/Thr214 (
      • Goto S.
      • Yao Z.
      • Proud C.G.
      The C-terminal domain of Mnk1a plays a dual role in tightly regulating its activity.
      ). To explore the potential functional interdependence of Thr209/Thr214 and Ser353 phosphorylation, we studied specific MNK1a phospho-mutants in U2OS cells in combination with pharmacological stimulation rather than metabolic stress, as this allows separation of upstream signaling events. AICAR treatment, alone or in combination with TPA (M/SAPK activator), consistently and highly induced pACC1, whereas TPA treatment alone only modestly induced pACC1 (Fig. 2E). TPA, with or without AICAR, massively increased MNK1-Thr(P)209/Thr(P)214 (MNK1a-pTpT) (Fig. 2E); these observations are in good agreement with reported upstream signaling events connecting M/SAPKs to MNK1a (
      • Waskiewicz A.J.
      • Flynn A.
      • Proud C.G.
      • Cooper J.A.
      Mitogen-activated protein kinases activate the serine/threonine kinases Mnk1 and Mnk2.
      ). Combination treatment with TPA and AICAR resulted in increased MNK1a-Ser(P)353, but only in the case of MNK1aWT and MNK1aK→M (K78M; kinase-dead). Reduced Ser353 phosphorylation in U2OS MNK1aK→M cells may be explained by consistently lower expression of the MNK1aK→M mutant (Fig. 2E). As expected, the T209A/T214A (MNK1aT2→A2) mutant was no longer phosphorylated at the mutated phospho-sites, and neither IP MNK1aT2→A2 nor MNK1aK→M mutant kinases phosphorylated eIF4E (Fig. 2E). Remarkably, MNK1aT2→A2 was no longer phosphorylated at Ser353, whereas conversely, Ser353 mutation (S→A or S→D) did not block the ability of Thr209/Thr214 to become phosphorylated (Fig. 2E). IP MNK1aWT from AICAR only-treated cells led to only marginally increased peIF4E, in support of the need for regulatory co-signaling by M/SAPKs (Fig. 2E). In contrast, and in good agreement with the observations above (cf. Fig. 2D), MNK1aS353D consistently displayed enhanced peIF4E under AICAR only- or TPA only-treated conditions; moreover, eIF4E phosphorylation by MNK1aS353D was already enhanced under non-stimulated conditions (Fig. 2E). Of note, pACC1 was not altered by MNK1a mutation. The combined data suggest that MNK1a selectively acts in the signaling downstream of AMPK, leading to increased eIF4E phosphorylation, and point toward a hierarchical order of regulatory events within MNK1a: M/SAPK-mediated MNK1a-Thr209/Thr214 phosphorylation precedes AMPK-dependent MNK1a-Ser353 phosphorylation.

