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In Vitro Methods to Study AMPK

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AMP-activated Protein Kinase

Part of the book series: Experientia Supplementum ((EXS,volume 107))

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Abstract

AMPK studies in cell-free and cellular systems have significantly contributed to recent progress in the AMPK field. Biochemical characterization, structure determination and elucidation of AMPK-dependent signalling events benefit from application of state-of-the-art tools and methodology. This chapter provides a synopsis of recombinant protein expression systems and biochemical and cell-based study methods. We summarize three different expression systems for AMPK production: bacteria, insect cells and mammalian cells. In addition, kinase activity measurement, kinase substrate identification and determination of physical interaction with AMPK are discussed. The last part of this chapter focuses on the use of pharmacological activation, inhibition, and molecular genetic tools to study AMPK involvement in cellular signalling pathways.

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References

  • Aguilar V et al (2007) S6 kinase deletion suppresses muscle growth adaptations to nutrient availability by activating AMP kinase. Cell Metab 5(6):476–87

    Article  CAS  PubMed  Google Scholar 

  • Anderson KA et al (2008) Hypothalamic CaMKK2 contributes to the regulation of energy balance. Cell Metab 7(5):377–88

    Article  CAS  PubMed  Google Scholar 

  • Andrzejewski S et al (2014) Metformin directly acts on mitochondria to alter cellular bioenergetics. Cancer Metab 2:12

    Article  PubMed  PubMed Central  Google Scholar 

  • Banko MR et al (2011) Chemical genetic screen for AMPKalpha2 substrates uncovers a network of proteins involved in mitosis. Mol Cell 44(6):878–92

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Bullen JW et al (2014) Cross-talk between two essential nutrient-sensitive enzymes: O-GlcNAc transferase (OGT) and AMP-activated protein kinase (AMPK). J Biol Chem 289(15):10592–606

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Bungard D et al (2010) Signaling kinase AMPK activates stress-promoted transcription via histone H2B phosphorylation. Science 329(5996):1201–5

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Calabrese MF et al (2014) Structural basis for AMPK activation: natural and synthetic ligands regulate kinase activity from opposite poles by different molecular mechanisms. Structure 22(8):1161–72

    Article  CAS  PubMed  Google Scholar 

  • Carling D et al (1989) Purification and characterization of the AMP-activated protein kinase. Copurification of acetyl-CoA carboxylase kinase and 3-hydroxy-3-methylglutaryl-CoA reductase kinase activities. Eur J Biochem 186(1-2):129–36

    Article  CAS  PubMed  Google Scholar 

  • Chakrabarti KR et al (2015) Pharmacologic regulation of AMPK in breast cancer affects cytoskeletal properties involved with microtentacle formation and re-attachment. Oncotarget 6(34):36292–36307

    PubMed  PubMed Central  Google Scholar 

  • Cool B et al (2006) Identification and characterization of a small molecule AMPK activator that treats key components of type 2 diabetes and the metabolic syndrome. Cell Metab 3(6):403–16

    Article  CAS  PubMed  Google Scholar 

  • Davies SP, Carling D, Hardie DG (1989) Tissue distribution of the AMP-activated protein kinase, and lack of activation by cyclic-AMP-dependent protein kinase, studied using a specific and sensitive peptide assay. Eur J Biochem 186(1-2):123–8

    Article  CAS  PubMed  Google Scholar 

  • Davies SP et al (1994) Purification of the AMP-activated protein kinase on ATP-gamma-sepharose and analysis of its subunit structure. Eur J Biochem 223(2):351–7

    Article  CAS  PubMed  Google Scholar 

  • Ducommun S et al (2015) 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 27(5):978–88

    Article  CAS  PubMed  Google Scholar 

  • Dyck JR et al (1996) Regulation of 5'-AMP-activated protein kinase activity by the noncatalytic beta and gamma subunits. J Biol Chem 271(30):17798–803

    Article  CAS  PubMed  Google Scholar 

  • Foretz M et al (2010) Metformin inhibits hepatic gluconeogenesis in mice independently of the LKB1/AMPK pathway via a decrease in hepatic energy state. J Clin Invest 120(7):2355–69

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Guo D et al (2009) The AMPK agonist AICAR inhibits the growth of EGFRvIII-expressing glioblastomas by inhibiting lipogenesis. Proc Natl Acad Sci U S A 106(31):12932–7

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Gwinn DM et al (2008) AMPK phosphorylation of raptor mediates a metabolic checkpoint. Mol Cell 30(2):214–26

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Ha J et al (1994) Critical phosphorylation sites for acetyl-CoA carboxylase activity. J Biol Chem 269(35):22162–8

