title: The interaction between AMPKβ2 and the PP1-targeting subunit R6 is dynamically regulated by intracellular glycogen content.
abstract: AMP-activated protein kinase (AMPK) is a metabolic stress-sensing kinase. We previously showed that glucose deprivation induces autophosphorylation of AMPKβ at Thr-148, which prevents the binding of AMPK to glycogen. Furthermore, in MIN6 cells, AMPKβ1 binds to R6 (PPP1R3D), a glycogen-targeting subunit of protein phosphatase type 1 (PP1), thereby regulating the glucose-induced inactivation of AMPK. In the present study, we further investigated the interaction of R6 with AMPKβ and the possible dependency on Thr-148 phosphorylation status. Yeast two-hybrid (Y2H) analyses and co-immunoprecipitation (IP) of the overexpressed proteins in human embryonic kidney (HEK) 293T) cells revealed that both AMPKβ1 and AMPK-β2 wild-type (WT) isoforms bind to R6. The AMPKβ-R6 interaction was stronger with the muscle-specific AMPKβ2-WT and required association with the substrate-binding motif of R6. When HEK293T cells or C2C12 myotubes were cultured in high-glucose medium, AMPKβ2-WT and R6 weakly interacted. In contrast, glycogen depletion significantly enhanced this protein interaction. Mutation of AMPKβ2 Thr-148 prevented the interaction with R6 irrespective of the intracellular glycogen content. Treatment with the AMPK activator oligomycin enhanced the AMPKβ2-R6 interaction in conjunction with increased Thr-148 phosphorylation in cells grown in low-glucose medium. These data are in accordance with R6 binding directly to AMPKβ2 when both proteins detach from the diminishing glycogen particle, which is simultaneous with increased AMPKβ2 Thr-148 autophosphorylation. Such a model points to a possible control of AMPK by PP1-R6 upon glycogen depletion in muscle.
authors.affiliation: Department of Molecular Genetics, CARIM School for Cardiovascular Diseases, Maastricht University, 6200 MD Maastricht, The Netherlands.
authors.forename: Yvonne
authors.initials: Y
authors.lastname: Oligschlaeger
authors.affiliation: Department of Molecular Genetics, CARIM School for Cardiovascular Diseases, Maastricht University, 6200 MD Maastricht, The Netherlands.
authors.forename: Marie
authors.initials: M
authors.lastname: Miglianico
authors.affiliation: Department of Molecular Genetics, GROW School for Oncology & Developmental Biology, Maastricht University, 6200 MD Maastricht, The Netherlands.
authors.forename: Vivian
authors.initials: V
authors.lastname: Dahlmans
authors.affiliation: Instituto de Biomedicina de Valencia, Consejo Superior de Investigaciones Cientificas (CSIC) and Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), 46010 Valencia, Spain.
authors.forename: Carla
authors.initials: C
authors.lastname: Rubio-Villena
authors.affiliation: Department of Molecular Genetics, CARIM School for Cardiovascular Diseases, Maastricht University, 6200 MD Maastricht, The Netherlands.
authors.forename: Dipanjan
authors.initials: D
authors.lastname: Chanda
authors.affiliation: Instituto de Biomedicina de Valencia, Consejo Superior de Investigaciones Cientificas (CSIC) and Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), 46010 Valencia, Spain.
authors.forename: Maria Adelaida
authors.initials: MA
authors.lastname: Garcia-Gimeno
authors.affiliation: Department of Molecular Genetics, CARIM School for Cardiovascular Diseases, Maastricht University, 6200 MD Maastricht, The Netherlands.
authors.forename: Will A
authors.initials: WA
authors.lastname: Coumans
authors.affiliation: Department of Molecular Genetics, CARIM School for Cardiovascular Diseases, Maastricht University, 6200 MD Maastricht, The Netherlands.
authors.forename: Yilin
authors.initials: Y
authors.lastname: Liu
authors.affiliation: Department of Molecular Genetics, GROW School for Oncology & Developmental Biology, Maastricht University, 6200 MD Maastricht, The Netherlands.
authors.forename: J Willem
authors.initials: JW
authors.lastname: Voncken
authors.affiliation: Department of Molecular Genetics, CARIM School for Cardiovascular Diseases, Maastricht University, 6200 MD Maastricht, The Netherlands.
authors.forename: Joost J F P
authors.initials: JJ
authors.lastname: Luiken
authors.affiliation: Department of Molecular Genetics, CARIM School for Cardiovascular Diseases, Maastricht University, 6200 MD Maastricht, The Netherlands.
authors.forename: Jan F C
authors.initials: JF
authors.lastname: Glatz
authors.affiliation: Instituto de Biomedicina de Valencia, Consejo Superior de Investigaciones Cientificas (CSIC) and Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), 46010 Valencia, Spain.
authors.forename: Pascual
authors.initials: P
authors.lastname: Sanz
authors.affiliation: Department of Molecular Genetics, CARIM School for Cardiovascular Diseases, Maastricht University, 6200 MD Maastricht, The Netherlands d.neumann@maastrichtuniversity.nl.
authors.forename: Dietbert
authors.initials: D
authors.lastname: Neumann
mesh_terms: D055372:AMP-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism*; D019943:Amino Acid Substitution; D006003:Glycogen / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D020125:Mutation, Missense; D010766:Phosphorylation; D011485:Protein Binding; D054645:Protein Phosphatase 1 / Q000235:genetics / Q000378:metabolism*
chemical_list: D006003:Glycogen; C569522:Prkab2 protein, mouse; D055372:AMP-Activated Protein Kinases; C585576:PPP1R3D protein, human; D054645:Protein Phosphatase 1
keywords: AMP-activated protein kinase (AMPK); R6; glycogen metabolism; glycogen targeting; phosphorylation; protein–protein interaction