Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letter
  • Published:

Structural insight into the autoinhibition mechanism of AMP-activated protein kinase

Nature volume 459, pages 1146–1149 (2009)Cite this article

Abstract

The AMP-activated protein kinase (AMPK) is characterized by its ability to bind to AMP, which enables it to adjust enzymatic activity by sensing the cellular energy status and maintain the balance between ATP production and consumption in eukaryotic cells1,2. It also has important roles in the regulation of cell growth and proliferation, and in the establishment and maintenance of cell polarity3. These important functions have rendered AMPK an important drug target for obesity, type 2 diabetes and cancer treatments4. However, the regulatory mechanism of AMPK activity by AMP binding remains unsolved. Here we report the crystal structures of an unphosphorylated fragment of the AMPK α-subunit (KD-AID) from Schizosaccharomyces pombe that contains both the catalytic kinase domain and an autoinhibitory domain (AID), and of a phosphorylated kinase domain from Saccharomyces cerevisiae (Snf1-pKD). The AID binds, from the ‘backside’, to the hinge region of its kinase domain, forming contacts with both amino-terminal and carboxy-terminal lobes. Structural analyses indicate that AID binding might constrain the mobility of helix αC, hence resulting in an autoinhibited KD-AID with much lower kinase activity than that of the kinase domain alone. AMP activates AMPK both allosterically and by inhibiting dephosphorylation5,6. Further in vitro kinetic studies demonstrate that disruption of the KD-AID interface reverses the autoinhibition and these AMPK heterotrimeric mutants no longer respond to the change in AMP concentration. The structural and biochemical data have shown the primary mechanism of AMPK autoinhibition and suggest a conformational switch model for AMPK activation by AMP.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Structure and activity of the AMPK kinase domain and KD-AID.
Figure 2: The AID immobilizes the open conformation of kinase domain.
Figure 3: Disruption of KD-AID interface abolishes AID inhibition.
Figure 4: Effect of KD-AID interface mutants on AMP-regulation of AMPK holoenzyme.

Similar content being viewed by others

Accession codes

Primary accessions

Protein Data Bank

Data deposits

The structural and atomic coordinates have been deposited in the Protein Data Bank under accession codes 3H4J for S. pombe KD-AID and 3DAE for S. cerevisiae Snf1-pKD.

References

  1. Carling, D. The AMP-activated protein kinase cascade—a unifying system for energy control. Trends Biochem. Sci. 29, 18–24 (2004)

    Article  CAS  Google Scholar 

  2. Kahn, B. B., Alquier, T., Carling, D. & Hardie, D. G. AMP-activated protein kinase: ancient energy gauge provides clues to modern understanding of metabolism. Cell Metab. 1, 15–25 (2005)

    Article  CAS  Google Scholar 

  3. Hardie, D. G. AMP-activated/SNF1 protein kinases: conserved guardians of cellular energy. Nature Rev. Mol. Cell Biol. 8, 774–785 (2007)

    Article  CAS  Google Scholar 

  4. Hardie, D. G. AMP-activated protein kinase as a drug target. Annu. Rev. Pharmacol. Toxicol. 47, 185–210 (2007)

    Article  CAS  Google Scholar 

  5. Yeh, L. A., Lee, K. H. & Kim, K. H. Regulation of rat liver acetyl-CoA carboxylase. Regulation of phosphorylation and inactivation of acetyl-CoA carboxylase by the adenylate energy charge. J. Biol. Chem. 255, 2308–2314 (1980)

    CAS  Google Scholar 

  6. Davies, S. P., Helps, N. R., Cohen, P. T. & Hardie, D. G. 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 (1995)

    Article  CAS  Google Scholar 

  7. Crute, B. E., Seefeld, K., Gamble, J., Kemp, B. E. & Witters, L. A. Functional domains of the α1 catalytic subunit of the AMP-activated protein kinase. J. Biol. Chem. 273, 35347–35354 (1998)

    Article  CAS  Google Scholar 

  8. Pang, T. et al. Conserved α-helix acts as autoinhibitory sequence in AMP-activated protein kinase α subunits. J. Biol. Chem. 282, 495–506 (2007)

