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. 2018 Jun 5;115(23):E5279-E5288.
doi: 10.1073/pnas.1801287115. Epub 2018 May 21.

Coordination of the Leucine-Sensing Rag GTPase Cycle by leucyl-tRNA Synthetase in the mTORC1 Signaling Pathway

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Free PMC article

Coordination of the Leucine-Sensing Rag GTPase Cycle by leucyl-tRNA Synthetase in the mTORC1 Signaling Pathway

Minji Lee et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

A protein synthesis enzyme, leucyl-tRNA synthetase (LRS), serves as a leucine sensor for the mechanistic target of rapamycin complex 1 (mTORC1), which is a central effector for protein synthesis, metabolism, autophagy, and cell growth. However, its significance in mTORC1 signaling and cancer growth and its functional relationship with other suggested leucine signal mediators are not well-understood. Here we show the kinetics of the Rag GTPase cycle during leucine signaling and that LRS serves as an initiating "ON" switch via GTP hydrolysis of RagD that drives the entire Rag GTPase cycle, whereas Sestrin2 functions as an "OFF" switch by controlling GTP hydrolysis of RagB in the Rag GTPase-mTORC1 axis. The LRS-RagD axis showed a positive correlation with mTORC1 activity in cancer tissues and cells. The GTP-GDP cycle of the RagD-RagB pair, rather than the RagC-RagA pair, is critical for leucine-induced mTORC1 activation. The active RagD-RagB pair can overcome the absence of the RagC-RagA pair, but the opposite is not the case. This work suggests that the GTPase cycle of RagD-RagB coordinated by LRS and Sestrin2 is critical for controlling mTORC1 activation, and thus will extend the current understanding of the amino acid-sensing mechanism.

Keywords: GTPase-activating protein; Rag GTPase; Sestrin2; leucyl-tRNA synthetase; mTORC1.

Conflict of interest statement

Conflict of interest statement: Paul Schimmel and M.G. have coauthored papers, most recently in 2014.

