Kazyken, D. | Search bioRxiv

bioRxiv2021

Alkaline intracellular pH activates AMPK-mTORC2 signaling to promote cell survival during growth factor limitation

mTORC2 controls cell metabolism and promotes cell survival, yet its upstream regulation by diverse cellular cues remains poorly defined. While considerable evidence indicates that mTORC1 but not mTORC2 responds dynamically to amino acid levels, several studies reported activation of mTORC2 signaling by amino acids, a paradox that remains unresolved. Following amino acid starvation, we noted that addition of a commercial amino acid solution but not re-feeding with DMEM containing amino acids increased mTORC2 signaling. Interestingly, the pH of the amino acid solution was [~] 10. These key observations enabled us to discover that alkaline intracellular pH (pHi) represents a previously unknown activator of mTORC2. Using a fluorescent pH-sensitive dye (cSNARF-1-AM) coupled to live-cell imaging, we demonstrate that alkaline extracellular pH (pHe) increases intracellular pHi, which increases mTORC2 catalytic activity and downstream signaling to Akt. Alkaline pHi also activates AMPK, a sensor of energetic stress. Functionally, alkaline pHi attenuates apoptosis caused by growth factor withdrawal, which requires AMPK in part and mTOR in full. Collectively, these findings reveal that alkaline pHi increases AMPK-mTORC2 signaling to promote cell survival during growth factor limitation. As elevated pHi represents an under-appreciated hallmark of cancer cells, alkaline pH sensing by AMPK-mTORC2 may contribute to tumorigenesis. One Sentence SummaryAlkaline intracellular pH activates mTORC2

cancer biology↗

bioRxiv2021

The innate immune kinase TBK1 directly increases mTORC2 activity and downstream signaling to Akt

TBK1 (TANK-binding kinase 1) responds to microbial pathogens to initiate cellular responses critical for host innate immune defense. We found previously that TBK1 phosphorylates mTOR (mechanistic target of rapamycin) (on S2159) to increase mTOR complex 1 (mTORC1) activity and signaling in response to the growth factor EGF and the viral dsRNA mimetic poly(I:C). mTORC1 and the less well studied mTORC2 respond to diverse cues to control cellular metabolism, proliferation, and survival. Here we demonstrate that TBK1 activates mTOR complex 2 (mTORC2) directly to increase Akt phosphorylation at physiological levels of protein expression. We find that TBK1 phosphorylates mTOR S2159 within mTORC2 in vitro, phosphorylates mTOR S2159 in cells, and interacts with mTORC2 in cells. By studying MEFs lacking TBK1, as well as MEFs, macrophages, and mice bearing an Mtor S2159A knock-in allele (MtorA/A), we show that TBK1 and mTOR S2159 phosphorylation increase mTORC2 catalytic activity and promote mTOR-dependent downstream signaling to Akt in response to several growth factors and poly(I:C). While microbial-derived stimuli activate TBK1, we find that growth factors fail to activate TBK1 or increase mTOR S2159 phosphorylation in MEFs. Thus, we propose that basal TBK1 activity cooperates with growth factors in parallel to increase mTORC2 (and mTORC1) signaling. Collectively, these results reveal crosstalk between TBK1 and mTOR complexes (mTORCs), key nodes within two major signaling systems. As TBK1 and mTORCs have each been linked to tumorigenesis and metabolic disorders, these kinases may work together in a direct manner in a variety of physiological and pathological settings. One Sentence SummaryThe innate immune kinase TBK1 directly activates mTORC2

biochemistry↗