MINIREVIEW BRCA1 16 years later: nuclear import and export processes Marilyn E. Thompson Department of Pharmaceutical Sciences, School of Pharmacy, Belmont University, Nashville, TN, USA Introduction The importance of regulated nuclear transport pro- cesses in modulating protein function has become increasingly evident over the past several years. Energy-dependent, receptor-mediated mechanisms are required for proteins in excess of 40 kDa to cross the nuclear envelope [1]. This suggests that in order for many proteins to reside in their compartment of func- tion, the nucleus, they must first be recognized by import receptors, termed importins or karyopherins, which effectuate the translocation of the cargo protein across the membrane. Likewise, removal of the protein from the nuclear compartment involves its binding to nuclear export receptors (exportins). This translocation event would preclude the functional activity of the nuclear protein. Breast cancer susceptibility gene 1 protein product (BRCA1) is involved in multiple nuclear located func- tions. At an apparent relative molecular mass of approximately 220 kDa, it can only traverse the nuclear envelope by an active process. However, in more recent years, it has been realized that BRCA1 can also be exported from the nucleus as well. In this Keywords BRCA1-associated RING domain protein 1 (BARD1); breast cancer susceptibility gene 1 (BRCA1); nuclear export; nuclear export sequence; nuclear import; nuclear localization signal; RING finger Correspondence M. E. Thompson, Department of Pharmaceutical Sciences, School of Pharmacy, Belmont University, 1900 Belmont Blvd., Nashville, TN 37212, USA Fax: +615 460 6537 Tel: +615 460 8121 E-mail: marilyn.odom@belmont.edu Website: http://www.belmont.edu/ pharmacy/ (Received 7 July 2009, revised 27 April 2010, accepted 26 May 2010) doi:10.1111/j.1742-4658.2010.07733.x Over the past several years, the importance of regulated nuclear transport processes for tumor suppressors has become evident. Proteins with a molecular mass greater than 40 kDa can enter the nucleus only by active transport across the nuclear membrane. The most common pathway by which this occurs is via the importin alpha ⁄ beta pathway, whereby the cargo protein binds importin alpha. This heterodimer binds importin beta and the heterotrimer passes through nuclear pores at the expense of GTP. Breast cancer susceptibility gene 1 (BRCA1) is one such protein. As a mediator of transcription and DNA repair, two exclusively nuclear func- tions, BRCA1, at 220 kDa, can enter the nucleus only via active transport mechanisms. In addition to the classical importin alpha ⁄ beta pathway, BRCA1 can also enter the nucleus in a piggyback mechanism with BRCA1-associated RING domain protein 1 (BARD1). The interaction between BRCA1 and BARD1 is also important in the retention of BRCA1 in the nucleus. This is important because BRCA1 also undergoes active nuclear export. BRCA1 is also involved in apoptotic processes. Whether this occurs within the nucleus or cytoplasm is still unclear; thus, the conse- quences of BRCA1 nuclear export have not been clearly elucidated. This review will discuss the literature regarding the subcellular localization of BRCA1, with particular emphasis on its nuclear import and export processes. Abbreviations BARD1, BRCA1-associated RING domain protein 1; BRCA1, breast cancer susceptibility gene 1; NLS, nuclear localization signal; TRAIL, tumor necrosis factor apoptosis inducing ligand. 3072 FEBS Journal 277 (2010) 3072–3078 ª 2010 The Author Journal compilation ª 2010 FEBS review, the literature regarding BRCA1 nuclear import and export with potential functional implications of these processes will be discussed. Historical overview of cellular localization of BRCA1 BRCA1 was first identified and sequenced in 1994. At that time, the only notable structural feature of the protein documented was a C3HC4 sequence at its amino terminus. This was recognized as a zinc finger domain, which is common to many transcription fac- tors [2]. This suggested that BRCA1 served in this capacity and led to the assumption