Analysis of the Role of the AMP-Activated Protein Kinase in the Response to Cellular Stress

  • D. Grahame Hardie
  • Ian P. Salt
  • Stephen P. Davies
Part of the Methods in Molecular Biology™ book series (MIMB, volume 99)


The AMP-activated protein kinase (AMPK) is the central component of a protein kinase cascade that is activated by cellular stresses causing ATP depletion and has been referred to as a “fuel gauge” or “metabolic sensor” of the eukaryotic cell (1,2). The kinase is activated by phosphorylation by an upstream protein kinase termed AMP-activated protein kinase kinase (AMPKK) (3). Elevation of 5’-AMP activates the cascade by a complex mechanism involving binding of the nucleotide to both the upstream kinase (AMP-activated protein kinase kinase, AMPKK) and the downstream kinase, AMPK (See Subheading 1.2.). These effects of AMP are also antagonized by high concentrations (mM) of ATP. The AMP: ATP ratio in the cell varies approximately as the square of the ADP:ATP ratio, due to the action of adenylate kinase which maintains its reaction (2ADP ’ ATP + AMP) close to equilibrium at all times. Therefore, any cellular stress that affects the ability of the cell to maintain a high ATP: ADP ratio (normally approx 10:1 in an unstressed cell) leads to activation of the AMPK cascade. Cellular stresses can do this either by inhibiting ATP production or by increasing ATP consumption, and stresses shown to cause AMPK activation include heat shock (4), various mitochondrial inhibitors such as arsenite, antimycin A, dinitrophenol, and azide (4,5), ischemia/hypoxia in heart muscle (6), and exercise in skeletal muscle (7). ATP can also be depleted, and AMPK activated, by incubation of cells with high concentrations of certain sugars which trap cellular phosphate, such as fructose (8) and 2-deoxyglucose (9). Detachment of cultured cells from their substrate by trypsinization has also been reported to increase cellular AMP:ATP and to inhibit lipid synthesis, consistent with the activation of AMPK (10). Downstream targets for the system include biosynthetic pathways that are inhibited, thus conserving ATP, and catabolic pathways tht are activated, thus generating more ATP (1,2). Although most of the currently known targets for the system are metabolic enzymes, the yeast homolog of AMPK (i.e., the SNF1 complex) regulates gene expression (2). At least one isoform of AMPK is partly localized to the nucleus (see Subheading 1.1.), and it seems very likely that the mammalian system will also turn out to regulate gene expression.