      Discussion

      In this study, we report that MNK1a is a direct AMPK target. We provide biochemical and genetic evidence that MNK1a-Ser353 is phosphorylated in vitro and in vivo in an AMPK-dependent manner and that AMPK and MNK1a physically interact. Finally, we show that MNK1a-Ser(P)353 correlates with increased eIF4E phosphorylation in vitro and in vivo.
      Members of the MAPK-activated protein kinase (MK) family fulfill multiple biological roles in cellular responses to extracellular cues (e.g. mitogenic stimulation and cellular stress). MNK1a, like other MAPKAPKs (MAP kinase-activated protein kinases), is tightly regulated by M/SAPKs, including ERK and P38 (
      • Roux P.P.
      • Blenis J.
      ERK and p38 MAPK-activated protein kinases: a family of protein kinases with diverse biological functions.
      ,
      • Ueda T.
      • Watanabe-Fukunaga R.
      • Fukuyama H.
      • Nagata S.
      • Fukunaga R.
      Mnk2 and Mnk1 are essential for constitutive and inducible phosphorylation of eukaryotic initiation factor 4E but not for cell growth or development.
      ). The most well characterized biochemical connection in regard to MNK1 is eIF4E-Ser209 phosphorylation (
      • Wang X.
      • Flynn A.
      • Waskiewicz A.J.
      • Webb B.L.
      • Vries R.G.
      • Baines I.A.
      • Cooper J.A.
      • Proud C.G.
      The phosphorylation of eukaryotic initiation factor eIF4E in response to phorbol esters, cell stresses, and cytokines is mediated by distinct MAP kinase pathways.
      ), although the exact relevance of MNK1-mediated eIF4E phosphorylation remains unclear (
      • Joshi S.
      • Platanias L.C.
      Mnk kinase pathway: Cellular functions and biological outcomes.
      ). MNK1a mutation appears to affect phosphorylation of eIF4E, not of ACC1, suggesting a selective role for MNK1a as a signaling intermediate in the regulation of protein synthesis, downstream of AMPK.
      Based on a previously proposed regulatory model of MNK1a, our data suggest a physiological role for Ser353 phosphorylation in regulating MNK1a activity. MNK1a is activated upon T-loop phosphorylation, but also undergoes a conformational change to a more “open” structure (
      • Goto S.
      • Yao Z.
      • Proud C.G.
      The C-terminal domain of Mnk1a plays a dual role in tightly regulating its activity.
      ). Our MNK1a mutant analysis indicating that Thr209/Thr214 phosphorylation is a prerequisite for Ser353 phosphorylation appears congruent with this phospho-event being dependent on conformational change. Interestingly, an α helical structure from the first part of the unique MNK1a C-terminal region was predicted to suppress basal T-loop phosphorylation and activity (
      • Goto S.
      • Yao Z.
      • Proud C.G.
      The C-terminal domain of Mnk1a plays a dual role in tightly regulating its activity.
      ); Ser353 is located at the start of this region. Based on existing and current data, we propose a functional extension to the MNK1a activation model: T-loop phosphorylation by canonical M/SAPK pathway activity precedes AMPK-mediated phosphorylation of MNK1a at Ser353 (Fig. 2F).
      Our observations suggest that MNK1a-Ser353 phosphorylation by AMPK is not required for MNK1a activation per se, but affects and/or possibly directs kinase activity, upon a conformational change induced by M/SAPK. MNK1-Ser(P)353 may serve to fine-tune MNK1-eIF4E-mediated protein synthesis (mRNA translation) via AMPK. This finding has potential therapeutic implications for chronic or acute human conditions, including metabolic syndrome-associated disorders and cancer. Abnormal regulation of protein synthesis is known to drive tumor cell proliferation and survival. Importantly, both AMPK and MNK1a have been independently put forward as potential druggable targets (
      • Hardie D.G.
      AMPK: a target for drugs and natural products with effects on both diabetes and cancer.
      ,
      • Proud C.G.
      Mnks, eIF4E phosphorylation and cancer.
      ).

      Experimental Procedures

      Expression Vectors

      For bacterial expression of MNK1a protein, glutathione S-transferase(GST)-tagged MNK1a (pGEX-6P1) was kindly provided by C. G. Proud (Southampton, UK) (
      • Goto S.
      • Yao Z.
      • Proud C.G.
      The C-terminal domain of Mnk1a plays a dual role in tightly regulating its activity.
      ). Active, hexahistidine-tagged (His6) AMPK was produced using a single hexacistronic expression vector encoding three AMPK and three LKB1 subunits as described (
      • Oligschlaeger Y.
      • Miglianico M.
      • Chanda D.
      • Scholz R.
      • Thali R.F.
      • Tuerk R.
      • Stapleton D.I.
      • Gooley P.R.
      • Neumann D.
      The recruitment of AMP-activated protein kinase to glycogen is regulated by autophosphorylation.
      ). Expression of kinase-dead AMPKα1D157A mutant was described earlier (
      • Neumann D.
      • Suter M.
      • Tuerk R.
      • Riek U.
      • Wallimann T.
      Co-expression of LKB1, MO25α and STRADα in bacteria yield the functional and active heterotrimeric complex.
      ). Point mutations were introduced using QuikChange (Agilent). To generate eukaryotic expression vector, MNK1a cDNAs were subcloned in-frame with an N-terminal double polyoma (2PY) tag into the BamHI and SalI sites of a retroviral pBabe vector (
      • Morgenstern J.P.
      • Land H.
      Advanced mammalian gene transfer: high titre retroviral vectors with multiple drug selection markers and a complementary helper-free packaging cell line.
      ). Amphotropic retrovirus was produced as described (
      • Kinsella T.M.
      • Nolan G.P.
      Episomal vectors rapidly and stably produce high-titer recombinant retrovirus.
      ); transduced U2OS and MEF cells were selected in medium containing 4 μg/ml puromycin.

