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A systems study reveals concurrent activation of AMPK and mTOR by amino acids

View Article: PubMed Central - PubMed

ABSTRACT

Amino acids (aa) are not only building blocks for proteins, but also signalling molecules, with the mammalian target of rapamycin complex 1 (mTORC1) acting as a key mediator. However, little is known about whether aa, independently of mTORC1, activate other kinases of the mTOR signalling network. To delineate aa-stimulated mTOR network dynamics, we here combine a computational–experimental approach with text mining-enhanced quantitative proteomics. We report that AMP-activated protein kinase (AMPK), phosphatidylinositide 3-kinase (PI3K) and mTOR complex 2 (mTORC2) are acutely activated by aa-readdition in an mTORC1-independent manner. AMPK activation by aa is mediated by Ca2+/calmodulin-dependent protein kinase kinase β (CaMKKβ). In response, AMPK impinges on the autophagy regulators Unc-51-like kinase-1 (ULK1) and c-Jun. AMPK is widely recognized as an mTORC1 antagonist that is activated by starvation. We find that aa acutely activate AMPK concurrently with mTOR. We show that AMPK under aa sufficiency acts to sustain autophagy. This may be required to maintain protein homoeostasis and deliver metabolite intermediates for biosynthetic processes.

No MeSH data available.


Related in: MedlinePlus

Amino acids (aa) activate mTORC1 and AMPK independently of mTORC2.(a) Simulated response of p70-S6K-pT389, Akt-pS473, Akt-pT308 and AMPK-pT172 to aa induction in a system with mTORC2 inhibition (mTORC2 activity 10 to 100%; experimental equivalent: mSin1 knockdown). (b) Aa induce p70-S6K-pT389 and AMPK-pT172, when mTORC2 is inhibited by mSin1 knockdown (shSin1). Representative immunoblot results of aa-stimulated C2C12 cells in the absence or presence of shSin1 (4 days). Data are representative of three experiments. (c) Quantitative representations of simulated (mTORC2 inhibition: reduction to 40%, as shown in a) and experimentally determined dynamics of p70-S6K-pT389, AMPK-pT172, Akt-pS473 and Akt-pT308 upon stimulation with aa with mTORC2 inhibition using shSin1. Data are the mean and s.e.m., N=3. Statistical significance between control and treatment in experimental quantitations across the time course was detected by repeated measures analysis of variance (ANOVA). Exp Ctrl, experimental control condition (shControl); Exp Inhib, experimental mTORC2 inhibition (shSin1); Sim Ctrl, simulated control condition; Sim Inhib, simulated mTORC2 perturbation.
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f4: Amino acids (aa) activate mTORC1 and AMPK independently of mTORC2.(a) Simulated response of p70-S6K-pT389, Akt-pS473, Akt-pT308 and AMPK-pT172 to aa induction in a system with mTORC2 inhibition (mTORC2 activity 10 to 100%; experimental equivalent: mSin1 knockdown). (b) Aa induce p70-S6K-pT389 and AMPK-pT172, when mTORC2 is inhibited by mSin1 knockdown (shSin1). Representative immunoblot results of aa-stimulated C2C12 cells in the absence or presence of shSin1 (4 days). Data are representative of three experiments. (c) Quantitative representations of simulated (mTORC2 inhibition: reduction to 40%, as shown in a) and experimentally determined dynamics of p70-S6K-pT389, AMPK-pT172, Akt-pS473 and Akt-pT308 upon stimulation with aa with mTORC2 inhibition using shSin1. Data are the mean and s.e.m., N=3. Statistical significance between control and treatment in experimental quantitations across the time course was detected by repeated measures analysis of variance (ANOVA). Exp Ctrl, experimental control condition (shControl); Exp Inhib, experimental mTORC2 inhibition (shSin1); Sim Ctrl, simulated control condition; Sim Inhib, simulated mTORC2 perturbation.

Mentions: We next analysed if the observed responses of Akt-pS473, Akt-pT308 and AMPK-pT172 to aa were positively connected to each other, for example, via feedback mechanisms, or if they represent separate aa inputs to the mTOR network. We inhibited mTORC2 in silico (Fig. 4a) and experimentally (Fig. 4b), using an inducible mSin1 knockdown (shSin1) C2C12 cell line and recorded time-course data upon aa stimulation. The model simulation predicted that if the four inputs (mTORC1, mTORC2, PI3K and AMPK) were unrelated, mTORC2 inhibition (reflected by a reduction of Akt-pS473) should not affect aa stimulation of p70-S6K-pT389, AMPK-pT172 or Akt-pT308 (Fig. 4a). Indeed, we observed that upon mTORC2 inhibition by shSin1, Akt-pS473 was reduced, whereas p70-S6K-pT389 and AMPK-pT172 remained responsive to aa (Fig. 4b,c; Supplementary Fig. 17). In contrast to our simulation, Akt-pT308 was reduced by mTORC2 inhibition. This may be due to the fact that this readout not only reflects PI3K activity, but that Akt-T308 phosphorylation efficiency depends also in part on the phosphorylation at S473 (refs 32, 33). For completeness, a comparison between the prediction of the model without p70-S6K-pT229 module and experimental data upon mTORC2 inhibition is provided in Supplementary Fig. 18, showing consistent results.


