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Results for “Nuclear Localization Signals”

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PubMed2002

Complex nuclear localization signals in the matrix protein of vesicular stomatitis virus.

The matrix (M) protein of vesicular stomatitis virus (VSV) functions from within the nucleus to inhibit bi-directional nucleocytoplasmic transport. Here, we show that M protein can be imported into the nucleus by an active transport mechanism, even though it is small enough (approximately 27 kDa) to diffuse through nuclear pore complexes. We map two distinct nuclear localization signal (NLS)-containing regions of M protein, each of which is capable of directing the nuclear localization of a heterologous protein. One of these regions, comprising amino acids 47-229, is also sufficient to inhibit nucleocytoplasmic transport. Two amino acids that are conserved among the matrix proteins of vesiculoviruses are important for nuclear localization, but are not essential for the inhibitory activity of M protein. Thus, different regions of M protein function for nuclear localization and for inhibitory activity.

Active Transport, Cell Nucleus↗

PubMed1995

Isolation of a yeast protein kinase that is activated by the protein encoded by SRP1 (Srp1p) and phosphorylates Srp1p complexed with nuclear localization signal peptides.

Srp1p, the protein encoded by SRP1 of Saccharomyces cerevisiae, is a nuclear-pore-associated protein. Its Xenopus homolog, importin, was recently shown to be an essential component required for nuclear localization signal (NLS)-dependent binding of karyophilic proteins to the nuclear envelope [Gorlich, D., Prehn, S., Laskey, R. A. & Hartman, E. (1994) Cell 79, 767-778]. We have discovered a protein kinase whose activity is stimulated by Srp1p (Srp1p fused to glutathione S-transferase and expressed in Escherichia coli) and is detected by phosphorylation of Srp1p and of a 36-kDa protein, a component of the protein kinase complex. The enzyme, called Srp1p kinase, is a protein-serine kinase and was found in extracts in two related complexes of approximately 180 kDa and 220 kDa. The second complex, when purified, contained four protein components including the 36-kDa protein. We observed that, upon purification of the kinase, phosphorylation of Srp1p became very weak, while activation of phosphorylation of the 36-kDa protein by Srp1p remained unaltered. Significantly, NLS peptides and the nuclear proteins we have tested greatly stimulated phosphorylation of Srp1p, suggesting that Srp1p, complexed with karyophilic proteins carrying an NLS, is the in vivo substrate of this protein kinase.

Amino Acid Sequence↗

PubMed1997

Intracellular distribution and identification of the nuclear localization signals of two plant heat-stress transcription factors.

Similar to heat-stress transcription factors (HSFs) from non-plant sources, HSFA1 and HSFA2 from tomato (Lycopersicon esculentum Mill) contain two conserved clusters of basic amino acid residues (K/R1 and K/R2) which might serve as nuclear localization signal (NLS) motifs. Mutation of either one of them and functional testing of the corresponding proteins in a transient expression assay using tobacco (Nicotiana plumbaginifolia L:) protoplasts gave the following results. Whereas K/R1, positioned in all HSFs at the C-terminus of the DNA-binding domain, had no influence on nuclear import, the K/R1 mutants were impaired in their interaction with the DNA (band-shift assays). In contrast to this, mutants of the K/R2 motif, found 15-20 amino acid residues C-terminal of the oligomerization domain (HR-A/B region), had wild-type activity in DNA-binding but were defective in nuclear import. Thus, for the related tomato HSFA1 and HSFA2 the K/R2 cluster represents the only NLS motif, and in this function it cannot be replaced by K/R1.

Amino Acid Sequence↗

PubMed2005

Synthesis of acridine-nuclear localization signal (NLS) conjugates and evaluation of their impact on lipoplex and polyplex-based transfection.

We report on the synthesis of various acridine (Acr)-spacer-nuclear localization signal (NLS) peptide conjugates and explore whether their use as NLS-labeling agent of plasmidic DNA could improve gene nuclear import and expression into cells when mediated by synthetic DNA complexes. As the conditions of successful use of the NLS properties to enhance gene transfer are not clear, and with the aim of detecting and defining the requirements of NLS-enhanced transfection, we investigated gene delivery and expression into various cell lines with various DNA complexes (lipoplexes or polyplexes) that were formulated for various N/P ratios from various preformed Acr-spacer-NLS/DNA complexes (1:1, 5:1 and 10:1 molar ratio). For the in vitro transfection assays, the lipoplexes and polyplexes were formulated from the preformed Acr-spacer-NLS/DNA complexes and dioctadecylamidoglycylspermine (DOGS)/dioleylphosphatidylethanolamine (DOPE) 1:1 mol and branched polyethyleneimine (PEI) 25 kDa, respectively, which are very efficient in vitro gene transfer systems. We show by fluorescence experiments that part of the acridine-NLS-conjugates remains intercalated within the plasmid for most of the N/P lipoplexes and polyplexes investigated. We show that, as several other studies performed with NLS-conjugates that are not covalently linked to DNA, the expression of the transgene is in most cases not improved upon complexation of plasmidic DNA with NLS-intercalating conjugates prior to its formulation as lipoplexes or polyplexes.