that BRCA1 was a nuclear protein. In support of this assumption, several laboratories reported the nuclear localization of BRCA1. Scully et al. [3] indicated that BRCA1 was present predominantly within the nucleus of a variety of breast and ovarian tumor-derived cell lines. This countered an earlier report by Chen et al. [4], who sug- gested that BRCA1 was indeed localized to the nucleus in nonmalignant cells; however, in the tumor cell lines examined by this group, BRCA1 was concluded to aberrantly localize within the cytoplasm. Many locali- zation studies [5–8] subsequent to these continued to implicate BRCA1, as well as its variants, as nuclear proteins. For example, Thomas et al. [6] demonstrated by both immunofluorescence and biochemical fraction- ation techniques that in some breast, ovarian and cer- vical cancer cell lines, BRCA1 was vastly nuclear. Immunoblot analysis of subcellular fractions from two breast cancer cell lines, MDA-MB-469 and MCF-7, did indicate the presence of BRCA1 in the cytoplasm. However, the cytoplasmic levels were substantially less than in the nucleus. Whether the presence of BRCA1 in the cytoplasm was attributable to newly translated protein prior to being transported into the nucleus or due to another molecular event was not addressed. Rather, this study substantiated the report by Scully et al. [3] that regardless of the malignant state of the cells, the proportion of BRCA1 localizing to the nucleus was much higher than that outside the nucleus. The discrepancies in the literature regarding the nuclear ⁄ non-nuclear distribution of BRCA1 in malig- nant cells generated much concern and discussion over the validity of the data. Perhaps the most significant contribution towards resolving this cause ce ´ le ` bre was provided by Wilson et al. [5]. In an exhaustive analysis of 19 different BRCA1 IgGs generated against various epitopes, this group demonstrated that the presence of a BRCA1 signal outside the nucleus could be the result of experimental artifact. Analysis of the antibodies for their utility in recognizing BRCA1 via immunoprecipi- tations, immunoblotting, immunohistochemistry and immunocytochemistry revealed that certain variables, such as antibody concentration and method of fixation, influenced the detection of a signal. This highly cited paper was instrumental in reconciling that BRCA1 was predominantly localized to the nucleus in both malig- nant and nonmalignant cells. In 1997, the Rao laboratory [8] first indicated a reg- ulatory aspect to BRCA1 nuclear ⁄ non-nuclear distri- bution. Prior to this, Scully et al. [9] had proposed that the intranuclear localization of BRCA1 was influ- enced by the cell cycle-dependent phosphorylation state of the protein. However, this dictated whether or not BRCA1 localized into nuclear foci or not within the nucleus and did not address the non-nuclear pres- ence of BRCA1. The Rao laboratory suggested that BRCA1 nuclear ⁄ non-nuclear distribution could be dictated by the presence of serum growth factors. Although they did not attribute this to phosphoryla- tion per se, their report did suggest that BRCA1 did not spontaneously traverse the nuclear envelope. Nuclear import of BRCA1 Although it was appreciated early after the identifica- tion of its gene that BRCA1 was a predominantly nuclear protein, the mechanism of its import into the nucleus was not described until 1997. Two reports doc- umenting BRCA1 nuclear import were published within a short time of each other. First, Thakur et al. [10] compared the subcellular localization of full-length BRCA1 with that of a BRCA1 variant lacking exon 11, resulting in the expression of a 97 kDa protein. This comparison demonstrated the presence of a nuclear localization signal (NLS) within this exon as the variant was unable to localize to the nucleus as the wild-type could. Considering potential changes in the secondary and tertiary structures of the protein result- ing from the loss of half of the amino acids, the authors were able to demonstrate it was the loss of a distinct sequence that precluded the variant from entering the nucleus. Two NLSs