Alkaline intracellular pH (pHi) increases mTORC1 and mTORC2 signaling through PI3K to promote protein synthesis and cell survival
Authors: Kazyken Dubek , Lentz Stephen , Wadley Maxwell , Fingar Diane C. 
Published: May 2022

The conserved kinase mTOR (mechanistic target of rapamycin) regulates cell metabolism and promotes cell growth, proliferation, and survival in response to diverse environmental cues (e.g., nutrients; growth factors; hormones). mTOR forms the catalytic core of two multiprotein complexes, mTORC1 and mTORC2, which possess unique downstream targets and cellular functions. While mTORC1 and mTORC2 often respond to distinct upstream cues, they share a requirement for PI3K in their activation by growth factors. While many studies agree that amino acids activate mTORC1 but not mTORC2, several studies reported paradoxical activation of mTORC2 by amino acids. We noted that stimulating amino acid starved cells with a commercial mixture of amino acids increased mTORC2‐dependent Akt S473 phosphorylation rapidly while re‐feeding cells with complete DMEM containing amino acids failed to do so. Interestingly, we found the pH of the commercial amino acid mixture to be ~ pH 10. Upon controlling for pH, stimulating starved cells with amino acids at pH 10 but not 7.4 increased mTORC2 signaling. Moreover, DMEM at alkaline pH was sufficient to increase mTORC2 catalytic activity and signaling. Using a fluorescent pH‐sensitive dye (cSNARF‐1‐AM) coupled to ratio‐metric live cell imaging, we confirmed that alkaline extracellular pH (pHe) translated into a rapid increase in intracellular pH (pHi). Moreover, blunting this increase with a pharmacological inhibitor of an H+ transporter attenuated the increase in mTORC2 signaling by pHe. Alkaline pHi also activated AMPK, a canonical sensor of energetic stress that promotes mTORC2 signaling, as reported previously by us. Functionally, we found that alkaline pHi attenuated apoptosis caused by growth factor withdrawal through activation of AMPK‐mTORC2 signaling. These results indicate that alkaline pHi augments mTORC2 signaling to promote cell survival, in part through AMPK. In the course of this work, we noted that pHi increased phosphorylation of several downstream targets of PI3K (e.g., Akt P‐T308 and P‐S473; S6K1 P‐T389 and P‐T229; PRAS40 P‐T246; Tsc2 P‐S939), suggesting that PI3K itself responds to changes in pHi. Indeed, alkaline pHi increased PI‐3’,4’,5’‐P3 levels in a manner sensitive to the PI3K inhibitor BYL‐719. Thus, alkaline pHi elevates PI3K activity, which increases both mTORC1 and mTORC2 signaling. Mechanistically, we found that activation of PI3K by alkaline pHi induced dissociation of Tsc2 from lysosomal membranes, thereby relieving TSC‐mediated suppression of Rheb, a mTORC1‐activating GTPase. Functionally, we found that activation of PI3K by alkaline pHi increased mTORC1‐mediated 4EBP1 phosphorylation, which initiates cap‐dependent translation by eIF4E. Alkaline pHi also increased mTORC1‐driven protein synthesis. Taken together, these findings reveal alkaline pHi as a previously unrecognized activator of PI3K‐mTORC1/2 signaling that promotes protein synthesis and cell survival. As elevated pHi represents an under‐appreciated hallmark of cancer cells, these findings suggest that by alkaline pHi sensing by the PI3K‐mTOR axis and AMPK‐mTORC2 axes may contribute to tumorigenesis.