The inhibitory SAPS3-AMPK interaction detected in HEK293 cells is not detectable in muscle or liver from humans or mice.

It has been proposed that the regulatory Sit4-associated protein subunit 3 (SAPS3) of protein phosphatase 6 (PP6C) acts as an AMP-activated protein kinase (AMPK) inhibitor by recruiting PP6C to dephosphorylate AMPKα-T172. While we confirm this interaction in HEK293 cells, we find limited evidence for a SAPS3-AMPK interaction in metabolically perturbed liver and skeletal muscle from humans and mice. Across fasting, high-fat diet feeding and exercise conditions, co-immunoprecipitation assays failed to detect endogenous SAPS3-AMPK and PP6C-AMPK interactions. These findings challenge the physiological relevance of SAPS3/PP6C as regulators of AMPK in mature tissues and highlight the need for further investigation into the regulation of AMPK by protein phosphatases in vivo.