Fig. 4.
Chemical validation of LRS role in the control of RagD GTPase and mTORC1. a HA-RagD WT or HA-ARF1 was transfected into SW620 cells. After 24 h, the cells were incubated with 100 μCi/ml 32P-orthophosphate for 8 h, starved for leucine for 90 min, and then re-stimulated with leucine for 10 min. Cells were treated with 10 μM BC-LI-0186 during leucine starvation and re-stimulation. HA-RagD or HA-ARF1 was immunoprecipitated with anti-HA antibody and the bound nucleotides were eluted and analyzed by TLC. GDP% means GDP/(GDP + GTP) × 100. b Effect of BC-LI-0186 and BC-LI-0198 on the leucine-induced change of GTP hydrolysis of RagD. GTP-agarose bead pull-down assays were used to monitor the GTP-bound RagD or ARF1. After cells were treated with 10 μM BC-LI-0186 for 1 h, cell lysates were pulled down with GTP-agarose beads and the precipitated proteins with the beads were analyzed by immunoblotting with anti-RagD and anti-ARF1 antibodies. c Effect of LRS WT or S974A overexpression on S6K phosphorylation inhibited by BC-LI-0186. Inducible LRS WT or S974A-overexpressed SW620 cells were treated with 10 μM BC-LI-0186 for 1 h. Cell lysates were analyzed by immunoblotting with anti-p-S6K (T389), anti-S6K, anti-LRS, and anti-actin antibodies. d Effect of LRS WT or S974A overexpression on BC-LI-0186-induced growth inhibition (d) and cell death (e) (see Supplementary Fig. 5a). The data in Supplementary Fig. 5a were quantified and displayed as bar graphs. The error bars represent mean ± S.D. (n = 3). f Effect of RagDGDP on the BC-LI-0186-dependent inhibition of GTP hydrolysis of RagD and S6K phosphorylation. SW620 cells were transfected with GDP mutant of RagD (S77L) and then starved for leucine for 1 h and re-stimulated with leucine for 10 min in the presence or absence of 10 μM BC-LI-0186. Cell lysates were pulled down with GTP-agarose beads and the precipitated proteins with the beads were analyzed by immunoblotting with anti-RagD antibody. g Effect of RagBGTP/RagDGDP on rapamycin or BC-LI-0186-dependent inhibition of S6K phosphorylation. SW620 cells were co-transfected with GDP mutant of RagD (S77L) and GTP mutant of RagB (Q99L) and then starved for leucine for 1 h and re-stimulated with leucine for 10 min in the presence or absence of 10 μM BC-LI-0186. h Effect of LRS overexpression on the kinetic changes of RagD and S6K phosphorylation that was arrested by BC-LI-0186 pretreatment. SW620 cells were treated with 10 μM BC-LI-0186 for 1 h. Then, cells were incubated with BC-LI-0186-free media for the indicated times. Cell lysates were pulled down with GTP-agarose beads and the precipitated proteins with the beads were analyzed by immunoblotting with anti-RagD or ARF1 antibodies (upper). Cell lysates were analyzed by immunoblotting with their specific antibodies (lower). The error bars represent mean ± S.D. (n = 3)