Cytoplasmic mislocalization of BRCA1 caused by cancer-associated mutations in the BRCT domain
Introduction
The BRCA1 tumor suppressor gene is mutated in the germ-line of women who suffer a genetic predisposition to breast and ovarian cancer [1], [2]. BRCA1 mutations are found in approximately 50% of patients with inherited breast cancer, and up to 90% of families with breast and ovarian cancer susceptibility [1], [2]. The BRCA1 protein comprises 1863 amino acids [1], and has proposed roles in DNA repair, transcriptional activation, cell cycle regulation and apoptosis [3], [4], [5]. BRCA1 has two main types of protein interaction domains that are frequently targeted by genetic mutations: an N-terminal RING finger domain and two tandem BRCT domains at the C-terminal end. The RING domain mediates an enzymatic ubiquitin E3 ligase activity when BRCA1 forms a stable heterodimer with its binding partner, BARD1 [6], [7]. The C-terminal BRCT domains are thought to mediate the transcriptional activity of BRCA1 [8], [9]. As a tumor suppressor, inherited mutations affect certain vital functions of BRCA1. The most frequent published mutations occur within the BRCT domains and these have been reported to adversely affect BRCA1 nuclear functions including DNA repair [10], [11] and transcriptional activity [9], [12], [13].
Several years ago, Chen et al. [14] published controversial findings which claimed that BRCA1 was mislocalized almost exclusively to the cytoplasm in breast cancer tissues, but remained nuclear in normal tissue and in other cancer cell types. However, this finding was not validated by others [15], and it was later concluded that antibody specificity was the main problem, with most agreeing that BRCA1 in general displayed a “primarily” nuclear localization in different breast cancer cell lines and in histological breast tumor tissue slices [16]. Nonetheless, BRCA1 subcellular localization is well known to vary between nucleus and cytoplasm in both patient tumor samples [17], [18] and in breast cancer cell lines [19], [20]. Our laboratory recently discovered that BRCA1 is a nuclear-cytoplasmic shuttling protein, and that its nuclear localization is regulated by the combined action of nuclear localization (NLS; [21], [22]) and nuclear export signals (NES; Ref. [23]). In most cases, however, cellular and ectopically expressed BRCA1 are primarily nuclear due to nuclear import mediated by the two NLSs and interaction with the RING domain binding protein, BARD1, which can carry BRCA1 into the nucleus and trap it there by masking its nuclear export signal [20].
We decided to revisit the question of BRCA1 subcellular localization, and more specifically to determine whether any of the more commonly studied gene mutations affect BRCA1 localization by regulating nuclear import or export. We demonstrate that out of the ten mutations tested, only the five which target the BRCT domains altered BRCA1 localization, causing it to be excluded from the nucleus. This nuclear exclusion was not due to increased nuclear export, but to reduced nuclear import. Similar findings were observed for both the overexpressed and endogenous forms of the BRCT mutant, BRCA1(5382insC). Since the BARD-dependent import pathway [20] is still active for the BRCT mutants, their overall nuclear entry is reduced but not fully negated. Our new findings may explain some of the disparity in the literature concerning BRCA1 localization and movement.
Section snippets
BRCA1 mutations within the BRCT domain cause cytoplasmic mislocalization
To assess the impact of BRCA1 cancer mutations on subcellular localization, we first used PCR-based mutagenesis to create a series of single-site or truncated BRCA1 mutant cDNAs corresponding to naturally occurring breast and ovarian cancer-linked mutations [24], [25], [26], [27]. These mutant BRCA1 cDNAs were cloned into the mammalian expression vector, pFLAG-CMV2 (see Ref. [23]), for transfection and expression in human breast cancer cells. As summarized in Fig. 1, many of the BRCA1 mutations
Discussion
In this study, we identified a specific subset of C-terminal mutations (those localized to the BRCT domain) that cause the partial or near-complete nuclear exclusion of BRCA1. Similar findings were made for both transiently expressed and endogenous BRCA1. The observed cytoplasmic mislocalization may partly explain why BRCT mutations reduce BRCA1 nuclear DNA repair [10], [11], replication [27] and transcriptional activities [8], [9], [28], [29]. Significantly, even when the mutant
Acknowledgements
We thank Dr. Jeff Holt and Richard Baer for providing BRCA1 and BARD1 plasmids and antibodies. This work was supported in part by grants to BRH from the National Health and Medical Research Council of Australia and the CureCancer Australia Foundation.
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Present address: Department of Medical Oncology, Academic Hospital Vrije Universiteit Amsterdam, 1081HV, Amsterdam, The Netherlands.