AMP-activated protein kinase (AMPK) is a metabolic stress-sensing kinase. We previously showed that glucose deprivation induces autophosphorylation of AMPK beta at Thr-148, which prevents the binding of AMPK to glycogen. Furthermore, in MIN6 cells, AMPK beta 1 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 AMPK beta 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 AMPK beta 1 and AMPK-beta 2 wild-type (WT) isoforms bind to R6. The AMPK beta-R6 interaction was stronger with the muscle-specific AMPK beta 2-WT and required association with the substrate-binding motif of R6. When HEK293T cells or C2C12 myotubes were cultured in high-glucose medium, AMPK beta 2-WT and R6 weakly interacted. In contrast, glycogen depletion significantly enhanced this protein interaction. Mutation of AMPK beta 2 Thr-148 prevented the interaction with R6 irrespective of the intracellular glycogen content. Treatment with the AMPK activator oligomycin enhanced the AMPK beta 2-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 AMPK beta 2 when both proteins detach from the diminishing glycogen particle, which is simultaneous with increased AMPK beta 2 Thr-148 autophosphorylation. Such a model points to a possible control of AMPK by PP1-R6 upon glycogen depletion in muscle.
- AMP-activated protein kinase (AMPK)
- glycogen metabolism
- glycogen targeting
- protein-protein interaction