Fig 2: Model of the multifaceted response to ammonium.Ammonium triggers mTORC2 activation which phosphorylates and activates AKT in integrinβ1, YES1 (Src family kinase), and FAK (Focal adhesion kinase) dependent manner. In addition, activated AKT phosphorylates and inhibits TSC2 in the TSC complex and PRAS40, relieving the TSC-mediated repression of Rheb and Raptor-PRAS40 interaction respectively, thereby increasing mTORC1 signaling. Active mTORC1 phosphorylates and inhibits 4E-BP1. Once phosphorylated by mTORC1, 4E-BP1 releases eIF4E and stimulates translation initiation. We further propose that ammonium activates, yet to identify, phosphatases mediating the dephosphorylation of p70S6K1. In parallel to mTORC2 and mTORC1 stimulation, ammonium also promotes ERK signaling.

Fig 3: STAT6 interacts with Rheb and inhibits mTOR signaling under hypoxic conditions. a Schematic of inhibition of mTOR signaling under hypoxic conditions. b C U87MG cells transfected with non-targeting control siRNA (CONsi) or pooled siRNA targeting STAT6 (STAT6si) were exposed to 0% O2 for 18 h, followed by immunoblotting for STAT6 and p-AKT (b), and quantification of BNIP3, PML, and REDD1 mRNA by RT-qPCR (c). d Immunoblot of p-TSC2, TSC2, and STAT6 in cells exposed to 0% O2 for the indicated periods (left) and summary data (right) showing p-TSC2 levels, expressed as fold changes relative to that in CONsi-transfected cells exposed to 21% O2. e Immunoblot of Rheb in mTOR immunoprecipitates (IPs) from U87MG cells transfected with CONsi or STAT6si and exposed to 21% or 0% O2 for 10 h. f Immunoblot of STAT6 in mTOR IPs from U87MG cells exposed to 0% O2 for 6 h. g Immunoblot of Rheb in STAT6 IPs from U87MG cells exposed to 0% O2 for 10 h. IgG IPs were used as a negative control. h Immunoblot of Rheb in STAT6 IPs from U87MG cells exposed to 0% O2 for the indicated periods. i Immunoblot of Rheb in STAT6 IPs from U87MG cells exposed 0% O2 for 3 h (H3h), followed by reoxygenation for 12 h (H3h/R). j Top, schematic of STAT6 deletion mutants. α-H, a-helicase domain; DBD, DNA-binding domain; LD, linker domain; SH2: src homology 2 domain; TAD, transactivation domain. FL, full length; d1-d4, deletion of residues 1–260 (d1), 261–430 (d2), 431–522(d3), or 523–622 (d4), respectively. Bottom, immunoblot of Rheb in Myc-tagged STAT6 (Myc-STAT6) IPs from U373MG cells transfected with the indicated Myc-STAT6 deletions and exposed to 0% O2 for 6 h

