BARD1 Induces BRCA1 Intranuclear Foci Formation by Increasing RING-dependent BRCA1 Nuclear Import and Inhibiting BRCA1 Nuclear Export

Abstract

BRCA1 is a tumor suppressor with several important nuclear functions. BRCA1 has no known cytoplasmic functions. We show here that the two previously identified nuclear localization signals (NLSs) are insufficient for nuclear localization of BRCA1 due to the opposing action of an NH 2 -terminal nuclear export signal. In transfected breast cancer cells, BRCA1 nuclear localization requires both the NLSs and NH 2 -terminal RING domain region; mutating either of these sequences shifts BRCA1 to the cytoplasm. The BRCA1 RING element mediates nuclear import via association with BARD1, and this is not affected by cancer-associated RING mutations. Moreover, BARD1 directly masks the BRCA1 nuclear export signal, and the resulting block to nuclear export is requisite for efficient import and nuclear localization of ectopic and endogenous BRCA1. Our results explain why BRCA1 exon 11 splice variants, which lack the NLSs but retain the RING domain, are frequently detected in the nucleus and in nuclear foci in vivo. In fact, co-expression of BARD1 promoted formation of DNA damage-induced nuclear foci comprising ectopic wild-type or NLS-deficient BRCA1, implicating BARD1 in nuclear targeting of BRCA1 for DNA repair. Our identification of BARD1 as a BRCA1 nuclear chaperone has regulatory implications for its reported effects on BRCA1 protein stability, ubiquitin ligase activity, and DNA repair.The tumor suppressor, BRCA1, was the first susceptibility gene linked to breast and ovarian cancer (1). Germ-line mutations of BRCA1 are found in ϳ50% of patients with inherited breast cancer and up to 90% of families with breast and ovarian cancer susceptibility (1, 2). The role of BRCA1 as a tumor suppressor is not fully defined, although accumulated evidence suggests that BRCA1 plays a role in transcriptional regulation (3), cell cycle control (4, 5), and cell survival responses to DNA damage (6 -8).BRCA1 is a large gene of 24 exons that encodes a 1,863-amino acid protein (1). The BRCA1 protein contains several protein-interaction domains: an NH 2 -terminal RING domain common to many regulatory proteins (1), two tandem copies of the BRCT (BRCA1 carboxyl terminus) motif at the COOH terminus (9), and both nuclear import (10, 11) and export signals (12). The BRCT domain is found in a variety of proteins, including 53BP1, RAD9, RAD4, Crb2, and RAP1, all of which are associated with cell cycle regulation and DNA repair (13). The BRCT motifs of BRCA1 appear to be critical for its transcription activation function (3, 14), and cancer mutations in this COOH-terminal region impair transcriptional activity (3,15). This is likely due to altered association with specific proteins, such as the RNA polymerase II holoenzyme, which normally interacts with the COOH terminus of BRCA1 (16). The NH 2 -terminal RING domain of BRCA1 mediates association with proteins including BARD1 (17) and BAP1 (18). BARD1 is similar in primary structure to BRCA1, in that it also contains an NH 2 -terminal RING finger and two COOHterminal BRCT domains (17). BRCA1 and BARD1 i...

Functional consequences of cyclin D1/BRCA1 interaction in breast cancer cells

“…Asynchronous MCF-7 cells, hydroxyurea (HU; early S phase blocker) and nocodazole (G 2 /M blocker)-treated cells were fixed and fluorescently stained for both cyclin D1 and BRCA1 proteins (Figure 2). BRCA1 was detected in both the nucleus and cytoplasm of untreated cells, previously reported for MCF-7 cells (Fabbro et al, 2002). Cyclin D1 staining was also found in both the cytoplasm and nucleus (Figure 2, untreated cells).…”

BRCA1 gene in breast cancer

“…Another study suggests that BARD1 may also act to facilitate nuclear entry of BRCA1 and block its exit (by covering up the BRCA1 nuclear export signal) (Fabbro et al, 2002). It has been suggested that the ubiquitin protein ligase activity of the BRCA1/BARD1 contributes to many of the biologic functions of the BRCA1 protein, including its breast and ovarian cancer suppressor activity (Baer and Ludwig, 2002).…”

Phosphatidylinositol 3-kinase/Akt signaling enhances nuclear localization and transcriptional activity of BRCA1

“…Interaction with importin α can occur through two nuclear localization signals in BRCA1 located at amino acids 503-508 and 606-615 [14], but studies suggest that only the interaction at amino acid residues 503-508 is physiologically important in targeting BRCA1 to the nucleus [15]. The interaction of BARD1 with BRCA1 also results in the translocation of BRCA1 into the nucleus via a piggyback mechanism [16,17]. The nuclear export of BRCA1 occurs via two leucine-rich nuclear export sequences located at amino acid residues 81-99 [17] and 22-30 [18].…”