were identified at amino acid residues 501–507 (KCKRKRR; referred to as NLS1) and 607–614 (KKNRLRRK; referred to as NLS2). Both sequences are located within exon 11, the largest of the exons in BRCA1 (Fig. 1). Deletion muta- genesis demonstrated that only the sequence between residues 501 and 507 was vital for BRCA1 nuclear import. Although these data indicated that BRCA1 entered the nucleus via a basic arginine-lysine-rich nuclear M. E. Thompson BRCA1 nuclear import and export FEBS Journal 277 (2010) 3072–3078 ª 2010 The Author Journal compilation ª 2010 FEBS 3073 localization sequence; they did not clearly demonstrate interaction with import machinery. Chen et al. [11] carried the work to the next step and demonstrated the interaction of NLS1 (amino acids 501–508) with importin alpha. This clearly marked BRCA1 as a pro- tein that enters the nucleus via the classical importin alpha ⁄ beta pathway (Fig. 2). An alternative nuclear import pathway for BRCA1 Although BRCA1 does interact with importin alpha to be actively transported across the nuclear envelope, Wang et al. [8] observed that splice variants lacking exon 11, where both NLS are located, could also be RING finger domain (aa 1–109) Transcriptional activation domain (aa 1560–1863) 1 1863 Nuclear export sequences (aa 22–39 and aa 81–99) Nuclear localization signals (aa 503–508 and aa 607–614) BRCT domains (aa 1640–1729 and aa 1760–1821) Fig. 1. Schematic of key structural ⁄ functional elements of BRCA1. Nuclear targeting sequences of BRCA1 are shown in relationship to the RING finger domain, BRCA1 C Terminus (BRCT) and transcriptional activation domains. BRCA1 α β BRCA1 α β BRCA1 α β BRCA1 α β BRCA1 α BRCA1 α β α α BRCA1 BARD 1 BRCA1 BARD 1 α β β BRCA1 BARD 1 α β BRCA1 BARD 1 α β BRCA1 BARD 1 α 1 2 3 4 5 6 i ii iii iv v vi Extracellular Cytoplasm Nucleus Fig. 2. Schematic representing the two mechanisms for BRCA1 nuclear import. The depiction on the left represents BRCA1 binding directly to the import machinery. (1) BRCA1 binds importin alpha, which subsequently (2) binds importin beta. (3) The heterotrimer crosses the nuclear envelope in a GTP-dependent manner (hydrolysis of GTP to GDP not shown). Inside the nucleus, the trimer dissociates (4, 5) and importin alpha and beta are exported from the nucleus (6) to be used again. The depiction on the right side represents BRCA1 binding to BARD in order to enter the nucleus. The BARD1:BRCA1 complex binds to the import machinery by first binding to importin alpha (i). The BRCA1:BARD1:importin alpha complex binds importin beta (ii). This complex enters the nucleus in an energy-dependent manner (iii). Inside the nucleus, importins beta (iv) and alpha (v) dissociate from the BARD1:BRCA1 complex and are recycled. BRCA1 nuclear import and export M. E. Thompson 3074 FEBS Journal 277 (2010) 3072–3078 ª 2010 The Author Journal compilation ª 2010 FEBS found in the nucleus. This led to the supposition that there was an alternative pathway by which BRCA1 entered the nucleus. Fabbro et al. [12] reported that the non-NLS-dependent mechanism for BRCA1 locali- zation to the nucleus required its RING finger domain. Furthermore, the extent of nuclear localization of the variants correlated with the level of BRCA1-associated RING domain protein 1 (BARD1) expression. Given that BRCA1 and BARD1 bind via their RING finger domains, it was postulated that BRCA1 can enter the nucleus in a piggyback mechanism with BARD1. With the demonstration of two independent mecha- nisms for BRCA1 nuclear import, studies turned to investigating potential regulation of this translocation event. It had already been proposed that phosphoryla- tion, potentially due to the presence of serum growth factors, modulated BRCA1 localization, although no specific phosphoacceptor sites were initially identified. However, in 1999, Altiok et al. [13] published that the administration of heregulin b1 to T47D human breast cancer cells resulted in Akt-mediated phosphorylation of BRCA1 on serine 508, a site adjacent to NLS1. However, no functional consequences of this phos- phorylation were determined. Hinton et