TSC2 regulates lysosome biogenesis via a non-canonical RAGC and TFEB-dependent mechanism

Nicola Alesi,Damir Khabibullin,Heng-Jia Liu,Elizabeth P Henske,Manrico Morroni,Shawn M Ferguson,Elie W Akl,Wei Shi,Harilaos Filippakis,Hilaire C Lam,Srinivas R Viswanathan,Emma R Garner,Anna S Nidhiry

doi:10.1038/s41467-021-24499-6

Abstract

Tuberous Sclerosis Complex (TSC) is caused by TSC1 or TSC2 mutations, resulting in hyperactivation of the mechanistic target of rapamycin complex 1 (mTORC1). Transcription factor EB (TFEB), a master regulator of lysosome biogenesis, is negatively regulated by mTORC1 through a RAG GTPase-dependent phosphorylation. Here we show that lysosomal biogenesis is increased in TSC-associated renal tumors, pulmonary lymphangioleiomyomatosis, kidneys from Tsc2+/− mice, and TSC1/2-deficient cells via a TFEB-dependent mechanism. Interestingly, in TSC1/2-deficient cells, TFEB is hypo-phosphorylated at mTORC1-dependent sites, indicating that mTORC1 is unable to phosphorylate TFEB in the absence of the TSC1/2 complex. Importantly, overexpression of folliculin (FLCN), a GTPase activating protein for RAGC, increases TFEB phosphorylation at the mTORC1 sites in TSC2-deficient cells. Overexpression of constitutively active RAGC is sufficient to relocalize TFEB to the cytoplasm. These findings establish the TSC proteins as critical regulators of lysosomal biogenesis via TFEB and RAGC and identify TFEB as a driver of the proliferation of TSC2-deficient cells.

Highlights

Tuberous Sclerosis Complex (TSC) is caused by TSC1 or TSC2 mutations, resulting in hyperactivation of the mechanistic target of rapamycin complex 1
Because mechanistic target of rapamycin complex 1 (mTORC1) is hyperactive in TSC, Transcription factor EB (TFEB) should be predominantly cytoplasmic in TSC-deficient cells
To understand the mechanisms driving high lysosome abundance in TSC, we focused on the MiTF family of transcription factors (TFEB, TFE3, MITF, TFEC), known regulators of lysosome biogenesis[10,11,13]

Summary

Introduction

Tuberous Sclerosis Complex (TSC) is caused by TSC1 or TSC2 mutations, resulting in hyperactivation of the mechanistic target of rapamycin complex 1 (mTORC1). Overexpression of folliculin (FLCN), a GTPase activating protein for RAGC, increases TFEB phosphorylation at the mTORC1 sites in TSC2deficient cells. Overexpression of constitutively active RAGC is sufficient to relocalize TFEB to the cytoplasm These findings establish the TSC proteins as critical regulators of lysosomal biogenesis via TFEB and RAGC and identify TFEB as a driver of the proliferation of TSC2deficient cells. Germline mutations in FLCN cause the hereditary cancer syndrome Birt-Hogg-Dube (BHD)[25,26], which shares some clinical phenotypes with TSC including benign skin tumors, cystic lung disease, and renal cell carcinoma (RCC). It has recently been discovered that renal tumorigenesis in BHD is TFEB-dependent[24] This supports the concept that the regulation of TFEB is the critical mechanistic link between tumorigenesis in TSC and BHD, diseases in which there is some clinical similarity, and further highlights the possibility that TFEB may be a primary driver of tumorigenesis in TSC. Our findings indicate that TFEB is a critical disease-relevant target of the TSC proteins

Methods

Results

Conclusion