Essential Role of Human Leukocyte Antigen-encoded Proteasome Subunits in NF-κB Activation and Prevention of Tumor Necrosis Factor-α-induced Apoptosis

Takuma Hayashi,Denise Faustman

doi:10.1074/jbc.275.7.5238

Abstract

The multisubunit proteasome complex is the principal mediator of nonlysosomal protein degradation. The proteasome subunit varies minimally between cells with the exception of LMP2, LMP7, and LMP10 subunits in rodent and human cells. LMP2 and LMP7 subunits are encoded by the human lymphocyte antigen region, and they optimize proteolytic mediated antigen presentation. The proteasome is also important for the function of transcription factor nuclear factor-kappaB (NF-kappaB). It is required for NF-kappaB subunits p50 and p52 generation and catalyzes degradation of phosphorylated IkappaBalpha. These proteasome-mediated reactions have now been shown to be defective in T2 cells, a human lymphocyte cell line that lacks both LMP2 and LMP7. Although T2 cells contain normal expression of p100 and p105, the abundance of p50 and p52 was greatly reduced. Tumor necrosis factor-alpha (TNF-alpha) induced normal phosphorylation of IkappaBalpha but failed to induce degradation of phosphorylated IkappaBalpha. Both DNA binding assays and luciferase assays revealed that TNF-alpha-induced NF-kappaB activation is defective in T2 cells. Unlike parental cells, T2 cells were susceptible to TNF-alpha-induced apoptosis. These data indicate human leukocyte antigen-linked proteasome subunits are essential for NF-kappaB activation and protection of cells from TNF-alpha-induced apoptosis.

Highlights

Nuclear factor-␬B (NF-␬B)1/Rel superfamily is a transcription factor that contributes to the ability of the immune system to respond rapidly to foreign antigens [1,2,3,4,5,6]
In human lymphocytes, LMP2 and LMP7 are required for the generation and activation of nuclear factor-␬B (NF-␬B) as well as for prevention of TNF-␣-induced apoptosis
Impaired Activation of NF-␬B in T2 Cells—We first investigated the effect that deletions of the HLA-linked Lmp2 and Lmp7 proteasome subunit genes might have on NF-␬B function by Electrophoretic Mobility Shift Assay (EMSA) with the nuclear extracts prepared from T2 cells

Summary

Introduction

Nuclear factor-␬B (NF-␬B)1/Rel superfamily is a transcription factor that contributes to the ability of the immune system to respond rapidly to foreign antigens [1,2,3,4,5,6]. Further studies confirmed anti-p52 antibodies selectively reduce the mobility of the DNA-protein complex formed by the nuclear extract of TNF-␣-treated control Molt-4 cells with an oligonucleotide probe to H2TF1 ␬B corresponding to the ␬B binding motif of the MHC class I gene enhancer (data not shown).

Results

Conclusion