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
Comparative Study
. 2010 Aug;299(2):C335-44.
doi: 10.1152/ajpcell.00039.2010. Epub 2010 Apr 28.

Phosphatidic acid mediates activation of mTORC1 through the ERK signaling pathway

Jeremiah N Winter  1 , Todd E Fox, Mark Kester, Leonard S Jefferson, Scot R Kimball
Affiliations
Comparative Study

Phosphatidic acid mediates activation of mTORC1 through the ERK signaling pathway

Jeremiah N Winter et al. Am J Physiol Cell Physiol. 2010 Aug.

Abstract

The mammalian target of rapamycin (mTOR) assembles into two distinct multiprotein complexes known as mTORC1 and mTORC2. Of the two complexes, mTORC1 acts to integrate a variety of positive and negative signals to downstream targets that regulate cell growth. The lipid second messenger, phosphatidic acid (PA), represents one positive input to mTORC1, and it is thought to act by binding directly to mTOR, thereby enhancing the protein kinase activity of mTORC1. Support for this model includes findings that PA binds directly to mTOR and addition of PA to the medium of cells in culture results in activation of mTORC1. In contrast, the results of the present study do not support a model in which PA activates mTORC1 through direct interaction with the protein kinase but, instead, show that the lipid promotes mTORC1 signaling through activation of the ERK pathway. Moreover, rather than acting directly on mTORC1, the results suggest that exogenous PA must be metabolized to lysophosphatidic acid (LPA), which subsequently activates the LPA receptor endothelial differentiation gene (EDG-2). Finally, in contrast to previous studies, the results of the present study demonstrate that leucine does not act through phospholipase D and PA to activate mTORC1 and, instead, show that the two mediators act through parallel upstream signaling pathways to activate mTORC1. Overall, the results demonstrate that leucine and PA signal through parallel pathways to activate mTORC1 and that PA mediates its effect through the ERK pathway, rather than through direct binding to mTOR.

