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
. 2011 Feb 25;286(8):6128-42.
doi: 10.1074/jbc.M110.166991. Epub 2010 Dec 3.

Amino acids activate mammalian target of rapamycin complex 2 (mTORC2) via PI3K/Akt signaling

Irantzu Tato  1 , Ramon Bartrons, Francesc Ventura, Jose Luis Rosa
Affiliations

Amino acids activate mammalian target of rapamycin complex 2 (mTORC2) via PI3K/Akt signaling

Irantzu Tato et al. J Biol Chem. .

Abstract

The activity of mammalian target of rapamycin (mTOR) complexes regulates essential cellular processes, such as growth, proliferation, or survival. Nutrients such as amino acids are important regulators of mTOR complex 1 (mTORC1) activation, thus affecting cell growth, protein synthesis, and autophagy. Here, we show that amino acids may also activate mTOR complex 2 (mTORC2). This activation is mediated by the activity of class I PI3K and of Akt. Amino acids induced a rapid phosphorylation of Akt at Thr-308 and Ser-473. Whereas both phosphorylations were dependent on the presence of mTOR, only Akt phosphorylation at Ser-473 was dependent on the presence of rictor, a specific component of mTORC2. Kinase assays confirmed mTORC2 activation by amino acids. This signaling was functional, as demonstrated by the phosphorylation of Akt substrate FOXO3a. Interestingly, using different starvation conditions, amino acids can selectively activate mTORC1 or mTORC2. These findings identify a new signaling pathway used by amino acids underscoring the crucial importance of these nutrients in cell metabolism and offering new mechanistic insights.