    Article  CAS  Google Scholar 

  9. Hawley, S. A. et al. 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, 27879–27887 (1996)

    Article  CAS  Google Scholar 

  10. Scott, J. W. et al. CBS domains form energy-sensing modules whose binding of adenosine ligands is disrupted by disease mutations. J. Clin. Invest. 113, 274–284 (2004)

    Article  CAS  Google Scholar 

  11. Iseli, T. J. et al. AMP-activated protein kinase β subunit tethers α and γ subunits via its C-terminal sequence (186–270). J. Biol. Chem. 280, 13395–13400 (2005)

    Article  CAS  Google Scholar 

  12. McBride, A., Ghilagaber, S., Nikolaev, A. & Hardie, D. G. The glycogen-binding domain on the AMPK β subunit allows the kinase to act as a glycogen sensor. Cell Metab. 9, 23–34 (2009)

    Article  CAS  Google Scholar 

  13. Cool, B. et al. Identification and characterization of a small molecule AMPK activator that treats key components of type 2 diabetes and the metabolic syndrome. Cell Metab. 3, 403–416 (2006)

    Article  CAS  Google Scholar 

  14. Sanders, M. J. et al. 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, 32539–32548 (2007)

    Article  CAS  Google Scholar 

  15. Göransson, O. et al. Mechanism of action of A-769662, a valuable tool for activation of AMP-activated protein kinase. J. Biol. Chem. 282, 32549–32560 (2007)

    Article  Google Scholar 

  16. Scott, J. W. et al. Thienopyridone drugs are selective activators of AMP-activated protein kinase β1-containing complexes. Chem. Biol. 15, 1220–1230 (2008)

    Article  CAS  Google Scholar 

  17. Townley, R. & Shapiro, L. Crystal structures of the adenylate sensor from fission yeast AMP-activated protein kinase. Science 315, 1726–1729 (2007)

    Article  ADS  CAS  Google Scholar 

  18. Amodeo, G. A., Rudolph, M. J. & Tong, L. Crystal structure of the heterotrimer core of Saccharomyces cerevisiae AMPK homologue SNF1. Nature 449, 492–495 (2007)

    Article  ADS  CAS  Google Scholar 

  19. Xiao, B. et al. Structural basis for AMP binding to mammalian AMP-activated protein kinase. Nature 449, 496–500 (2007)

    Article  ADS  CAS  Google Scholar 

  20. Rudolph, M. J., Amodeo, G. A., Bai, Y. & Tong, L. Crystal structure of the protein kinase domain of yeast AMP-activated protein kinase Snf1. Biochem. Biophys. Res. Commun. 337, 1224–1228 (2005)

    Article  CAS  Google Scholar 

  21. Nayak, V. et al. Structure and dimerization of the kinase domain from yeast Snf1, a member of the Snf1/AMPK protein family. Structure 14, 477–485 (2006)

    Article  CAS  Google Scholar 

  22. Nolen, B., Taylor, S. & Ghosh, G. Regulation of protein kinases: controlling activity through activation segment conformation. Mol. Cell 15, 661–675 (2004)

    Article  CAS  Google Scholar 

  23. Pellicena, P. & Kuriyan, J. Protein–protein interactions in the allosteric regulation of protein kinases. Curr. Opin. Struct. Biol. 16, 702–709 (2006)

    Article  CAS  Google Scholar 

  24. Marx, A. et al. Structural variations in the catalytic and ubiquitin-associated domains of microtubule-associated protein/microtubule affinity regulating kinase (MARK) 1 and MARK2. J. Biol. Chem. 281, 27586–27599 (2006)

    Article  CAS  Google Scholar 

  25. Jaleel, M. et al. The ubiquitin-associated domain of AMPK-related kinases regulates conformation and LKB1-mediated phosphorylation and activation. Biochem. J. 394, 545–555 (2006)

    Article  CAS  Google Scholar 

  26. Huse, M. & Kuriyan, J. The conformational plasticity of protein kinases. Cell 109, 275–282 (2002)

    Article  CAS  Google Scholar 

  27. Deindl, S. et al. Structural basis for the inhibition of tyrosine kinase activity of ZAP-70. Cell 129, 735–746 (2007)