      Recombinant Protein Purification and Cell-free Kinase Assays

      Proteins were expressed in Rosetta 2 (DE3) Escherichia coli cells (Merck Millipore) as described (
      • Oligschlaeger Y.
      • Miglianico M.
      • Chanda D.
      • Scholz R.
      • Thali R.F.
      • Tuerk R.
      • Stapleton D.I.
      • Gooley P.R.
      • Neumann D.
      The recruitment of AMP-activated protein kinase to glycogen is regulated by autophosphorylation.
      ). His6-AMPK and GST-MNK1a protein were purified using nickel-Sepharose HP (GE Healthcare) and glutathione-Sepharose 4B (GE Healthcare), respectively. AMPK protein was eluted in elution buffer (50 mm NaH2PO4, 30% glycerol, 0.5 m sucrose, 250 mm imidazole, pH 8) MNK1a protein was eluted with freshly prepared buffer (50 mm Tris-HCl, 10 mm reduced glutathione, pH 8).
      Aliquots of resin-bound MNK1a protein (10 μg) were incubated in 50 μl of kinase buffer (50 mm Hepes, pH 7.4, 5 mm MgCl2, 0.04 mm AMP, 1 mm DTT, 200 μm ATP) with 1 μg of active AMPK protein and 4 μCi of [γ-32P]ATP for 15 min at 37 °C. Beads were collected by centrifugation (5 min, 800 × g), washed, and suspended in cleavage buffer (50 mm Hepes, pH 7.5, 150 mm NaCl, 0.5 mm EDTA, 1 mm DTT). For proteolytic removal of GST tags, resin-bound protein was incubated with PreScission protease (GE Healthcare) for 4 h at 4 °C with constant mixing. Beads were pelleted (5 min, 800 × g); supernatant was transferred to a fresh tube. Samples were heated in SDS-PAGE sample buffer (5 min, 95 °C). Samples were analyzed by SDS-PAGE (9% gels) electrophoresis followed by Coomassie Brilliant Blue (CBB) staining and/or autoradiography.

      Mass Spectrometry

      GST-MNK1a and AMPK proteins were incubated with or without ATP in vitro as described above. Samples were separated by SDS-PAGE; MNK1a bands were excised and in-gel digested as described (
      • von Kriegsheim A.
      • Preisinger C.
      • Kolch W.
      Mapping of signaling pathways by functional interaction proteomics.
      ). Extracted tryptic peptides were desalted using homemade C18 columns, resuspended in 10% formic acid, and analyzed by reversed phase nanoLC-MS/MS (Ultimate 3000 and Orbitrap Elite; Thermo Scientific). Peptides were trapped on a precolumn for 10 min (Acclaim PepMap100, C18, 5 μm, 100 Å, 300-μm inner diameter × 5 mm, Thermo Scientific) in Buffer A (0.1% formic acid in water) and separated on an analytical column (Acclaim PepMap100, C18, 5 μm, 100 Å, 75-μm inner diameter × 25 cm) using a 70-min gradient (0–10 min, 5% buffer B (80% acetonitrile, 0.1% formic acid); 10–45 min, 10–45% buffer B; 45–47 min, 45–99% buffer B; 47–53 min, 99% buffer B; 53–70 min, 5% buffer B) at 250 nl/min. The mass spectrometer was operated in data-dependent mode with a 20-s dynamic exclusion range. Full-scan MS spectra were acquired in the Orbitrap (range: m/z 350–1500) with a resolution of 120,000 and an automatic gain control of 1E6 ions. Collision-induced dissociation fragmentation in the ion trap was performed on the top five precursors of each full scan employing a collision energy of 35%. Analysis of raw data was done by using MaxQuant (version 1.4.1.2 (
      • Cox J.
      • Mann M.
      MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification.
      )). The spectra were searched against the human Swiss-Prot database version 06/2014 using the Andromeda search engine using default mass tolerance settings (
      • Cox J.
      • Neuhauser N.
      • Michalski A.
      • Scheltema R.A.
      • Olsen J.V.
      • Mann M.
      Andromeda: a peptide search engine integrated into the MaxQuant environment.
      ). Trypsin was set as the protease (two missed cleavages allowed). Fixed modification: carbamidomethylation (Cys); variable modifications: oxidation (Met), phosphorylation (Ser, Thr, Tyr), and N-terminal protein acetylation. False discovery rate was set to 0.01 for peptides, proteins, and modification sites. The minimum peptide score for modified peptides was set to 40, and the minimum peptide length was seven amino acids (aa).

