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Calmodulin-dependent protein kinase kinase-beta activates AMPK without forming a stable complex: synergistic effects

Sarah Fogarty1, Simon A Hawley, Kevin A Green

  • 1Division of Molecular Physiology, College of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, U.K.

The Biochemical Journal
|December 5, 2009
PubMed

Insights

Calcium/calmodulin-dependent protein kinase kinase-beta (CaMKKbeta) does not activate AMP-activated protein kinase (AMPK) related kinases, but AMP enhances Ca2+-dependent AMPK activation by inhibiting dephosphorylation. This study clarifies upstream kinase roles in AMPK activation.

Area of Science:

  • Cellular signaling pathways
  • Kinase regulation
  • Metabolic control

Background:

  • AMP-activated protein kinase (AMPK) activation at Thr172 is crucial for cellular energy homeostasis.
  • LKB1 and CaMKKbeta are known upstream kinases catalyzing AMPK phosphorylation.
  • AMPK-related kinases share conserved phosphorylation sites, suggesting potential overlapping regulation.

Purpose of the Study:

  • To investigate the role of CaMKKbeta in phosphorylating and activating AMPK and AMPK-related kinases.
  • To examine the interaction between CaMKKbeta and AMPK subunits.
  • To elucidate the effect of AMP on AMPK activation in LKB1-deficient cells.

Main Methods:

  • Cell-based assays using intact cells.
  • Cell-free kinase assays.
  • Western blotting to detect protein phosphorylation.
  • AMP concentration manipulation in HeLa cells.

Main Results:

  • CaMKKbeta did not phosphorylate or activate most tested AMPK-related kinases, with a minor effect on BRSK1 by CaMKKalpha.
  • No stable complex formation was observed between AMPK subunits and CaMKKbeta.
  • In LKB1-deficient HeLa cells, increased AMP did not affect basal AMPK phosphorylation but enhanced Ca2+-induced activation.

Conclusions:

  • CaMKKbeta is not a general upstream kinase for AMPK-related kinases.
  • AMP's effect on AMPK activation is primarily through inhibiting dephosphorylation, independent of the upstream kinase.
  • AMPK activation mechanisms are complex and involve interplay between upstream kinases and AMP levels.

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