title: β-subunit myristoylation functions as an energy sensor by modulating the dynamics of AMP-activated Protein Kinase.
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.
authors.affiliation: Department of Biochemistry &Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, 30 Flemington Road, The University of Melbourne, Parkville, Victoria 3010, Australia.
authors.forename: Nada
authors.initials: N
authors.lastname: Ali
authors.affiliation: Metabolic Signalling Laboratory, St. Vincent's Institute of Medical Research, University of Melbourne, 41 Victoria Parade, Fitzroy, Victoria 3065, Australia.
authors.forename: Naomi
authors.initials: N
authors.lastname: Ling
authors.affiliation: Department of Biological Sciences, National University of Singapore, 14 Science Dr 4, 117543, Singapore.
authors.forename: Srinath
authors.initials: S
authors.lastname: Krishnamurthy
authors.affiliation: Metabolic Signalling Laboratory, St. Vincent's Institute of Medical Research, University of Melbourne, 41 Victoria Parade, Fitzroy, Victoria 3065, Australia.
authors.forename: Jonathan S
authors.initials: JS
authors.lastname: Oakhill
authors.affiliation: Department of Protein Chemistry and Metabolism, St. Vincent's Institute of Medical Research, University of Melbourne, 41 Victoria Parade, Fitzroy, Victoria 3065, Australia.
authors.forename: John W
authors.initials: JW
authors.lastname: Scott
authors.affiliation: Department of Physiology, The University of Melbourne, Cnr Grattan Street and Royal Parade, Parkville, Victoria 3010, Australia.
authors.forename: David I
authors.initials: DI
authors.lastname: Stapleton
authors.affiliation: Department of Protein Chemistry and Metabolism, St. Vincent's Institute of Medical Research, University of Melbourne, 41 Victoria Parade, Fitzroy, Victoria 3065, Australia.|Mary MacKillop Institute for Health Research, Australian Catholic University, Victoria Parade, Fitzroy, Victoria 3065, Australia.
authors.forename: Bruce E
authors.initials: BE
authors.lastname: Kemp
authors.affiliation: Department of Biological Sciences, National University of Singapore, 14 Science Dr 4, 117543, Singapore.
authors.forename: Ganesh Srinivasan
authors.initials: GS
authors.lastname: Anand
authors.affiliation: Department of Biochemistry &Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, 30 Flemington Road, The University of Melbourne, Parkville, Victoria 3010, Australia.
authors.forename: Paul R
authors.initials: PR
authors.lastname: Gooley
mesh_terms: D055372:AMP-Activated Protein Kinases / Q000378:metabolism*; D003903:Deuterium / Q000378:metabolism; D006801:Humans; D006859:Hydrogen / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods; D010766:Phosphorylation / Q000502:physiology; D000072417:Protein Domains; D021122:Protein Subunits / Q000378:metabolism*
chemical_list: D021122:Protein Subunits; D006859:Hydrogen; D003903:Deuterium; D055372:AMP-Activated Protein Kinases