FBXO3 Protein Promotes Ubiquitylation and Transcriptional Activity of AIRE (Autoimmune Regulator)

Wei Shao,Kristina Zumer,Koh Fujinaga,B Matija Peterlin

doi:10.1074/jbc.m116.724401

Abstract

The autoimmune regulator (AIRE) is a transcription factor which is expressed in medullary thymic epithelial cells. It directs the expression of otherwise tissue-specific antigens, which leads to the elimination of autoreactive T cells during development. AIRE is modified post-translationally by phosphorylation and ubiquitylation. In this report we connected these modifications. AIRE, which is phosphorylated on two specific residues near its N terminus, then binds to the F-box protein 3 (FBXO3) E3 ubiquitin ligase. In turn, this SCF(FBXO3) (SKP1-CUL1-F box) complex ubiquitylates AIRE, increases its binding to the positive transcription elongation factor b (P-TEFb), and potentiates its transcriptional activity. Because P-TEFb is required for the transition from initiation to elongation of transcription, this interaction ensures proper expression of AIRE-responsive tissue-specific antigens in the thymus.

Highlights

The autoimmune regulator (AIRE)2 is a transcription factor (TF) that regulates central tolerance in the thymus [1,2,3,4]
AIRE is phosphorylated on two residues, threonine at position 68 (Thr68) and serine at position 156 (Ser-156), which when mutated to alanine abrogated AIRE transcriptional activity [11]
In this study we identified and validated F-box protein 3 (FBXO3) as the E3 ubiquitin ligase that modifies AIRE post-translationally and increases its activity on tissue-specific antigens (TSAs) genes

Summary

Experimental Procedures

Cell Culture and Transfection—HEK 293T (293T) cells and 1C6 mTECs were cultured in Dulbecco’s modified Eagle’s medium with 10% fetal calf serum at 37 °C and 5% CO2. Transfection of plasmid DNA was performed using Lipofectamine 2000 (Life Technologies) according to the manufacturer’s. Cells were washed by PBS and fixed in 4% formaldehyde for 30 min at room temperature. They were incubated with 0.2% Triton X-100 in PBS for 5 min. Whole cell lysates were incubated with the appropriate primary antibodies or antibody-conjugated beads overnight at 4 °C. After incubations with primary antibodies, lysates were centrifuged, and supernatants were incubated with Protein Aor G-Sepharose beads for 2 h at 4 °C. The lysate was sonicated as above described, and the lysates were centrifuged and precleared by protein A-Sepharose beads (Invitrogen). FBXO3 and its number of peptides are written in bold letters

Gene symbol

Results

Discussion