YWHA/14-3-3 proteins recognize phosphorylated TFEB by a noncanonical mode for controlling TFEB cytoplasmic localization

Yang Xu; Jinqi Ren; Xiaolong He; Han Chen; Taotao Wei; Wei Feng

doi:10.1080/15548627.2019.1569928

YWHA/14-3-3 proteins recognize phosphorylated TFEB by a noncanonical mode for controlling TFEB cytoplasmic localization

Autophagy. 2019 Jun;15(6):1017-1030. doi: 10.1080/15548627.2019.1569928. Epub 2019 Jan 27.

Authors

Yang Xu^{1

2}, Jinqi Ren¹, Xiaolong He^{1

2}, Han Chen^{1

2}, Taotao Wei^{1

2}, Wei Feng^{1

2}

Affiliations

¹ a National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics , Chinese Academy of Sciences , Beijing , China.
² b College of Life Sciences , University of Chinese Academy of Sciences , Beijing , China.

Abstract

As a master regulator of the macroautophagy/autophagy-lysosomal pathway, TFEB (transcription factor EB) plays a prominent role in regulating neurodegenerative diseases and cancer. The transcription activity of TFEB is tightly controlled by phosphorylation and dephosphorylation. Phosphorylated S211 (p-S211) of TFEB can be recognized by YWHA/14-3-3 proteins for TFEB cytoplasmic localization. Here, we characterized the interactions between phosphorylated TFEB and YWHA/14-3-3 proteins and determined the structures of YWHA/14-3-3 proteins in complex with a TFEB p-S211-peptide. Although the critical arginine for YWHA/14-3-3 recognition is missing in the N terminus of the TFEB p-S211-peptide, the C-terminal additional hydrophobic residues of the peptide unexpectedly occupy nearly half of the target-binding groove of YWHA/14-3-3 proteins, which compensates for the N-terminal defect and is distinct from the canonical YWHA/14-3-3-binding mode. Mutations of essential residues in the interaction interface between TFEB and YWHA/14-3-3 proteins disrupted their interactions and severely impaired the cytoplasmic localization of TFEB, which altered the expression of TFEB target genes and affected autophagy. Thus, YWHA/14-3-3 proteins recognize phosphorylated TFEB by a noncanonical mode for controlling TFEB cytoplasmic localization and its activity. Abbreviation: ACTB: actin beta; ALP: autophagy-lysosomal pathway; ATP6V1H: ATPase H⁺ transporting V1 subunit H; bHLH: basic helix-loop-helix; CLEAR: coordinated lysosomal expression and regulation; Co-IP: co-immunoprecipitation; CTSB: cathepsin B; CTSD: cathepsin D; LAMP1: lysosomal associated membrane protein 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MITF: melanocyte inducing transcription factor; NLS: nuclear localization signal; TFEB: transcription factor EB; YWHA/14-3-3: tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein.

Keywords: Autophagy; X-ray crystallography; autophagy-lysosomal pathway; protein interaction; structural biology; transcription factor.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

14-3-3 Proteins / chemistry*
14-3-3 Proteins / genetics
14-3-3 Proteins / metabolism*
Amino Acid Motifs / genetics
Autophagy / drug effects
Autophagy / genetics*
Basic Helix-Loop-Helix Leucine Zipper Transcription Factors / chemistry
Basic Helix-Loop-Helix Leucine Zipper Transcription Factors / genetics
Basic Helix-Loop-Helix Leucine Zipper Transcription Factors / metabolism*
Cell Nucleus / drug effects
Cell Nucleus / genetics
Cell Nucleus / metabolism
Cytosol / drug effects
Cytosol / metabolism*
HeLa Cells
Humans
Lysosomes / drug effects
Lysosomes / genetics
Lysosomes / metabolism
Phosphorylation

Substances

14-3-3 Proteins
Basic Helix-Loop-Helix Leucine Zipper Transcription Factors
TFEB protein, human

Grants and funding

This work was supported by the National Key R&D Program of China under grant 2017YFA0503501 and 2017YFA0205501; the National Major Basic Research Program of China under grant 2014CB910202, and the National Natural Science Foundation of China under grants 31470746, 31770786, 31600608, 31600622 and 31470814.