title: Probing the enzyme kinetics, allosteric modulation and activation of α1- and α2-subunit-containing AMP-activated protein kinase (AMPK) heterotrimeric complexes by pharmacological and physiological activators.
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.
authors.affiliation: Department of World Wide Medicinal Chemistry, Pfizer Global Research and Development, Eastern Point Road, Groton, CT 06340, U.S.A.
authors.forename: Francis
authors.initials: F
authors.lastname: Rajamohan
authors.affiliation: CVMED Research unit, Pfizer Global Research and Development, 620 Memorial Drive, Cambridge, MA 02139, U.S.A.
authors.forename: Allan R
authors.initials: AR
authors.lastname: Reyes
authors.affiliation: The Department of Pharmacokinetics Dynamics and Metabolism, Pfizer Global Research and Development, Eastern Point Road, Groton, CT 06340, U.S.A.
authors.forename: Richard K
authors.initials: RK
authors.lastname: Frisbie
authors.affiliation: Department of World Wide Medicinal Chemistry, Pfizer Global Research and Development, Eastern Point Road, Groton, CT 06340, U.S.A.
authors.forename: Lise R
authors.initials: LR
authors.lastname: Hoth
authors.affiliation: Department of World Wide Medicinal Chemistry, Pfizer Global Research and Development, Eastern Point Road, Groton, CT 06340, U.S.A.
authors.forename: Parag
authors.initials: P
authors.lastname: Sahasrabudhe
authors.affiliation: Department of World Wide Medicinal Chemistry, Pfizer Global Research and Development, Eastern Point Road, Groton, CT 06340, U.S.A.
authors.forename: Rachelle
authors.initials: R
authors.lastname: Magyar
authors.affiliation: CVMED Research unit, Pfizer Global Research and Development, 620 Memorial Drive, Cambridge, MA 02139, U.S.A.
authors.forename: James A
authors.initials: JA
authors.lastname: Landro
authors.affiliation: Department of World Wide Medicinal Chemistry, Pfizer Global Research and Development, Eastern Point Road, Groton, CT 06340, U.S.A.
authors.forename: Jane M
authors.initials: JM
authors.lastname: Withka
authors.affiliation: Department of World Wide Medicinal Chemistry, Pfizer Global Research and Development, Eastern Point Road, Groton, CT 06340, U.S.A.
authors.forename: Nicole L
authors.initials: NL
authors.lastname: Caspers
authors.affiliation: Department of World Wide Medicinal Chemistry, Pfizer Global Research and Development, Eastern Point Road, Groton, CT 06340, U.S.A.
authors.forename: Matthew F
authors.initials: MF
authors.lastname: Calabrese
authors.affiliation: CVMED Research unit, Pfizer Global Research and Development, 620 Memorial Drive, Cambridge, MA 02139, U.S.A.
authors.forename: Jessica
authors.initials: J
authors.lastname: Ward
authors.affiliation: Department of World Wide Medicinal Chemistry, Pfizer Global Research and Development, Eastern Point Road, Groton, CT 06340, U.S.A. ravi.g.kurumbail@pfizer.com.
authors.forename: Ravi G
authors.initials: RG
authors.lastname: Kurumbail
mesh_terms: D055372:AMP-Activated Protein Kinases / Q000737:chemistry*; D000249:Adenosine Monophosphate / Q000737:chemistry; D000494:Allosteric Regulation; D001713:Biphenyl Compounds; D004789:Enzyme Activation; D020536:Enzyme Activators / Q000737:chemistry; D057075:Enzyme Assays; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry; D007700:Kinetics; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry; D011753:Pyrones / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D013876:Thiophenes / Q000737:chemistry
chemical_list: D001713:Biphenyl Compounds; D020536:Enzyme Activators; D007527:Isoenzymes; D021122:Protein Subunits; D011753:Pyrones; D011994:Recombinant Proteins; D013876:Thiophenes; D000249:Adenosine Monophosphate; C526183:PRKAB2 protein, human; D055372:AMP-Activated Protein Kinases; C526180:PRKAA1 protein, human; C512408:4-hydroxy-3-(4-(2-hydroxyphenyl)phenyl)-6-oxo-7H-thieno(2,3-b)pyridine-5-carbonitrile
keywords: AMP-activated protein kinase (AMPK); allosteric activation; heterotrimeric complex; kinase activity; metabolic sensor; recombinant protein