Fig 2: A Phenotypic Screen with Tsc2-KD Neurons Identifies Hsp90 as Drug Target for mTORC1 through Regulation of PI3K/Akt Signaling Components(A) mTORC1 screen workflow. Tsc2-sh neurons were treated with screen compounds for 24 h between DIV19 and DIV20.(B) Z score correlation between compound Replica 1 and Replica 2. Percent of GFP+pS6+ neurons was converted to Z score. Z score of -1.8 (p < 0.05) was marked by the red lines. Hits were considered compounds with a Z score < -1.8 in the dotted black box. Compounds used for follow-up experiments are rapamycin (in red), GA (in green), and 17-AGG (in blue); all other compounds are labeled in gray.(C) Zoom of hits in the dotted black box (B).(D) List of hits, Z scores, and compound concentration in the library.(E and F) Validation of GA and 17-AGG by dose-response using the mTORC1HCA. Tsc2-sh neurons were treated with vehicle and with a dose-curve of GA (E) or 17-AGG (F) for 24 h between DIV19 and DIV20. Data are GFP+ ps6+ neurons expressed as average percentage of vehicle-treated Tsc2-sh neurons (n = 7–16 biological replica/condition; one-way ANOVA eith Dunnetts multiple comparison test, **p < 0.01, ***p < 0.005, ****p < 0.0001). Error bars indicate ± SEM.(G) Western blot of protein lysates from ctrl-sh and Tsc2-sh treated with vehicle or with 17-AGG dose-curve.(H–J) Quantification of pS6 (H), total IGF-IRb (I), and p-IGF-IRß (J) expressed as average fold changes of vehicle-treated ctrl-sh neurons. Western blot data were normalized using GAPDH as loading control. Data quantification relative to vehicle-treated Tsc2-sh neurons (n = 3–4 experiment/condition; one-way ANOVA with Dunnett’s multiple comparison test, *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.0001). Error bars indicate ± SEM.(K) Hsp90 inhibition results in IGF-IRß degradation through the proteasome. Wild-type cortical neurons were treated with vehicle and with 4-µM 17-AGG alone or in the presence of 100-nM bortezomib (BTZ, 24 h at DIV20).(L) Quantification of western blot (n = 5; one-way ANOVA with Tukey’s multiple comparison test, *p < 0.05, **p < 0.005). Western blot data were normalized using GAPDH as loading control. Data are mean ± SEM.See also Figure S3 and Table S3.

Fig 3: In vitro methylation assay of PRMT1-mediated methylation of TSC2 at R1457 and R1459. (A) Predicted arginine methylation sites in TSC2 determined using the iPTMnet search tool. (B) Methylation at predicted sites analysed by incubating GST-TSC2 fusion peptides with [3H]-SAM and recombinant PRMT1. Methylation was detected by autoradiography. The CBB staining for each GST-TSC2 peptide was quantified by ImageJ. (C) Confirmation of TSC2 methylation at R1457 and R1459 of TSC2 by analysis of methylation of the wild-type GST-TSC2 (aa 1432–1469) and mutated peptides having single (R1457A or R1459A) or double (R1457A/R1459A) arginine to alanine substitutions performed as described for (C). CBB: Coomassie Brilliant Blue. The CBB staining for each GST-TSC2 peptide was quantified by ImageJ.

Fig 4: PRMT1 binds and methylates intact TSC2. (A) Autoradiography and western blot analysis. Methylation of intact TSC2 in 293 T cells expressing wild-type TSC2-overexpressing CHX (40 µg/µL) and chloramphenicol (40 µg/µL) cultured in medium containing [3H]-Met with or without the PRMT1 inhibitor ADOX/MTA (20 µM). TSC2 was immunoprecipitated (IP) and TSC2 methylation was detected by autoradiography. Levels of precipitated TSC2 were assessed by western blotting (IB; left panel). The effect of MTA was confirmed by measuring total protein methylation (upper panel), and TSC2 expression was confirmed by IB (right panel). (B) Lysates from 293 T cells treated with complete media (comp) or methionine deprivation media (?Met) were immunoprecipitated and immunoblotted with an anti-TSC2 antibody or anti-dimethyl arginine antibody (ADMA). (C) Lysates from 293 T cells transfected with Myc-PRMT1 and HA-TSC2 were immunoprecipitated with anti-HA antibody and subsequently immunoblotted with anti-Myc antibody. (D) Binding of TSC2 to PRMT1 was confirmed using 293 T cells transfected with Myc-PRMT1 and HA-TSC2. Cell lysates were immunoprecipitated with an anti-HA antibody and subsequently immunoblotted with an anti-Myc antibody. (E) Lysates from 293 T cells were immunoprecipitated with the anti-PRMT1 antibody or anti-TSC2 antibody and subsequently immunoblotted with anti-TSC2 antibody or anti-dimethyl arginine antibody.

Fig 5: Proposed model of PRL-3 activation of mTORC1.PRL-3 activates mTORC1 activity through (1) Akt/TSC2/Rheb hyperactivation, and (2) enhanced binding to Rag GTPases and lysosomal recruitment. PRL-3-driven mTOR activation ultimately results in increased motility, invasion and MMP-2 secretion of cancer cells.