Acridines↗

PubMed2003

Synergy of silent and hot spot mutations in importin beta reveals a dynamic mechanism for recognition of a nuclear localization signal.

Molecular recognition of the importin beta-binding (IBB) domain of importin alpha by importin beta is critical for the nuclear import of protein cargoes containing a classical nuclear localization signal. We have studied the function of four conserved tryptophans of importin beta (Trp-342, Trp-430, Trp-472, and Trp-864) located at the binding interface with the IBB domain by systematic alanine substitution mutagenesis. We found that Trp-864 is a mutational hot spot that significantly affects IBB-binding and import activity, whereas residues Trp-342, Trp-430, and Trp-472 are mutationally silent when analyzed individually. Interestingly, the combination of the hot spot at residue Trp-864 with mutations in the other three tryptophans gives rise to a striking synergy that diminishes IBB domain binding by up to approximately 1000-fold and, in turn, abolishes import activity. We propose that importin beta uses the tryptophans to select and stabilize a helical conformation of the IBB domain, which, in turn, conveys specific, high affinity binding.

Active Transport, Cell Nucleus↗

PubMed2002

Acute glucose starvation activates the nuclear localization signal of a stress-specific yeast transcription factor.

In yeast, environmental conditions control the transcription factor Msn2, the nuclear accumulation and function of which serve as a sensitive indicator of nutrient availablity and environmental stress load. We show here that the nuclear localization signal (NLS) of Msn2 is a direct target of cAMP-dependent protein kinase (cAPK). Genetic analysis suggests that Msn2-NLS function is inhibited by phosphorylation and activated by dephosphorylation. Msn2-NLS function is unaffected by many stress conditions that normally induce nuclear accumulation of full-length Msn2. The Msn2-NLS phosphorylation status is, however, highly sensitive to carbohydrate fluctuations during fermentative growth. Dephosphorylation occurs in >2 min after glucose withdrawal but the effect is reversed rapidly by refeeding with glucose. This response to glucose depletion is due to changes in cAPK activity rather than an increase in protein phosphatase activity. Surprisingly, the classical glucose-sensing systems are not connected to this rapid response system. Our results further imply that generic stress signals do not cause short-term depressions in cAPK activity. They operate on Msn2 by affecting an Msn5-dependent nuclear export and/or retention mechanism.

Base Sequence↗

PubMed1998

A Ty1 integrase nuclear localization signal required for retrotransposition.

Ty1 retrotransposition in Saccharomyces cerevisiae requires integrase (IN)-mediated insertion of Ty1 cDNA into the host genome. The transposition components are assembled in the cytoplasm and must cross the nuclear envelope to reach the genomic target, since, unlike animal cell nuclear membranes, the yeast cell nuclear membrane remains intact throughout the cell cycle. We have identified a bipartite nuclear localization signal (NLS) in IN required for Ty1 transposition (Ty1 IN) that directs IN to the nucleus. Mutations in the NLS that specifically abolish nuclear localization inactivate transpositional integration but do not affect reverse transcription, protein processing, or catalytic activity in vitro. No additional Ty1-encoded proteins are required for IN nuclear localization. Intragenic complementation experiments suggest that Ty1 IN functions as a multimer and contains two distinct domains, one required for integration and the other for nuclear localization. Nuclear targeting of the preintegration complex by an IN NLS may prove to be a general strategy used by retrotransposons and retroviruses that infect nondividing cells.

Amino Acid Sequence↗

PubMed2005

Insertion/deletion polymorphism in the BRCA2 nuclear localization signal.

Mutations in human BRCA2 confer an increased risk of female breast cancer. In this study, we found a novel insertion/deletion polymorphism (10204insAAA causing amino acid change M3332IK) in canine BRCA2, which is located in the putative second nuclear localization signal (NLS2) and C-terminal Rad51-binding region. The nuclear localization of the insAAA C-terminus was more efficient than localization of the delAAA sequence when NLS1 was mutated. Strong, comparable Rad51 binding was observed for both the insAAA and delAAA C-termini. Dogs with the insertion/deletion polymorphism will provide a new model for studying the function of BRCA2.

