The Recruitment of AMP-activated Protein Kinase to Glycogen Is Regulated by Autophosphorylation

Oligschlaeger, Yvonne; Miglianico, Marie; Chanda, Dipanjan; Scholz, Roland; Thali, Ramon F.; Tuerk, Roland; Stapleton, David; Gooley, Paul R.; Neumann, Dietbert

doi:10.1074/jbc.m114.633271

Cited by 38 publications

(40 citation statements)

References 60 publications

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“…More recently, we demonstrated that autophosphorylation at the β-threonine-148 (Thr-148) residue, centrally located in the CBM, prevents AMPK from binding to carbohydrates such as glycogen (22). As we show here that R6 also interacts with AMPKβ2, we next investigated whether Thr-148 is required for AMPKβ2/R6 interaction.…”

Section: Ampkβ2 Thr-148 Mutant Shows Reduced Interaction With R6mentioning

confidence: 99%

“…Intracellular glycogen content was measured, as previously described (22). Briefly, HEK293T cells (nontransfected or transfected) or stably-infected C2C12 myotubes were lysed in potassium hydroxide (30 %) and boiled at 70 °C for 30 min.…”

Section: Biochemical Intracellular Glycogen Measurementmentioning

confidence: 99%

“…Briefly, retroviral systems and Phoenix helper-free retrovirus producer cell lines were used as published before (26)(27)(28). Amphotropic retroviral supernatants were produced as previously described (22). Briefly, 24 -48 h after calcium phosphate/DNA transfection of producer cells, supernatants were harvested, filtered (0.45 micron filters; Corning, Germany) and used for infection of C2C12 cells in presence of 4 µg/ml polybrene (Sigma).…”

Section: Retroviral Infectionsmentioning

confidence: 99%

“…Immunoprecipitation procedures were performed as previously described (22). Briefly, exogenous myc-AMPKα1 was immunoprecipitated using anti-myc-tag antibody (9B11, Cell Signaling Technology, Beverly, MA), exogenous R6 was immunoprecipitated using anti-FLAG-tag antibody (F3165, Sigma), and endogenous AMPK was immunoprecipitated using a combination of AMPKα1 and AMPKα2 antibodies raised in sheep (kindly provided by Prof. dr. G. Hardie), followed by incubation with protein G-Sepharose beads (GE Healthcare).…”

Section: Immunoprecipitation and Western Blottingmentioning

confidence: 99%

“…Briefly, exogenous myc-AMPKα1 was immunoprecipitated using anti-myc-tag antibody (9B11, Cell Signaling Technology, Beverly, MA), exogenous R6 was immunoprecipitated using anti-FLAG-tag antibody (F3165, Sigma), and endogenous AMPK was immunoprecipitated using a combination of AMPKα1 and AMPKα2 antibodies raised in sheep (kindly provided by Prof. dr. G. Hardie), followed by incubation with protein G-Sepharose beads (GE Healthcare). Western blot analysis was carried out using the following primary antibodies: myc-tag, tAMPKα, AMPKβ1, AMPKβ2, phospho-AMPK-T172 (all from Cell Signaling Technology, Beverly, MA), FLAG-tag (F3165, Sigma), R6 (AP13440a, Abgent), and phospho-AMPKβ2-T148 (22). R6 was detected using FLAG-tag (F3165, Sigma), unless otherwise stated.…”

Section: Immunoprecipitation and Western Blottingmentioning

confidence: 99%

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The interaction between AMPKβ2 and the PP1-targeting subunit R6 is dynamically regulated by intracellular glycogen content