      Cell Culture, Chemicals, and Antibodies

      Human osteosarcoma U2OS cells and immortal mouse embryo fibroblasts (AMPKWT and AMPKdKO MEFs (
      • Laderoute K.R.
      • Amin K.
      • Calaoagan J.M.
      • Knapp M.
      • Le T.
      • Orduna J.
      • Foretz M.
      • Viollet B.
      5′-AMP-activated protein kinase (AMPK) is induced by low-oxygen and glucose deprivation conditions found in solid-tumor microenvironments.
      )) were cultured in DMEM (25 mm glucose; Gibco), supplemented with 10% (v/v) heat-inactivated fetal calf serum (Bodinco BV, Alkmaar, The Netherlands) and penicillin/streptomycin (Invitrogen). Cellular AMPK activation was achieved with AICAR (1.5 mm; Sigma) or A769662 (100 μm; Tocris Biosciences) for 45–60 min following 16 h of serum starvation (DMEM, 25 mm glucose). For M/SAPK activation, TPA (100 μm) was used following serum starvation. For glucose deprivation, DMEM medium without glucose (Gibco) was used. Antibodies used are: total MNK1a (tMNK1a) (#2195), pMNK1 (Thr209/Thr214, #2111), AMPK-Thr(P)172 (#2535), AMPK (#2532), eIF4E-Ser(P)209 (#9741), and pERK (#9101; Cell Signaling Technology); pACC (#07-303; Merck Millipore); β-actin (bACT, #0869100; MP Biomedicals); and eIF4E (#610269; BD Biosciences). Rabbit polyclonal MNK1a Ser353 phosphorylation-specific antibodies were custom-ordered (Genosphere Biotechnologies, Paris, France).

      Immunoprecipitation and Immunoblotting

      Cells were lysed in mild lysis buffer (250 mm NaCl, 0.1% IGEPAL (Nonidet P-40), 5 mm EDTA, 50 mm HEPES, pH 7.0) supplemented with 5 mm benzamidine, 5 μg/ml antipain, 5 μg/ml leupeptin, 5 μg/ml aprotinin, 1 mm sodium vanadate, 10 mm sodium fluoride, 10 mm pyrophosphate, 10 mm β-glycerophosphate, 0.5 mm DTT, and 1 mm PMSF. Extracts were sonicated on ice and centrifuged; 3% of the supernatant was stored as input (−80 °C). Endogenous AMPK was IP using a mix of AMPKα1 and AMPKα2 antibodies (sheep polyclonal; kindly provided by D. G. Hardie, Dundee, Scotland, UK); 2PY-tagged MNK1a (2PY-MNK1a) was IP using PY antiserum (Covance). For immunoprecipitation, 500 μg of protein lysate were incubated with primary antibody under constant mixing at 4 °C overnight, followed by incubation with protein G-Sepharose beads (4 h, 4 °C). Immune complexes were collected by centrifugation and analyzed by immunoblotting as described (
      • Oligschlaeger Y.
      • Miglianico M.
      • Chanda D.
      • Scholz R.
      • Thali R.F.
      • Tuerk R.
      • Stapleton D.I.
      • Gooley P.R.
      • Neumann D.
      The recruitment of AMP-activated protein kinase to glycogen is regulated by autophosphorylation.
      ). Signals were detected using enhanced chemiluminescence (ECL; Pierce). Band intensities were quantitated with the Quantity One software (Bio-Rad). Data were statistically analyzed by performing two-tailed paired t tests using Microsoft Excel.

      eIF4E Kinase Assay

      350 μg of U2OS 2PY-MNK1a cell lysates were incubated with PY antibody (2 h, 4 °C); 40 μl of protein G slurry were added for 1.5 h (4 °C). Beads were pelleted (5 min, 800 × g) and washed twice with mild lysis buffer, twice with 0.5 m LiCl, and twice with kinase buffer as described previously (
      • Goto S.
      • Yao Z.
      • Proud C.G.
      The C-terminal domain of Mnk1a plays a dual role in tightly regulating its activity.
      ). Beads were re-suspended in 50 μl of kinase buffer and incubated (15 min, 37 °C) with 1 μg of recombinant GST-eIF4E. The reaction was terminated by adding SDS-PAGE sample buffer (5 min, 95 °C). Samples were analyzed by SDS-PAGE (9% gels) and autoradiography. 200 μg of cell lysates were processed in parallel with immunoprecipitation antibody as controls.

