Allosteric Modulation of AMPK Enzymatic Activity: In Vitro Characterization.

AMP-activated protein kinase (AMPK) is a heterotrimeric serine/threonine protein kinase found in nearly all eukaryotes that functions as a master energy sensor in cells. During times of cell stress and changes in the AMP/ATP ratio, AMPK becomes activated and phosphorylates a multitude of protein substrates involved in various cellular processes such as metabolism, cell growth and autophagy. The endogenous ligand AMP is known to bind to the γ-subunit and activates the enzyme via three distinct mechanisms (1) enhancing phosphorylation by upstream kinases of Thr172 in the activation loop (a site critical for AMPK activity), (2) protecting Thr172 from dephosphorylation by phosphatases, and (3) allosteric activation of the kinase activity. Given the important regulatory role for AMPK in various cellular processes and the multiple known modes of activation, there is great interest in identifying small-molecule activators of this kinase and a need for assays to identify and characterize compounds. Here we describe several assay formats that have been used for identifying and characterizing small-molecule AMPK activators.