Oligschlaeger

Miglianico

Dahlmans

et al. 2016

Biochemical Journal

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AMP-activated protein kinase (AMPK) is a metabolic stress-sensing kinase. We previously showed that glucose deprivation induces autophosphorylation of AMPKβ at threonine-148 (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 1 (PP1), thereby regulating the glucose-induced inactivation of AMPK. Here, we further investigated the interaction of R6 with AMPKβ and the possible dependency on Thr-148 phosphorylation status. Yeast two-hybrid analyses and co-immunoprecipitation of the overexpressed proteins in HEK293T cells revealed that both AMPKβ1 and β2 wild-type (WT) isoforms bind to R6. The AMPKβ/R6 interaction was stronger with the muscle-specific β2-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 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 to increased AMPKβ2 Thr-148 autophosphorylation. Such model points to a possible control of AMPK by PP1-R6 upon glycogen depletion in muscle. SUMMARY STATEMENTBreakdown of intracellular glycogen enhances interaction of the AMPKβ2 subunit and the R6 glycogentargeting subunit of the protein phosphatase-1 (PP1), which occurs in conjunction with increased β2-threonine-148 phosphorylation. SHORT TITLEGlycogen-dependent regulation of AMPKβ2/R6 interaction

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Section: Ampkβ2 Thr-148 Mutant Shows Reduced Interaction With R6mentioning

confidence: 99%

Section: Biochemical Intracellular Glycogen Measurementmentioning

confidence: 99%

Section: Retroviral Infectionsmentioning

confidence: 99%

Section: Immunoprecipitation and Western Blottingmentioning

confidence: 99%

Section: Immunoprecipitation and Western Blottingmentioning

confidence: 99%

See 3 more Smart Citations

The interaction between AMPKβ2 and the PP1-targeting subunit R6 is dynamically regulated by intracellular glycogen content

Oligschlaeger

Miglianico

Dahlmans

et al. 2016

Biochemical Journal

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Unravelling the Carbohydrate‐Binding Preferences of the Carbohydrate‐Binding Modules of AMP‐Activated Protein Kinase

et al. 2018

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The β subunit of adenosine monophosphate (AMP)-activated protein kinase (AMPK), which exists as two isoforms (β1 and β2) in humans, has a carbohydrate-binding module (CBM) that interacts with glycogen. Although the β1- and β2-CBMs are structurally similar, with strictly conserved ligand-contact residues, they show different carbohydrate affinities. β2-CBM shows the strongest affinity for both branched and unbranched oligosaccharides and it has recently been shown that a Thr insertion into β2-CBM (Thr101) forms a pocket to accommodate branches. This insertion does not explain why β2-CBM binds all carbohydrates with stronger affinity. Herein, it is shown that residue 134 (Val for β2 and Thr for β1), which does not come into contact with a carbohydrate, appears to account for the affinity difference. Characterisation by NMR spectroscopy, however, suggests that mutant β2-Thr101Δ/Val134Thr differs from that of β1-CBM, and mutant β1-Thr101ins/Thr134Val differs from that of β2-CBM. Furthermore, these mutants are less stable to chemical denaturation, relative to that of wild-type β-CBMs, which confounds the affinity analyses. To support the importance of Thr101 and Val134, the ancestral CBM has been constructed. This CBM retains Thr101 and Val134, which suggests that the extant β1-CBM has a modest loss of function in carbohydrate binding. Because the ancestor bound carbohydrate with equal affinity to that of β2-CBM, it is concluded that residue 134 plays an indirect role in carbohydrate binding.

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Relationship between genetic variation at PPP1R3B and levels of liver glycogen and triglyceride

et al. 2018

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The Recruitment of AMP-activated Protein Kinase to Glycogen Is Regulated by Autophosphorylation

Cited by 38 publications

References 60 publications

The interaction between AMPKβ2 and the PP1-targeting subunit R6 is dynamically regulated by intracellular glycogen content

The interaction between AMPKβ2 and the PP1-targeting subunit R6 is dynamically regulated by intracellular glycogen content

Unravelling the Carbohydrate‐Binding Preferences of the Carbohydrate‐Binding Modules of AMP‐Activated Protein Kinase

Relationship between genetic variation at PPP1R3B and levels of liver glycogen and triglyceride

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