Figures

Fig. 1.
Fig. 1.
Correlation of LRS expression with hyperactive mTORC1 in cancer cells. (AC) Boxplots showing the expression level of LARS in colon adenoma (A; P = 2.37E−8), colon carcinoma (B; P = 6.23E−10), and rectosigmoid adenocarcinoma (C; P = 1.07E−6). LARS data were extracted from the Oncomine database and expressed as log2 median-centered intensity. (D) Heatmap of gene expression of LARS and MTOR pathway genes in TCGA dataset (n = 433). (E) Fold change of the selected genes LARS, MTOR, RHEB, RPTOR, RRAGA, RRAGB, RRAGC, RRAGD, and TSC1 in mTOR pathway genes. (F) Immunostaining of LRS in colorectal tumor and normal tissues with anti-LRS antibody. Representative images for strong, weak, or negative LRS staining are shown. Case numbers indicate different patients. (G) Intensity scores of LRS staining in colorectal tumor or normal tissues are shown as circle graphs. Scores 3 (purple) or 2 (green), 1 (red), and 0 (blue) stand for strong, weak, and negative LRS staining, respectively (n = 117). (H) Consecutive tissue images were stained for LRS or S6 phosphorylation. Representative images for strong, weak, or negative LRS and S6 phosphorylation staining are shown. (Scale bar, 100 μm.) (I) Total number is shown as a table with low and high staining for LRS or S6 phosphorylation. (J) Correlation between cellular levels of LRS and S6K phosphorylation shown in SI Appendix, Fig. S1G is displayed as a scatterplot and evaluated by a Pearson correlation coefficient. (K) Correlation between cellular levels of LRS and 4E-BP1 phosphorylation shown in SI Appendix, Fig. S1G is displayed as a scatterplot and evaluated by a Pearson correlation coefficient. (L) Heatmap of the protein intensity ratio of tumor/normal tissues shown in SI Appendix, Fig. S1J. Blue indicates the ratio of tumor/normal tissues is below 0.8. Red indicates the ratio is higher than 1.2, and gray indicates the ratio is between 0.8 and 1.2.
Fig. 2.
Fig. 2.
Distinct roles of LRS and Sestrin2 in the Rag GTPase–mTORC1 axis. (A) SW620 cells transfected with HA-RagDWT, RagBWT, or ARF1WT were labeled with 100 μCi/mL [32P]orthophosphate for 8 h, starved of amino acids or leucine for 90 min, and then restimulated with amino acids or leucine for 10 min. The bound nucleotides of the precipitated HA-tagged proteins were eluted and analyzed by TLC (Upper). GDP (%) and GTP (%) indicate GDP/(GDP + GTP) × 100 and GTP/(GDP + GTP) × 100, respectively (Lower). IP, immunoprecipitation. (B) Specificity of the GTP-conjugated agarose bead method. GTPγS or GDPβS (100 μM) was used to confirm the binding specificity of the beads. (C) HA-RagDGTP (DGTP, Q121L), RagDGDP (DGDP, S77L), RagCGTP (CGTP, Q120L), RagCGDP (CGDP, S75L), RagBGTP (BGTP, Q99L), RagBGDP (BGDP, T54L), RagAGTP (AGTP, Q66L), or RagAGDP (AGDP, T21L) was transfected into SW620 cells. HA-tagged proteins were immunoprecipitated and then the precipitated proteins were analyzed by immunoblotting with the indicated antibodies. (D and E) Effect of LRS, Sestrin2, or EPRS knockdown (D) and effect of LRS, Sestrin2, or EPRS overexpression (E) on Rag GTPases and S6K phosphorylation. (F and G) DOX-inducible LRS SW620 cells were untreated (Con) or treated with DOX (LRS). Cells were incubated with 20 μM BC-LI-0186 for 90 min and then deprived of BC-LI-0186 for the indicated times. Relative intensity of GTP-loaded RagD (RagDGTP) (F) or GTP-loaded RagB (RagBGTP) (G) in SI Appendix, Fig. S2D was normalized to ARF1 and quantified with respect to 0 min. (H and I) DOX-inducible sh-LRS SW620 cells were untreated (Con) or treated with DOX (sh-LRS). Relative intensity of RagDGTP (H) or RagBGTP (I) in SI Appendix, Fig. S2E was quantified. (J and K) SW620 cells were transfected with control or Sestrin2 cDNA for 24 h. Relative intensity of RagDGTP (J) or RagBGTP (K) in SI Appendix, Fig. S2F was quantified. (L and M) SW620 cells were transfected with control or Sestrin1/2 siRNA, incubated with 20 μM BC-LI-0186 for 90 min, and then deprived of BC-LI-0186 for the indicated times. Relative intensity of RagDGTP (L) or RagBGTP (M) in SI Appendix, Fig. S2G was quantified.
Fig. 3.
Fig. 3.
Kinetics of the Rag GTPase cycle in amino acid signaling. (A and B) SW620 cells were starved of amino acids (A) or leucine (B) for 90 min and restimulated with amino acids or leucine for 13 min. Relative intensities of RagDGTP, RagBGTP, RagCGTP, RagAGTP, and p-S6K in SI Appendix, Fig. S3 A and B are shown. (C and D) SW620 cells were starved of amino acids (C) or leucine (D) for 100 min. Relative intensities of RagDGTP, RagBGTP, RagCGTP, RagAGTP, and p-S6K in SI Appendix, Fig. S3 C and D are shown. (E) Schematic representation for the kinetic model of the RagD–RagB GTPase cycle during leucine signaling. (F) Effects of an inactive (RagDGTP–RagBGDP), intermediate (RagDGDP–RagBGDP), or active (RagDGDP–RagBGTP) pair on Rag GTPases and S6K phosphorylation. (G and H) SW620 cells were starved of glutamine for 100 min and restimulated with glutamine for 60 min (G) or starved of glutamine for 100 min (H). Relative intensities of RagDGTP, RagBGTP, RagCGTP, RagAGTP, and p-S6K in SI Appendix, Fig. S3 E and F are shown. (I and J) SW620 cells were starved of arginine for 100 min and restimulated with arginine for 60 min (I) or starved of arginine for 100 min (J). Relative intensities of RagDGTP, RagBGTP, RagCGTP, RagAGTP, and p-S6K in SI Appendix, Fig. S3 G and H are shown.
