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Control of TSC2-Rheb signaling axis by arginine regulates mTORC1 activity.

Carroll B, Maetzel D, Maddocks OD, Otten G, Ratcliff M, Smith GR, Dunlop EA, Passos JF, Davies OR, Jaenisch R, Tee AR, Sarkar S, Korolchuk VI - Elife (2016)

Bottom Line: Herein, we demonstrate that arginine acts independently of its metabolism to allow maximal activation of mTORC1 by growth factors via a mechanism that does not involve regulation of mTORC1 localization to lysosomes.Dependence on arginine is maintained once hESCs are differentiated to fibroblasts, neurons, and hepatocytes, highlighting the fundamental importance of arginine-sensing to mTORC1 signaling.Together, our data provide evidence that different growth promoting cues cooperate to a greater extent than previously recognized to achieve tight spatial and temporal regulation of mTORC1 signaling.

View Article: PubMed Central - PubMed

Affiliation: Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle upon Tyne, United Kingdom.

ABSTRACT
The mammalian target of rapamycin complex 1 (mTORC1) is the key signaling hub that regulates cellular protein homeostasis, growth, and proliferation in health and disease. As a prerequisite for activation of mTORC1 by hormones and mitogens, there first has to be an available pool of intracellular amino acids. Arginine, an amino acid essential during mammalian embryogenesis and early development is one of the key activators of mTORC1. Herein, we demonstrate that arginine acts independently of its metabolism to allow maximal activation of mTORC1 by growth factors via a mechanism that does not involve regulation of mTORC1 localization to lysosomes. Instead, arginine specifically suppresses lysosomal localization of the TSC complex and interaction with its target small GTPase protein, Rheb. By interfering with TSC-Rheb complex, arginine relieves allosteric inhibition of Rheb by TSC. Arginine cooperates with growth factor signaling which further promotes dissociation of TSC2 from lysosomes and activation of mTORC1. Arginine is the main amino acid sensed by the mTORC1 pathway in several cell types including human embryonic stem cells (hESCs). Dependence on arginine is maintained once hESCs are differentiated to fibroblasts, neurons, and hepatocytes, highlighting the fundamental importance of arginine-sensing to mTORC1 signaling. Together, our data provide evidence that different growth promoting cues cooperate to a greater extent than previously recognized to achieve tight spatial and temporal regulation of mTORC1 signaling.

No MeSH data available.


