Li, JingwenMadsen, Agnete B.Knudsen, Jonas R.Henríquez-Olguín, CarlosPersson, Kaspar W.Li, ZhenchengRaun, Steffen H.Li, TianjiaoKiens, BenteWojtaszewski, Jørgen F. P.Richter, Erik A.Nogara, LeonardoBlaauw, BertOgasawara, RikiJensen, Thomas E.2025-12-052025-12-052025-01-31FASEB Journal, Vol. 39, N° 2 (2025) p. 1-130892-6638https://hdl.handle.net/20.500.12254/7392The kinases AMPK, and mTOR as part of either mTORC1 or mTORC2, are major orchestrators of cellular growth and metabolism. Phosphorylation of mTOR Ser1261 is reportedly stimulated by both insulin and AMPK activation and a regulator of both mTORC1 and mTORC2 activity. Intrigued by the possibilities that Ser1261 might be a convergence point between insulin and AMPK signaling in skeletal muscle, we investigated the regulation and function of this site using a combination of human exercise, transgenic mouse, and cell culture models. Ser1261 phosphorylation on mTOR did not respond to insulin in any of our tested models, but instead responded acutely to contractile activity in human and mouse muscle in an AMPK activity-dependent manner. Contraction-stimulated mTOR Ser1261 phosphorylation in mice was decreased by Raptor muscle knockout (mKO) and increased by Raptor muscle overexpression, yet was not affected by Rictor mKO, suggesting most of Ser1261 phosphorylation occurs within mTORC1 in skeletal muscle. In accordance, HEK293 cells mTOR Ser1261Ala mutation strongly impaired phosphorylation of mTORC1 substrates but not mTORC2 substrates. However, neither mTORC1 nor mTORC2-dependentenAtribución-NoComercial-CompartirIgual 3.0 Chile (CC BY-NC-SA 3.0 CL)mTORAMPKPhosphorylationArticlehttps://orcid.org/0000-0002-9315-9365https://doi.org/10.1096/fj.202402064R