Post-translational regulation of TFEB/3 activity

Thesis

The ability of cells to sense and adapt to changes in their environment is critical for their survival. Cancer cells acquire the ability to promote cell growth and proliferation independently of growth factors, whilst retaining the ability to sense and adapt to changes in nutrient availability. During tumorigenesis, poor vascularisation results in the promotion of catabolic processes, such as autophagy. Transcription factor EB (TFEB), a member of the microphthalmia/transcription factor E (MiT/TFE) family has been coined as the master regulator of autophagy and lysosomal biogenesis. In response to nutrient deprivation, TFEB is dephosphorylated and rapidly accumulates in the nucleus to promote expression of the CLEAR network. In tumours that exhibit high levels of TFEB/3-dependent autophagy, TFEB is constitutively nuclear and hyperactivated, contributing to cancer pathogenesis. Conversely, in neurodegenerative diseases, such as Alzheimer’s disease, lysosomal function and autophagy are often impaired. Over-expression or activation of TFEB can ameliorate disease pathogenesis through promoting intracellular clearance.

In this study, we show that TFEB accumulates in the nucleus in response to both amino acid and glucose depletion which is readily reversed upon nutrient replenishment. Furthermore, glucose deprivation does not inhibit mTORC1 but instead, activates the mTORC2-AKT signalling pathway. We demonstrated that GSK3β can phosphorylate TFEB/MITF on Ser138/Ser69 but only when Ser142/Ser73 is first phosphorylated by ERK2 or mTORC1, highlighting that this dual phosphorylation event is required for nuclear export of TFEB.

Additionally, we performed two reciprocal, high-throughput image-based screens to explore the regulatory mechanisms which govern TFEB subcellular localisation. The first screen allowed us to identify 50 FDA-approved drugs that could promote nuclear accumulation of TFEB as well as increase lysosomal staining. We identified pimozide as a potential candidate for drug re-purposing for neurodegenerative diseases as it displayed neuroprotection in cell-based models of neurodegenerative disease. The second screen identified 10 kinase inhibitors from the PKISI/II library (GSK) that may have cancer therapeutic potential, being able to attenuate nuclear accumulation of TFEB in response to glucose and/or amino acid deprivation. GW869516X was able to partially rescue mTORC1 activity whilst inhibiting mTORC2 signalling in response to amino acid and glucose starvation respectively. Additionally, GW869516X inhibited AMPK signalling and can directly bind to AMPK. Overall, this work contributes to our understanding of how TFEB activity is regulated through dynamic phosphorylation events. The results obtained from our screens provide a reservoir of drugs that can modulate TFEB subcellular localisation, warranting further investigation.

Authors: Andrews, S

• DOI: 10.5287/ora-jb7k2na4g
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: MITF; TFEB; TFE3
• Subjects: Melanoma