Related in: MedlinePlus

Arginine contributes to mTORC1 activity via TSC2/Rheb signaling axis.(A–C) Arginine deprivation specifically perturbs growth factor-mediated mTORC1 activity. HeLa cells (A, B) and wild-type MEFs (C) were incubated with either a complete amino acid mixture or starved of individual amino acids as indicated, in the presence or absence of EGF (100 ng/ml) (A) or insulin (10 μg/ml) (B, C). Lysates were subjected to immunoblotting for phosphorylation of S6K. (D) Arginine starvation activates autophagy. HeLa cells were pre-treated with chloroquine for 3 hr prior to being starved of the indicated amino acid (in the presence of dFCS) for 1 hr. Cells were fixed and stained with antibodies against LC3. The number of autophagosomes per cell was quantified. (E–G) Perturbation of growth factor signaling by arginine starvation is not dependent on SLC38A9/Rag GTPase protein complexes. HeLa cells were transfected with constitutive active heterodimer of RagB (Q99L), RagC (S75L), and HA-tagged S6K and deprived of amino acids as indicated, either in the presence or in the absence of dFCS. Lysates were analyzed by immunoblot for phosphorylation of S6K (E). (F) HeLa cells were transfected with active or inactive Rag GTPases and incubated in the presence or absence of amino acids. Cells were fixed and stained with antibodies against mTOR and V5 or HA-tagged Rag GTPases to confirm their localization and effect on mTOR localization. (G) HeLa cells were subjected to siRNA against SLC38A9 (100 nM) or scramble control (Scr) for 96 hr. Cells were then analyzed for mTORC1 activity in cells; first, cells were starved and recovered with either arginine or leucine or starved either in the presence or absence of dFCS. Cell lysates were analyzed for phospho-S6K, phospho-S6 and 4E-BP1 and SLC38A9. HeLa cells (H) and wild-type MEFs (I) were incubated with amino acid and dFCS mixtures as indicated. Lysates were analyzed for phosphorylation of TSC2 at serine 939. Of note, there is some increased phosphorylation in cells completely starved of amino acids, arginine and leucine. It has been noted previously that amino acid deprivation can contribute to feed-back mechanisms that involve IRS1 and re-activation of Akt (see review [Manning, 2004]). As there was no specificity to arginine starvation, this observation was not explored further. HeLa cells (J, K) were incubated with amino acid and dFCS mixtures as indicated. Lysates were analyzed for phosphorylation of Akt at serine 473 (J) or threonine 308 (K). HeLa cells (L) and wild-type MEFs (M) were incubated with amino acid and dFCS mixtures as indicated. Lysates were analyzed for phosphorylation of AMPK. As a positive control, cells were starved of glucose. (N, O) Loss of TSC2 renders mTORC1 insensitive to arginine deprivation. TSC2+/+ and TSC2-/- MEFs were subjected to leucine or arginine starvation. Where indicated, recovery was carried out by the re-addition of arginine or leucine. Lysates were assessed for phosphorylation of ULK1 by immunoblotting (N). Alternatively, TSC2+/+ and TSC2-/- MEFs were subjected to amino acid or arginine starvation. Lysates were analyzed for phosphorylation of S6K and ULK1 (O). Scale bars: 10 μm. Nuclei are visualized in merge images with TO-PRO-3 iodide. All graphs with statistics represent an average of at least three independent experiments and error bars represent s.e.m. Graphs not displaying statistics represent an average of two independent experiments and error bars represent standard deviation. *p<0.05, **p<0.01, ***p<0.005. SE: short exposure; LE: long exposure; NS: not significant.DOI:http://dx.doi.org/10.7554/eLife.11058.006
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fig1s3: Arginine contributes to mTORC1 activity via TSC2/Rheb signaling axis.(A–C) Arginine deprivation specifically perturbs growth factor-mediated mTORC1 activity. HeLa cells (A, B) and wild-type MEFs (C) were incubated with either a complete amino acid mixture or starved of individual amino acids as indicated, in the presence or absence of EGF (100 ng/ml) (A) or insulin (10 μg/ml) (B, C). Lysates were subjected to immunoblotting for phosphorylation of S6K. (D) Arginine starvation activates autophagy. HeLa cells were pre-treated with chloroquine for 3 hr prior to being starved of the indicated amino acid (in the presence of dFCS) for 1 hr. Cells were fixed and stained with antibodies against LC3. The number of autophagosomes per cell was quantified. (E–G) Perturbation of growth factor signaling by arginine starvation is not dependent on SLC38A9/Rag GTPase protein complexes. HeLa cells were transfected with constitutive active heterodimer of RagB (Q99L), RagC (S75L), and HA-tagged S6K and deprived of amino acids as indicated, either in the presence or in the absence of dFCS. Lysates were analyzed by immunoblot for phosphorylation of S6K (E). (F) HeLa cells were transfected with active or inactive Rag GTPases and incubated in the presence or absence of amino acids. Cells were fixed and stained with antibodies against mTOR and V5 or HA-tagged Rag GTPases to confirm their localization and effect on mTOR localization. (G) HeLa cells were subjected to siRNA against SLC38A9 (100 nM) or scramble control (Scr) for 96 hr. Cells were then analyzed for mTORC1 activity in cells; first, cells were starved and recovered with either arginine or leucine or starved either in the presence or absence of dFCS. Cell lysates were analyzed for phospho-S6K, phospho-S6 and 4E-BP1 and SLC38A9. HeLa cells (H) and wild-type MEFs (I) were incubated with amino acid and dFCS mixtures as indicated. Lysates were analyzed for phosphorylation of TSC2 at serine 939. Of note, there is some increased phosphorylation in cells completely starved of amino acids, arginine and leucine. It has been noted previously that amino acid deprivation can contribute to feed-back mechanisms that involve IRS1 and re-activation of Akt (see review [Manning, 2004]). As there was no specificity to arginine starvation, this observation was not explored further. HeLa cells (J, K) were incubated with amino acid and dFCS mixtures as indicated. Lysates were analyzed for phosphorylation of Akt at serine 473 (J) or threonine 308 (K). HeLa cells (L) and wild-type MEFs (M) were incubated with amino acid and dFCS mixtures as indicated. Lysates were analyzed for phosphorylation of AMPK. As a positive control, cells were starved of glucose. (N, O) Loss of TSC2 renders mTORC1 insensitive to arginine deprivation. TSC2+/+ and TSC2-/- MEFs were subjected to leucine or arginine starvation. Where indicated, recovery was carried out by the re-addition of arginine or leucine. Lysates were assessed for phosphorylation of ULK1 by immunoblotting (N). Alternatively, TSC2+/+ and TSC2-/- MEFs were subjected to amino acid or arginine starvation. Lysates were analyzed for phosphorylation of S6K and ULK1 (O). Scale bars: 10 μm. Nuclei are visualized in merge images with TO-PRO-3 iodide. All graphs with statistics represent an average of at least three independent experiments and error bars represent s.e.m. Graphs not displaying statistics represent an average of two independent experiments and error bars represent standard deviation. *p<0.05, **p<0.01, ***p<0.005. SE: short exposure; LE: long exposure; NS: not significant.DOI:http://dx.doi.org/10.7554/eLife.11058.006

