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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 September 17, 2019
Citation Information: J Clin Invest. 2019;129(12):5584-5599. https://doi.org/10.1172/JCI128287.
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Research Article Metabolism Oncology

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

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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 6

mTORC1 promotes lysosomal biogenesis and activity.

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mTORC1 promotes lysosomal biogenesis and activity.
(A) Immunoblot analys...
(A) Immunoblot analyses of lysosomal proteins in lysosomal fractions of cellular lysates, showing increased expression of lysosomal proteins in Tsc1-cre keratinocytes (left panel) and Rheb1 S16H Tg keratinocytes (middle panel) compared with controls, and decreased expression of lysosomal proteins in Rptor-cre keratinocytes (right panel) compared with controls. Lysosomal marker Rragc was unaltered across genotypes and used as a loading control. (B) Confocal microscopy analyses of LAMP2 immunostaining demonstrates decreased presence of LAMP2 in Rptor-cre keratinocytes compared with empty controls. Scale bar: 100 μm. (C) Confocal microscopy analyses and double immunostaining for LAMP1/Rptor demonstrates decreased presence of Lamp1 and Rptor in Rptor-cre keratinocytes compared with empty controls. Scale bar: 100 μm. (D) Quantification of LAMP1 fluorescence intensity showing a decrease in mean LAMP1 fluorescence in Rptor-cre keratinocytes compared with controls. The area of LAMP1 was measured using Image J and normalized to the number of nuclei (r = 3, n > 1000). Error bars represent SD, P = 0.0003 by Student’s t test. (E) Lysosomal activity, as measured by fluorometric analyses of cathepsin B activity using the Magic Red Cathepsin B kit, is decreased in Rptor-cre keratinocytes compared with controls (r = 4, error bars represent SD; P = 0.007 by Student’s t test).

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