Fig 4: PIs inhibit XR-elevated mTORC1 signaling via stabilizing DEPTOR(A) Western blot analysis of the levels of mTORC1 signaling pathway components and regulators in FaDu and Detroit 562 cells treated with PIs (DMSO as vehicle control) plus XR (NR as control) as in Figure S4B. (B, D–F, H) FaDu cells were treated with PT33 (20 nM), BTZ (10 nM), rapamycin (RAPA, 100 nM), or PT33 (20 nM) + rapamycin (100 nM) for 12 h (DMSO as vehicle control) and subsequently exposed to XR (6-Gy). (B) Western blot analysis of DEPTOR levels in FaDu and Detroit 562 cells treated with BTZ (top) or PT33 (bottom), followed by XR for indicated times. (Left) Representative images; (right) DEPTOR grayscale intensity normalized to GAPDH at the indicated times after XR treatment. (C) Western blot analysis of the DEPTOR levels in FaDu and Detroit 562 cells, at 2 h with CHX (50 μM) and PT33 (20 nM) treatment, subsequently exposed to XR, and collected cell lysis for the indicated times. (Left) Representative images. (Right) DEPTOR grayscale intensity normalized to GAPDH at indicated times after XR treatment. (D and E) Co-IP and Western blot analyses for detecting the K48-linked polyubiquitinated levels of DEPTOR. (D) Cells were transfected with HA-ubiquitin (K48-specific) and FLAG-DEPTOR followed by XR treatment, and collected cell lysis for the indicated times. IP with FLAG, IB with HA. (E) Cells were transfected with HA-ubiquitin (K48 specific) followed by XR treatment, and collected cell lysis for the indicated times. IP with HA, IB with endogenous DEPTOR. (F) Co-IP and Western blot analyses of the interaction between β-TrCP/CKI/mTOR and DEPTOR in FLAG-DEPTOR or Myc-β-TrCP overexpression cells treated with XR, and collected cell lysis for 6 h, NR as control. (G) 2D adherent colony formation assays in DEPTOR-KO and TSC2-KO cells exposed to a single 3- or 6-Gy dose of XR (mock or NC cells were used as control). (Top) Cell survival rates. (Bottom) The ratio of DEPTOR-KO or TSC2-KO to NC colony number. (H) Western blot analysis of the levels of mTORC1 signaling pathway components in DEPTOR-KO and TSC2-KO cells pretreated with BTZ, PT33, or rapamycin and subsequently exposed to XR, and collected cell lysis for 6 h. (I) 2D adherent colony formation assays for cell survival rate in DEPTOR-KO or WT FaDu cells pretreated with PT33 (10 nM), and consequently exposed to a single 3-Gy dose of XR treatment. (J) Cell viability was measured using the CCK-8 assay, and half-maximal inhibitory concentration (IC50) values were calculated in DEPTOR-KO or WT FaDu cells treated with PT33 plus XR-3Gy (NR as control). All statistical data are presented as means ± SDs (n = 3; B, G, and J: 1-way ANOVA; C and I: 2-sample t test). ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001; N.S., not significant.

Fig 5: Deficiency for Crk and Crkl results in aberrant glucose metabolism and Igf1 signaling.(A) Glucose availability in culture media throttled dose-dependent phosphorylation of ribosomal protein S6 (pS6-S240/244), as well as that of Akt (pAKT-S473), TSC2 (pTSC2-T1462), and p70 S6 kinase (pS6K-T389) as shown in immunoblots. MEFs were cultured with indicated concentrations of glucose for 24 h after a 24-h period of glucose restriction at a concentration of 0.1 mM, the lowest glucose concentration that MEFs can tolerate in the presence of 10% dialyzed FBS (Fig S8). As a point of reference, basal DMEM includes 5 mM glucose, whereas a high-glucose formula includes 25 mM glucose. (B) The glucose metabolism inhibitor 2-deoxy-D-glucose (2DG) induced a cell blebbing phenotype in a dose-dependent manner, and Crk/Crkl deficiency significantly exacerbated the frequency of the phenotype in single cell analysis. The 2DG concentrations are indicated under the x-axis. The frequency of the blebbing phenotype was determined as described in the Materials and Methods section and Fig S9. Large numbers of cells (n) were imaged in a high-content imaging apparatus, and individual cells were analyzed through a series of MATLAB scripts. Beneath the bar graph is a matrix table for P-values by Fisher’s exact tests adjusted for multiple comparisons (FDR). (C) Crk/Crkl deficiency increased Igf1 mRNA levels. The bar graph represents levels of Igf1 messages upon Crk/Crkl deficiency induction in MEFs, relative to that of their controls without deficiency induction. As a control, fold change of Rplp0 (60S ribosomal protein p0) is also shown. Two non-overlapping primer sets 1 and 2 (ps1 and ps2) were used to confirm up-regulation approximately at an order of magnitude greater. Error bars indicate standard deviations (n = 3). Red-dotted line shows the level in control samples set at a relative fold change of 1. (D) Crk/Crkl deficiency resulted in muted response to exogenous Igf1 over a range of doses in phosphorylation of p70 S6 kinase (S6K) and ribosomal protein S6. In contrast, Akt phosphorylation was induced by Igf1, suggesting a role of Crk and Crkl in a regulatory mechanism on S6K and S6. MEFs were cultured without serum for the last 3 h of 48 h post deficiency induction, then were stimulated with Igf1 at different concentrations indicated for 15 min. (E) Crk and Crkl played an important role in S6K and S6 phosphorylation on which both Igf1 and fibronectin (FN) cooperate as shown in immunoblots probed with different antibodies. Note that FN alone did not activate Akt (as seen in pAkt-S473), whereas Igf1 did. After 3 h serum starvation, MEFs were re-plated on FN or poly-L-lysine in serum-free medium for 60 min followed by incubation with Igf1 for 15 min before harvest.