Mentions: Several observations argue for a mechanism of arginine action different to that of leucine. First, deprivation of arginine but not leucine or isoleucine, significantly perturbed the growth factor-dependent input into mTORC1. This is evident from suppressed phosphorylation of ribosomal protein S6 kinase 1 (S6K1), eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) and ULK1, with a concomitant increase in autophagy (Figure 1A–C and Figure 1—figure supplement 3A–D). Deprivation of arginine in the presence of growth factors limits mTORC1 activation to a level similar to that resulting from serum starvation. Addition of growth factors to leucine or isoleucine-deprived cells, however, permits maximal mTORC1 activation (Figure 1A–C and Figure 1—figure supplement 3A–D). Amino acids, particularly leucine, are known to signal to mTORC1 via the V-ATPase/Ragulator/Rag GTPase protein complexes and have been comprehensively demonstrated to control mTORC1 localization to the lysosome and its activity (Carroll et al., 2014). We observed that arginine did not affect mTOR localization in any cell line tested. While complete amino acid starvation and, to a lesser extent, leucine starvation, caused redistribution of mTORC1 to the cytoplasm, arginine deprivation led to a strong suppression of mTORC1 without a significant reduction in co-localization of mTOR with the lysosomal marker Lamp1 in HeLa, MEFs, and HEK293T cells (Figure 1D). Furthermore, overexpression of constitutively active Rag heterodimer (Sancak et al., 2008) did not completely rescue the effect of arginine starvation in the absence or presence of growth factors, thus suggesting that arginine may affect mTORC1 both via Rag-dependent and Rag-independent mechanisms (Figure 1—figure supplement 3E,F). Finally, the knockdown of membrane transporter, SLC38A9 that has recently been implicated in Rag GTPase-dependent lysosomal recruitment and activation of mTORC1 (Wang et al., 2015; Rebsamen et al., 2015; Jung et al., 2015) did partially perturb the recovery of mTORC1 following arginine starvation; however, there was no effect on the response of mTORC1 to arginine starvation either in the absence or presence of growth factors (Figure 1—figure supplement 3G).10.7554/eLife.11058.003Figure 1.Arginine is a permissive factor for growth factor-dependent mTORC1 activity and acts via TSC2-Rheb axis.


