The metabolic waste ammonium regulates mTORC2 and mTORC1 signaling

Abstract

Two structurally and functionally distinct mammalian TOR complexes control cell growth and metabolism in physiological and pathological contexts including cancer. Upregulated glutaminolysis is part of the metabolic reprogramming occurring in cancer, providing fuels for growth but also liberating ammonium, a potent neurotoxic waste product. Here, we identify ammonium as a novel dose-dependent signal mediating rapid mTORC2 activation and further regulating mTORC1. We show that ammonium induces rapid RICTOR-dependent phosphorylation of AKT-S473, a process requiring the PI3K pathway and further involving the Src-family kinase YES1, the FAK kinase and the ITGβ1 integrin. Release of calcium from the endoplasmic reticulum store triggers rapid mTORC2 activation, similar to ammonium-induced activation, the latter being conversely prevented by calcium chelation.Moreover, in analogy to growth factors, ammonium triggers the AKT-dependent phosphoinhibition of the TSC complex and of PRAS40, two negative regulators of mTORC1. Consistent with mTORC1 stimulation, ammonium induces the inhibitory phosphorylation of 4EBP1, a negative regulator of protein biogenesis. Ammonium however dually impacts on the phosphorylation of p70S6K1 triggering a transient AKT-independent decrease in the phosphorylation of this second mTORC1 readout. Finally, we reveal ammonium as a dose-dependent stimulator of proliferation. This study underscores an mTORC2 and mTORC1 response to the so-called ammonium waste.

Figure 1. Ammonium induces dose and time dependent AKT phosphorylation.

(A) A phospho-proteins kinase array was probed with lysates of MCF-7 cells treated or not with NH₄Cl (5 mM) for 30 minutes. The dots corresponding to pAKT, pERK and p70S6K1 are indicated. (B) MCF-7, U2OS, HepG2, SW480 or BJ-1 cells were treated with NH₄Cl (5 mM) for the indicate time. (C) MCF-7 cells were treated with NH₄Cl (5 mM) for different time intervals. (D and E) MCF-7 cells were treated with the indicated NH₄Cl concentrations for 30 minutes. (F) MCF-7 cells were treated with NH₄OH (5 mM) for different time intervals. (G) MCF-7 cells were treated with the indicated NH₄OH concentrations for 30 minutes. Lysates were immunoblotted with the indicated antibodies. Uncropped images of immunoblots are shown in Supplementary Figure S1.

Figure 2. Ammonium-induced AKT phosphorylation is mTORC2-dependent.

(A) MCF-7 cells were pretreated with DMSO, 50 μM LY29004 or 1 μM Wortmannin for 2 hours prior to NH₄Cl (5 mM) addition for 30 minutes. (B) MCF-7 cells were transfected with scramble or RICTOR siRNA for 72 hours prior to NH₄Cl (5 mM) addition for 30 minutes. (C) MCF-7 cells were treated with NH₄Cl (5 mM) for 4 or 6 hours. (D) MCF-7 cells were treated with NH₄Cl (5 mM) for the indicated time intervals. (E) MCF-7 cells were transfected with scramble or RICTOR siRNA for 72 hours prior to NH₄Cl (5 mM) addition for 30 minutes. Lysates were immunoblotted with the indicated antibodies. Uncropped images of immunoblots are shown in Supplementary Figure S1.

Figure 3. Ammonium-induced mTORC2 activation is dependent on YES1, FAK kinases and integrin β1.

(A,B) MCF-7 cells were pretreated with DMSO, 10 μM Gefitinib, or 10 μM U0126 for 2 hours prior to NH₄Cl (5 mM) addition for 30 minutes. (C) MCF-7 cells were pretreated with DMSO or 10 μM PP2 for 2 hours prior to NH₄Cl (5 mM) addition for 30 or 60 minutes. (D,E) MCF-7 cells were transfected with scramble, YES1 or SRC siRNA for 72 hours prior to NH₄Cl (5 mM) addition for 30 minutes. (F,G) MCF-7 cells were pretreated with DMSO or 10 μM PF573228 for 2 hours prior to NH₄Cl (5 mM) addition for 30 minutes. (H,I) MCF-7 cells were transfected with scramble, integrin β1 (ITGB1) or ILK siRNA for 72 hours prior to NH₄Cl (5 mM) addition for 30 minutes. Lysates were immunoblotted with the indicated antibodies. Uncropped images of immunoblots are shown in Supplementary Figure S1.

Figure 4. Ammonium-induced mTORC2 activation is modulated by calcium.

(A,B) MCF-7 cells were pretreated with DMSO or 50 μM BAPTA for 1 hour prior to NH₄Cl (5 mM) addition for 30 minutes. (C) MCF-7 cells were treated with Thapsigargin (TG) for the indicated times. (D) MCF-7 cells were transfected with scramble or RICTOR siRNA for 72 hours prior to TG addition for 30 minutes. Lysates were immunoblotted with the indicated antibodies. Uncropped images of immunoblots are shown in Supplementary Figure S1.

Figure 5. Ammonium induced mTORC1 kinase activation in AKT dependent manner.

(A) MCF-7 cells were treated with NH₄Cl (5 mM) for different time intervals. (B) MCF-7 cells were treated with NH₄OH (5 mM) for different time intervals. (C) MCF-7 cells were pretreated with DMSO or 1 μM MK2206 for 10 minutes prior to NH₄Cl (5 mM) addition for different time intervals. Lysates were immunoblotted with the indicated antibodies. Uncropped images of immunoblots are shown in Supplementary Figure S1.

Figure 6. Ammonium stimulates cell proliferation in a dose-dependent manner.

(A) MCF-7 cells were treated with the indicated NH₄Cl concentrations for 3 or 5 days and proliferation was determined by crystal violet staining. (B) Cells were either in culture medium with 2 mM glutamine or glutamine-free medium and supplemented with the indicated concentration of NH₄Cl. Cell growth was determined by crystal violet staining.

Figure 7. 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.