    CAS  PubMed  Google Scholar 

  • Hardie DG (2011) AMPK and autophagy get connected. EMBO J 30(4):634–5

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Hardie DG, Ross FA, Hawley SA (2012) AMPK: a nutrient and energy sensor that maintains energy homeostasis. Nature reviews. Mol Cell Biol 13(4):251–62

    CAS  Google Scholar 

  • Harhaji-Trajkovic L et al (2009) AMPK-mediated autophagy inhibits apoptosis in cisplatin-treated tumour cells. J Cell Mol Med 13(9B):3644–54

    Article  CAS  PubMed  Google Scholar 

  • Hawley SA et al (1996) Characterization of the AMP-activated protein kinase kinase from rat liver and identification of threonine 172 as the major site at which it phosphorylates AMP-activated protein kinase. J Biol Chem 271(44):27879–87

    Article  CAS  PubMed  Google Scholar 

  • Hawley SA et al (2003) Complexes between the LKB1 tumor suppressor, STRAD alpha/beta and MO25 alpha/beta are upstream kinases in the AMP-activated protein kinase cascade. J Biol 2(4):28

    Article  PubMed  PubMed Central  Google Scholar 

  • Hawley SA et al (2005) Calmodulin-dependent protein kinase kinase-beta is an alternative upstream kinase for AMP-activated protein kinase. Cell Metab 2(1):9–19

    Article  CAS  PubMed  Google Scholar 

  • Hawley SA et al (2010) Use of cells expressing gamma subunit variants to identify diverse mechanisms of AMPK activation. Cell Metab 11(6):554–65

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Hawley SA et al (2012) The ancient drug salicylate directly activates AMP-activated protein kinase. Science 336(6083):918–22

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Hoogendijk AJ et al (2013) AMP-activated protein kinase activation by 5-aminoimidazole-4-carbox-amide-1-beta-d-ribofuranoside (AICAR) reduces lipoteichoic acid-induced lung inflammation. J Biol Chem 288(10):7047–52

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Hurley RL et al (2005) The Ca2+/calmodulin-dependent protein kinase kinases are AMP-activated protein kinase kinases. J Biol Chem 280(32):29060–6

    Article  CAS  PubMed  Google Scholar 

  • Inoki K, Zhu T, Guan KL (2003) TSC2 mediates cellular energy response to control cell growth and survival. Cell 115(5):577–90

    Article  CAS  PubMed  Google Scholar 

  • Iseli TJ et al (2008) AMP-activated protein kinase subunit interactions: beta1:gamma1 association requires beta1 Thr-263 and Tyr-267. J Biol Chem 283(8):4799–807

    Article  CAS  PubMed  Google Scholar 

  • Jarvis DL (2009) Baculovirus-insect cell expression systems. Methods Enzymol 463:191–222

    Article  CAS  PubMed  Google Scholar 

  • Kawaguchi T et al (2002) Mechanism for fatty acid "sparing" effect on glucose-induced transcription: regulation of carbohydrate-responsive element-binding protein by AMP-activated protein kinase. J Biol Chem 277(6):3829–35

    Article  CAS  PubMed  Google Scholar 

  • Khan KH (2013) Gene expression in mammalian cells and its applications. Adv Pharm Bull 3(2):257–63

    PubMed  PubMed Central  Google Scholar 

  • Kim TK, Eberwine JH (2010) Mammalian cell transfection: the present and the future. Anal Bioanal Chem 397(8):3173–8

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Klaus A et al (2013) Glutathione S-transferases interact with AMP-activated protein kinase: evidence for S-glutathionylation and activation in vitro. PLoS One 8(5), e62497

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Koay A et al (2010) AMPK beta subunits display isoform specific affinities for carbohydrates. FEBS Lett 584(15):3499–503

    Article  CAS  PubMed  Google Scholar 

  • Kukimoto-Niino M et al (2011) Crystal structure of the Ca(2)(+)/calmodulin-dependent protein kinase kinase in complex with the inhibitor STO-609. J Biol Chem 286(25):22570–9

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Lee KM et al (2011) Functional modulation of AMP-activated protein kinase by cereblon. Biochim Biophys Acta 1813(3):448–55

    Article  CAS  PubMed  Google Scholar 

  • Li Y et al (2011) AMPK phosphorylates and inhibits SREBP activity to attenuate hepatic steatosis and atherosclerosis in diet-induced insulin-resistant mice. Cell Metab 13(4):376–88

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Liu X et al (2014) The AMPK inhibitor compound C is a potent AMPK-independent antiglioma agent. Mol Cancer Ther 13(3):596–605

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Ma H, Deacon S, Horiuchi K (2008) The challenge of selecting protein kinase assays for lead discovery optimization. Expert Opin Drug Discov 3(6):607–621