Control of TSC2-Rheb signaling axis by arginine regulates mTORC1 activity.

Carroll B, Maetzel D, Maddocks OD, Otten G, Ratcliff M, Smith GR, Dunlop EA, Passos JF, Davies OR, Jaenisch R, Tee AR, Sarkar S, Korolchuk VI - Elife (2016)

Arginine contributes to mTORC1 activity via TSC2/Rheb signaling axis.(A–C) Arginine deprivation specifically perturbs growth factor-mediated mTORC1 activity. HeLa cells (A, B) and wild-type MEFs (C) were incubated with either a complete amino acid mixture or starved of individual amino acids as indicated, in the presence or absence of EGF (100 ng/ml) (A) or insulin (10 μg/ml) (B, C). Lysates were subjected to immunoblotting for phosphorylation of S6K. (D) Arginine starvation activates autophagy. HeLa cells were pre-treated with chloroquine for 3 hr prior to being starved of the indicated amino acid (in the presence of dFCS) for 1 hr. Cells were fixed and stained with antibodies against LC3. The number of autophagosomes per cell was quantified. (E–G) Perturbation of growth factor signaling by arginine starvation is not dependent on SLC38A9/Rag GTPase protein complexes. HeLa cells were transfected with constitutive active heterodimer of RagB (Q99L), RagC (S75L), and HA-tagged S6K and deprived of amino acids as indicated, either in the presence or in the absence of dFCS. Lysates were analyzed by immunoblot for phosphorylation of S6K (E). (F) HeLa cells were transfected with active or inactive Rag GTPases and incubated in the presence or absence of amino acids. Cells were fixed and stained with antibodies against mTOR and V5 or HA-tagged Rag GTPases to confirm their localization and effect on mTOR localization. (G) HeLa cells were subjected to siRNA against SLC38A9 (100 nM) or scramble control (Scr) for 96 hr. Cells were then analyzed for mTORC1 activity in cells; first, cells were starved and recovered with either arginine or leucine or starved either in the presence or absence of dFCS. Cell lysates were analyzed for phospho-S6K, phospho-S6 and 4E-BP1 and SLC38A9. HeLa cells (H) and wild-type MEFs (I) were incubated with amino acid and dFCS mixtures as indicated. Lysates were analyzed for phosphorylation of TSC2 at serine 939. Of note, there is some increased phosphorylation in cells completely starved of amino acids, arginine and leucine. It has been noted previously that amino acid deprivation can contribute to feed-back mechanisms that involve IRS1 and re-activation of Akt (see review [Manning, 2004]). As there was no specificity to arginine starvation, this observation was not explored further. HeLa cells (J, K) were incubated with amino acid and dFCS mixtures as indicated. Lysates were analyzed for phosphorylation of Akt at serine 473 (J) or threonine 308 (K). HeLa cells (L) and wild-type MEFs (M) were incubated with amino acid and dFCS mixtures as indicated. Lysates were analyzed for phosphorylation of AMPK. As a positive control, cells were starved of glucose. (N, O) Loss of TSC2 renders mTORC1 insensitive to arginine deprivation. TSC2+/+ and TSC2-/- MEFs were subjected to leucine or arginine starvation. Where indicated, recovery was carried out by the re-addition of arginine or leucine. Lysates were assessed for phosphorylation of ULK1 by immunoblotting (N). Alternatively, TSC2+/+ and TSC2-/- MEFs were subjected to amino acid or arginine starvation. Lysates were analyzed for phosphorylation of S6K and ULK1 (O). Scale bars: 10 μm. Nuclei are visualized in merge images with TO-PRO-3 iodide. All graphs with statistics represent an average of at least three independent experiments and error bars represent s.e.m. Graphs not displaying statistics represent an average of two independent experiments and error bars represent standard deviation. *p<0.05, **p<0.01, ***p<0.005. SE: short exposure; LE: long exposure; NS: not significant.DOI:http://dx.doi.org/10.7554/eLife.11058.006
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fig1s3: Arginine contributes to mTORC1 activity via TSC2/Rheb signaling axis.(A–C) Arginine deprivation specifically perturbs growth factor-mediated mTORC1 activity. HeLa cells (A, B) and wild-type MEFs (C) were incubated with either a complete amino acid mixture or starved of individual amino acids as indicated, in the presence or absence of EGF (100 ng/ml) (A) or insulin (10 μg/ml) (B, C). Lysates were subjected to immunoblotting for phosphorylation of S6K. (D) Arginine starvation activates autophagy. HeLa cells were pre-treated with chloroquine for 3 hr prior to being starved of the indicated amino acid (in the presence of dFCS) for 1 hr. Cells were fixed and stained with antibodies against LC3. The number of autophagosomes per cell was quantified. (E–G) Perturbation of growth factor signaling by arginine starvation is not dependent on SLC38A9/Rag GTPase protein complexes. HeLa cells were transfected with constitutive active heterodimer of RagB (Q99L), RagC (S75L), and HA-tagged S6K and deprived of amino acids as indicated, either in