Abstract

Transcription factor EB (TFEB) is a master regulator of lysosomal biogenesis and autophagy that plays a key role in the regulation of cellular clearance pathways. TFEB is regulated via a complex array of post-translational modifications (PTMs), but the exact molecular mechanism that regulates TFEB stability has remained elusive. Here, we show that TFEB levels are critically regulated by a defined phosphorylation-ubiquitination cascade. A human kinome screen identifies IKK (inhibitor of κB kinase) as a TFEB modifier, and a combination of phosphorylation assays, mass spectrometry analyses, and site-specific mutagenesis unveils a previously unrecognized TFEB phospho-degron (423SPFPSLS429) as the target of IKK. We show that the phosphorylation event triggers ubiquitination of adjacent lysine residues (K430 and K431) by the E3 ligase β-TrCP2 (β-Transducin repeat-containing protein 2), thereby tagging TFEB for degradation. Modified TFEB constructs that abolish these PTMs show much increased stability and expression levels but remain equally sensitive to autophagy- or stress-related stimuli while maintaining the capability to promote the expression of TFEB target genes and the clearance of Alzheimer’s associated tau in a cellular model of disease. Our results therefore uncover an IKK/β-TrCP2 phosphorylation-ubiquitination cascade as a major mechanism that governs TFEB stability independently of other TFEB regulators.

Committee Chair

Marco Sardiello

Degree

Doctor of Philosophy (PhD)

Author's Department

Biology & Biomedical Sciences (Molecular Genetics & Genomics)

Author's School

Graduate School of Arts and Sciences

Document Type

Dissertation

Date of Award

12-20-2023

Language

English (en)

Author's ORCID

https://orcid.org/0000-0002-9268-3907

Download

Included in

Biology Commons

Share

COinS