Alleles↗

PubMed2005

Identification of a functional bipartite nuclear localization signal in the tumor suppressor parafibromin.

Parafibromin is a putative tumor suppressor encoded by HRPT2, mutations in which have been implicated in the familial tumor syndrome hyperparathyroidism jaw tumor syndrome (HPT-JT), and sporadic parathyroid carcinoma. Recently, parafibromin has been shown to be an accessory factor for RNA polymerase II as part of the human Paf 1 complex, suggesting, as has been shown for its yeast homologue (Cdc 73), that it may have a role as an important regulator of transcription. Parafibromin has also been shown to interact with a histone methyltransferase complex that methylates histone H3 and to inhibit proliferation when overexpressed in mammalian cell lines. Despite these findings, the cellular localization of parafibromin has been controversial, with reports of both nuclear and nucleocytoplasmic localization. We have expressed wild-type and mutant parafibromin tagged with enhanced green fluorescent protein and have identified a functional bipartite nuclear localization signal (NLS) at residues 125-139 (nucleotides 373-417), KRAADEVLAEAKKPR, that is evolutionarily conserved and critical for the nuclear localization of parafibromin. We have also shown that the C-terminal arm of this bipartite NLS plays the primary role in nuclear localization. In support of these findings, specific HRPT2 mutations identified in HPT-JT or sporadic parathyroid carcinoma predicted to truncate parafibromin upstream of or within this NLS disrupt nuclear localization.

Amino Acid Sequence↗

PubMed1999

Autoinhibition by an internal nuclear localization signal revealed by the crystal structure of mammalian importin alpha.

Importin alpha is the nuclear import receptor that recognizes classical monopartite and bipartite nuclear localization signals (NLSs). The structure of mouse importin alpha has been determined at 2.5 A resolution. The structure shows a large C-terminal domain containing armadillo repeats, and a less structured N-terminal importin beta-binding domain containing an internal NLS bound to the NLS-binding site. The structure explains the regulatory switch between the cytoplasmic, high-affinity form, and the nuclear, low-affinity form for NLS binding of the nuclear import receptor predicted by the current models of nuclear import. Importin beta conceivably converts the low- to high-affinity form by binding to a site overlapping the autoinhibitory sequence. The structure also has implications for understanding NLS recognition, and the structures of armadillo and HEAT repeats.

Amino Acid Sequence↗

PubMed2002

The NPK1 mitogen-activated protein kinase kinase kinase contains a functional nuclear localization signal at the binding site for the NACK1 kinesin-like protein.

The tobacco mitogen-activated protein kinase kinase kinase NPK1 localizes to the equatorial region of phragmoplasts by interacting with kinesin-like protein NACK1. This leads to activation of NPK1 kinase at late M phase, which is necessary for cell plate formation. Until now, its localization during interphase has not been reported. We investigated the subcellular localization of NPK1 in tobacco-cultured BY-2 cells at interphase using indirect immunofluorescence microscopy and fusion to green fluorescent protein (GFP). Fluorescence of anti-NPK1 antibodies and GFP-fused NPK1 were detected only in the nuclei of BY-2 cells at interphase. Examination of the amino acid sequence of NPK1 showed that at the carboxyl-terminal region in the regulatory domain, which contains the binding site of NACK1, NPK1 contained a cluster of basic amino acids that resemble a bipartite nuclear localization signal (NLS). Amino acid substitution mutations in the critical residues in putative NLS caused a marked reduction in nuclear localization of NPK1 in BY-2 cells, indicating that this sequence is functional in tobacco BY-2 cells. We also found that the 64-amino acid sequence at the carboxyl terminus that contains NLS sequence is essential for interaction with NACK1, and that mutations in the NLS sequence prevented NPK1 from interacting with NACK1. Thus, the amino acid sequence at the carboxyl-terminal region of NPK1 has dual functions for nuclear localization during interphase and binding NACK1 in M phase.

Active Transport, Cell Nucleus↗

PubMed1995

Nuclear localization signal of HIV-1 as a novel target for therapeutic intervention.

