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

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 ␣1) 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 ␣ 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.

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“…Cells were grown at 30°C in yeast extract peptone dextrose (YPD) broth. Cell lysates were prepared with lysis buffer (150 mM NaCl, 1% Nonidet P-40, 1% deoxycholate, 0.1% SDS, 50 mM TrisHCl, pH 7.5, and protease inhibitors) or trichloroacetic acid (TCA) precipitated 63 . Whole proteins were extracted and resolved by 10% (except Fig.…”

213Bi-induced death of HSC45-M2 gastric cancer cells is characterized by G2 arrest and up-regulation of genes known to prevent apoptosis but induce necrosis and mitotic catastrophe

“…This complex consisting of MRE11, RAD50, and NBS1 forms nuclear foci that are required for DNA double strand break repair. Irradiation of HeLa cells (15 Gy) resulted in increased nuclear activity of MRN and DNA repair (36). Interestingly, incubation of HSC45-M2 cells with the a-emitter 213 Bi decreased KPNA2 expression, implying a reduction of MRN-mediated double strand break repair.…”

“…Down-regulation of these genes is in accordance with the occurrence multinucleated/mitotic catastrophe cells. KPNA2 also mediates nuclear localization of the MRN complex in response to radiation (36). This complex consisting of MRE11, RAD50, and NBS1 forms nuclear foci that are required for DNA double strand break repair.…”

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“…35 Blyth et al 13 suggested that deletion of KPNA2 accounted for the phenotypic characteristics of their patient (short stature, microcephaly, syndactyly, clinodactyly and intellectual disability), which could not be explained by Carney Complex. 13,36 In patient 4, the contribution of KCNJ2 (Inward rectifier potassium channel 2, subfamily J), for which heterozygous mutations are reported to be responsible for Long QT syndrome type 7 also known as Andersen syndrome (OMIM no. 170390), cannot be excluded.…”