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Targeted dephosphorylation of TFEB promotes its nuclear translocation.

iScience (2024-07-31)
Jin-Feng Zhao, Natalia Shpiro, Gajanan Sathe, Abigail Brewer, Thomas J Macartney, Nicola T Wood, Florentina Negoita, Kei Sakamoto, Gopal P Sapkota
ABSTRACT

Reversible phosphorylation of the transcription factor EB (TFEB) coordinates cellular responses to metabolic and other stresses. During nutrient replete and stressor-free conditions, phosphorylated TFEB is primarily localized to the cytoplasm. Stressor-mediated reduction of TFEB phosphorylation promotes its nuclear translocation and context-dependent transcriptional activity. In this study, we explored targeted dephosphorylation of TFEB as an approach to activate TFEB in the absence of nutrient deprivation or other cellular stress. Through an induction of proximity between TFEB and several phosphatases using the AdPhosphatase system, we demonstrate targeted dephosphorylation of TFEB in cells. Furthermore, by developing a heterobifunctional molecule BDPIC (bromoTAG-dTAG proximity-inducing chimera), we demonstrate targeted dephosphorylation of TFEB-dTAG through induced proximity to bromoTAG-PPP2CA. Targeted dephosphorylation of TFEB-dTAG by bromoTAG-PPP2CA with BDPIC at the endogenous levels is sufficient to induce nuclear translocation and some transcriptional activity of TFEB.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Monoclonal ANTI-FLAG® M2-Peroxidase (HRP) antibody produced in mouse, clone M2, purified immunoglobulin, buffered aqueous glycerol solution
Sigma-Aldrich
Anti-phospho TFEB (Ser142), from rabbit, purified by affinity chromatography
Millipore
ANTI-FLAG® M2 Affinity Gel, purified immunoglobulin, buffered aqueous glycerol solution