Skip to Main content Skip to Navigation
Journal articles

Dissecting the role of 5'-AMP for allosteric stimulation, activation, and deactivation of AMP-activated protein kinase.

Abstract : AMP-activated protein kinase (AMPK) is a heterotrimeric protein kinase that is crucial for cellular energy homeostasis of eukaryotic cells and organisms. Here we report on the activation of AMPK alpha1beta1gamma1 and alpha2beta2gamma1 by their upstream kinases (Ca(2+)/calmodulin-dependent protein kinase kinase-beta and LKB1-MO25alpha-STRADalpha), the deactivation by protein phosphatase 2Calpha, and on the extent of stimulation of AMPK by its allosteric activator AMP, using purified recombinant enzyme preparations. An accurate high pressure liquid chromatography-based method for AMPK activity measurements was established, which allowed for direct quantitation of the unphosphorylated and phosphorylated artificial peptide substrate, as well as the adenine nucleotides. Our results show a 1000-fold activation of AMPK by the combined effects of upstream kinase and saturating concentrations of AMP. The two AMPK isoforms exhibit similar specific activities (6 mumol/min/mg) and do not differ significantly by their responsiveness to AMP. Due to the inherent instability of ATP and ADP, it proved impossible to assay AMPK activity in the absolute absence of AMP. However, the half-maximal stimulatory effect of AMP is reached below 2 microm. AMP does not appear to augment phosphorylation by upstream kinases in the purified in vitro system, but deactivation by dephosphorylation of AMPK alpha-subunits at Thr-172 by protein phosphatase 2Calpha is attenuated by AMP. Furthermore, it is shown that neither purified NAD(+) nor NADH alters the activity of AMPK in a concentration range of 0-300 microm, respectively. Finally, evidence is provided that ZMP, a compound formed in 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside-treated cells to activate AMPK in vivo, allosterically activates purified AMPK in vitro, but compared with AMP, maximal activity is not reached. These data shed new light on physiologically important aspects of AMPK regulation.
Document type :
Journal articles
Complete list of metadatas

https://www.hal.inserm.fr/inserm-00390888
Contributor : Sarah Hamant <>
Submitted on : Wednesday, June 3, 2009 - 12:05:12 AM
Last modification on : Wednesday, July 15, 2020 - 9:10:04 AM

Links full text

Identifiers

Collections

LBFA | UGA

Citation

Marianne Suter, Uwe Riek, Roland Tuerk, Uwe Schlattner, Theo Wallimann, et al.. Dissecting the role of 5'-AMP for allosteric stimulation, activation, and deactivation of AMP-activated protein kinase.. Journal of Biological Chemistry, American Society for Biochemistry and Molecular Biology, 2006, 281 (43), pp.32207-16. ⟨10.1074/jbc.M606357200⟩. ⟨inserm-00390888⟩

Share

Metrics

Record views

532