BACKGROUND: Human immunodeficiency virus type 1 (HIV-1) is a lentivirus and shares with other members of this retroviral subfamily the ability to replicate in nondividing cells, in particular, cells of the monocyte/macrophage lineage. This feature relies on the presence of a specific nuclear localization signal (NLS) within the viral matrix protein (MA p17), which to some degree can be complemented by the activity of the viral vpr gene product. The MA p17 NLS ensures efficient transportation of the viral preintegration complex into the nucleus of an infected macrophage and confers persistence of HIV-1 in quiescent T cells, and therefore presents an attractive target for therapeutic intervention. MATERIALS AND METHODS: Nuclear localization signals (NLS) in general and the HIV-1 MA p17 NLS in particular are characterized by a stretch of positively charged amino acids including one or more lysine residues. A series of compounds potentially capable of binding and reacting with lysine by forming Schiff base adducts was synthesized. Our special consideration was to make compounds that would preferentially bind to two closely contiguous amino functions, as opposed to isolated single lysine residues. We assumed that this approach might specifically target the compound to NLS while affecting other regions less, thus reducing nonspecific cytotoxicity. Antiviral activity was assessed in primary monocytes and in peripheral blood lymphocytes (PBL) infected with HIV-1ADA strain. Viral replication was monitored by reverse transcriptase (RT) activity in the supernatant. Efficiency of nuclear importation of the viral preintegration complex was estimated by the formation of 2-LTR circle forms of HIV-1 DNA and also by in situ PCR techniques. RESULTS: Arylene bis(methyl ketone) compounds with a nitrogenous third subsituent, especially a pyrimidinic side-chain, inhibited HIV-1 replication in human monocytes at an IC50 as low as 1 nM. These compounds did not block HIV-1 replication in peripheral blood lymphocyte cultures. The inhibitory effect observed in monocyte cultures appeared in the context of markedly reduced nuclear importation of viral DNA in the presence of the drug. No cytotoxic effects of the compounds was observed in vitro at concentrations as high as 10 microM. An amidinohydrazone derivative of the most active compound was about 100 times less active than the parent, indicating that carbonyl groups were instrumental in the antiviral effect. CONCLUSIONS: These early results suggest that retroviral replication in nondividing cells is susceptible to pharmaceutical intervention targeted against the NLS activity of HIV-1 proteins in the viral preintegration complex. The compounds described efficiently block translocation of viral DNA to the nuclei of infected primary monocytes, and inhibit viral replication. This inhibition is effective only in nondividing cells and is not seen in proliferating cultures, such as activated PBLs. Thus, drugs that target HIV-1 NLS may be useful to specifically block the macrophage arm of HIV infection and could thereby be of value in treating macrophage-specific manifestations of HIV disease, such as HIV-1 dementia. In combination with other drugs, potential therapeutics exploiting this target may also help to control the progression of HIV-1 infection and disease.

Antiviral Agents↗

PubMed1995

Human prothymosin alpha inhibits division of yeast Saccharomyces cerevisiae cells, while its mutant lacking nuclear localization signal does not.

Effect of human prothymosin alpha and its mutant over-produced in S. cerevisiae on yeast cell division was studied. Wild-type prothymosin alpha appeared to block division of yeast cells. Its inhibitory action could be abolished by deletion of the last nine carboxy-terminal amino acids of prothymosin alpha containing nuclear localization signal, thus pointing to the nucleus as a compartment, where prothymosin alpha performs its action.

Amino Acid Sequence↗

PubMed2004

A single subunit of a heterotrimeric CCAAT-binding complex carries a nuclear localization signal: piggy back transport of the pre-assembled complex to the nucleus.

An unresolved question concerns the nuclear localization of the heterotrimeric CCAAT-binding complex, which is evolutionarily conserved in eukaryotic organisms including fungi, plants and mammals. All three subunits are necessary for DNA binding. In the filamentous fungus Aspergillus nidulans the corresponding complex was designated AnCF (A.nidulans CCAAT-binding factor). AnCF consists of the HapB, HapC and HapE subunits. Here, by using various green fluorescent protein constructs, a nuclear localization signal sequence (NLS) of the HapB protein was identified, outside of the evolutionarily conserved domain. HapB-EGFP was transported into the nucleus in both DeltahapC and DeltahapE strains, indicating that its NLS interacts with the import machinery independently of the other Hap subunits. In contrast, HapC-EGFP did not enter the nucleus in the absence of HapE or HapB. A similar finding was made for HapE-EGFP, which did not localize to the nucleus in the absence of HapC or HapB. Addition of the HapB-NLS to either HapC or HapE led to nuclear localization of the respective protein fusions, indicating that both HapC and HapE lack a functional NLS. Furthermore, these data strongly suggest that HapC and HapE have first to form a heterodimer and can be transported only as a heterodimer via the HapB protein into the nucleus. Therefore, the HapB subunit is the primary cargo for the import machinery, while HapC and HapE are transported to the nucleus only as a heterodimer and in complex with HapB via a piggy back mechanism. This enables the cell to provide equimolar concentrations of all subunits to the nucleus.