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • McGee SL et al (2008) AMP-activated protein kinase regulates GLUT4 transcription by phosphorylating histone deacetylase 5. Diabetes 57(4):860–7

    Article  CAS  PubMed  Google Scholar 

  • Michell BJ et al (1996) Isoform-specific purification and substrate specificity of the 5'-AMP-activated protein kinase. J Biol Chem 271(45):28445–50

    Article  CAS  PubMed  Google Scholar 

  • Mihaylova MM, Shaw RJ (2011) The AMPK signalling pathway coordinates cell growth, autophagy and metabolism. Nat Cell Biol 13(9):1016–23

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Neumann D et al (2003) Mammalian AMP-activated protein kinase: functional, heterotrimeric complexes by co-expression of subunits in Escherichia coli. Protein Expr Purif 30(2):230–7

    Article  CAS  PubMed  Google Scholar 

  • Neumann D et al (2007) Co-expression of LKB1, MO25alpha and STRADalpha in bacteria yield the functional and active heterotrimeric complex. Mol Biotechnol 36(3):220–31

    Article  CAS  PubMed  Google Scholar 

  • Ning J, Xi G, Clemmons DR (2011) Suppression of AMPK activation via S485 phosphorylation by IGF-I during hyperglycemia is mediated by AKT activation in vascular smooth muscle cells. Endocrinology 152(8):3143–54

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Oakhill JS et al (2010) beta-Subunit myristoylation is the gatekeeper for initiating metabolic stress sensing by AMP-activated protein kinase (AMPK). Proc Natl Acad Sci U S A 107(45):19237–41

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Oligschlaeger Y et al (2015) The recruitment of AMP-activated protein kinase to glycogen is regulated by autophosphorylation. J Biol Chem 290(18):11715–28

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Rajamohan F et al (2010) Escherichia coli expression, purification and characterization of functional full-length recombinant alpha2beta2gamma3 heterotrimeric complex of human AMP-activated protein kinase. Protein Expr Purif 73(2):189–97

    Article  CAS  PubMed  Google Scholar 

  • Ramanathan L et al (2010) Purification and characterization of truncated human AMPK alpha 2 beta 2 gamma 3 heterotrimer from baculovirus-infected insect cells. Protein Expr Purif 70(1):13–22

    Article  CAS  PubMed  Google Scholar 

  • Riek U et al (2008) Structural properties of AMP-activated protein kinase: dimerization, molecular shape, and changes upon ligand binding. J Biol Chem 283(26):18331–43

    Article  CAS  PubMed  Google Scholar 

  • Riek U et al (2009) A versatile multidimensional protein purification system with full internet remote control based on a standard HPLC system. Biotechniques 46(6):ix–xii

    Article  PubMed  Google Scholar 

  • Salt IP et al (1998a) AMP-activated protein kinase is activated by low glucose in cell lines derived from pancreatic beta cells, and may regulate insulin release. Biochem J 335(Pt 3):533–9

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Salt I et al (1998b) AMP-activated protein kinase: greater AMP dependence, and preferential nuclear localization, of complexes containing the alpha2 isoform. Biochem J 334(Pt 1):177–87

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Sanders MJ et al (2007) Defining the mechanism of activation of AMP-activated protein kinase by the small molecule A-769662, a member of the thienopyridone family. J Biol Chem 282(45):32539–48

    Article  CAS  PubMed  Google Scholar 

  • Sanz P, Rubio T, Garcia-Gimeno MA (2013) AMPKbeta subunits: more than just a scaffold in the formation of AMPK complex. FEBS J 280(16):3723–33

    Article  CAS  PubMed  Google Scholar 

  • Scott JW et al (2004) CBS domains form energy-sensing modules whose binding of adenosine ligands is disrupted by disease mutations. J Clin Invest 113(2):274–84

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Scott JW et al (2014) Small molecule drug A-769662 and AMP synergistically activate naive AMPK independent of upstream kinase signaling. Chem Biol 21(5):619–27

    Article  CAS  PubMed  Google Scholar 

  • Shaw RJ et al (2004a) The tumor suppressor LKB1 kinase directly activates AMP-activated kinase and regulates apoptosis in response to energy stress. Proc Natl Acad Sci U S A 101(10):3329–35

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Shaw RJ et al (2004b) The LKB1 tumor suppressor negatively regulates mTOR signaling. Cancer Cell 6(1):91–9

    Article  CAS  PubMed  Google Scholar 

  • Stahmann N et al (2006) Thrombin activates AMP-activated protein kinase in endothelial cells via a pathway involving Ca2+/calmodulin-dependent protein kinase kinase beta. Mol Cell Biol 26(16):5933–45