the presence or in the absence of dFCS. Lysates were analyzed by immunoblot for phosphorylation of S6K (E). (F) HeLa cells were transfected with active or inactive Rag GTPases and incubated in the presence or absence of amino acids. Cells were fixed and stained with antibodies against mTOR and V5 or HA-tagged Rag GTPases to confirm their localization and effect on mTOR localization. (G) HeLa cells were subjected to siRNA against SLC38A9 (100 nM) or scramble control (Scr) for 96 hr. Cells were then analyzed for mTORC1 activity in cells; first, cells were starved and recovered with either arginine or leucine or starved either in the presence or absence of dFCS. Cell lysates were analyzed for phospho-S6K, phospho-S6 and 4E-BP1 and SLC38A9. HeLa cells (H) and wild-type MEFs (I) were incubated with amino acid and dFCS mixtures as indicated. Lysates were analyzed for phosphorylation of TSC2 at serine 939. Of note, there is some increased phosphorylation in cells completely starved of amino acids, arginine and leucine. It has been noted previously that amino acid deprivation can contribute to feed-back mechanisms that involve IRS1 and re-activation of Akt (see review [Manning, 2004]). As there was no specificity to arginine starvation, this observation was not explored further. HeLa cells (J, K) were incubated with amino acid and dFCS mixtures as indicated. Lysates were analyzed for phosphorylation of Akt at serine 473 (J) or threonine 308 (K). HeLa cells (L) and wild-type MEFs (M) were incubated with amino acid and dFCS mixtures as indicated. Lysates were analyzed for phosphorylation of AMPK. As a positive control, cells were starved of glucose. (N, O) Loss of TSC2 renders mTORC1 insensitive to arginine deprivation. TSC2+/+ and TSC2-/- MEFs were subjected to leucine or arginine starvation. Where indicated, recovery was carried out by the re-addition of arginine or leucine. Lysates were assessed for phosphorylation of ULK1 by immunoblotting (N). Alternatively, TSC2+/+ and TSC2-/- MEFs were subjected to amino acid or arginine starvation. Lysates were analyzed for phosphorylation of S6K and ULK1 (O). Scale bars: 10 μm. Nuclei are visualized in merge images with TO-PRO-3 iodide. All graphs with statistics represent an average of at least three independent experiments and error bars represent s.e.m. Graphs not displaying statistics represent an average of two independent experiments and error bars represent standard deviation. *p<0.05, **p<0.01, ***p<0.005. SE: short exposure; LE: long exposure; NS: not significant.DOI:http://dx.doi.org/10.7554/eLife.11058.006
Mentions: Several observations argue for a mechanism of arginine action different to that of leucine. First, deprivation of arginine but not leucine or isoleucine, significantly perturbed the growth factor-dependent input into mTORC1. This is evident from suppressed phosphorylation of ribosomal protein S6 kinase 1 (S6K1), eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) and ULK1, with a concomitant increase in autophagy (Figure 1A–C and Figure 1—figure supplement 3A–D). Deprivation of arginine in the presence of growth factors limits mTORC1 activation to a level similar to that resulting from serum starvation. Addition of growth factors to leucine or isoleucine-deprived cells, however, permits maximal mTORC1 activation (Figure 1A–C and Figure 1—figure supplement 3A–D). Amino acids, particularly leucine, are known to signal to mTORC1 via the V-ATPase/Ragulator/Rag GTPase protein complexes and have been comprehensively demonstrated to control mTORC1 localization to the lysosome and its activity (Carroll et al., 2014). We observed that arginine did not affect mTOR localization in any cell line tested. While complete amino acid starvation and, to a lesser extent, leucine starvation, caused redistribution of mTORC1 to the cytoplasm, arginine deprivation led to a strong suppression of mTORC1 without a significant reduction in co-localization of mTOR with the lysosomal marker Lamp1 in HeLa, MEFs, and HEK293T cells (Figure 1D). Furthermore, overexpression of constitutively active Rag heterodimer (Sancak et al., 2008) did not completely rescue the effect of arginine starvation in the absence or presence of growth factors, thus suggesting that arginine may affect mTORC1 both via Rag-dependent and Rag-independent mechanisms (Figure 1—figure supplement 3E,F). Finally, the knockdown of membrane transporter, SLC38A9 that has recently been implicated in Rag GTPase-dependent lysosomal recruitment and activation of mTORC1 (Wang et al., 2015; Rebsamen et al., 2015; Jung et al., 2015) did partially perturb the recovery of mTORC1 following arginine starvation; however, there was no effect on the response of mTORC1 to arginine starvation either in the absence or presence of growth factors (Figure 1—figure supplement 3G).10.7554/eLife.11058.003Figure 1.Arginine is a permissive factor for growth factor-dependent mTORC1 activity and acts via TSC2-Rheb axis.