Fig. 4.
Fig. 4.
Dominant role of the RagD–RagB heterodimer in leucine signaling. (A and B) Interaction of endogenous RagD with RagB. SW620 cell lysates were subjected to immunoprecipitation with anti-RagD, -RagC, or -Rab1A antibodies (A) or with anti-RagB, -RagA, or -Rab1A antibodies (B). Coimmunoprecipitation was confirmed by immunoblotting with the indicated antibodies. (C) SW620 cells transfected with control or siRNA against RagA, B, C, or D were starved of leucine for 90 min and restimulated for 10 min. The proteins precipitated with GTP-conjugated agarose beads were analyzed by immunoblotting with the indicated antibodies. (D and E) SW620 cells were transfected with Myc-RagDGTP, Myc-RagDGDP, Myc-RagCGTP, or Myc-RagCGDP (D) or with Myc-RagBGTP, Myc-RagBGDP, Myc-RagAGTP, or Myc-RagAGDP (E). The proteins precipitated with GTP-conjugated agarose beads were analyzed by immunoblotting with the indicated antibodies. (F) Dox-inducible sh-LRS SW620 cells were untreated (Con) or treated with DOX (sh-LRS). Cells were starved of leucine for 90 min and restimulated with leucine for 10 min. Cell lysates were incubated with GTP-conjugated agarose beads in the presence of 100 μM GTPγS or GDPβS. (G) Effect of BC-LI-0186 on the leucine-induced change of Rag GTPases. Cells were treated with 20 μM BC-LI-0186 for 1 h and then cell lysates were incubated with GTP-conjugated agarose beads, and the precipitated proteins were analyzed by immunoblotting with the indicated antibodies. (H and I) Dominant effect of the RagD–RagB pair on S6K phosphorylation. Normalized protein intensity graph of SI Appendix, Fig. S4C (H) or SI Appendix, Fig. S4D (I). Phosphorylated S6K was normalized to total S6K and quantified with respect to 10 or 12 min of the control group, respectively. (J) SW620 cells were transfected with si-RagA/si-RagC or si-RagD/si-RagB. After 24 h, cells were retransfected with active Rag GTPase (Myc-RagCGDP–HA-RagAGTP or Myc-RagDGDP–HA-RagBGTP). Cells were starved of leucine for 90 min and restimulated for 10 min. Cell lysates were immunoblotted with the indicated antibodies. (K and L) SW620 cells harboring Dox-inducible sh-LRS (K) or LRS (L) were untreated (Con) or treated with DOX. Cells were transfected with active Rag GTPase (Myc-RagDGDP–HA-RagBGTP or Myc-RagCGDP–HA-RagAGTP) (K) or inactive Rag GTPase (Myc-RagDGTP–HA-RagBGDP or Myc-RagCGTP–HA-RagAGDP) (L). Cells were starved of leucine for 90 min and restimulated for 10 min. Cell lysates were analyzed with the indicated antibodies.
Fig. 5.
Fig. 5.
Coordination of Rag GTPase by LRS and Sestrin2. (A and B) SW620 cells with inducible LRS overexpression were untreated (Con) or treated with DOX (LRS) and transfected with control or Sestrin1/2 siRNA (A) or with control or Sestrin2 cDNA (B). Cells were then starved of leucine for 90 min and restimulated with leucine for 10 min. The proteins precipitated with GTP-conjugated agarose beads were analyzed by immunoblotting with the indicated antibodies. (C and D) SW620 cells with inducible LRS shRNA were untreated (Con) or treated with DOX (sh-LRS) and transfected with control or Sestrin1/2 siRNA (C) or with control or Sestrin2 cDNA (D). Cells were then starved of leucine for 90 min and restimulated with leucine for 10 min. The proteins precipitated with GTP-conjugated agarose beads were analyzed by immunoblotting with the indicated antibodies. (E) Effect of LRS and Sestrin2 overexpression on cell growth. SW620 cells with inducible LRS overexpression were untreated (Con) or treated with DOX (LRS) and transfected with control or Sestrin2 cDNA. Cell growth was quantified and displayed as bar graphs. The error bars represent mean ± SD (n = 3). (F) Effect of LRS and Sestrin2 knockdown on cell growth. SW620 cells with inducible LRS knockdown were untreated (Con) or treated with DOX (sh-LRS) and transfected with control or Sestrin1/2 siRNA. Cell growth was quantified and displayed as bar graphs. The error bars represent mean ± SD (n = 3). (G) Size distributions of cells transfected with control, si-LRS, si-Sestrin1/2, or a combination of si-LRS and si-Sestrin1/2. Representative data from three independent experiments (Upper). Cell size distributions (Forward scatter-FSC of G1 cells) were quantified (Lower). The error bars represent mean ± SD (n = 3).
Fig. 6.
Fig. 6.