A systems study reveals concurrent activation of AMPK and mTOR by amino acids
Amino acids (aa) activate mTORC1 and AMPK independently of mTORC2.(a) Simulated response of p70-S6K-pT389, Akt-pS473, Akt-pT308 and AMPK-pT172 to aa induction in a system with mTORC2 inhibition (mTORC2 activity 10 to 100%; experimental equivalent: mSin1 knockdown). (b) Aa induce p70-S6K-pT389 and AMPK-pT172, when mTORC2 is inhibited by mSin1 knockdown (shSin1). Representative immunoblot results of aa-stimulated C2C12 cells in the absence or presence of shSin1 (4 days). Data are representative of three experiments. (c) Quantitative representations of simulated (mTORC2 inhibition: reduction to 40%, as shown in a) and experimentally determined dynamics of p70-S6K-pT389, AMPK-pT172, Akt-pS473 and Akt-pT308 upon stimulation with aa with mTORC2 inhibition using shSin1. Data are the mean and s.e.m., N=3. Statistical significance between control and treatment in experimental quantitations across the time course was detected by repeated measures analysis of variance (ANOVA). Exp Ctrl, experimental control condition (shControl); Exp Inhib, experimental mTORC2 inhibition (shSin1); Sim Ctrl, simulated control condition; Sim Inhib, simulated mTORC2 perturbation.
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Related In: Results  -  Collection

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f4: Amino acids (aa) activate mTORC1 and AMPK independently of mTORC2.(a) Simulated response of p70-S6K-pT389, Akt-pS473, Akt-pT308 and AMPK-pT172 to aa induction in a system with mTORC2 inhibition (mTORC2 activity 10 to 100%; experimental equivalent: mSin1 knockdown). (b) Aa induce p70-S6K-pT389 and AMPK-pT172, when mTORC2 is inhibited by mSin1 knockdown (shSin1). Representative immunoblot results of aa-stimulated C2C12 cells in the absence or presence of shSin1 (4 days). Data are representative of three experiments. (c) Quantitative representations of simulated (mTORC2 inhibition: reduction to 40%, as shown in a) and experimentally determined dynamics of p70-S6K-pT389, AMPK-pT172, Akt-pS473 and Akt-pT308 upon stimulation with aa with mTORC2 inhibition using shSin1. Data are the mean and s.e.m., N=3. Statistical significance between control and treatment in experimental quantitations across the time course was detected by repeated measures analysis of variance (ANOVA). Exp Ctrl, experimental control condition (shControl); Exp Inhib, experimental mTORC2 inhibition (shSin1); Sim Ctrl, simulated control condition; Sim Inhib, simulated mTORC2 perturbation.
Mentions: We next analysed if the observed responses of Akt-pS473, Akt-pT308 and AMPK-pT172 to aa were positively connected to each other, for example, via feedback mechanisms, or if they represent separate aa inputs to the mTOR network. We inhibited mTORC2 in silico (Fig. 4a) and experimentally (Fig. 4b), using an inducible mSin1 knockdown (shSin1) C2C12 cell line and recorded time-course data upon aa stimulation. The model simulation predicted that if the four inputs (mTORC1, mTORC2, PI3K and AMPK) were unrelated, mTORC2 inhibition (reflected by a reduction of Akt-pS473) should not affect aa stimulation of p70-S6K-pT389, AMPK-pT172 or Akt-pT308 (Fig. 4a). Indeed, we observed that upon mTORC2 inhibition by shSin1, Akt-pS473 was reduced, whereas p70-S6K-pT389 and AMPK-pT172 remained responsive to aa (Fig. 4b,c; Supplementary Fig. 17). In contrast to our simulation, Akt-pT308 was reduced by mTORC2 inhibition. This may be due to the fact that this readout not only reflects PI3K activity, but that Akt-T308 phosphorylation efficiency depends also in part on the phosphorylation at S473 (refs 32, 33). For completeness, a comparison between the prediction of the model without p70-S6K-pT229 module and experimental data upon mTORC2 inhibition is provided in Supplementary Fig. 18, showing consistent results.

View Article: PubMed Central - PubMed

ABSTRACT

Amino acids (aa) are not only building blocks for proteins, but also signalling molecules, with the mammalian target of rapamycin complex 1 (mTORC1) acting as a key mediator. However, little is known about whether aa, independently of mTORC1, activate other kinases of the mTOR signalling network. To delineate aa-stimulated mTOR network dynamics, we here combine a computational–experimental approach with text mining-enhanced quantitative proteomics. We report that AMP-activated protein kinase (AMPK), phosphatidylinositide 3-kinase (PI3K) and mTOR complex 2 (mTORC2) are acutely activated by aa-readdition in an mTORC1-independent manner. AMPK activation by aa is mediated by Ca2+/calmodulin-dependent protein kinase kinase β (CaMKKβ). In response, AMPK impinges on the autophagy regulators Unc-51-like kinase-1 (ULK1) and c-Jun. AMPK is widely recognized as an mTORC1 antagonist that is activated by starvation. We find that aa acutely activate AMPK concurrently with mTOR. We show that AMPK under aa sufficiency acts to sustain autophagy. This may be required to maintain protein homoeostasis and deliver metabolite intermediates for biosynthetic processes.

No MeSH data available.


Related in: MedlinePlus