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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, α1β1γ 1, α1β2γ 1, α1β2γ 3, α2β1γ 1, α2β2γ1 and α2β2γ 3 using known activators, A769662 and AMP. The α1-containing complexes exhibited higher specific activities and lowerKm 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 (pThr172/174). 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.. [ABSTRACT FROM AUTHOR]

Pseudokinases are classified by the lack of one or several of the highly conserved motifs involved in nucleotide (nt) binding or catalytic activity of protein kinases (PKs). Pseudokinases represent ∼10% of the human kinome and they are found in all evolutionary classes of kinases. It has become evident that pseudokinases, which were initially considered somewhat peculiar dead kinases, are important components in several signalling cascades. Furthermore, several pseudokinases have been linked to human diseases, particularly cancer, which is raising interest for therapeutic approaches towards these proteins. The ATP-binding pocket is a well-established drug target and elucidation of the mechanism and properties of nt binding in pseudokinases is of significant interest and importance. Recent studies have demonstrated that members of the pseudokinase family are very diverse in structure as well as in their ability and mechanism to bind nts or perform phosphoryl transfer reactions. This diversity also precludes prediction of pseudokinase function, or the importance of nt binding for said function, based on primary sequence alone. Currently available data indicate that ∼40% of pseudokinases are able to bind nts, whereas only few are able to catalyse occasional phosphoryl transfer. Pseudokinases employ diverse mechanisms to bind nts, which usually occurs at low, but physiological, affinity. ATP binding serves often a structural role but in most cases the functional roles are not precisely known. In the present review, we discuss the various mechanisms that pseudokinases employ for nt binding and how this often low-affinity binding can be accurately analysed. [ABSTRACT FROM AUTHOR]