al. [14] subse- quently demonstrated that the exposure of cells to the growth factor resulted in an increase in nuclear accu- mulation of BRCA1. The cells were serum starved prior to the administration of heregulin. Interestingly, these conditions resulted in much of the BRCA1 local- izing outside the nucleus, in contrast to the observa- tions of Wang et al. [8], who reported that in the absence of serum, NIH3T3 fibroblasts, Hs578BST, HBL-100, ZR75-1, CAMA breast tumor cells and NIH OVCAR3 ovarian cancer cells exhibited a mostly nuclear presence of BRCA1, which redistributed to the cytoplasm upon serum addition. The far-reaching biological implications of Akt-med- iated regulation of BRCA1 are not understood. The ability of Akt to enhance the nuclear presence of BRCA1 superficially seems to be dichotomous. BRCA1 has many tumor suppressive associated func- tions within the nucleus. Thus, it would be confound- ing for Akt, a cell survival kinase, to facilitate its nuclear presence. However, numerous reports docu- ment a role for BRCA1 in apoptosis. Some of these data suggest that this function is mediated through cytoplasmic BRCA1. Indeed, several reports have doc- umented an association of BRCA1 with the cytoplas- mic enzyme, acetyl CoA carboxylase [15–17]. This interaction, which is mediated through the BRCA1 C Terminus domains of BRCA1, regulated lipogenesis in mammary epithelial cell lines. Specifically, BRCA1 interacted with the phosphorylated, inactive form of acetyl CoA carboxylase and interfered with its dephos- phorylation [16]. The decrease in functional, dephos- phorylated acetyl CoA carboxylase resulted in limited synthesis of palmitic acid and this led to apoptosis [17,18]. Also, noteworthy is the work of Dizin et al. [19], who demonstrated that cytoplasmic BRCA1 is proteolytically processed into a 90 kDa fragment that exhibits the apoptotic activity of the protein. These results may explain the otherwise confounding scenario of Akt-mediated nuclear accumulation of BRCA1. Other factors shown to regulate BRCA1 localization include hypoxia and tumor necrosis factor apoptosis inducing ligand (TRAIL). Fitzgerald et al. [20] demon- strated that exposure of several malignant mammary cell lines to hypoxia resulted in an enhancement of nuclear BRCA1, with concomitant decreases in cyto- plasmic levels. Although earlier reports had suggested that hypoxia decreased BRCA1 expression [21,22], this was not evident under the conditions used by Fitzger- ald et al. [20]. Additionally, this effect was not observed in nonmalignant cells, but was mimicked by the administration of TRAIL to the cells. The expres- sion of BRCA1 facilitated TRAIL-induced apoptosis. The idea that TRAIL induction of apoptosis is enhanced by nuclear BRCA1 would suggest that under those conditions it is nuclear BRCA1 that augments apoptosis. One plausible mechanism by which this may occur is that BRCA1 could be transactivating genes involved in the apoptotic process. However, this hypothesis has yet to be tested. Nuclear export of BRCA1 The nuclear ⁄ non-nuclear distribution of BRCA1 has been reported by numerous laboratories. This includes several reports of BRCA1 association with microtubules and centrosomes. However, the non-nuclear presence of BRCA1 was not specifically addressed until 2000, when Rodriguez & Henderson [23] published that BRCA1 underwent receptor-mediated nuclear export. These investigators delineated amino acid residues 81–99 as a Rev-like nuclear export sequence. This type sequence, which binds to CRM1 nuclear export receptor to facili- tate translocation of the cargo protein across the nuclear membrane at the expense of GTP, is defined by a characteristic pattern of hydrophobic amino acids. The consensus sequence is L-X 1-3 -L-X 2-4 -L-X-L. In addition to the presence of this motif at residues 81–99, BRCA1 has three other regions (amino acids 22–30, 591–600, 783–793) that also fit the consensus sequence. Of these, only residues 22–30 have been shown to have functional nuclear export activity [24]. The presence of two distinct nuclear export sequences is not novel. Other M. E. Thompson