Ubiquitin-specific Peptidase 21 Inhibits Tumor Necrosis Factor α-induced Nuclear Factor κB Activation via Binding to and Deubiquitinating Receptor-interacting Protein 1

Gufeng Xu,Xiaojie Tan,Hongmei Wang,Wenjing Sun,Yi Shi,Susan Burlingame,Xue Gu,Guangwen Cao,Ting Zhang,Jun Qin,Jianhua Yang

doi:10.1074/jbc.m109.042689

Abstract

Ubiquitination and deubiquitination of receptor-interacting protein 1 (RIP1) play an important role in the positive and negative regulation of the tumor necrosis factor alpha (TNFalpha)-induced nuclear factor kappaB (NF-kappaB) activation. Using a combination of functional genomic and proteomic approaches, we have identified ubiquitin-specific peptidase 21 (USP21) as a deubiquitinase for RIP1. USP21 is constitutively associated with RIP1 and deubiquitinates RIP1 in vitro and in vivo. Notably, knockdown of USP21 in HeLa cells enhances TNFalpha-induced RIP1 ubiquitination, IkappaB kinase beta (IKKbeta), and NF-kappaB phosphorylation, inhibitor of NF-kappaB alpha (IkappaB alpha) phosphorylation and ubiquitination, as well as NF-kappaB-dependent gene expression. Therefore, our results demonstrate that USP21 plays an important role in the down-regulation of TNFalpha-induced NF-kappaB activation through deubiquitinating RIP1.

Highlights

Transcription factor nuclear factor ␬B (NF-␬B)3 plays an important role in controlling the expression of survival factors, cytokines, and proinflammatory molecules in a broad range of cellular responses [1,2,3]
In the TNF␣-induced NF-␬B signal transduction pathway, the Lys63linked polyubiquitination of RIP1 protein mediated by TNF receptor-associated factor 2 (TRAF2) E3 ligase is essential for TNF␣-induced IKK/NF-␬B activation, whereas phosphorylation of the I␬B proteins by activated IKK leads to their Lys48-linked polyubiquitination, which labels it for its degradation by the 26 S proteasome [21]
We found that USP21 wild type but not C221A mutant proteins catalyze the deubiquitination of both Lys48- and Lys63-linked ubiquitin

Summary

Introduction

Transcription factor nuclear factor ␬B (NF-␬B)3 plays an important role in controlling the expression of survival factors, cytokines, and proinflammatory molecules in a broad range of cellular responses [1,2,3]. In Vitro Deubiquitination Assay—Wild-type or mutant Myctagged USP21 proteins were immunoprecipitated from the transfected HEK-293T cell lysates prepared with Nonidet P-40 lysis buffer (25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 10% (v/v) glycerol, 0.5% (v/v) Nonidet P-40, 1 mM PMSF, and 1 mM DTT).

Results

Conclusion