Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2016 Jan 1;351(6268):43-8.
doi: 10.1126/science.aab2674. Epub 2015 Oct 8.

Sestrin2 is a leucine sensor for the mTORC1 pathway

Rachel L Wolfson  1 , Lynne Chantranupong  1 , Robert A Saxton  1 , Kuang Shen  1 , Sonia M Scaria  2 , Jason R Cantor  1 , David M Sabatini  3
Affiliations

Sestrin2 is a leucine sensor for the mTORC1 pathway

Rachel L Wolfson et al. Science. .

Abstract

Leucine is a proteogenic amino acid that also regulates many aspects of mammalian physiology, in large part by activating the mTOR complex 1 (mTORC1) protein kinase, a master growth controller. Amino acids signal to mTORC1 through the Rag guanosine triphosphatases (GTPases). Several factors regulate the Rags, including GATOR1, aGTPase-activating protein; GATOR2, a positive regulator of unknown function; and Sestrin2, a GATOR2-interacting protein that inhibits mTORC1 signaling. We find that leucine, but not arginine, disrupts the Sestrin2-GATOR2 interaction by binding to Sestrin2 with a dissociation constant of 20 micromolar, which is the leucine concentration that half-maximally activates mTORC1. The leucine-binding capacity of Sestrin2 is required for leucine to activate mTORC1 in cells. These results indicate that Sestrin2 is a leucine sensor for the mTORC1 pathway.