PubMed Disclaimer

Figures

FIGURE 1.
FIGURE 1.
Akt is phosphorylated at Thr-308 in response to amino acids. A–D, HeLa cells were deprived of serum overnight and stimulated with 200 nm insulin for 30 min, or after serum withdrawal, they were deprived for 2 h of amino acids (AA) with DPBS medium, followed by stimulation with 1× amino acids for 30 min or the indicated times (C). In B, MCF7 or HEK293 cells were treated with amino acids as in A. Where indicated, the cells were pretreated with 20 nm rapamycin (R), 100 nm wortmannin (W), 5 μm U0126 (U), or 10 μm SB 203580 (S) for 60 min before insulin or amino acid stimulation. Cellular lysates were analyzed by Western blot with the indicated antibodies. Molecular mass markers are indicated on the left. Quantification and statistical analyses (right panels) were performed as described under “Experimental Procedures.” These data are representative of at least three independent experiments.
FIGURE 2.
FIGURE 2.
Amino acids induce Akt phosphorylation at Ser-473. HeLa (A–E), MCF7 (B), or HEK293 (B) cells were treated with insulin or amino acids (AA) as in Fig. 1. D, cells deprived of amino acids as in A were stimulated with amino acids (1×), betaine (10 mm), or γ-amino-n-butyric acid (AIB, 10 mm) for 30 min. Where indicated, the cells were pretreated with 20 nm rapamycin (R), 100 nm wortmannin (W), 5 μm U0126 (U), or 10 μm SB 203580 (S) for 60 min before insulin or amino acid stimulation. Cellular lysates were analyzed by Western blot with the indicated antibodies. *, anti-P-T389-S6K1 antibody recognized phosphorylated RSK or MSK in amino acid-stimulated cells (39). Molecular mass markers are indicated on the left. These data are representative of three independent experiments. Quantification and statistical analyses (right panels) were performed as described under “Experimental Procedures.”
FIGURE 3.
FIGURE 3.
Amino acids induce Akt phosphorylations via class I PI3K. HeLa cells were treated with insulin or amino acids as in Fig. 1. Cells were pretreated with 100 nm wortmannin (W), 1 μm class I PI3K isoform-specific inhibitors (α, β, and δ) (panel A), or with 100 mm wortmanin (W), 1 μm class I PI3K isoform-specific inhibitor (α), or 10 mm hVps34 class III PI3K-specific inhibitor 3-methyladenine (3MA) for 60 min before insulin or amino acid stimulation. Molecular mass markers are indicated on the left. These data are representative of three independent experiments. Quantification and statistical analyses (lower panels) were performed as described under “Experimental Procedures.”
FIGURE 4.
FIGURE 4.
mTOR and rictor are required for the phosphorylation of Akt Ser-473 induced by amino acids. HeLa cells were transfected with either mTOR siRNA (A), rictor siRNA (B), or nontargeting (NT) control siRNA (A and B) 48 h before serum deprivation. Deprivation and stimulation with insulin or amino acids were performed as in Fig. 1. Cellular lysates were analyzed by Western blot with the indicated antibodies. *, phospho-RSK/MSK band (see Fig. 2). Molecular mass markers are indicated on the left. Quantification and statistical analyses (right panels) were performed as described under “Experimental Procedures.” These data are representative of three independent experiments.
FIGURE 5.
FIGURE 5.
Amino acids induce phosphorylation of Akt Ser-473 through mTORC2. HeLa cells were treated as in Fig. 1. Postnuclear supernatants (PNS) were immunoprecipitated (IP) with anti-mTOR (A), anti-raptor (B), or anti-rictor (C) antibodies. Immunocomplexes were resuspended in kinase buffer and incubated with purified FLAG-HA-Akt1 (substrate) in the presence or absence of ATP for 40 min at 37 °C. The reactions were stopped in ice with SDS/PAGE buffer and analyzed by Western blot with the indicated antibodies. Band intensities from three independent experiments were quantified and normalized relative to mTOR (A), raptor (B), or rictor (C). The data represent the ratio of P-S473-FLAG-HA-Akt1 phosphorylation and are expressed as the means ± S.E. of percentage of respective control.
FIGURE 6.
FIGURE 6.
Amino acids inactivate FOXO3a independently of JNK activity. HeLa cells were deprived of serum overnight and stimulated with 50 ng/ml PDGF for 30 min, or after serum withdrawal, they were deprived for 2 h of amino acids (AA) with DPBS medium, followed by stimulation with 1× amino acids for 30 min. Where indicated, the cells were pretreated with 100 nm wortmannin (W) and 10 μm Akti-1/2 (panel A), or with 100 nm wortmanin (W) and 10 μm SP600125 (SP) for 60 min before amino acid stimulation. Cellular lysates were analyzed by Western blot with the indicated antibodies. Molecular mass markers are indicated on the left. Quantification and statistical analyses (right panels) were performed as described under “Experimental Procedures.” In this figure, the significance (***, p < 0.001) in the statistical analyses is indicated by †. These data are representative of three independent experiments.
FIGURE 7.
FIGURE 7.
Amino acid signaling mechanisms depend on starvation conditions. After serum deprivation, HeLa (A, C, and D) or HEK293 (B) cells were deprived for 2 h of amino acids using either DPBS or DMEM(−AA) medium (DMEM without serum and without amino acids). After this, the cells were stimulated with 1× amino acids for 30 min. Where indicated, the cells were pretreated with 20 nm rapamycin (R), 100 nm wortmannin (W), or 10 mm hVps34 class III PI3K-specific inhibitor 3-methyladenine (3MA) for 60 min before amino acid stimulation. Cellular lysates were analyzed by Western blot with the indicated antibodies. *, phospho-RSK/MSK band (see Fig. 2). Molecular mass markers are indicated on the left. Quantification and statistical analyses (supplemental Fig. S2) were performed as described under “Experimental Procedures.” These data are representative of three independent experiments.
FIGURE 8.
FIGURE 8.
mTOR dependence in the signaling by amino acids. HeLa cells were transfected with either mTOR siRNA or nontargeting (NT) control siRNA 48 h before serum deprivation. Starvation and stimulation by amino acids were as in Fig. 7. Cellular lysates were analyzed by Western blot with the indicated antibodies. *, phospho-RSK/MSK band (see Fig. 2). Molecular mass markers are indicated on the left. Quantification and statistical analyses (right panels) were performed as described under “Experimental Procedures.” These data are representative of three independent experiments.
FIGURE 9.
FIGURE 9.
Amino acid signaling depends on DPBS starvation time. HeLa cells were deprived of serum overnight. After serum withdrawal, they were deprived with DPBS medium for the indicated times. Finally the cells were stimulated with 1× amino acids for 30 min. Cellular lysates were analyzed by Western blot with the indicated antibodies. Molecular mass markers are indicated on the left. Quantification and statistical analyses (right panels) were performed as described under “Experimental Procedures.” These data are representative of at least three independent experiments.
FIGURE 10.
FIGURE 10.
Amino acid supplementation leads to activation of mTORC1 and mTORC2. HeLa cells were supplemented with 1× amino acids for 30 min after 48 h in DMEM complete medium. Cellular lysates were analyzed by Western blot with the indicated antibodies. Molecular mass markers are indicated on the left. Quantification and statistical analyses (right panels) were performed as described under “Experimental Procedures.” These data are representative of at least three independent experiments.
FIGURE 11.
FIGURE 11.
A putative model of how amino acids may stimulate cell proliferation and survival pathways. When cells are supplemented with amino acids, kinase activities of mTORC1 (measured as the phosphorylation of their substrates: 4E-BP1 at Thr-37/Thr-46 and S6K1 at Thr-389) and mTORC2 (measured as the phosphorylation of its substrate: Akt at Ser-473) are stimulated. Previous amino acid starvation with DMEM(−AA) or DPBS selects the further activation of mTORC1 or mTORC2 when amino acids are added. Under these conditions, mTORC1 activation is dependent on the class III PI3K (hVps34), and mTORC2 activation is dependent on the α isoform of the class I PI3K. Additionally, Akt Thr-308 phosphorylation is also dependent of the α isoform of the class I PI3K and of the presence of mTOR but independent of mTORC1 and mTORC2 complexes (not shown). Both starvation conditions stimulate autophagy, which can be inhibited either by mTORC1-dependent (71, 72) and mTORC1-independent mechanisms (39). The dashed lines indicate that several steps may be involved. Activation or inhibition is indicated with arrows or blunt-ended arrows, respectively.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Wullschleger S., Loewith R., Hall M. N. (2006) Cell. 124, 471–484 - PubMed
    1. Laplante M., Sabatini D. M. (2009) J. Cell Sci. 122, 3589–3594 - PMC - PubMed
    1. Kim D. H., Sarbassov D. D., Ali S. M., King J. E., Latek R. R., Erdjument-Bromage H., Tempst P., Sabatini D. M. (2002) Cell. 110, 163–175 - PubMed
    1. Dann S. G., Selvaraj A., Thomas G. (2007) Trends Mol Med. 13, 252–259 - PubMed
    1. Peterson T. R., Laplante M., Thoreen C. C., Sancak Y., Kang S. A., Kuehl W. M., Gray N. S., Sabatini D. M. (2009) Cell. 137, 873–886 - PMC - PubMed

Publication types

MeSH terms