mTORC1 feedback to AKT modulates lysosomal biogenesis through MiT/TFE regulation
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
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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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 4

mTORC1 accelerates EGF-induced EGFR degradation.

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mTORC1 accelerates EGF-induced EGFR degradation. (A) Starved empty and R...
(A) Starved empty and Rptor-cre keratinocytes were stimulated with EGF (50 ng/mL) for the indicated times and immunoblotted for EGFR. (B) EGFR degradation curves. Error bars represent SD. Immunoblots are representative of 3 independent experiments. Empty and Rptor-cre lysates were run on the same gel, separated by a molecular weight marker. (C) Starved WT and Tsc1-cKO keratinocytes were stimulated with EGF (1.5 ng/mL) for the indicated times and immunoblotted for EGFR. (D) EGFR degradation curves. Error bars represent SD. Immunoblots are representative of 3 independent experiments. (E) The Gene Set Enrichment Analysis (GSEA) Enrichment Score Plot depicting the Rptor-cKO versus Rptor WT fold changes of 360 lysosomal genes (from the mouse Lysosome Gene Database [mLGDB]) subset compared with those of all assayed transcripts. The green line is the enrichment score, reflecting the degree of lysosomal genes’ overrepresentation among the Rptor-cKO downregulated (left side) and upregulated (right side) genes. (F) Lysosomal proteins, including those containing a CLEAR-binding motif, are decreased in Rptor-cre keratinocytes compared with empty controls, by immunoblot analyses. Rptor, Ctsd, SQSTM1, Rab7 Laptm4b, and actin (far right panel) are noncontemporaneous immunoblots from the same biological replicate. Densitometry quantification of representative immunoblots from 4 independent experiments are provided in Supplemental Figure 4A.