Abstract
AMP-activated protein kinase (AMPK) is a serine/threonine protein kinase that serves as a pleotropic regulator of whole body energy homoeostasis. AMPK exists as a heterotrimeric complex, composed of a catalytic subunit (α) and two regulatory subunits (β and γ), each present as multiple isoforms. In the present study, we compared the enzyme kinetics and allosteric modulation of six recombinant AMPK isoforms, α1β1γ1, α1β2γ1, α1β2γ3, α2β1γ1, α2β2γ1 and α2β2γ3 using known activators, A769662 and AMP. The α1-containing complexes exhibited higher specific activities and lower Km values for a widely used peptide substrate (SAMS) compared with α2-complexes. Surface plasmon resonance (SPR)-based direct binding measurements revealed biphasic binding modes with two distinct equilibrium binding constants for AMP, ADP and ATP across all isoforms tested. The α2-complexes were ∼25-fold more sensitive than α1-complexes to dephosphorylation of a critical threonine on their activation loop (pThr(172/174)). However, α2-complexes were more readily activated by AMP than α1-complexes. Compared with β1-containing heterotrimers, β2-containing AMPK isoforms are less sensitive to activation by A769662, a synthetic activator. These data demonstrate that ligand induced activation of AMPK isoforms may vary significantly based on their AMPK subunit composition. Our studies provide insights for the design of isoform-selective AMPK activators for the treatment of metabolic diseases.
Highlights
AMP-activated protein kinase (AMPK) serves as a metabolic fuel gauge, sensing the energy status of the cell and whole organism [1]
Previous structural studies with truncated mammalian AMPK regulatory core revealed the presence of four potential nt-binding sites on the γ -subunit, three of which were occupied by AMP whereas site 2 was empty [20,21]
Activation of the AMPK complexes was achieved by treating with calmodulin-dependent protein kinase β (CaMKKβ) and the extent of α-Thr172/174 phosphorylation was estimated by MS analysis
Summary
AMP-activated protein kinase (AMPK) serves as a metabolic fuel gauge, sensing the energy status of the cell and whole organism [1]. Phosphorylation of a critical threonine (Thr172/174) on its activation loop (Thr174 for α1 and Thr172 for α2) within the kinase domain of the α-subunit either by calcium/calmodulindependent protein kinase β (CaMKKβ) or by liver kinase B (LKB1), leads to a significant increase (>500-fold higher) in the kinase activity of AMPK [12,13,14]. Recent work has shown that β-myristoylation plays a gatekeeper role in signal initiation by allowing upstream kinases to fully phosphorylate and activate AMPK in response to increases in cellular AMP concentration which is absent from recombinant AMPK expressed in Escherichia coli [19]. Previous structural studies with truncated mammalian AMPK regulatory core revealed the presence of four potential nt-binding sites on the γ -subunit, three of which were occupied by AMP (sites 1, 3 and 4) whereas site 2 was empty [20,21].
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