Identification of a Novel Cytoplasmic Protein That Specifically Binds to Nuclear Localization Signal Motifs

Abstract

Active transport of proteins into the nucleus is mediated by interaction between the classical nuclear localization signals (NLSs) of the targeted proteins and the NLS receptor (importin) complex. This nuclear transport system is highly regulated and conserved in eukaryotes and is essential for cell survival. Using a fragment of BRCA1 containing the two NLS motifs as a bait for yeast two-hybrid screening, we have isolated four clones, one of which is importin ␣. Here we characterize one of the other clones identified, BRAP2, which is a novel gene and expressed as a 2-kilobase mRNA in human mammary epithelial cells and some but not all tissues of mice. The isolated full-length cDNA encodes a novel protein containing 600 amino acid residues with pI 6.04. Characteristic motifs of C2H2 zinc fingers and leucine heptad repeats are present in the middle and C-terminal regions of the protein, respectively. BRAP2 also shares significant homology with a hypothetical protein from yeast Saccharomyces cerevisiae, especially in the zinc finger region. Antibodies prepared against the C-terminal region of BRAP2 fused to glutathione S-transferase specifically recognize a cellular protein with a molecular size of 68 kDa, consistent with the size of the in vitro translated protein. Cellular BRAP2 is mainly cytoplasmic and binds to the NLS motifs of BRCA1 with similar specificity to that of importin ␣ in both two-hybrid assays in yeast and glutathione S-transferase pull-down assays in vitro. Other motifs such as the SV40 large T antigen NLS motif and the bipartite NLS motif found in mitosin are also recognized by BRAP2. Similarly, the yeast homolog of BRAP2 also binds to these NLS motifs in vitro. These results imply that BRAP2 may function as a cytoplasmic retention protein and play a role in regulating transport of nuclear proteins.The passage of macromolecules between the nucleus and the cytoplasm occurs through nuclear pores. Small macromolecules can diffuse through the nuclear pores at a rate inversely proportional to their mass. Proteins with molecular masses greater than 40 -60 kDa are actively transported through the nuclear pores. To be transported into the nucleus, the protein must either contain a nuclear localization signal or, if not, be bound to another protein that does (1, 2). This process requires at least four different factors acting in two distinct steps. The first step is mediated by importin ␣ (also termed karyopherin ␣) and importin ␤ (also termed karyopherin ␤). The ␣ subunit is primarily responsible for NLS 1 recognition, whereas the ␤ subunit appears to mediate docking to the nuclear pore complex. The second translocation step requires the small G protein Ran/TC4 and an interacting partner, p15 (3-13).The presence of a nuclear localization signal may not be sufficient to direct nuclear import. The target efficiency of NLS motifs can be modified by the presence of multiple NLS motifs within a protein, by modifications of the flanking sequences, and by the accessibility of the NLSs to the import machinery (1...

Activation of the ERK signal transduction pathway by Epstein–Barr virus immediate-early protein Rta

“…Confocal microscopy showed that Rta and BRAP2 co-localized in the cytoplasm (Fig. 3), which is consistent with the knowledge that BRAP2 is a cytoplasmic protein (Asada et al, 2004;Li et al, 1998).…”

“…As BRAP2 interacts with the NLS in BRCA1 and p21 to retain these two proteins in the cytoplasm (Asada et al, 2004;Li et al, 1998), we investigated whether expression of BRAP2 also retained Rta in the cytoplasm. To accomplish this, 293(maxi-EBV) cells were transfected with pHA-BRAP2 and treated with TPA and sodium butyrate.…”

Amphiphysin IIb-1, a novel splicing variant of amphiphysin II, regulates p73β function through protein-protein interactions

“…Depending on the timing or signal for nuclear translocation, p73b protein is translocated into the nucleus, where it exerts transcriptional activity and cell death. Consistent with our proposal, several investigators suggest that one of the inactivation mechanisms of the tumor suppressor involves cytoplasmic sequestration (Moll et al, 1992;Darzynkiewicz et al, 1992;Li et al, 1998). In the case of p53, the tumor-suppressor protein is found in the cytoplasm in tumor tissue from breast cancer, and neuroblastoma Moll et al, 1996).…”

Validation of eight genetic risk factors in East Asian populations replicated the association of BRAP with coronary artery disease

“…Considering the interaction between BRAP protein and Breast Cancer 1 protein (BRCA1), 21 there was a possibility that a significant association of CAD with BRAP in the Korean population might be obtained because of a selection bias in the controls, because Korean cancer patients without CAD were included in the control panel in addition to the random healthy individuals. However, there was no significant difference in the minor allele frequencies of the BRAP SNP among patients with stomach cancer (0.189, n¼230), colon cancer (0.143, n¼147), lung cancer (0.201, n¼87) and hepatocellular carcinoma (0.190, n¼71), compared with the random healthy individuals (0.191, n¼178), suggesting that the BRAP SNP was a genetic risk factor for CAD and was not related to the cancer phenotype in the Korean population.…”