Localization of BRCA1 and a Splice Variant Identifies the Nuclear Localization Signal

Abstract

Inherited mutations in BRCA1 confer susceptibility to breast and ovarian neoplasms. However, the function of BRCA1 and the role of BRCA1 in noninherited cancer remain unknown. Characterization of alternately spliced forms of BRCA1 may identify functional regions; thus, we constructed expression vectors of BRCA1 and a splice variant lacking exon 11, designated BRCA1⌬672-4095. Immunofluorescence studies indicate nuclear localization of BRCA1 but cytoplasmic localization of BRCA1⌬672-4095. Two putative nuclear localization signals (designated NLS1 and NLS2) were identified in exon 11; immunofluorescence studies indicate that only NLS1 is required for nuclear localization. RNA analysis indicates the expression of multiple, tissue-specific forms of BRCA1 RNAs; protein analysis with multiple antibodies suggests that at least three BRCA1 isoforms are expressed, including those lacking exon 11. The results suggest that BRCA1 is a nuclear protein and raise the possibility that splicing is one form of regulation of BRCA1 function by alteration of the subcellular localization of expressed proteins.BRCA1 was isolated by positional cloning methods as a gene linked to breast cancer in families with a pattern of autosomal dominant inheritance of the disease (21). Single dominant susceptibility alleles are thought to account for 5 to 10% of all breast cancers, and BRCA1 germ line mutations are widely held to be responsible for approximately 50% of all inherited breast cancers. Inherited BRCA1 mutations are also thought to be responsible for the disease in 80 to 90% of all families with breast-ovarian cancer syndrome (9). Women inheriting mutations in BRCA1, most of which are truncating mutations that result in nonfunctional or unstable proteins (8), have an 85% chance of developing breast cancer in their lifetime (9). An on-line database now provides a listing of known BRCA1 mutations (http:// www. nchgr. nih. gov/ Intramural_research/ Lab_transfer/Bic/index.html), but little is known about the regulation of this gene or the function of its protein product. Unfortunately, mutations in BRCA1 are distributed evenly over the gene, providing little in the way of clues for localizing critical functional regions.BRCA1 is a large gene, with a coding region of 5.5 kb and a total mRNA of approximately 8.0 kb. There is little identifiable homology to known genes, with the exception of a short region in the 5Ј end (spanning exons 2 to 5) that encodes a RING finger with a typical Cys 3 -His-Cys 4 structure. This is a zincbinding motif that is found in a family of transcription factors and may be a protein-protein interaction site. Initial reports also provided evidence for a complex pattern of alternate splicing and the potential for translation of a number of BRCA1 protein isoforms (21). BRCA1 fits the model of a classic tumor suppressor gene, a hypothesis supported by recent work demonstrating that expression of BRCA1 inhibits growth of breast and ovarian cancer cell lines and MCF7-based tumor development in nude mice (16). Additional da...

BRCA1 physically associates with p53 and stimulates its transcriptional activity

“…This conserved domain has not been noted previously to play a role in BRCA1 function. It is notable that this region is missing from several alternatively spliced forms of BRCA1 (Thakur et al, 1997).…”

“…An N-terminal segment of BRCA1 exon 11 extending from residues 224 ± 500 is su cient for its interaction with p53. Interestingly, exon 11 is deleted in several naturally occurring isoforms of BRCA1 (Thakur et al, 1997), leading to speculation that the inability of the BRCA1Dexon11 mutant to bind to p53 may be a regulatory mechanism for BRCA1. Our ®ndings also suggest that BRCA1 binding to p53 may not be mediated by RAD51, RNA polymerase II holoenzyme or BARD1 since they bind to di erent regions on BRCA1 (Scully et al, 1997a,c;Wu et al, 1996).…”

“…The Luciferase T7 control DNA was obtained from Promega. Wild-type and mutant BRCA1 (Dexon11, D500 ± 1863, D1312 ± 1863, P1749R, Y1853insA and Q1756insC) were described previously (Thakur et al, 1997;Somasundaram et al, 1997). The p53 cDNA was ampli®ed using the primers 5'-AAGCTTGCCACCATGGAGGAGCCGCAGTCA-3' and 5'-ATGCGGCCGCTCAGTCTGAGTCAG-3' and the human p53 cDNA as template.…”

“…Evidence implicates a role for BRCA1 in the control gene expression. BRCA1 contains a nuclear localization signal (Thakur et al, 1997), a C-terminal domain that transactivates gene expression when fused to a heterologous DNA binding domain (Chapman and Verma, 1996;Monteiro et al, 1996) and BRCA1 has been found as a component of RNA polymerase II (Scully et al, 1997a).…”

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

“…BRCA1 interacts with importin α and can be actively imported into the nucleus via the importin α/β pathway. 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].…”