Nuclear Targeting and Cell Cycle Regulatory Function of Human BARD1
Journal of Biological Chemistry
The BARD1 gene is mutated in a subset of breast and ovarian cancers, implicating BARD1 as a potential tumor suppressor. BARD1 gains a ubiquitin E3 ligase activity when heterodimerized with BRCA1, but the only known BRCA1-independent BARD1 function is a p53-dependent proapoptotic activity stimulated by nuclear export to the cytoplasm. We described previously the nuclear-cytoplasmic shuttling of BARD1, and in this study, we identify the transport sequences that target BARD1 to the nucleus and
... the nucleus and show that they are essential for BARD1 regulation of the cell cycle. We used deletion mapping and mutagenesis to define two active nuclear localization signals (NLSs) present in human BARD1 that are not conserved in rodent BARD1. Site-directed mutagenesis of the primary bipartite NLS abolished BARD1 nuclear import and caused its cytoplasmic accumulation. Using flow cytometry and 5-bromo-2-deoxyuridine incorporation assays, we discovered that transiently expressed BARD1 can elicit a p53-independent cell cycle arrest in G 1 phase, and that this was abrogated by mutation of the BARD1 NLS but not by mutation of the nuclear export signal. Thus, BARD1 regulation of the cell cycle is a nuclear event and may be linked to its induced expression during mitosis and its possible involvement in the DNA damage checkpoint. BARD1 is the major cellular binding partner of the breast and ovarian cancer susceptibility protein, BRCA1 (1). BARD1 itself was reported to be mutated in a subset of breast and ovarian cancer patients, and BARD1 germ line mutations were identified in breast cancer patients negative for BRCA1 or BRCA2 gene alterations, suggesting a possible tumor-suppressive function of BARD1 (2-4). This hypothesis is supported by findings that the repression of BARD1 expression in murine mammary epithelial cells by antisense RNA resulted in a premalignant phenotype (5) and the observation that BARD1 induced apoptosis via a pathway depending on p53 but not requiring BRCA1 (6). BARD1 plays a key role in regulating BRCA1 stability, localization and function, and several lines of evidence indicate that the BARD1-BRCA1 heterodimer is the physiologically relevant form of BRCA1. The two proteins are coordinately expressed in a variety of different tissues and cell types during Xenopus laevis development, and they stabilize one another (7-9). Furthermore, the phenotypes of either BARD1 or BRCA1 null mice, as well as BARD1:BRCA1 double null mice, display striking similarities in their accumulation of chromosomal abnormalities and early embryonic death due to severe cell proliferation defects (9, 10). BARD1 and BRCA1 are predominantly found in the cell nucleus, where they co-localize in S phase-specific dots (11). Following DNA damage, they redistribute within the nucleus into discrete foci that also co-stain with DNA repair-associated factors such as BRCA2 and Rad51 (12). BARD1 and BRCA1 also co-fractionate with several DNA repair-associated nuclear protein complexes, suggesting a role for the two proteins in DNA repair and replication (13). BRCA1 possesses E3 ubiquitin ligase activity, which is ablated by tumor-derived point mutations within the RING motif (14, 15) but greatly enhanced by complex formation with BARD1 (8, 16 -18). Recently, it was shown for the first time in vivo that BARD1-BRCA1 can induce the formation of and colocalize with ubiquitin conjugates during replication and DNA repair (19). Both BARD1 and BRCA1 contain nuclear export sequences (NES), 1 which enable the proteins to shuttle between nucleus and cytoplasm (20, 21). We showed recently that BARD1 nuclear export stimulates its apoptotic activity (21), and a comparable finding was made by Jefford et al. (22), who observed a correlation between cytoplasmic staining of BARD1 and apoptosis. The co-expression of BRCA1 inhibited both BARD1 nuclear export and apoptotic activity (21). Whereas two nuclear localization signals (NLSs) have been identified in BRCA1 (23, 24) , no NLS has yet been defined for BARD1. In this study, we mapped three active nuclear localization signals in BARD1, of which two are not conserved in the rodent BARD1 protein. Furthermore, we show that BARD1 itself can elicit a G 1 phase cell cycle arrest when overexpressed and that this activity was dependent on its NLS-mediated nuclear localization. This discovery is reminiscent of previous findings for BRCA1 (25) and reveals that the two proteins not only bind and regulate one another but perhaps can substitute for each other in several functions, including cell cycle arrest (this study), apoptosis (6, 21), genomic stability (9), and homologous DNA repair/recombination (26). Collectively, these findings support a role for BARD1 in tumor suppression. MATERIALS AND METHODS Cell Culture and Transfections-MCF-7 breast cancer cells and U2OS and Saos-2 osteosarcoma cells were grown under standard conditions in Dulbecco's modified Eagle's medium supplemented with 10% 1 The abbreviations used are: NES, nuclear export sequence(s); YFP, yellow fluorescence protein; WT, wild type; PBS, phosphate-buffered saline; NLS, nuclear localization sequence; BrdUrd, 5-bromo-2deoxyuridine.