BRCA1 nuclear import and export FEBS Journal 277 (2010) 3072–3078 ª 2010 The Author Journal compilation ª 2010 FEBS 3075 tumor suppressors, including p53 [25,26], also have mul- tiple functional nuclear export sequences. Few reports have documented a regulatory compo- nent to BRCA1 nuclear export [12,27–30]. Fabbro et al. [12] suggested that the interaction of BRCA1 with BARD1 resulted in inhibition of BRCA1 nuclear export. Comparing the localization of wild-type BRCA1 with BRCA1 mutant construct in which the amino terminal nuclear export sequence at amino acids 81–99 was transferred to the carboxy terminus, this group showed that co-expression of BARD1 resulted in nuclear accumulation of wild-type BRCA1, but had no inhibitory effect on the nuclear export of the mutant. The authors concluded that BARD1 masks the amino terminal BRCA1 nuclear export sequence and, therefore, export could be regulated by BARD1 expression. Thus, BARD1 is important for both the nuclear import and nuclear retention of BRCA1. This laboratory subsequently assigned a physiologi- cal relevance to BARD1-mediated nuclear retention of BRCA1. Data support the idea that nuclear retention of BRCA1 decreases BRCA1-dependent apoptosis [27]. The data indicating that the recovery of BRCA1 nuclear export support those of others who have sug- gested that the apoptotic function of BRCA1 is due to its cytoplasmic localization. Another study revealing a novel regulatory event for BRCA1 nuclear export was reported by Glover-Collins & Thompson [28]. In this report, the authors were able to demonstrate that nuclear export of BRCA1 occurred in a cell cycle-dependent manner, resulting in BRCA1 transiently concentrating in a perinuclear loca- tion during the early part of S phase. This relocation was blocked by the introduction of the calcium chela- tor, BAPTA-AM, and was mimicked by the calcium ionophore, A23187. The biological relevance of this calcium-dependent modulation is not yet understood. However, progression of the mammalian cell cycle has been shown to be dependent on calcium signaling and a calcium transient occurs at the G1 ⁄ S boundary [29]. These findings may suggest that the transient nuclear export of BRCA1 during early S phase is a conse- quence of the G1 ⁄ S calcium peak. Conclusion In this review we have discussed one aspect of BRCA1 regulation – its nuclear to non-nuclear distribution. BRCA1 is one of many tumor suppressors that undergo active nuclear import and export processes (see Table 1 for targeting sequences). It would seem almost wasteful to expend the energy for opposing transport mechanisms. However, the dynamic equilib- rium between BRCA1 nuclear import and export could provide a fine level of regulation of its nuclear and⁄ or cytoplasmic functions. Tight regulatory control of BRCA1 localization has far-reaching implications. Numerous mutations with different functional classifications have been docu- mented in BRCA1 [31]. Several reported patient muta- tions probably affect either the nuclear import or export of BRCA1 and result in a disturbance of the nuclear to non-nuclear distribution and function. Pharmacologi- cally, this could potentially be exploited. Data suggest that histone deacetylase [32] and poly ADP ribose poly- merase [33,34] inhibitors are more effective in patients who are BRCA1 negative. Theoretically, if BRCA1 localization can be pharmacologically manipulated to render it nonfunctional, the use of histone deacetylase and poly ADP ribose polymerase inhibitors could bene- fit a broader range of patients. 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Thompson 3078 FEBS Journal 277 (2010) 3072–3078 ª 2010 The Author Journal compilation ª 2010 FEBS . activation domains. BRCA1 α β BRCA1 α β BRCA1 α β BRCA1 α β BRCA1 α BRCA1 α β α α BRCA1 BARD 1 BRCA1 BARD 1 α β β BRCA1 BARD 1 α β BRCA1 BARD 1 α β BRCA1 BARD 1 α 1 2 3 4 5 6 i ii iii iv v vi Extracellular Cytoplasm Nucleus Fig this Keywords BRCA1- associated RING domain protein 1 (BARD1); breast cancer susceptibility gene 1 (BRCA1) ; nuclear export; nuclear export sequence; nuclear import; nuclear localization