NHE1-dependent regulation of cytosolic pH and the impact on mTORC1 activation

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Author
Date
2021Type
- Doctoral Thesis
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yes
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Abstract
Mammalian cells regulate and maintain their cytosolic pH to a narrow, slightly alkaline, pH range. Although fluctuations are small, changes in cytosolic pH has been shown to regulate cell growth and cell cycle progression through unknown mechanisms. These processes rely on growth factor signaling, for example via the Ser/Thr protein kinase Akt, and the availability of nutrients such as glucose. Interestingly, we show that both glucose and Akt are potent regulators of cytosolic pH, which in most cell types depends on the sodium/hydrogen-exchanger isoform 1 (NHE1). Akt has been shown monophosphorylate NHE1 directly at Ser648 in vitro, but the functional impact on NHE1 is unclear. To further investigate, we overexpressed wild type NHE1 (NHE1 WT) an NHE1 mutant (NHE1 S648A), which is nonphosphorylatable at the Akt phosphorylation site, in human cell cultures genetically depleted of endogenous NHE1 by siRNA. Expression of NHE1 S648A produced a cell proliferation defect compared to NHE1 WT but we could not detect changes in steady-state cytosolic pH or NHE1 localization within the cell. Using an antibody against phosphorylated Akt motives, we show that Akt does not phosphorylate NHE1 immunoprecipitated from cells. We thus propose a hypothetical model for indirect ATP-dependent regulation of NHE1 by Akt through glucose metabolism. Upon pharmacological inhibition of NHEs or NHE1 depletion, cytosolic pH drops, leading to G1 cell arrest and attenuation of mTORC1 activity, a major cell growth regulator. We discovered that NHE-inhibition prevents Cyclin D1 transcription and loss of CDK4/6 activity required for G1 to S-phase transition. Cytosolic pH was found to regulate the binding of histone acetyltransferase P300/CBP to transcription factor CREB1 at the Cyclin D1 promoter site. P300/CBP binding to CREB1 occurs at elevated cytosolic pH and acts as a pH sensor possibly through the protonation state of a phosphoserine on CREB1. Further, we discovered that cytosolic pH does not regulate mTORC1 through the canonical energy-sensing AMPK or growth factor-activated Akt signaling. Both AMPK and Akt regulate mTORC1 through phosphorylation of TSC2. Active TSC2 inhibits Rheb, which is needed for full mTORC1 activation. Interestingly, we could show that cytosolic pH regulates mTORC1 by the same mechanism as for cell cycle progression. We found that NHE1 inhibition regulates CDK-dependent inhibitory phosphorylation of TSC2 through Cyclin D1 expression. Our preliminary data show that overexpression of hyperactivated Rheb rendered mTORC1 insensitive to NHE1 inhibition. Our findings highlight cytosolic pH as a potent regulator of both cell growth and proliferation. Indeed, dysregulation of cytosolic pH has become a hallmark of cancer cells, which generally display elevated cytosolic pH compared to normal cells. We further show that elevated cytosolic pH increases Cyclin D1 expression in malignant pleural mesotheliomas (MPMs) and is important for activating mTORC1 in MPM cell lines. Targeting elevated cytosolic pH as a cancer treatment could be of value for future therapeutic development. Show more
Permanent link
https://doi.org/10.3929/ethz-b-000514036Publication status
publishedExternal links
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Contributors
Examiner: Dechant, Reinhard Christoph
Examiner: Peter, Matthias
Examiner: Staub, Olivier
Examiner: Christofori, Gerhard
Publisher
ETH ZurichOrganisational unit
03532 - Barral, Yves / Barral, Yves
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yes
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