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Updated: Jun 1, 2025
Oligopeptide Competition Assay for Phosphorylation Site Determination
Published on: May 18, 2017
mTOR Ser1261 is an AMPK-dependent phosphosite in mouse and human skeletal muscle not required for mTORC2 activity
Jingwen Li1,2, Agnete B Madsen1, Jonas R Knudsen1
1August Krogh Section for Human and Molecular Physiology, Department of Nutrition, Exercise and Sports, University of Copenhagen, Copenhagen, Denmark.
Abstract:
The 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-dependent phosphorylations were affected in muscle-specific kinase-dead AMPK mice with no detectable mTOR Ser1261 phosphorylation in skeletal muscle. Thus, mTOR Ser1261 is an exercise but not insulin-responsive AMPK-dependent phosphosite in human and murine skeletal muscle, playing an unclear role in mTORC1 regulation but clearly not required for mTORC2 activity.
Insights
Exercise, not insulin, activates mTOR Ser1261 phosphorylation in skeletal muscle via AMPK. This site
Area of Science:
- Cellular metabolism and signaling pathways
- Muscle physiology and exercise science
Background:
- AMP-activated protein kinase (AMPK) and the mechanistic target of rapamycin (mTOR) are key regulators of cellular growth and metabolism.
- mTOR, existing in complexes mTORC1 and mTORC2, plays a critical role in cellular processes.
- Phosphorylation of mTOR at Serine 1261 (Ser1261) has been suggested to be influenced by both insulin and AMPK, potentially acting as a convergence point.
Purpose of the Study:
- To investigate the regulation and function of mTOR Ser1261 phosphorylation in skeletal muscle.
- To determine if Ser1261 serves as a convergence point for insulin and AMPK signaling in muscle.
- To elucidate the role of Ser1261 phosphorylation within the mTORC1 and mTORC2 complexes.
Main Methods:
- Utilized human exercise studies, transgenic mouse models (including Raptor muscle knockout/overexpression and kinase-dead AMPK models), and HEK293 cell culture.
- Assessed mTOR Ser1261 phosphorylation in response to insulin and contractile activity.
- Examined the impact of genetic modifications on mTORC1 and mTORC2 substrate phosphorylation.
Main Results:
- mTOR Ser1261 phosphorylation was not stimulated by insulin but acutely responded to muscle contractile activity in an AMPK-dependent manner.
- Contraction-stimulated Ser1261 phosphorylation was modulated by Raptor levels, indicating involvement of mTORC1.
- HEK293 cells with an mTOR Ser1261Ala mutation showed impaired mTORC1 substrate phosphorylation but not mTORC2 substrate phosphorylation.
- Muscle-specific kinase-dead AMPK mice lacking detectable Ser1261 phosphorylation showed no changes in mTORC1 or mTORC2 activity.
Conclusions:
- mTOR Ser1261 is an exercise-responsive, AMPK-dependent phosphosite in skeletal muscle, independent of insulin.
- Phosphorylation at Ser1261 appears to occur predominantly within the mTORC1 complex in skeletal muscle.
- While Ser1261 is regulated by AMPK, it is not essential for mTORC1 or mTORC2 activity in this context.
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