Alkaline intracellular pH (pHi) activates AMPK–mTORC2 signaling to promote cell survival during growth factor limitation

D Kazyken,S.I Lentz,D.C Fingar

doi:10.1016/j.jbc.2021.101100

D Kazyken, S.I Lentz + Show 1 more

Open Access

https://doi.org/10.1016/j.jbc.2021.101100

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Abstract

The mechanistic target of rapamycin (mTOR) complex 2 (mTORC2) signaling controls cell metabolism, promotes cell survival, and contributes to tumorigenesis, yet its upstream regulation remains poorly defined. Although considerable evidence supports the prevailing view that amino acids activate mTOR complex 1 but not mTORC2, several studies reported paradoxical activation of mTORC2 signaling by amino acids. We noted that after amino acid starvation of cells in culture, addition of an amino acid solution increased mTORC2 signaling. Interestingly, we found the pH of the amino acid solution to be alkaline, ∼pH 10. These observations led us to discover and demonstrate here that alkaline intracellular pH (pHi) represents a previously unknown activator of mTORC2. Using a fluorescent pH-sensitive dye (cSNARF1-AM) coupled with live-cell imaging, we demonstrate that culturing cells in media at an alkaline pH induces a rapid rise in the pHi, which increases mTORC2 catalytic activity and downstream signaling to the pro-growth and pro-survival kinase Akt. Alkaline pHi also activates AMPK, a canonical sensor of energetic stress. Functionally, alkaline pHi activates AMPK-mTOR signaling, which attenuates apoptosis caused by growth factor withdrawal. Collectively, these findings reveal that alkaline pHi increases mTORC2- and AMPK-mediated signaling to promote cell survival during conditions of growth factor limitation, analogous to the demonstrated ability of energetic stress to activate AMPK–mTORC2 and promote cell survival. As an elevated pHi represents an underappreciated hallmark of cancer cells, we propose that the alkaline pHi stress sensing by AMPK–mTORC2 may contribute to tumorigenesis by enabling cancer cells at the core of a growing tumor to evade apoptosis and survive.

Highlights

The mechanistic target of rapamycin comprises the catalytic core of two distinct multiprotein complexes, mTOR complex 1 and mTOR complex 2
When mouse embryonic fibroblasts (MEFs) incubated in Dulbecco’s PBS (D-PBS)/dialyzed fetal bovine serum (FBS) (dFBS) were stimulated with the alkaline amino acid solution or refed with D-PBS/dFBS adjusted to pH 9.5, we found that alkaline extracellular pH increased mTOR complex 2 (mTORC2) signaling in a manner partly dependent on AMPK (Fig. 4, A and B)
This study identifies the alkaline intracellular pH (pHi) as a previously unrecognized activator of AMPK and mTORC2 that attenuates apoptosis during growth factor limitation (Fig. 4F)

Summary

Introduction

The mechanistic target of rapamycin (mTOR) comprises the catalytic core of two distinct multiprotein complexes, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). While studying the mTORC1 response to amino acids, we noted that after starving cells of amino acids in D-PBS (which contains glucose) with or without dialyzed fetal bovine serum (FBS) (dFBS), addition of a commercial amino acid solution increased mTORC2 signaling, as measured by the sensitivity of Akt S473 phosphorylation to the mTOR inhibitor Torin 1 (Fig. 1A, left and right panels).

Results

Conclusion