Importin KPNA2 Is Required for Proper Nuclear Localization and Multiple Functions of NBS1

Shun-Fu Tseng,Chun-Yu Chang,Kou-Juey Wu,Shu-Chun Teng

doi:10.1074/jbc.m508425200

Abstract

Nijmegen breakage syndrome (NBS) is a chromosomal-instability syndrome associated with cancer predisposition, radiosensitivity, microcephaly, and growth retardation. The NBS gene product, NBS1, is a component of the MRE11-RAD50-NBS1 (MRN) complex, a central player associated with double strand break (DSB) repair. In response to radiation, NBS1 is phosphorylated by ATM, and the MRN complex relocalizes to form punctate nuclear foci for DNA repair. NBS1 controls both the nuclear localization of the MRN complexes and radiation-induced focus formation. We report here that the KPNA2 (importin alpha1) is important for the normal nuclear localization of the MRN complex and its proper formation of the nuclear foci. KPNA2 is the only member of the importin alpha family that physically interacts with NBS1, and the KPNA2-mediated nucleus localization sequence (NLS) is mapped to amino acid residues 461-467 of NBS1 that is sufficient for both the interaction with KPNA2 and the proper nuclear localization. Inhibition of KPNA2 or blockage of the KPNA2 interaction with NBS1 results in a reduction of radiation-induced nuclear focus accumulation, DSB repair, and cell cycle checkpoint signaling of NBS1. Collectively, our results strongly suggest that an interaction with KPNA2 contributes to nuclear localization and multiple tumor suppression functions of the NBS1 complex.

Highlights

Tional complementation studies identified a single gene, NBS1, which is mutated in most patients with Nijmegen breakage syndrome (NBS) [6, 7]
We found that abilities of NBS1 in nuclear focus formation, double strand break (DSB) repair, and DSB-dependent checkpoint signaling were compromised in a KPNA2i background or in strains expressing NBS1 mutants that prevent their interaction with KPNA2
Two-hybrid screenings in yeast have allowed us to identify a new physical and functional interaction between NBS1 and KPNA2 importin ␣, a family member of proteins involved in the active transport of cargo proteins containing an nucleus localization sequence (NLS) from the cytoplasm to the nucleus

Summary

Introduction

Tional complementation studies identified a single gene, NBS1, which is mutated in most patients with NBS [6, 7]. In NBS cells, which express truncated nibrin, MRE11 and RAD50 still interact, but complexes containing these two proteins are confined to the cytoplasm and cannot form nuclear foci [9]. This suggests that one of the major roles of NBS1 is to carry MRE11 and RAD50 into nucleus. Some nuclear proteins are delivered into the nucleus with the aid of the adaptor function of importin ␣, most proteins that carry cargoes directly through nuclear pore complex are members of the importin ␤ family [25, 29]. Our data fit the model that KPNA2 plays a role as an import receptor of an NBS1-containing cytoplasmic MRN complex

Methods

Results

Conclusion