Amino Acid Sequence↗

PubMed2003

Importin alpha/beta and Ran-GTP regulate XCTK2 microtubule binding through a bipartite nuclear localization signal.

The small GTPase Ran is essential for spindle assembly. Ran is proposed to act through its nuclear import receptors importin alpha and/or importin beta to control the sequestration of proteins necessary for spindle assembly. To date, the molecular mechanisms by which the Ran pathway functions remain unclear. Using purified proteins, we have reconstituted Ran-regulated microtubule binding of the C-terminal kinesin XCTK2, a kinesin important for spindle assembly. We show that the tail of XCTK2 binds to microtubules and that this binding is inhibited in the presence of importin alpha and beta (alpha/beta) and restored by addition of Ran-GTP. The bipartite nuclear localization signal (NLS) in the tail of XCTK2 is essential to this process, because mutation of the NLS abolishes importin alpha/beta-mediated regulation of XCTK2 microtubule binding. Our data show that importin alpha/beta directly regulates the activity of XCTK2 and that one of the molecular mechanisms of Ran-regulated spindle assembly is identical to that used in classical NLS-driven nuclear transport.

Animals↗

PubMed2001

Identification of Nash1, a novel protein containing a nuclear localization signal, a sterile alpha motif, and an SH3 domain preferentially expressed in mast cells.

By using a serial analysis of gene expression (SAGE), we have identified a novel full-length cDNA that is preferentially expressed in human cord blood-derived mast cells. The predicted protein showed unique primary structure with a nuclear localization signal (NLS), a sterile alpha motif (SAM), and a Src homology 3 domain (SH3) (termed Nash1). Nash1 was mapped to human chromosome 21q11.1 and highly expressed in spleen, liver, peripheral blood, and mast cell lines. In consistent with the presence of NLS, Nash1 was localized in the nucleus. Interestingly, screening gene databases for Nash1-related sequences revealed the existence of a Nash1-related gene termed SLY that was preferentially detected in lymphoid cells. We also found at least two additional candidates for this gene family in the database. These findings suggested that Nash1 and Nash1-related proteins consisted of a novel family of signaling/adaptor proteins, and Nash1 might function as a signaling component of mast cells, possibly in the nucleus.

Adaptor Proteins, Vesicular Transport↗

PubMed1999

Phenotype of HIV-1 lacking a functional nuclear localization signal in matrix protein of gag and Vpr is comparable to wild-type HIV-1 in primary macrophages.

Human immunodeficiency virus type 1 (HIV-1) is considered to infect nondividing cells because nuclear localization signals (NLS) in matrix (MA, p17(Gag)) and Vpr allow active nuclear transport of the preintegration complex. Previous studies demonstrated that HIV-1 reverse transcription is successful only in cells with proliferative potential, thus restricting HIV-1 replication to cycling cells. To sort out this apparent discrepancy we compared the phenotype of a chimeric HIV-1 variant lacking a functional Vpr and MA-NLS (R7. deltaVpr.deltaNLS), and previously described to lack replicative capacity in macrophages and growth-arrested cells, with a chimera lacking a functional Vpr (R7.deltaVpr). Both variants replicated efficiently in primary macrophages, with only minimal differences in the kinetics of reverse transcription, integration, or p24 production. In agreement with our previous observation, elongation of reverse transcription was restricted to the proliferating subpopulation of macrophages. Replication of R7.deltaVpr and R7.deltaVpr.deltaNLS could also be demonstrated in aphidicolin-treated macrophages, indicating efficient nuclear transport in G1/S phase-arrested cells. In conclusion, our results confirm the dependency of the process of HIV-1 reverse transcriptase on cell proliferation in primary macrophages and exclude an important role of MA-NLS and Vpr in macrophage infection.

Bromodeoxyuridine↗

PubMed2000

Screening of Genes Encoding Nuclear Localization Signal in a Mouse Embryonic cDNA Library.

A genetic screening system was established previously for the cloning of gene fragments encoding nuclear localization signals (NLS). In the current study, the system was improved and its ability to distinguish nuclear and cytoplasm-localized proteins was verified by inserting a known NLS-encoding gene. A mouse 10.5-d pc embryonic cDNA library was then inserted into the cloning site of the screening vector and 22 out of 10(4) clones were shown to be positive after screening using the system. The cDNA inserts of some of these clones were sequenced, and typical and untypical NLS were identified. The nuclear localizing activity of the cDNA inserts was further verified using the GFP fusion protein system. So, the system is useful in the cDNA library screening of genes encoding NLS-bearing proteins.

Journal Article↗