NBA1/MERIT40 and BRE Interaction Is Required for the Integrity of Two Distinct Deubiquitinating Enzyme BRCC36-containing Complexes

Xin Hu,Jin Ah Kim,Andy Castillo,Michael Huang,Jianxin Liu,Bin Wang

doi:10.1074/jbc.m110.200857

Abstract

BRCC36-deubiquitinating enzyme (DUB) forms two different complexes through interactions with two different adaptor proteins Abraxas and ABRO1 in cells. Abraxas mainly localizes in the nucleus, mediating the interaction of BRCC36 with BRCA1. ABRO1 is mainly localized in the cytoplasm. Because it lacks the BRCA1-interacting motif, the ABRO1 complex does not interact with BRCA1. Both BRCC36-containing complexes contain common components including BRE and NBA1/MERIT40. Here, we found that the two complexes are assembled in a similar manner and NBA1 and BRE interaction is critical for maintaining the integrity of both of the complexes. Knockdown of NBA1 or BRE leads to decreased levels of components of the two BRCC36-containing complexes. We provided evidence that NBA1 interacts with BRE through a C-terminal conserved motif of the NBA1 protein and a C-terminal UEV domain of the BRE protein. Furthermore, the NBA1-BRE interaction is required for cellular resistance to ionizing irradiation and NBA1's role in recruiting BRCA1 to DNA damage sites. Together, these studies reveal critical interactions required for the formation and function of BRCC36-containing DUB complexes.

Highlights

Health, NCI Howard Temin Award (1K01CA116275-01) and a Sidney Kimmel Foundation Scholar Award
Two Distinct BRCC36-deubiquitinating Complexes Are Formed in a Similar Manner through Abraxas and Its Paralog ABRO1 Protein—From tandem affinity purification (TAP) and mass spectrometry analysis of NBA1-associated proteins, we found that NBA1 associates with a protein that is a paralog of the Abraxas protein, termed ABRO1 [16]
We found that while Abraxas/ Rap80 are mostly localized in the nuclear chromatin fraction, ABRO1 is mainly localized in the cytoplasm (Fig. 1D)

Summary

Introduction

NCI Howard Temin Award (1K01CA116275-01) and a Sidney Kimmel Foundation Scholar Award Rap80 contains two ubiquitin-interacting motif (UIM) domains that bind to K63linked Ub chains, which is critical for BRCA1 recruitment to DNA damage sites [3]. To investigate whether ABRO1 binds to NBA1, BRE, and BRCC36 in a similar manner as Abraxas protein, we made deletion mutants of ABRO1 and tested their binding to different components of the BRCA1 A complex.

Results

Conclusion