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

The AMP-activated protein kinase (AMPK) is a ubiquitous mammalian protein k inase important in the adaptation of cells to metabolic stress. The enzyme is a heterotrimer, consisting of a catalytic a subunit and regulatory beta and gamma subunits, each of which is a member of a larger isoform family. T he enzyme is allosterically regulated by AMP and by phosphorylation of the a subunit. The beta subunit is post-translationally modified by myristoylat ion and multi-site phosphorylation. In the present study, we have examined the impact of post-translational modification of the beta -1 subunit on enz yme activity, heterotrimer assembly and subcellular localization, using sit e-directed mutagenesis and expression of subunits in mammalian cells. Remov al of the myristoylation site (G2A mutant) results in a 4-fold activation o f the enzyme and relocalization of the beta subunit from a particulate extr anuclear distribution to a more homogenous cell distribution. Mutation of t he serine-108 phosphorylation site to alanine is associated with enzyme inh ibition, but no change in cell localization. In contrast, the phosphorylati on site mutations, SS24,25AA and S182A, while having no effects on enzyme a ctivity, are associated with nuclear redistribution of the subunit. Taken t ogether, these results indicate that both myristoylation and phosphorylatio n of the beta subunit of AMPK modulate enzyme activity and subunit cellular localization, increasing the complexity of AMPK regulation.