PubMed Disclaimer

Figures

Fig. 1.
Fig. 1.
Lysophosphatidic acid (LPA) treatment increases phospholipase D (PLD) activity and phosphatidic acid (PA) content. A: Rat2 cells were deprived of serum and leucine for 2 h before addition of 22 μM LPA, which approximated its concentration in serum (48). Values are means ± SE of 3 experiments; in each experiment, 3 samples per condition were independently analyzed. *P < 0.0001 vs. deprived (−) condition or leucine replacement. B–D: 18:1-18:1 PA, 18:0-18:1 PA, and 16:0-18:1 PA content was assessed by mass spectrometry. Values are means ± SE (n = 3). *P < 0.002 vs. deprived (−).
Fig. 2.
Fig. 2.
Rapamycin and 1-butanol, but not tert-butanol, attenuate LPA-mediated activation of mammalian target of rapamycin (mTOR) complex 1 (mTORC1). Cells were maintained in DMEM containing 10% FBS or deprived of serum and leucine for 2 h. 1-Butanol (1-But) or tert-butanol (t-But; A and B) or rapamycin (Rapa, C) was added to the medium 30 min prior to harvest. S6 protein kinase 1 (S6K1) hyperphosphorylation was assessed as proportion of the protein present in β-, γ-, and δ-forms relative to the total amount of the protein (α + β + γ + δ; A) or phosphorylation on Thr389 using an antibody specific for the phosphorylated form of the protein (C). Eukaryotic initiation factor 4E-binding protein (4E-BP1) hyperphosphorylation was assessed as proportion of the protein present in the γ-form relative to the total amount of the protein (α + β + γ; B). For representative blots, all samples were run on the same gel, but not in contiguous lanes. Noncontiguous lanes are separated by white lines. Values are means ± SE of 3 experiments; in each experiment, 3 samples per condition were independently analyzed. In A and B, *P < 0.003 vs. cells deprived of serum and leucine; †P < 0.008 vs. LPA. In C, *P < 0.0001 vs. cells deprived of serum and leucine; †P < 0.0001 vs. LPA.
Fig. 3.
Fig. 3.
Extracellular PA acts through the LPA receptor to activate mTORC1. Cells were deprived of serum and leucine (A) or incubated in medium containing 0.5% serum without leucine (B and C) for 2 h before addition of PA or LPA. Cells were harvested 30 min (A and B) or 2 h (A–C) later. When present, Ki16425 was added to the medium 30 min prior to PA. S6K1 phosphorylation on Thr389 was assessed as described in Fig. 2 legend. Representative blots are shown. Values are means ± SE of 3 experiments; in each experiment, 3 samples per condition were independently analyzed. In B, *P < 0.002 vs. cells incubated in medium containing 0.5% serum without leucine at the corresponding time point; †P < 0.035 vs. PA at 30 min. In C, *P < 0.05 vs. cells deprived of serum and leucine; †P < 0.005 vs. PA.
Fig. 4.
Fig. 4.
LPA and PA act through the same signaling pathway to activate mTORC1. Cells were incubated in medium containing 0.5% serum without leucine for 2 h before addition of LPA and/or PA. A: ERK1/2 phosphorylation on Thr202/Tyr204 was measured by Western blot analysis using an antibody specific for the phosphorylated form of the protein. B: S6K1 phosphorylation was assessed as described in Fig. 3 legend. Values are means ± SE of 3 experiments; in each experiment, 3 samples per condition were independently analyzed. Representative blots are shown. *P < 0.0005 vs. cells incubated in medium containing 0.5% serum without leucine. †P < 0.02; #P < 0.04 vs. PA alone.
Fig. 5.
Fig. 5.
LPA, but not leucine, activates mTORC1 signaling through the MAP kinase pathway. Cells were incubated in medium containing 0.5% serum without leucine for 2 h. Leucine, LPA, PD-98059, and/or U-0126 was added to the medium, and S6K1 phosphorylation on Thr389 and ERK1/2 phosphorylation on Thr202/Tyr204 were assessed as described in Fig. 3 and 4 legends, respectively. A and B: means ± SE of 5 experiments. In each experiment, 2 samples per condition were independently analyzed. C and D: means ± SE of 3 experiments. In each experiment, 3 samples per condition were independently analyzed. Representative blots are shown. *P < 0.02 vs. cells treated in medium containing 0.5% serum without leucine. †P < 0.011 vs. cells treated with LPA alone.
Fig. 6.
Fig. 6.
Leucine acts in an additive manner with LPA or insulin to activate mTORC1 signaling. Cells were incubated in medium containing 0.5% serum without leucine for 2 h. Leucine and/or LPA (A) or leucine and/or insulin (B) were added, and, after 2 h, S6K1 phosphorylation was assessed as described in Fig. 3 legend. Values are means ± SE of 3 experiments; in each experiment, 3 samples per condition were independently analyzed. Representative blots are shown. *P < 0.0005 vs. cells incubated in medium containing 0.5% serum without leucine. †P < 0.005 vs. cells treated with leucine alone. ‡P < 0.05 vs. cells treated with LPA alone or insulin alone.
Fig. 7.
Fig. 7.
Inhibition of MEK attenuates LPA-induced, but not leucine- or insulin-induced, activation of mTORC1 signaling. Cells were incubated in medium containing 0.5% serum without leucine for 2 h. Leucine, insulin, LPA, U-0126, and/or PD-98059 was added to the medium, and S6K1 phosphorylation was assessed as described in Fig. 3 legend. A and C: means ± SE of 5 experiments. In each experiment, 2 samples per condition were independently analyzed. B and D: means ± SE of 3 experiments. In each experiment, 3 samples per condition were independently analyzed. Representative blots are shown. *P < 0.045 vs. cells incubated in medium containing 0.5% serum without leucine. †P < 0.03 vs. cells treated with leucine alone. ‡P < 0.02 vs. cells treated with leucine and LPA.
Fig. 8.
Fig. 8.
Model for the PA-mediated activation of mTORC1 signaling. PA is hydrolyzed by phospholipase A (PLA), and resulting LPA activates the endothelial differentiation gene (EDG-2) receptor. Activation of the EDG-2 receptor results in upregulated signaling through the MEK-ERK pathway via 2 distinct mechanisms. One mechanism involves a G protein-mediated increase in PLD activity, leading to hydrolysis of phosphatidylcholine (PC) to choline and PA. PA then binds to Raf, allowing for activation of the ERK cascade. The second mechanism involves G proteins acting through Ras to activate the MEK-ERK pathway. Subsequently, ERK acts to inhibit tuberous sclerosis complex (TSC1/2), thereby increasing GTP loading on Ras homolog enriched in brain (Rheb) and, thus, upregulating mTORC1 activation. mTORC1 then phosphorylates downstream targets, such as S6K1 and eukaryotic initiation factor 4E-binding protein (4E-BP1), ultimately leading to increased mRNA translation. Leucine also activates mTORC1, although the mechanism involved in the effect is not understood.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Ballif BA, Roux PP, Gerber SA, MacKeigan JP, Blenis J, Gygi SP. Quantitative phosphorylation profiling of the ERK/p90 ribosomal S6 kinase-signaling cassette and its targets, the tuberous sclerosis tumor suppressors. Proc Natl Acad Sci USA 102: 667–672, 2005 - PMC - PubMed
    1. Baum JI, Kimball SR, Jefferson LS. Glucagon acts in a dominant manner to repress insulin-induced mammalian target of rapamycin complex 1 signaling in perfused rat liver. Am J Physiol Endocrinol Metab 297: E410–E415, 2009 - PMC - PubMed
    1. Bocckino SB, Wilson PB, Exton JH. Phosphatidate-dependent protein phosphorylation. Proc Natl Acad Sci USA 88: 6210–6213, 1991 - PMC - PubMed
    1. Bodine SC, Stitt TN, Gonzalez M, Kline WO, Stover GL, Bauerlein R, Zlotchenko E, Scrimgeour A, Lawrence JC, Glass DJ, Yancopoulous GD. Akt/mTOR pathway is a crucial regulator of skeletal muscle hypertrophy and can prevent muscle atrophy in vivo. Nat Cell Biol 3: 1014–1019, 2001 - PubMed
    1. Bolster DR, Kubica N, Crozier SJ, Williamson DL, Farrell PA, Kimball SR, Jefferson LS. Immediate response of mammalian target of rapamycin (mTOR)-mediated signalling following acute resistance exercise in rat skeletal muscle. J Physiol 553: 213–220, 2003 - PMC - PubMed

Publication types

MeSH terms

LinkOut - more resources