Localization of BRCA1 and a splice variant identifies the nuclear localization signal.

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 delta 672-4095. Immunofluorescence studies indicate nuclear localization of BRCA1 but cytoplasmic localization of BRCA1 delta 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.     

MDM2 splice variants predominantly localize to the nucleoplasm mediated by a COOH-terminal nuclear localization signal

Of the >40 alternative and aberrant splice variants of MDM2 that have been described to date, the majority has lost both the well-characterized nuclear localization signal (NLS1) and the nuclear export signal (NES) sequence. Because cellular localization of proteins provides insight regarding their potential function, we determined the localization of three different MDM2 splice variants. The splice variants chosen were the common variants MDM2-A and MDM2-B. In addition, MDM2-FB26 was chosen because it is one of the few variants described that contains the complete p53-binding site. All three splice variants predominantly localized to the nucleus. Nuclear localization of MDM2-A and MDM2-B was controlled by a previously uncharacterized nuclear localization signal (NLS2), whereas nucleoplasmic localization of MDM2-FB26 was mediated by NLS1. p53 and full-length MDM2 colocalized with the splice variants in the nucleus. MDM2-A and MDM2-B both contain a COOH-terminal RING finger domain, and interaction with full-length MDM2 through this domain was confirmed. MDM2-FB26 was the only splice variant evaluated that contained a p53-binding domain; however, interaction between MDM2-FB26 and p53 could not be shown. p14(ARF) did not colocalize with the splice variants and was predominantly expressed within the nucleoli. In summary, nuclear localization signals responsible for the nucleoplasmic distribution of MDM2 splice variants have been characterized. Colocalization and interaction of MDM2-A and MDM2-B with full-length MDM2 in the nucleus have important physiologic consequences, for example, deregulation of p53 activity.     

BARD1 induces BRCA1 intranuclear foci formation by increasing RING-dependent BRCA1 nuclear import and inhibiting BRCA1 nuclear export

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 NH2-terminal nuclear export signal. In transfected breast cancer cells, BRCA1 nuclear localization requires both the NLSs and NH2-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.     

Finding nuclear localization signals

A variety of nuclear localization signals (NLSs) are experimentally known although only one motif was available for database searches through PROSITE. We initially collected a set of 91 experimentally verified NLSs from the literature. Through iterated ‘in silico mutagenesis’ we then extended the set to 214 potential NLSs. This final set matched in 43% of all known nuclear proteins and in no known non-nuclear protein. We estimated that >17% of all eukaryotic proteins may be imported into the nucleus. Finally, we found an overlap between the NLS and DNA-binding region for 90% of the proteins for which both the NLS and DNA-binding regions were known. Thus, evolution seemed to have used part of the existing DNA-binding mechanism when compartmentalizing DNA-binding proteins into the nucleus. However, only 56 of our 214 NLS motifs overlapped with DNA-binding regions. These 56 NLSs enabled a de novo prediction of partial DNA-binding regions for ~800 proteins in human, fly, worm and yeast.     

The BRCA1 RING and BRCT domains cooperate in targeting BRCA1 to ionizing radiation-induced nuclear foci

BRCA1 accumulates in nuclear foci during S-phase and reassembles into DNA repair-associated foci after DNA damage, reflecting its role in genome maintenance. BRCA1 comprises a RING domain at the N terminus and a BRCT domain at the C terminus, through which it associates with DNA repair proteins. The key sequences that target BRCA1 to DNA damage-induced foci have not been identified. Here, we mapped the BRCA1 foci-targeting domains of yellow fluorescence protein (YFP)-tagged BRCA1 in MCF-7 breast cancer cells exposed to ionizing radiation (IR). Cancer mutations specific to the BRCT domain, but not the RING domain, abolished BRCA1 recruitment to IR-induced foci. The YFP-BRCT domain itself, however, localized poorly at IR-induced foci, and the RING domain and other sequences were negative. We discovered that only when the RING and BRCT domains were combined was foci targeting restored to levels observed for wild-type BRCA1. The RING-BRCT fusion co-localized at foci with the MDC1 DNA damage response factor and inhibited entry of endogenous BRCA1 into nuclear foci. Our results explain why exon 11-deficient BRCA1 splice variants are targeted to IR-induced foci even though they are incapable of repairing DNA damage. We propose that both RING and BRCT domains together target BRCA1 to large focal assemblies at DNA double-stranded breaks.     