      Author Contributions

      X. Z., J. W. V., and D. N. designed the study; X. Z., V. D., R. T., C. P., B. V., J. W. V., and D. N. contributed to data acquisition, analysis, and interpretation; X. Z., J. W. V., and D. N. drafted the manuscript; X. Z., and V. D., R. T., C. P., B. V., J. W. V., and D. N. provided final approval of the submitted version.

      Acknowledgments

      We thank R. Stead (Southampton, UK) and members of the Molecular Genetics Department (Maastricht University) for helpful comments and support. The Proteomics Facility is funded by the IZKF Aachen.

      References

        • Hardie D.G.
        • Ross F.A.
        • Hawley S.A.
        AMPK: a nutrient and energy sensor that maintains energy homeostasis.
        Nat. Rev. Mol. Cell Biol. 2012; 13: 251-262
        • Steinberg G.R.
        • Kemp B.E.
        AMPK in health and disease.
        Physiol. Rev. 2009; 89: 1025-1078
        • Stein S.C.
        • Woods A.
        • Jones N.A.
        • Davison M.D.
        • Carling D.
        The regulation of AMP-activated protein kinase by phosphorylation.
        Biochem. J. 2000; 345: 437-443
        • Woods A.
        • Vertommen D.
        • Neumann D.
        • Turk R.
        • Bayliss J.
        • Schlattner U.
        • Wallimann T.
        • Carling D.
        • Rider M.H.
        Identification of phosphorylation sites in AMP-activated protein kinase (AMPK) for upstream AMPK kinases and study of their roles by site-directed mutagenesis.
        J. Biol. Chem. 2003; 278: 28434-28442
        • Oligschlaeger Y.
        • Miglianico M.
        • Chanda D.
        • Scholz R.
        • Thali R.F.
        • Tuerk R.
        • Stapleton D.I.
        • Gooley P.R.
        • Neumann D.
        The recruitment of AMP-activated protein kinase to glycogen is regulated by autophosphorylation.
        J. Biol. Chem. 2015; 290: 11715-11728
        • Viollet B.
        • Foretz M.
        • Schlattner U.
        Bypassing AMPK phosphorylation.
        Chem. Biol. 2014; 21: 567-569
        • Dasgupta B.
        • Chhipa R.R.
        Evolving lessons on the complex role of AMPK in normal physiology and cancer.
        Trends Pharmacol. Sci. 2016; 37: 192-206
        • Li W.
        • Saud S.M.
        • Young M.R.
        • Chen G.
        • Hua B.
        Targeting AMPK for cancer prevention and treatment.
        Oncotarget. 2015; 6: 7365-7378
        • Mihaylova M.M.
        • Shaw R.J.
        The AMPK signalling pathway coordinates cell growth, autophagy and metabolism.
        Nat. Cell Biol. 2011; 13: 1016-1023
        • Munday M.R.
        Regulation of mammalian acetyl-CoA carboxylase.
        Biochem. Soc. Trans. 2002; 30: 1059-1064
        • Gwinn D.M.
        • Shackelford D.B.
        • Egan D.F.
        • Mihaylova M.M.
        • Mery A.
        • Vasquez D.S.
        • Turk B.E.
        • Shaw R.J.
        AMPK phosphorylation of raptor mediates a metabolic checkpoint.
        Mol. Cell. 2008; 30: 214-226
        • Huang J.
        • Manning B.D.
        The TSC1-TSC2 complex: a molecular switchboard controlling cell growth.
        Biochem. J. 2008; 412: 179-190
        • Ducommun S.
        • Deak M.
        • Sumpton D.
        • Ford R.J.
        • Núñez Galindo A.
        • Kussmann M.
        • Viollet B.
        • Steinberg G.R.
        • Foretz M.
        • Dayon L.
        • Morrice N.A.
        • Sakamoto K.
        Motif affinity and mass spectrometry proteomic approach for the discovery of cellular AMPK targets: identification of mitochondrial fission factor as a new AMPK substrate.
        Cell. Signal. 2015; 27: 978-988
        • Thali R.F.
        • Tuerk R.D.
        • Scholz R.
