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Published on: May 16, 2021
The interaction between AMPKβ2 and the PP1-targeting subunit R6 is dynamically regulated by intracellular glycogen
Yvonne Oligschlaeger1, Marie Miglianico1, Vivian Dahlmans2
1Department of Molecular Genetics, CARIM School for Cardiovascular Diseases, Maastricht University, 6200 MD Maastricht, The Netherlands.
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.
Insights
AMP-activated protein kinase (AMPK) interacts with R6, a glycogen-binding protein. This interaction is regulated by AMPKβ Thr-148 phosphorylation and glycogen levels, suggesting a role in muscle metabolic stress.
Area of Science:
- Biochemistry
- Molecular Biology
- Cellular Metabolism
Background:
- AMP-activated protein kinase (AMPK) is a key regulator of cellular energy homeostasis.
- AMPK activity is modulated by phosphorylation, including autophosphorylation at Thr-148 on the AMPKβ subunit.
- R6 (PPP1R3D) is a glycogen-targeting subunit of protein phosphatase type 1 (PP1) that interacts with AMPKβ1 in MIN6 cells.
Purpose of the Study:
- To investigate the interaction between R6 and AMPKβ isoforms (AMPKβ1 and AMPKβ2).
- To determine the dependency of the R6-AMPKβ interaction on AMPKβ Thr-148 phosphorylation status and intracellular glycogen content.
- To elucidate the role of the R6-AMPKβ interaction in the context of metabolic stress in muscle cells.
Main Methods:
- Yeast two-hybrid (Y2H) assays were used to assess protein-protein interactions.
- Co-immunoprecipitation (IP) experiments were performed in human embryonic kidney (HEK) 293T cells and C2C12 myotubes.
- Site-directed mutagenesis was employed to create non-phosphorylatable AMPKβ2 mutants at Thr-148.
Main Results:
- Both AMPKβ1 and AMPKβ2 wild-type (WT) isoforms bind to R6.
- The interaction between AMPKβ2-WT and R6 was stronger with the muscle-specific isoform and required R6's substrate-binding motif.
- Glycogen depletion significantly enhanced the AMPKβ2-R6 interaction, while mutation of AMPKβ2 Thr-148 abolished this interaction.
- AMPK activation by oligomycin increased both AMPKβ2-R6 interaction and Thr-148 phosphorylation under low-glucose conditions.
Conclusions:
- R6 directly binds to AMPKβ2, and this interaction is regulated by glycogen availability and AMPKβ Thr-148 phosphorylation.
- The data support a model where R6 interacts with AMPKβ2 upon dissociation from glycogen during glycogen depletion.
- This interaction, coupled with increased AMPKβ2 Thr-148 autophosphorylation, suggests a mechanism for PP1-R6-mediated control of AMPK in muscle under metabolic stress.
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