Post-translational modifications of the β-1 subunit of AMP-activated protein kinase affect enzyme activity and cellular localization

Abstract

The AMP-activated protein kinase (AMPK) is a ubiquitous mammalian protein kinase important in the adaptation of cells to metabolic stress. The enzyme is a heterotrimer, consisting of a catalytic α subunit and regulatory β and γ subunits, each of which is a member of a larger isoform family. The enzyme is allosterically regulated by AMP and by phosphorylation of the α subunit. The β subunit is post-translationally modified by myristoylation and multi-site phosphorylation. In the present study, we have examined the impact of post-translational modification of the β-1 subunit on enzyme activity, heterotrimer assembly and subcellular localization, using site-directed mutagenesis and expression of subunits in mammalian cells. Removal of the myristoylation site (G2A mutant) results in a 4-fold activation of the enzyme and relocalization of the β subunit from

Defining the Mechanism of Activation of AMP-activated Protein Kinase by the Small Molecule A-769662, a Member of the Thienopyridone Family

“…5). The basal activity of AMPK was reduced by ϳ45% in the ␤1 S108A mutant complex, similar to that reported in a previous study (39). H 2 O 2 activated the S108A mutant complex by a similar degree as the wild type, although as with the basal condition, the specific activity of the S108A mutant was ϳ45% that of the wild-type complex (Fig.…”

“…In addition, we found that mutation of Ser-108 to alanine within the ␤1 subunit completely abolished activation by A-769662, while only partially reducing AMP activation, providing further evidence that the mechanisms are different. Previously, Ser-108 was shown to undergo autophosphorylation (38,40), and it was reported that mutation of Ser-108 to alanine reduced AMPK activity by ϳ60% relative to the wild type, following expression in COS cells (39). In the same study it was reported that the reduction in activity was not correlated with reduced Thr-172 phosphorylation but might be explained by changes in subunit interaction in the AMPK heterotrimer (39).…”

“…Previously, Ser-108 was shown to undergo autophosphorylation (38,40), and it was reported that mutation of Ser-108 to alanine reduced AMPK activity by ϳ60% relative to the wild type, following expression in COS cells (39). In the same study it was reported that the reduction in activity was not correlated with reduced Thr-172 phosphorylation but might be explained by changes in subunit interaction in the AMPK heterotrimer (39). In this study, we also observed a marked reduction in the activity of AMPK harboring the S108A mutation compared with wild type, in both basal and H 2 O 2 -treated cells.…”

5′-AMP-Activated protein kinase in avian biology

“…Beta subunits contain an N-terminal myristoylation modification site (G 2 ) which acts to localize AMPK to cellular membranes, a C-terminal alphaygamma subunitbinding domain (ayg ID) and a central glycogen (carbohydrate) binding domain (GBD) (Figure 1) (Warden et al, 2001;Hudson et al, 2003;Polekhina et al, 2003;Iseli et al, 2005). Recently, the beta subunit glycogen-binding domain was reclassified as a member of the carbohydrate-binding module-containing family 48 based on the similarity of its amino acid sequence to other carbohydrate-binding proteins Parker et al, 2007).…”

p53 and metabolism: Old player in a new game

“…25 Apart from this, p53 can also induce AMPK β1, which is the regulatory subunit of AMPK and increases AMPK activity. 24,26 p53 also transactivates PTEN, which dephosphorylates phosphatidylinositol-3,4,5-trisphosphate (PIP3) resulting in downregulation of Akt pathway. 27 Another p53 target, IGF-BP3, inhibits IGF-1 binding to its receptor.…”