Ragulator mediation of the interplay between LRS and Sestrin2 in the Rag GTPase cycle. (A) The effect of Ragulator knockdown on Rag GTPases and S6K phosphorylation was compared in LRS-normal (Con) and -high (LRS) SW620 cells. SW620 cells with inducible LRS overexpression were untreated (Con) or treated with DOX (LRS) and transfected with control or LAMTOR2 siRNA. Cells were then starved of leucine for 90 min and restimulated with leucine for 10 min. The proteins precipitated with GTP-conjugated agarose beads were analyzed by immunoblotting with the indicated antibodies. (B) SW620 cells were transfected with si-control (si-con) or si-LAMTOR2 in combination with control (Con), Myc-RagDGDP, or Myc-RagDGTP. Cells were starved of leucine for 90 min and restimulated with leucine for 10 min. The proteins precipitated with GTP-conjugated agarose beads were analyzed by immunoblotting with the indicated antibodies. (C) The effect of Ragulator knockdown on Rag GTPases and S6K phosphorylation was compared in si-control (si-con) or si-Sestrin1/2–transfected SW620 cells. (D) The effect of LAMTOR2 knockdown and Sestrin1/2 knockdown on Rag GTPases and S6K phosphorylation were compared in LRS-normal (Con) and -high (LRS) SW620 cells. SW620 cells with inducible LRS overexpression were untreated (Con) or treated with DOX (LRS) and transfected with si-control, si-LAMTOR2, or si-Sestrin1/2 and si-LAMTOR2. Then, cells were starved of leucine for 90 min and restimulated with leucine for 10 min. The proteins precipitated with GTP-conjugated agarose beads were analyzed by immunoblotting with the indicated antibodies. (E) SW620 cells were transfected with si-control (si-con) or si-LAMTOR2 in combination with control (Con), Myc-RagBGTP, or Myc-RagBGDP. Cells were starved of leucine for 90 min and restimulated with leucine for 10 min. The proteins precipitated with GTP-conjugated agarose beads were analyzed by immunoblotting with the indicated antibodies. (F) Schematic representation for the coordination model of the Rag GTPase cycle by LRS, Ragulator, and Sestrin2.
Fig. 7.
Fig. 7.
GATOR complexes mediate Rag GTPase regulation by Sestrin2. (A) The effect of GATOR1 knockdown on Rag GTPases and S6K phosphorylation was compared in control (Con) or Sestrin2-transfected SW620 cells. SW620 cells transfected with si-DEPDC5 and Sestrin2 were starved of leucine for 90 min and restimulated with leucine for 10 min. The proteins precipitated with GTP-conjugated agarose beads were analyzed by immunoblotting with the indicated antibodies. (B) The effect of GATOR1 knockdown on Rag GTPases and S6K phosphorylation was compared in si-control (si-con) or si-Sestrin1/2–transfected SW620 cells. SW620 cells transfected with si-DEPDC5 and si-Sestrin1/2 were starved of leucine for 90 min and restimulated with leucine for 10 min. The proteins precipitated with GTP-conjugated agarose beads were analyzed by immunoblotting with the indicated antibodies. (C) The effect of GATOR2 knockdown on Rag GTPases and S6K phosphorylation was compared in control (Con) or Sestrin2-transfected SW620 cells. SW620 cells transfected with si-WDR24 and Sestrin2 were starved of leucine for 90 min and restimulated with leucine for 10 min. The proteins precipitated with GTP-conjugated agarose beads were analyzed by immunoblotting with the indicated antibodies. (D) The effect of GATOR2 knockdown on Rag GTPases and S6K phosphorylation was compared in control (si-con) or si-Sestrin1/2–transfected SW620 cells. SW620 cells transfected with si-WDR24 and si-Sestrin1/2 were starved of leucine for 90 min and restimulated with leucine for 10 min. The proteins precipitated with GTP-conjugated agarose beads were analyzed by immunoblotting with the indicated antibodies. (E) The effect of GATOR1 knockdown on Rag GTPases and S6K phosphorylation was compared in LRS-normal (Con) and -high (LRS) SW620 cells. SW620 cells with inducible LRS overexpression were untreated (Con) or treated with DOX (LRS) and transfected with si-DEPDC5. Cells were then starved of leucine for 90 min and restimulated with leucine for 10 min. The proteins precipitated with GTP-conjugated agarose beads were analyzed by immunoblotting with the indicated antibodies. (F) The effect of GATOR2 knockdown on Rag GTPases and S6K phosphorylation was compared in LRS-normal (Con) and -high (LRS) SW620 cells. SW620 cells with inducible LRS overexpression were untreated (Con) or treated with DOX (LRS) and transfected with si-WDR24. Cells were then starved of leucine for 90 min and restimulated with leucine for 10 min. The proteins precipitated with GTP-conjugated agarose beads were analyzed by immunoblotting with the indicated antibodies. (G) The effects of the knockdown of Sestrin1/2, LAMTOR2, or DEPDC5 on cell growth were compared in LRS-normal (Con) and -high (LRS) SW620 cells. The error bars represent mean ± SD (n = 3). (H) Proposed model for the Rag GTPase cycle controlled by LRS, Ragulator, and the Sestrin2–GATOR2–GATOR1 pathway during leucine signaling.

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