Bottom Line: Herein, we demonstrate that arginine acts independently of its metabolism to allow maximal activation of mTORC1 by growth factors via a mechanism that does not involve regulation of mTORC1 localization to lysosomes.Dependence on arginine is maintained once hESCs are differentiated to fibroblasts, neurons, and hepatocytes, highlighting the fundamental importance of arginine-sensing to mTORC1 signaling.Together, our data provide evidence that different growth promoting cues cooperate to a greater extent than previously recognized to achieve tight spatial and temporal regulation of mTORC1 signaling.

View Article: PubMed Central - PubMed

Affiliation: Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle upon Tyne, United Kingdom.

ABSTRACT
The mammalian target of rapamycin complex 1 (mTORC1) is the key signaling hub that regulates cellular protein homeostasis, growth, and proliferation in health and disease. As a prerequisite for activation of mTORC1 by hormones and mitogens, there first has to be an available pool of intracellular amino acids. Arginine, an amino acid essential during mammalian embryogenesis and early development is one of the key activators of mTORC1. Herein, we demonstrate that arginine acts independently of its metabolism to allow maximal activation of mTORC1 by growth factors via a mechanism that does not involve regulation of mTORC1 localization to lysosomes. Instead, arginine specifically suppresses lysosomal localization of the TSC complex and interaction with its target small GTPase protein, Rheb. By interfering with TSC-Rheb complex, arginine relieves allosteric inhibition of Rheb by TSC. Arginine cooperates with growth factor signaling which further promotes dissociation of TSC2 from lysosomes and activation of mTORC1. Arginine is the main amino acid sensed by the mTORC1 pathway in several cell types including human embryonic stem cells (hESCs). Dependence on arginine is maintained once hESCs are differentiated to fibroblasts, neurons, and hepatocytes, highlighting the fundamental importance of arginine-sensing to mTORC1 signaling. Together, our data provide evidence that different growth promoting cues cooperate to a greater extent than previously recognized to achieve tight spatial and temporal regulation of mTORC1 signaling.

No MeSH data available.


Related in: MedlinePlus