Dissection of the karyopherin alpha nuclear localization signal (NLS)-binding groove: functional requirements for NLS binding

Classical protein import, mediated by the binding of a classical nuclear localization signal (NLS) to the NLS receptor, karyopherin/importin alpha, is the most well studied nuclear transport process. Classical NLSs are either monopartite sequences that contain a single cluster of basic amino acids (Lys/Arg) or bipartite sequences that contain two clusters of basic residues separated by an unconserved linker region. We have created mutations in conserved residues in each of the three NLS-binding sites/regions in Saccharomyces cerevisiae karyopherin alpha (SRP1). For each mutant we have analyzed binding to both a monopartite and a bipartite NLS cargo in vitro. We have also expressed each karyopherin alpha mutant in vivo as the only cellular copy of the NLS receptor and examined the impact on cell growth and import of both monopartite and bipartite NLS-containing cargoes. Our results reveal the functional significance of specific residues within karyopherin alpha for NLS cargo binding. A karyopherin alpha variant with a mutation in the major NLS-binding site exhibits decreased binding to both monopartite and bipartite NLS cargoes, and this protein is not functional in vivo. However, we also find that a karyopherin alpha variant with a mutation in the minor NLS-binding site, which shows decreased binding only to bipartite NLS-containing cargoes, is also not functional in vivo. This suggests that the cell is dependent on the function of at least one bipartite NLS cargo that is imported into the nucleus by karyopherin alpha. Our experiments also reveal functional importance for the linker-binding region. This study provides insight into how changes in binding to cellular NLS sequences could impact cellular function. In addition, this work has led to the creation of conditional alleles of karyopherin alpha with well characterized defects in NLS binding that will be useful for identifying and characterizing novel NLS cargoes.     

Two short protein domains are responsible for the nuclear localization of the mouse spermine oxidase mu isoform

In mouse, at least two catalytically active splice variants (mSMOalpha and mSMOmicro) of the flavin-containing spermine oxidase enzyme are present. We have demonstrated previously that the cytosolic mSMOalpha is the major isoform, while the mSMOmicro enzyme is present in both nuclear and cytoplasmic compartments and has an extra protein domain corresponding to the additional exon VIa. By amino acid sequence comparison and molecular modeling of mSMO proteins, we identified a second domain that is necessary for nuclear localization of the mSMOmicro splice variant. A deletion mutant enzyme of this region was constructed to demonstrate its role in protein nuclear targeting by means of transient expression in the murine neuroblastoma cell line, N18TG2.     

The sequence [EKRKI(E/R)(K/L/R/S/T)] is a nuclear localization signal for importin 7 binding (NLS7)

BackgroundNuclear translocation of large proteins is mediated through specific protein carriers, collectively named karyopherins (importins, exportins and adaptor proteins). Cargo proteins are recognized by importins through specific motifs, known as nuclear localization signals (NLS). However, only the NLS recognized by importin α and transportin (M9 NLS) have been identified so farMethodsAn unsupervised in silico approach was used, followed by experimental validation.ResultsWe identified the sequence EKRKI(E/R)(K/L/R/S/T) as an NLS signal for importin 7 recognition. This sequence was validated in the breast cancer cell line T47D, which expresses importin 7. Finally, we verified that importin 7-mediated nuclear protein transport is affected by cargo protein phosphorylation.ConclusionsThe NLS sequence for importin 7 was identified and we propose this approach as an identification method of novel specific NLS sequences for β-karyopherin family members.General significanceElucidating the complex relationships of the nuclear transporters and their cargo proteins may help in laying the foundation for the development of novel therapeutics, targeting specific importins, with an immediate translational impact.     

Neurofibromin is actively transported to the nucleus

Mutations in the neurofibromatosis type 1 (NF1) tumor suppressor gene predispose individuals to a variety of benign and malignant tumors. Many tumor suppressors ‘shuttle’ between the nucleus and the cytoplasm, thus regulating their function. By expressing different NF1 constructs in COS-7 cells (encompassing exons 28–49 and fused to the green fluorescent protein), we identified a functional nuclear localization signal (NLS) in exon 43. Mutation of the NLS completely abolishes the nuclear entry of the NF1-derivative fusion protein. A highly expressed splice variant that lacks this NLS controls the localization and hence the function of neurofibromin. The localization of neurofibromin in the nucleus may provide novel clues to unknown functions for NF1.     

The Intracellular Mobility of Nuclear Import Receptors and NLS Cargoes

We have investigated classical nuclear localization sequence (NLS) mediated protein trafficking by measuring biomolecular dynamics within living cells using two-photon fluorescence correlation spectroscopy. By directly observing the behavior of specific molecules in their native cellular environment, it is possible to uncover functional details that are not apparent from traditional biochemical investigations or functional assays. We show that the intracellular mobility of NLS cargoes and their import receptor proteins, karyopherin-α and karyopherin-β, can be robustly measured and that quantitative comparison of intracellular diffusion coefficients provides new insights into nuclear transport mechanisms. Import cargo complexes are assembled throughout the cytoplasm, and their diffusion is slower than predicted by molecular weight due to specific interactions. Analysis of NLS cargo diffusion in the cytoplasm indicates that these interactions are likely disrupted by NLS cargo binding. Our results suggest that delivery of import receptors and NLS cargoes to nuclear pores may complement selective translocation through the pores as a functional mechanism for regulating transport of proteins into the nucleus.