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Steinberg GR, Kemp BE (2009) AMPK in health and disease. Physiol Rev 89(3):1025–78

    Article  CAS  PubMed  Google Scholar 

  • Sujobert P et al (2015) Co-activation of AMPK and mTORC1 Induces Cytotoxicity in Acute Myeloid Leukemia. Cell Rep 11(9):1446–57

    Article  CAS  PubMed  Google Scholar 

  • Suter M et al (2006) Dissecting the role of 5'-AMP for allosteric stimulation, activation, and deactivation of AMP-activated protein kinase. J Biol Chem 281(43):32207–16

    Article  CAS  PubMed  Google Scholar 

  • Suzuki T et al (2013) Inhibition of AMPK catabolic action by GSK3. Mol Cell 50(3):407–19

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Tamas P et al (2006) Regulation of the energy sensor AMP-activated protein kinase by antigen receptor and Ca2+ in T lymphocytes. J Exp Med 203(7):1665–70

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Tangeman L, Wyatt CN, Brown TL (2012) Knockdown of AMP-activated protein kinase alpha 1 and alpha 2 catalytic subunits. J RNAi Gene Silencing 8:470–8

    CAS  PubMed  PubMed Central  Google Scholar 

  • Taylor EB et al (2008) Discovery of TBC1D1 as an insulin-, AICAR-, and contraction-stimulated signaling nexus in mouse skeletal muscle. J Biol Chem 283(15):9787–96

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Thali RF et al (2010) Novel candidate substrates of AMP-activated protein kinase identified in red blood cell lysates. Biochem Biophys Res Commun 398(2):296–301

    Article  CAS  PubMed  Google Scholar 

  • Treebak JT et al (2006) AMPK-mediated AS160 phosphorylation in skeletal muscle is dependent on AMPK catalytic and regulatory subunits. Diabetes 55(7):2051–8

    Article  CAS  PubMed  Google Scholar 

  • Tuerk RD et al (2007) New candidate targets of AMP-activated protein kinase in murine brain revealed by a novel multidimensional substrate-screen for protein kinases. J Proteome Res 6(8):3266–77

    Article  CAS  PubMed  Google Scholar 

  • Turban S et al (2012) Defining the contribution of AMP-activated protein kinase (AMPK) and protein kinase C (PKC) in regulation of glucose uptake by metformin in skeletal muscle cells. J Biol Chem 287(24):20088–99

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Vingtdeux V et al (2010) AMP-activated protein kinase signaling activation by resveratrol modulates amyloid-beta peptide metabolism. J Biol Chem 285(12):9100–13

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Woods A et al (2003a) LKB1 is the upstream kinase in the AMP-activated protein kinase cascade. Curr Biol 13(22):2004–8

    Article  CAS  PubMed  Google Scholar 

  • Woods A et al (2003b) 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 278(31):28434–42

    Article  CAS  PubMed  Google Scholar 

  • Xiao B et al (2011) Structure of mammalian AMPK and its regulation by ADP. Nature 472(7342):230–3

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Xiao B et al (2013) Structural basis of AMPK regulation by small molecule activators. Nat Commun 4:3017

    PubMed  PubMed Central  Google Scholar 

  • Zhan YY et al (2012) The orphan nuclear receptor Nur77 regulates LKB1 localization and activates AMPK. Nat Chem Biol 8(11):897–904

    CAS  PubMed  Google Scholar 

  • Zhou J et al (2009) Inactivation of AMPK alters gene expression and promotes growth of prostate cancer cells. Oncogene 28(18):1993–2002

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Authors and Affiliations

  1. Department of Molecular Genetics, CARIM School for Cardiovascular Diseases, Maastricht University Medical Centre, 616, 6200 MD, Maastricht, The Netherlands

    Xiaoqing Zhu, J. Willem Voncken & Dietbert Neumann

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  1. Xiaoqing Zhu
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  2. J. Willem Voncken
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  3. Dietbert Neumann
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Correspondence to Dietbert Neumann .

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Editors and Affiliations

  1. Oral Medicine Department, Universidad de Sevilla, Sevilla, Spain

    Mario D. Cordero

  2. INSERM U1016 Institut Cochin CNRS UMR 8104, Université Paris Descartes, Paris, France

    Benoit Viollet

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Zhu, X., Voncken, J.W., Neumann, D. (2016). In Vitro Methods to Study AMPK. In: Cordero, M., Viollet, B. (eds) AMP-activated Protein Kinase. Experientia Supplementum, vol 107. Springer, Cham. https://doi.org/10.1007/978-3-319-43589-3_19

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