PubMed Disclaimer

Figures

Figure 1
Figure 1. Leucine, but not arginine, disrupts the Sestrin2-GATOR2 interaction in cells and in vitro
A) Binding of Sestrin2 to GATOR2 in HEK-293T cells stably expressing FLAG-WDR24 (a component of GATOR2). Cells were deprived of leucine, arginine, or all amino acids for 50 minutes. Where indicated, cells were re-stimulated with leucine, arginine, or all amino acids for 10 minutes and FLAG immunoprecipitates prepared from cell lysates. Immunoprecipitates and lysates were analyzed by immunoblotting for the indicated proteins. FLAG-metap2 served as a negative control. B) Effects of leucine and arginine on the Sestrin2-GATOR2 interaction in ice-cold detergents lysates of amino acid-starved cells. HEK-293T cells stably expressing FLAG-metap2 or FLAG-WDR24 were deprived of all amino acids for 50 minutes. Leucine or arginine was added to the culture media or cell lysates and FLAG immunoprecipitates prepared and analyzed as in (A). C) Effects of individual amino acids on the purified Sestrin2-GATOR2 complex. FLAG immunoprecipitates were prepared from HEK-293T cells stably expressing FLAG-metap2 or FLAG-WDR24 and deprived of all amino acids for 50 minutes. Indicated amino acids (300 µM) were added directly to the immunoprecipitates, which, after re-washing, were analyzed as in (A). D) Disruption of the purified Sestrin2-GATOR2 complex by leucine. Experiment was performed and analyzed as in (C) except that indicated concentrations of leucine or arginine were used. E) Disruption of the Sestrin2-GATOR2 interaction by isoleucine and methionine. Experiment was performed and analyzed as in (C) except that the indicated concentrations of isoleucine, methionine, leucine, or arginine were used.
Figure 2
Figure 2. Sestrin2 binds leucine with a Kd of 20 µM
A) Binding of radiolabeled leucine to Sestrin2, but not WDR24, GATOR2, or the control protein Rap2A. FLAG immunoprecipitates prepared from HEK-293T cells transiently expressing indicated proteins or complexes were used in binding assays with [3H]Leucine as described in the methods. Unlabeled leucine was added where indicated. Values are Mean ± SD for 3 technical replicates from one representative experiment. SDS-PAGE followed by Coomasie blue staining was used to analyze immunoprecipitates prepared in parallel to those included in the binding assays. Asterisks indicate breakdown products in the WDR24 and GATOR2 purifications. B) Leucine-binding capacities of Sestrin1 (two isoforms), Sestrin2, and Sestrin3. FLAG immunoprecipitates were prepared and binding assays performed and analyzed as in (A). C) Leucine binds to bacterially-produced Sestrin2, but not the RagA/RagC heterodimer. Leucine binding assays were performed as described in the methods and analyzed as in (A) with His- MBP-Sestrin2 or His-RagA/RagC bound to the Ni-NTA resin. D) Effects of leucine and arginine on the melting temperature of bacterially-produced Sestrin2 in a thermal shift assay. His-MBP-Sestrin2 was incubated with the Sypro orange dye with or without leucine or arginine. Upon heating the sample the change in fluorescence was captured and used to calculate melting temperatures (Tm) under the indicated conditions. Values are Mean ± SD from 3 replicates. E) Sestrin2 binds leucine with a Kd of 20 µM. FLAG-Sestrin2 immunoprecipitates prepared as in (A) were used in binding assays with 10 µM or 20 µM [3H]Leucine and indicated concentrations of unlabeled leucine. In the representative graph shown each point represents the normalized mean ± SD for n = 3 in an assay with 10 µM [3H]Leucine. The Kd was calculated from the results of six experiments (three with 10 µM and three with 20 µM [3H]Leucine). F) Methionine can compete the binding of leucine to Sestrin2. FLAG-Sestrin2 immunoprecipitates prepared as in (A) were used in binding assays with 10 µM [3H]Leucine and indicated concentrations of unlabeled methionine. In the graph shown each point represents the normalized mean ± SD for n = 3. The Ki was calculated using data from the three experiments. G) Isoleucine can compete the binding of leucine to Sestrin2. FLAG-Sestrin2 immunoprecipitates prepared as in (A) were used in binding assays with 10 µM [3H]Leucine and indicated concentrations of unlabeled isoleucine. In the graph shown each point represents the normalized mean ± SD for n = 3. The Ki was calculated using data from the three experiments.
Figure 3
Figure 3. Sestrin2 regulates mTORC1 through GATOR2
A) Effects of varying leucine concentrations on mTORC1 activity, as measured by the phosphorylation of S6K1. HEK-293T cells were deprived of leucine for 50 minutes and restimulated with leucine at the indicated concentrations for 10 minutes. Cell lysates were analyzed via immunoblotting for the indicated proteins and phosphorylation states. B) Effects of varying leucine concentrations on the Sestrin2-GATOR2 interaction. HEK-293T cells stably expressing the indicated proteins were starved as in (A) and FLAG immunoprecipitates were collected. The immunopurified complexes were treated with the indicated concentrations of leucine and then analyzed as in Figure 1C. C) Decreased GATOR2-binding capacity of the Sestrin2 S190W mutant. FLAG immunoprecipitates were prepared from HEK-293T cells transiently expressing the indicated proteins and were analyzed by immunoblotting for the indicated proteins. D) Determination of leucine-binding capacity of Sestrin2 S190W. Assays were performed and immunoprecipitates analyzed as in Figure 2A. E) In Sestrin1-3 triple null cells expressing Sestrin2 S190W the mTORC1 pathway cannot sense the absence of leucine. Wild-type HEK-293T cells and Sestrin1-3 triple null HEK-293T cells generated with the CRISPR/Cas9 system were used to express the indicated FLAG-tagged proteins. Cells were starved for leucine for 50 minutes and, where indicated, stimulated with leucine for 10 minutes and lysates analyzed via immunoblotting.
Figure 4
Figure 4. The capacity of Sestrin2 to bind leucine is required for the mTORC1 pathway to sense leucine
A) The Sestrin2 L261A and E451A mutants do not bind leucine. Binding assays were performed and immunoprecipitates analyzed as in Figure 2A. B) Leucine-insensitivity of the interactions of Sestrin2 L261A or E451A with GATOR2. FLAG immunoprecipitates were prepared from cells transiently expressing the indicated proteins. The immunoprecipitates were treated with the indicated concentrations of leucine and analyzed as in Figure 1C. C) In Sestrin1-3 triple null cells expressing Sestrin2 L261A or E451A the mTORC1 pathway cannot sense the presence of leucine. Cells were generated and analyzed as in Figure 3E. D) Model showing how amino acid inputs arising from multiple sensors in distinct compartments impinge on the Rag GTPases to control mTORC1 activity.
See this image and copyright information in PMC

Comment in

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Potier M, Darcel N, Tomé D. Protein, amino acids and the control of food intake. Current Opinion in Clinical Nutrition and Metabolic Care. 2009;12:54–58. - PubMed
    1. Panten U, Christians J, von Kriegstein E, Poser W, Hasselblatt A. Studies on the mechanism of L-leucine-and alpha-ketoisocaproic acid-induced insulin release from perifused isolated pancreatic islets. Diabetologia. 1974;10:149–154. - PubMed
    1. Greiwe JS, Kwon G, McDaniel ML, Semenkovich CF. Leucine and insulin activate p70 S6 kinase through different pathways in human skeletal muscle. American journal of physiology. Endocrinology and metabolism. 2001;281:E466–E471. - PubMed
    1. Nair KS, Schwartz RG, Welle S. Leucine as a regulator of whole body and skeletal muscle protein metabolism in humans. American Journal of Physiology -- Legacy Content. 1992;263:E928–E934. - PubMed
    1. Layman DK, Walker DA. Potential Importance of Leucine in Treatment of Obesity and the Metabolic Syndrome. 2006 - PubMed

Publication types

MeSH terms