Calmodulin-dependent protein kinase kinase-beta activates AMPK without forming a stable complex: synergistic effects of Ca2+ and AMP.
Activation of AMPK (AMP-activated protein kinase) by phosphorylation at Thr172 is catalysed by at least two distinct upstream kinases, i.e. the tumour suppressor LKB1, and CaMKKbeta (Ca2+/calmodulin-dependent protein kinase kinase-beta). The sequence around Thr172 is highly conserved between the two catalytic subunit isoforms of AMPK and the 12 AMPK-related kinases, and LKB1 has been shown to act upstream of all of them. In the present paper we report that none of the AMPK-related kinases tested could be phosphorylated or activated in intact cells or cell-free assays by CaMKKbeta, although we did observe a slow phosphorylation and activation of BRSK1 (brain-specific kinase 1) by CaMKKalpha. Despite recent reports, we could not find any evidence that the alpha and/or beta subunits of AMPK formed a stable complex with CaMKKbeta. We also showed that increasing AMP concentrations in HeLa cells (which lack LKB1) had no effect on basal AMPK phosphorylation, but enhanced the ability of agents that increase intracellular Ca2+ to activate AMPK. This is consistent with the effect of AMP on phosphorylation of Thr172 being due to inhibition of dephosphorylation, and confirms that the effect of AMP is independent of the upstream kinase utilized.
Fogarty
Sarah
S
Division of Molecular Physiology, College of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, U.K.
Hawley
Simon A
SA
Green
Kevin A
KA
Saner
Nazan
N
Mustard
Kirsty J
KJ
Hardie
D Grahame
DG
D055372:AMP-Activated Protein Kinases / Q000378:metabolism*; D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism / Q000502:physiology*; D054737:Calcium-Calmodulin-Dependent Protein Kinase Kinase / Q000378:metabolism*; D002460:Cell Line; D000242:Cyclic AMP / Q000494:pharmacology*; D006367:HeLa Cells; D006801:Humans; D047468:Immunoprecipitation; D007476:Ionophores / Q000494:pharmacology; D008969:Molecular Sequence Data; D010629:Phenformin / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding; D017386:Sequence Homology, Amino Acid
D007476:Ionophores; D000001:Calcimycin; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D010629:Phenformin; D000242:Cyclic AMP; D054737:Calcium-Calmodulin-Dependent Protein Kinase Kinase; D055372:AMP-Activated Protein Kinases; D002118:Calcium