AMPK Activation Adenylate Kinase Upstream Kinase Crude Lysate Polyethylene Glycol Precipitation 
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  1. 1.
    Hardie D. G. and Carling D. (1997) The AMP-activated protein kinase: fuel gauge of the mammalian cell? Eur. J. Biochem. 246, 259–273.PubMedCrossRefGoogle Scholar
  2. 2.
    Hardie D. G., Carling D., and Carlson M. (1998) The AMP-activated/SNF1 protein kinase subfamily: metabolic sensors of the eukaryotic cell? Ann. Rev. Biochem. 67, 821–855.PubMedCrossRefGoogle Scholar
  3. 3.
    Hawley S. A., Davison M., Woods A., Davies S. P., Beri R. K., Carling D., and Hardie D. G. (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 and activates AMP-activated protein kinase. J. Biol. Chem. 271, 27,879–27,887.PubMedCrossRefGoogle Scholar
  4. 4.
    Corton J. M., Gillespie J. G., and Hardie D. G. (1994) Role of the AMP-activated protein kinase in the cellular stress response. Current Biol. 4, 315–324.CrossRefGoogle Scholar
  5. 5.
    Witters L. A., Nordlund A. C., and Marshall L. (1991) Regulation of intracellular acetyl-CoA carboxylase by ATP depletors mimics the action of the 5′-AMP-activated protein kinase. Biochem. Biophys. Res. Comm. 181, 1486–1492.PubMedCrossRefGoogle Scholar
  6. 6.
    Kudo N., Barr A. J., Barr R. L., Desai S., and Lopaschuk G. D. (1995) High rates of fatty acid oxidation during reperfusion of ischemic hearts are associated with a decrease in malonyl-CoA levels due to an increase in 5′-AMP-activated protein kinase inhibition of acetyl-CoA carboxylase. J. Biol. Chem. 270, 17,513–17,520.PubMedCrossRefGoogle Scholar
  7. 7.
    Winder W. W., and Hardie D. G. (1996) Inactivation of acetyl-CoA carboxylase and activation of AMP-activated protein kinase in muscle during exercise. Am. J. Physiol. 270, E299–E304.PubMedGoogle Scholar
  8. 8.
    Moore F., Weekes J., and Hardie D. G. (1991) AMP triggers phosphorylation as well as direct allosteric activation of rat liver AMP-activated protein kinase. A sensitive mechanism to protect the cell against ATP depletion. Eur. J. Biochem. 199, 691–697.PubMedCrossRefGoogle Scholar
  9. 9.
    Sato R., Goldstein J. L., and Brown M. S. (1993) Replacement of Serine-871 of hamster 3-hydroxy-3-methylglutaryl CoA reductase prevents phosphorylation by AMP-activated protein kinase and blocks inhibition of sterol synthesis induced by ATP depletion. Proc. Natl. Acad. Sci. USA 90, 9261–9265.PubMedCrossRefGoogle Scholar
  10. 10.
    Page K. and Lange Y. (1997) Cell adhesion to fibronectin regulates membrane lipid biosynthesis through 5′-AMP-activated protein kinase. J. Biol. Chem. 272, 19,339–19,342.PubMedCrossRefGoogle Scholar
  11. 11.
    Carling D., Aguan K., Woods A., Verhoeven A. J. M., Beri R. K., Brennan C. H., Sidebottom C., Davison M. D., and Scott J. (1994) Mammalian AMP-activated protein kinase is homologous to yeast and plant protein kinases involved in the regulation of carbon metabolism. J. Biol. Chem. 269, 11,442–11,448.PubMedGoogle Scholar
  12. 12.
    Gao G., Widmer J., Stapleton D., Teh T., Cox T., Kemp B. E., and Witters L. A. (1995) Catalytic subunits of the porcine and rat 5′-AMP-activated protein kinase are members of the SNF1 protein kinase family. Biochim. Biophys. Acta 1266, 73–82.PubMedCrossRefGoogle Scholar
  13. 13.
    Stapleton D., Mitchelhill K. I., Gao G., Widmer J., Michell B. J., Teh T., House C. M., Fernandez C. S., Cox T., Witters L. A., and Kemp B. E. (1996) Mammalian AMP-activated protein kinase subfamily. J. Biol. Chem. 271, 611–614.PubMedCrossRefGoogle Scholar
  14. 14.
    Woods A., Salt I., Scott J., Hardie D. G., and Carling D. (1996) The αl and α2 isoforms of the AMP-activated protein kinase have similar activities in rat liver but exhibit differences in substrate specificity in vitro. FEBS Lett. 397, 347–351.PubMedCrossRefGoogle Scholar
  15. 15.
    Woods A., Cheung P. C. F., Smith F. C., Davison M. D., Scott J., Beri R. K., and Carling D. (1996) Characterization of AMP-activated protein kinase β and γ subunits: assembly of the heterotrimeric complex in vitro. J. Biol. Chem. 271, 10,282–10,290.PubMedCrossRefGoogle Scholar
  16. 16.
    Gao G., Fernandez S., Stapleton D., Auster A. S., Widmer J., Dyck J. R. B., Kemp B. E., and Witters L. A. (1996) Non-catalytic β-and γ-subunit isoforms of the 5′-AMP-activated protein kinase. J. Biol. Chem. 271, 8675–8681.PubMedCrossRefGoogle Scholar
