Abstract
The heterotrimeric AMP-activated protein kinase (AMPK), consisting of α, β and γ subunits, is a stress-sensing enzyme that is activated by phosphorylation of its activation loop in response to increases in cellular AMP. N-terminal myristoylation of the β-subunit has been shown to suppress Thr172 phosphorylation, keeping AMPK in an inactive state. Here we use amide hydrogen-deuterium exchange mass spectrometry (HDX-MS) to investigate the structural and dynamic properties of the mammalian myristoylated and non-myristoylated inactivated AMPK (D139A) in the presence and absence of nucleotides. HDX MS data suggests that the myristoyl group binds near the first helix of the C-terminal lobe of the kinase domain similar to other kinases. Our data, however, also shows that ATP.Mg2+ results in a global stabilization of myristoylated, but not non-myristoylated AMPK, and most notably for peptides of the activation loop of the α-kinase domain, the autoinhibitory sequence (AIS) and the βCBM. AMP does not have that effect and HDX measurements for myristoylated and non-myristoylated AMPK in the presence of AMP are similar. These differences in dynamics may account for a reduced basal rate of phosphorylation of Thr172 in myristoylated AMPK in skeletal muscle where endogenous ATP concentrations are very high.
Highlights
Liver kinase β1 (LKB1) and Ca2+/Calmodulin-dependent protein kinase β(CaMKKβ) kinases are two of the upstream kinases (AMPKKs) that catalyze the phosphorylation of AMPK at Thr[172] of the α-subunit kinase domain, leading to an increase in AMPK activity by several hundred-fold[24,25,26,27,28]
Under conditions of starvation the myristoylated β-subunit promotes AMPK association, phosphorylation and activation at cellular membranes, indicating a myristoyl-switch mechanism[15]. This mechanism is proposed to be accompanied by removal of the myristoyl group from the putative binding pocket to interact with the cell membrane[14,15,33,38]
In this study the HDX profiles of myristoylated and non-myristoylated AMPK characterized a peptide, 122 to 131, that suggests the binding site of the myristoyl group is near the C-terminal end of the first α-helix of the C-terminal lobe of the kinase domain (Fig. 2D)
Summary
Liver kinase β1 (LKB1) and Ca2+/Calmodulin-dependent protein kinase β(CaMKKβ) kinases are two of the upstream kinases (AMPKKs) that catalyze the phosphorylation of AMPK at Thr[172] of the α-subunit kinase domain, leading to an increase in AMPK activity by several hundred-fold[24,25,26,27,28]. The N-terminal myristoylation of the β-subunit has a binary function in AMPK regulation. It localizes and suppresses activity, as the removal of the myristoyl group by mutation of Gly[2] to Ala showed a wide distribution of AMPK into the cytoplasm and a four-fold increase in AMPK activity[14,15]. The mechanism of how myristoylation mediates AMPK inhibition is not fully understood It mediates Thr[172] phosphorylation in response to the metabolic stress signal from AMP, resulting in maximal AMPK activation. How the myristoyl group modulates activity of AMPK directly and facilitates allosteric interactions with AMP is unclear and represent a major challenge in understanding overall regulation of AMPK function. Myristoylation-dependent differences in the peptide residues known to have significant roles in interactions with nucleotides were investigated
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