HSCARG Regulates NF-κB Activation by Promoting the Ubiquitination of RelA or COMMD1

Min Lian,Xiaofeng Zheng

doi:10.1074/jbc.m809752200

Abstract

The redox sensor protein HSCARG translocates from the cytoplasm to the nucleus in response to decreased cellular NADPH or increased nitric oxide, and is involved in protein regulation. However, the regulatory mechanism of HSCARG has remained elusive. In this report, through a yeast two-hybrid screen, HSCARG was found to associate with the copper metabolism gene MURR1 domain containing protein 1 (COMMD1), an inhibitor of NF-kappaB, and negatively regulate COMMD1 by accelerating its ubiquitination and proteasome-dependent degradation. Interestingly, we observed that HSCARG also blocked basal and stimulus-coupled NF-kappaB activation by promoting ubiquitination and degradation of the NF-kappaB subunit RelA. Further analyses showed that in cells under normal conditions, HSCARG localized mainly in the cytoplasm and acted as a negative regulator of COMMD1, and was distributed in the nucleus in small quantities to inhibit NF-kappaB. Although in response to intracellular redox changes by dehydroepiandrosterone or S-nitroso-N-acetylpenicillamine treatment, a large amount of HSCARG translocated to the nucleus, which terminated NF-kappaB activation. Meanwhile, COMMD1 was restored due to decreased cytoplasmic HSCARG levels and negatively regulated NF-kappaB as well. Thus, NF-kappaB activation was terminated efficiently. Our results indicate that HSCARG plays critical roles in regulation of NF-kappaB in response to cellular redox changes by promoting ubiquitination and proteolysis of RelA or COMMD1.

Highlights

Nuclear factor NF-␬B is an important transcriptional factor that is involved in multiple biological processes, including immune responses, programmed cell death, and transcriptional regulation of viral replication [1,2,3]. ␬B-dependent gene transcription is regulated by the amount of the NF-␬B complex sequestered in the cytoplasm [4]
FLAG-HSCARG was observed in the HA-COMMD1 immunoprecipitate but not in the control, and no background precipitation was detected, demonstrating that there was a specific interaction between HSCARG and COMMD1
COMMD1 was co-precipitated with HSCARG using anti-HSCARG polyclonal antibody (pAb), HSCARG existed in the COMMD1 immunoprecipitate (Fig. 1B)

Summary

Introduction

Nuclear factor NF-␬B is an important transcriptional factor that is involved in multiple biological processes, including immune responses, programmed cell death, and transcriptional regulation of viral replication [1,2,3]. ␬B-dependent gene transcription is regulated by the amount of the NF-␬B complex sequestered in the cytoplasm [4]. I␬B plays a significant role in suppression of NF-␬B activation by shuttling nuclear NF-␬B back to the cytoplasm [2, 13, 14]. DHEA was found to potently inhibit NF-␬B activation by affecting NF-␬B itself and I␬B, at least partly through its effect on redox balance [23,24,25]. HSCARG is a NADPH sensor, which plays a regulatory role in cellular processes [28, 29]. In response to DHEA or SNAP treatment, HSCARG translocates from the cytoplasm to the nucleus [28, 29], suggesting that HSCARG may exert regulatory functions in the nucleus as well. We characterized a critical role for HSCARG in the regulation of NF-␬B in response to cellular redox changes.

Methods

Results

Conclusion