    Article  CAS  Google Scholar 

  28. Scott, J. W., Ross, F. A., Liu, J. K. & Hardie, D. G. Regulation of AMP-activated protein kinase by a pseudosubstrate sequence on the gamma subunit. EMBO J. 26, 806–815 (2007)

    Article  CAS  Google Scholar 

  29. Pang, T. et al. Small molecule antagonizes autoinhibition and activates AMP-activated protein kinase in cells. J. Biol. Chem. 283, 16051–16060 (2008)

    Article  CAS  Google Scholar 

  30. Sanders, M. J. et al. Investigating the mechanism for AMP activation of the AMP-activated protein kinase cascade. Biochem. J. 403, 139–148 (2007)

    Article  CAS  Google Scholar 

  31. Neumann, D., Woods, A., Carling, D., Wallimann, T. & Schlattner, U. Mammalian AMP-activated protein kinase: functional, heterotrimeric complexes by co-expression of subunits in Escherichia coli . Protein Expr. Purif. 30, 230–237 (2003)

    Article  CAS  Google Scholar 

  32. Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997)

    Article  CAS  Google Scholar 

  33. McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007)

    Article  CAS  Google Scholar 

  34. Adams, P. D. et al. PHENIX: building new software for automated crystallographic structure determination. Acta Crystallogr. D 58, 1948–1954 (2002)

    Article  Google Scholar 

  35. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D 60, 2126–2132 (2004)

    Article  Google Scholar 

  36. Woods, A. et al. 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, 28434–28442 (2003)

    Article  CAS  Google Scholar 

  37. Hawley, S. A. et al. Calmodulin-dependent protein kinase kinase-β is an alternative upstream kinase for AMP-activated protein kinase. Cell Metab. 2, 9–19 (2005)

    Article  CAS  Google Scholar 

  38. Roskoski, R. Assays of protein kinase. Methods Enzymol. 99, 3–6 (1983)

    Article  CAS  Google Scholar 

  39. Davies, S. P., Carling, D. & Hardie, D. G. Tissue distribution of the AMP-activated protein protein kinase, and lack of activation by cyclic-AMP-dependent protein kinase, studied using a specific and sensitive protein assay. Eur. J. Biochem. 186, 123–128 (1989)

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank Y. Shi and S. C. Lin for critical discussions and reading of the manuscript; J. Chai and Y. Dong for help with data collection and processing; L. Gu and J. Wang for advice on structure determination. This work is supported in part by MOST grants 2006CB503900 and 2007CB914400, and by NSFC grants 30425005 and 30770476.

Author Contributions L.C., Z.-H.J. and L.-S.Z. designed, performed and analysed most of the experiments. Y.-Y.Z. and S.-T.X. provided technical assistance. Z.-X.W. contributed to discussions. J.-W.W. led the team and wrote the paper.

Author information

Author notes

  1. Lei Chen, Zhi-Hao Jiao and Li-Sha Zheng: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Biological Sciences and Biotechnology, MOE Key Laboratory of Bioinformatics, Tsinghua University, Beijing 100084, China

    Lei Chen, Zhi-Hao Jiao, Yuan-Yuan Zhang, Shu-Tao Xie, Zhi-Xin Wang & Jia-Wei Wu

  2. Institute of Biophysics and Graduate University, Chinese Academy of Sciences, Beijing 100101, China

    Li-Sha Zheng & Zhi-Xin Wang

Authors
  1. Lei Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Zhi-Hao Jiao
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Li-Sha Zheng
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Yuan-Yuan Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Shu-Tao Xie
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Zhi-Xin Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Jia-Wei Wu
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Jia-Wei Wu.

Supplementary information

Supplementary Information

This file contains Supplementary Tables 1-2, Supplementary Figures 1-9 with extended Legends and Supplementary References. (PDF 4317 kb)

PowerPoint slides

Rights and permissions

Reprints and permissions

About this article

Cite this article

Chen, L., Jiao, ZH., Zheng, LS. et al. Structural insight into the autoinhibition mechanism of AMP-activated protein kinase. Nature 459, 1146–1149 (2009). https://doi.org/10.1038/nature08075

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature08075

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing