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

Abstract

We have developed a sensitive assay for the AMPactivated protein kinase kinase, the upstream component in the AMP-activated protein kinase cascade. Phosphorylation and activation of the downstream kinase by the upstream kinase absolutely requires AMP and is antagonized by high (millimolar) concentrations of ATP. We have purified the upstream kinase >1000-fold from rat liver; a variety of evidence indicates that the catalytic subunit may be a polypeptide of 58 kDa. The physical properties of the downstream and upstream kinases, e.g. catalytic subunit masses (63 versus 58 kDa) and native molecular masses (190 versus 195 kDa), are very similar. However, unlike the downstream kinase, the upstream kinase is not inactivated by protein phosphatases. The upstream kinase phosphorylates the downstream kinase at a single major site on the ␣ subunit, i.e. threonine 172, which lies in the "activation segment" between the DFG and APE motifs. This site aligns with activating phosphorylation sites on many other protein kinases, including Thr 177 on calmodulindependent protein kinase I. As well as suggesting a mechanism of activation of AMP-activated protein kinase, this finding is consistent with our recent report that the AMP-activated protein kinase kinase can slowly phosphorylate and activate calmodulin-dependent protein kinase I, at least in vitro (Hawley, S. A., Selbert, M.

Gene Expression Pattern in Human Brain Endothelial Cells in Response to Neisseria meningitidis

“…AMPK has been described to be activated during cellular and environmental stress and regulates energy and metabolic homeostasis of the cell (22,37). AMPK-␣ phosphorylation was detected after 24 h of infection in response to the MC58 and MC58 siaD strains (Fig.…”

LKB1, a novel serine/threonine protein kinase and potential tumour suppressor, is phosphorylated by cAMP-dependent protein kinase (PKA) and prenylated in vivo

“…This region has been shown to be important in substrate binding and contains the phosphorylated residues of the activation loop. AMPK kinase phosphorylates a threonine residue (Thr"(#) in this portion of the activation loop of AMPK, and causes a 20-fold activation [38]. Subdomain VIII of LKB1 exhibits amino acid sequence similarity with subdomain VIII of the AMPKs.…”

“Preconditioning” with latrepirdine, an adenosine 5′-monophosphate-activated protein kinase activator, delays amyotrophic lateral sclerosis progression in SOD1G93A mice

“…AMPK is a heterotrimeric protein composed of one catalytic subunit (α) and two regulatory subunits (β and γ), and is expressed in most tissues including the CNS (Culmsee et al 2001). AMPK is activated by phosphorylation of the α subunit at the threonine 172 site when there is a high AMP:ATP ratio (Hawley et al 1996). AMPK has also been shown to be activated in mutant SOD1-expressing primary mixed spinal cord cultures and in spinal cords of SOD1 G93A mice at post natal day (PND) 90 (Lim et al 2012, Perera et al 2014.…”