mTORC1 feedback to AKT modulates lysosomal biogenesis through MiT/TFE regulation
Kaushal Asrani, … , Michael Skaro, Tamara L. Lotan
Kaushal Asrani, … , Michael Skaro, Tamara L. Lotan
Published December 2, 2019; First published September 17, 2019
Citation Information: J Clin Invest. 2019;129(12):5584-5599. https://doi.org/10.1172/JCI128287.
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Categories: Research Article Metabolism Oncology

mTORC1 feedback to AKT modulates lysosomal biogenesis through MiT/TFE regulation

Abstract

The microphthalmia family of transcription factors (MiT/TFEs) controls lysosomal biogenesis and is negatively regulated by the nutrient sensor mTORC1. However, the mechanisms by which cells with constitutive mTORC1 signaling maintain lysosomal catabolism remain to be elucidated. Using the murine epidermis as a model system, we found that epidermal Tsc1 deletion resulted in a phenotype characterized by wavy hair and curly whiskers, and was associated with increased EGFR and HER2 degradation. Unexpectedly, constitutive mTORC1 activation with Tsc1 loss increased lysosomal content via upregulated expression and activity of MiT/TFEs, whereas genetic deletion of Rheb or Rptor or prolonged pharmacologic mTORC1 inactivation had the reverse effect. This paradoxical increase in lysosomal biogenesis by mTORC1 was mediated by feedback inhibition of AKT, and a resulting suppression of AKT-induced MiT/TFE downregulation. Thus, inhibiting hyperactive AKT signaling in the context of mTORC1 loss-of-function fully restored MiT/TFE expression and activity. These data suggest that signaling feedback loops work to restrain or maintain cellular lysosomal content during chronically inhibited or constitutively active mTORC1 signaling, respectively, and reveal a mechanism by which mTORC1 regulates upstream receptor tyrosine kinase signaling.

Authors

Kaushal Asrani, Sanjana Murali, Brandon Lam, Chan-Hyun Na, Pornima Phatak, Akshay Sood, Harsimar Kaur, Zoya Khan, Michaël Noë, Ravi K. Anchoori, C. Conover Talbot Jr., Barbara Smith, Michael Skaro, Tamara L. Lotan

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Figure 2

mTORC1 hyperactivation in Tsc1-cKO epidermis and keratinocytes downregulates EGFR and HER2 protein expression and activity.

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mTORC1 hyperactivation in Tsc1-cKO epidermis and keratinocytes downregul...
Immunoblotting of (A) WT and Tsc1-cKO epidermal lysates, (B) WT and Tsc1-cKO keratinocyte lysates, and (C) Tsc1fl/fl keratinocyte cultures infected with empty or adenoviral cre recombinase (Tsc1-cre) showing decreased EGFR and HER2 expression with Tsc1 loss (left panels). Immunoblots in B are noncontemporaneous from the same biological replicate, while those in C are contemporaneous and parallel from the same biological replicate. Densitometry quantification of immunoblots (right panels) (biological replicates r ≥ 4; P values are by Student’s t test). Error bars represent SD. (D) Immunoblotting following surface biotinylation and IP showing decreased membrane EGFR and HER2 in Tsc1-cKO keratinocyte lysates compared with WT controls. Na-K ATPase is used to normalize for membrane protein. (E) Immunoblotting of WT and Tsc1-cKO keratinocyte lysates, with or without mTORC1 inhibition using rapamycin (200 nm) or AZD8055 (500 nm), for p-S6, Tsc1, EGFR, and HER2 (left panel) and p-AKT (T308), p-AKT (S473) and p-ERK (right panel). Tsc1-cKO keratinocytes show an increase in p-S6 levels and downregulation of HER2, EGFR, p-AKT, and p-ERK which were rescued upon mTORC1 inhibition. p-S6 and total S6 are noncontemporaneous immunoblots from the same biological replicate. (F) Immunoblotting of serum-starved, EGF-stimulated WT and Tsc1-cKO keratinocyte lysates for EGFR, p-EGFR (Y1068), p-AKT (S473), and p-ERK. The intensity and duration of EGFR autophosphorylation and downstream signaling markers is decreased in Tsc1-cKO keratinocytes.