  17. 17.
    Thornton C., Snowden M. A., and Carling D. (1998) Identification of a novel AMP-activated protein kinase p subunit isoform which is highly expressed in skeletal muscle. J. Biol. Chem. 273, 12,443–12,450.PubMedCrossRefGoogle Scholar
  18. 18.
    Stapleton D., Woollatt E., Mitchelhill K. I., Nicholl J. K., Fernandez C. S., Michell B. J., Witters L. A., Power D. A., Sutherland G. R., and Kemp B. E. (1997) AMP-activated protein kinase isoenzyme family: subunit structure and chromosomal location. FEBS Lett. 409, 452–456.PubMedCrossRefGoogle Scholar
  19. 19.
    Salt I. P., Celler J. W., Hawley S. A., Prescott A., Woods A., Carling D., and Hardie D. G. (1998) AMP-activated protein kinase-greater AMP dependence, and preferential nuclear localization, of complexes containing the α2 isoform. Biochem. J. 334, 177–187.PubMedGoogle Scholar
  20. 20.
    Carling D., Clarke P. R., Zammit V. A., and Hardie D. G. (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, 129–136.PubMedCrossRefGoogle Scholar
  21. 21.
    Davies S. P., Helps N. R., Cohen P. T. W., and Hardie D. G. (1995) 5′-AMP inhibits dephosphorylation, as well as promoting phosphorylation, of the AMP-activated protein kinase. Studies using bacterially expressed human protein phosphatase-2Cα and native bovine protein phosphatase-2Ac. FEBS Lett. 377, 421–425.PubMedCrossRefGoogle Scholar
  22. 22.
    Davies S. P., Carling D., Munday M. R., and Hardie D. G. (1992) Diurnal rhythm of phosphorylation of rat liver acetyl-CoA carboxylase by the AMP-activated protein kinase, demonstrated using freeze-clamping. Effects of high fat diets. Eur. J. Biochem. 203, 615–623.PubMedCrossRefGoogle Scholar
  23. 23.
    Gillespie J. G., and Hardie D. G. (1992) Phosphorylation and inactivation of HMG-CoA reductase at the AMP-activated protein kinase site in response to fructose treatment of isolated rat hepatocytes. FEBS Lett. 306, 59–62.PubMedCrossRefGoogle Scholar
  24. 24.
    Corton J. M., Gillespie J. G., Hawley S. A., and Hardie D. G. (1995) 5-Aminoimidazole-4-carboxamide ribonucleoside: a specific method for activating AMP-activated protein kinase in intact cells? Eur. J. Biochem. 229, 558–565.PubMedCrossRefGoogle Scholar
  25. 25.
    Davies S. P., Carling D., and Hardie D. G. (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, 123–128.PubMedCrossRefGoogle Scholar
  26. 26.
    Dale S., Wilson W. A., Edelman A. M., and Hardie D. G. (1995) Similar substrate recognition motifs for mammalian AMP-activated protein kinase, higher plant HMG-CoA reductase kinase-A, yeast SNF1, and mammalian calmodulin-dependent protein kinase I. FEBS Lett. 361, 191–195.PubMedCrossRefGoogle Scholar
  27. 27.
    Henin N., Vincent M. F., Gruber H. E., and Van den Berghe G. (1995) Inhibition of fatty acid and cholesterol synthesis by stimulation of AMP-activated protein kinase. FASEB J. 9, 541–546.PubMedGoogle Scholar
  28. 28.
    Velasco G., Geelen M. J. H., and Guzman M. (1997) Control of hepatic fatty acid oxidation by 5′-AMP-activated protein kinase involves a malonyl-CoA-dependent and a malonyl-CoA-independent mechanism. Arch. Biochem. Biophys. 337, 169–175.PubMedCrossRefGoogle Scholar
  29. 29.
    Merrill G. M., Kurth E., Hardie D. G., and Winder W. W. (1997) AICAR decreases malonyl-CoA and increases fatty acid oxidation in skeletal muscle of the rat. Am. J. Physiol. 36, E1107–E1112.Google Scholar
  30. 30.
    Javaux F., Vincent M. F., Wagner D. R., and van den Berghe G. (1995) Celltype specificity of inhibition of glycolysis by 5-amino-4-imidazolecarboxamide riboside. Lack of effect in rabbit cardiomyocytes and human erythrocytes, and inhibition in FTO-2B rat hepatoma cells. Biochem J. 305, 913–919.PubMedGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2000

Authors and Affiliations

  • D. Grahame Hardie
    • 1
  • Ian P. Salt
    • 2
  • Stephen P. Davies
    • 3
  1. 1.Department of BiochemistryUniversity of Dundee
  2. 2.Division of BiochemistryInstitute of Biomedical and Life Sciences, University of GlasgowGlasgow
  3. 3.Department of BiochemistryUniversity of DundeeDundee

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