        • Yoho-Auchli Y.
        • Brunisholz R.A.
        • Neumann D.
        Novel candidate substrates of AMP-activated protein kinase identified in red blood cell lysates.
        Biochem. Biophys. Res. Commun. 2010; 398: 296-301
        • Tuerk R.D.
        • Thali R.F.
        • Auchli Y.
        • Rechsteiner H.
        • Brunisholz R.A.
        • Schlattner U.
        • Wallimann T.
        • Neumann D.
        New candidate targets of AMP-activated protein kinase in murine brain revealed by a novel multidimensional substrate-screen for protein kinases.
        J. Proteome Res. 2007; 6: 3266-3277
        • Banko M.R.
        • Allen J.J.
        • Schaffer B.E.
        • Wilker E.W.
        • Tsou P.
        • White J.L.
        • Villén J.
        • Wang B.
        • Kim S.R.
        • Sakamoto K.
        • Gygi S.P.
        • Cantley L.C.
        • Yaffe M.B.
        • Shokat K.M.
        • Brunet A.
        Chemical genetic screen for AMPKα2 substrates uncovers a network of proteins involved in mitosis.
        Mol. Cell. 2011; 44: 878-892
        • Moreno D.
        • Viana R.
        • Sanz P.
        Two-hybrid analysis identifies PSMD11, a non-ATPase subunit of the proteasome, as a novel interaction partner of AMP-activated protein kinase.
        Int. J. Biochem. Cell Biol. 2009; 41: 2431-2439
        • Mok J.
        • Im H.
        • Snyder M.
        Global identification of protein kinase substrates by protein microarray analysis.
        Nat. Protoc. 2009; 4: 1820-1827
        • Thali R.F.
        Exploring the Target Spectrum of AMP-activated Protein Kinase Ph.D dissertation, Diss. ETH No. 18981. ETH Zurich, Switzerland2010: 84-109
        • Hou J.
        • Lam F.
        • Proud C.
        • Wang S.
        Targeting Mnks for cancer therapy.
        Oncotarget. 2012; 3: 118-131
        • Goto S.
        • Yao Z.
        • Proud C.G.
        The C-terminal domain of Mnk1a plays a dual role in tightly regulating its activity.
        Biochem. J. 2009; 423: 279-290
        • Waskiewicz A.J.
        • Flynn A.
        • Proud C.G.
        • Cooper J.A.
        Mitogen-activated protein kinases activate the serine/threonine kinases Mnk1 and Mnk2.
        EMBO J. 1997; 16: 1909-1920
        • Waskiewicz A.J.
        • Johnson J.C.
        • Penn B.
        • Mahalingam M.
        • Kimball S.R.
        • Cooper J.A.
        Phosphorylation of the cap-binding protein eukaryotic translation initiation factor 4E by protein kinase Mnk1 in vivo.
        Mol. Cell. Biol. 1999; 19: 1871-1880
        • Wang X.
        • Flynn A.
        • Waskiewicz A.J.
        • Webb B.L.
        • Vries R.G.
        • Baines I.A.
        • Cooper J.A.
        • Proud C.G.
        The phosphorylation of eukaryotic initiation factor eIF4E in response to phorbol esters, cell stresses, and cytokines is mediated by distinct MAP kinase pathways.
        J. Biol. Chem. 1998; 273: 9373-9377
        • Pyronnet S.
        • Imataka H.
        • Gingras A.C.
        • Fukunaga R.
        • Hunter T.
        • Sonenberg N.
        Human eukaryotic translation initiation factor 4G (eIF4G) recruits Mnk1 to phosphorylate eIF4E.
        EMBO J. 1999; 18: 270-279
        • Bhat M.
        • Robichaud N.
        • Hulea L.
        • Sonenberg N.
        • Pelletier J.
        • Topisirovic I.
        Targeting the translation machinery in cancer.
        Nat. Rev. Drug Discov. 2015; 14: 261-278
        • Siddiqui N.
        • Sonenberg N.
        Signalling to eIF4E in cancer.
        Biochem. Soc. Trans. 2015; 43: 763-772
        • Klaus A.
        • Zorman S.
        • Berthier A.
        • Polge C.
        • Ramirez S.
        • Michelland S.
        • Sève M.
        • Vertommen D.
        • Rider M.
        • Lentze N.
        • Auerbach D.
        • Schlattner U.
        Glutathione S-transferases interact with AMP-activated protein kinase: evidence for S-glutathionylation and activation in vitro.
        PLoS ONE. 2013; 8: e62497
        • Göransson O.
        • McBride A.
        • Hawley S.A.
        • Ross F.A.
        • Shpiro N.
        • Foretz M.
        • Viollet B.
        • Hardie D.G.
        • Sakamoto K.
        Mechanism of action of A-769662, a valuable tool for activation of AMP-activated protein kinase.
        J. Biol. Chem. 2007; 282: 32549-32560
        • Guigas B.
        • Sakamoto K.
        • Taleux N.
        • Reyna S.M.
        • Musi N.
        • Viollet B.
        • Hue L.
        Beyond AICA riboside: in search of new specific AMP-activated protein kinase activators.
        IUBMB Life. 2009; 61: 18-26
        • Lu C.
        • Makala L.
        • Wu D.
        • Cai Y.
        Targeting translation: eIF4E as an emerging anticancer drug target.
        Expert Rev. Mol. Med. 2016; 18: e2
        • Roux P.P.
        • Blenis J.
        ERK and p38 MAPK-activated protein kinases: a family of protein kinases with diverse biological functions.
        Microbiol. Mol. Biol. Rev. 2004; 68: 320-344
        • Ueda T.
        • Watanabe-Fukunaga R.
        • Fukuyama H.
        • Nagata S.
        • Fukunaga R.
        Mnk2 and Mnk1 are essential for constitutive and inducible phosphorylation of eukaryotic initiation factor 4E but not for cell growth or development.
        Mol. Cell. Biol. 2004; 24: 6539-6549
        • Joshi S.
        • Platanias L.C.
        Mnk kinase pathway: Cellular functions and biological outcomes.
        World J. Biol. Chem. 2014; 5: 321-333
        • Hardie D.G.
        AMPK: a target for drugs and natural products with effects on both diabetes and cancer.
        Diabetes. 2013; 62: 2164-2172
        • Proud C.G.
        Mnks, eIF4E phosphorylation and cancer.
        Biochim. Biophys. Acta. 2015; 1849: 766-773
        • Neumann D.
        • Suter M.
        • Tuerk R.
        • Riek U.
        • Wallimann T.
        Co-expression of LKB1, MO25α and STRADα in bacteria yield the functional and active heterotrimeric complex.
        Mol. Biotechnol. 2007; 36: 220-231
        • Morgenstern J.P.
        • Land H.
        Advanced mammalian gene transfer: high titre retroviral vectors with multiple drug selection markers and a complementary helper-free packaging cell line.
        Nucleic Acids Res. 1990; 18: 3587-3596
        • Kinsella T.M.
        • Nolan G.P.
        Episomal vectors rapidly and stably produce high-titer recombinant retrovirus.
        Hum. Gene Ther. 1996; 7: 1405-1413
        • von Kriegsheim A.
        • Preisinger C.
        • Kolch W.
        Mapping of signaling pathways by functional interaction proteomics.
        Methods Mol. Biol. 2008; 484: 177-192
        • Cox J.
        • Mann M.
        MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification.
        Nat. Biotechnol. 2008; 26: 1367-1372
        • Cox J.
        • Neuhauser N.
        • Michalski A.
        • Scheltema R.A.
        • Olsen J.V.
        • Mann M.
        Andromeda: a peptide search engine integrated into the MaxQuant environment.
        J. Proteome Res. 2011; 10: 1794-1805
        • Laderoute K.R.
        • Amin K.
        • Calaoagan J.M.
        • Knapp M.
        • Le T.
        • Orduna J.
        • Foretz M.
        • Viollet B.
        5′-AMP-activated protein kinase (AMPK) is induced by low-oxygen and glucose deprivation conditions found in solid-tumor microenvironments.
        Mol. Cell. Biol. 2006; 26: 5336-5347