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Results for “Nuclear Localization Signals”

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PubMed2005

Conserved amino acids of the human immunodeficiency virus type 2 Vpx nuclear localization signal are critical for nuclear targeting of the viral preintegration complex in non-dividing cells.

The HIV-2 viral accessory protein Vpx is related to, but distinct from the Vpr protein of HIV-1. Vpx is packaged into virions and as a component of the viral preintegration complex (PIC) is required for efficient virus replication in non-dividing cells. We have previously reported that the minimal transferable region of Vpx that contained karyophilic properties was aa 65 to 72. Analysis of Vpx sequences from various HIV-2/SIV strains reveals that this region contains highly conserved amino acids, including two basic residues (K68, R70) and three tyrosines (Y66, Y69, Y71). Here, we demonstrate that mutation of the basic or tyrosine residues abolishes PIC nuclear import in arrested cells as assessed by PCR detection of viral integration. Examination of cell-free virus by Western blot indicated that all mutant proteins were incorporated into virions, suggesting that the lack of replication in arrested cells was not due to a loss of Vpx in target cells. Together, these studies map critical residues of the Vpx nuclear localization signal that are required for efficient infection of non-dividing cells.

Active Transport, Cell Nucleus↗

PubMed2006

A cytotoxic ribonuclease variant with a discontinuous nuclear localization signal constituted by basic residues scattered over three areas of the molecule.

Nuclear import of proteins is determined by specific signals that allow them to bind to receptors that mediate their energy-dependent transport through the nuclear pore. These signals are termed nuclear localization signals and do not constitute a specific consensus sequence. Among them, the most characterized correspond to monopartite and bipartite nuclear localization signals, which interact with the importin alpha/beta heterodimer. We previously described a cytotoxic variant of human pancreatic-ribonuclease that is actively transported into the nucleus. Here, we show that this protein interacts with importin alpha through different basic residues, including Lys1 and the arginine clusters 31-33 and 89-91. Although these residues are scattered along the sequence, they are close in the three-dimensional structure of the protein and their topological disposition strongly resembles that of a classical bipartite nuclear localization signal.

Active Transport, Cell Nucleus↗

PubMed2003

Alternative splicing disrupts a nuclear localization signal in spleen tyrosine kinase that is required for invasion suppression in breast cancer.

Spleen tyrosine kinase (Syk) is a candidate tumor (metastasis) suppressor that is highly expressed in mammary epithelial cells. Loss of Syk expression through promoter hypermethylation is associated with increased invasiveness in a subset of breast cancer. Here, we show that in addition to full-length Syk [Syk(L)], an alternatively spliced variant, Syk(S), is frequently expressed in breast cancer cells. Syk(S) is identical to Syk(L), except that it lacks 23 amino acid residues (deletion) within the interdomain B (IDB) of Syk. We also show that the aberrant expression of Syk(S) occurs frequently in primary breast tumors but never in matched normal mammary tissues, suggesting a contribution of Syk(S) to mammary tumor progression. Expression of Syk(L) suppressed breast cancer cell invasiveness. In contrast, Syk(S) expression did not affect the cell invasion potential. This differential phenotypic response is accompanied by their different subcellular localization. Immunocytochemical studies and nuclear and cytoplasmic fractionation experiments indicated that Syk(L) could enter the nucleus, whereas Syk(S) was located exclusively in the cytoplasm. Five basic residues in deletion were found to be critical in determining Syk(L) nuclear transport and invasion suppression activity; mutations completely excluded Syk(L) from the nucleus and blocked Syk(L)-inducible invasion suppression. Moreover, IDB acted as an autonomous nuclear localization signal to facilitate nuclear transport of a heterologous protein. Thus, the IDB of Syk(L) contains a nuclear localization signal that is responsible for Syk(L) nuclear translocation. The correlation of the nuclear localization and invasion suppression function of Syk(L) indicated that nuclear Syk possesses biological activities associated with tumor suppression in mammary epithelial cells.

Alternative Splicing↗

PubMed2003

NLSdb: database of nuclear localization signals.

NLSdb is a database of nuclear localization signals (NLSs) and of nuclear proteins. NLSs are short stretches of residues mediating transport of nuclear proteins into the nucleus. The database contains 114 experimentally determined NLSs that were obtained through an extensive literature search. Using 'in silico mutagenesis' this set was extended to 308 experimental and potential NLSs. This final set matched over 43% of all known nuclear proteins and matches no currently known non-nuclear protein. NLSdb contains over 6000 predicted nuclear proteins and their targeting signals from the PDB and SWISS-PROT/TrEMBL databases. The database also contains over 12 500 predicted nuclear proteins from six entirely sequenced eukaryotic proteomes (Homo sapiens, Mus musculus, Drosophila melanogaster, Caenorhabditis elegans, Arabidopsis thaliana and Saccharomyces cerevisiae). NLS motifs often co-localize with DNA-binding regions. This observation was used to also annotate over 1500 DNA-binding proteins. NLSdb can be accessed via the web site: http://cubic.bioc.columbia.edu/db/NLSdb/.

Active Transport, Cell Nucleus↗

PubMed2006

Morbillivirus nucleoprotein possesses a novel nuclear localization signal and a CRM1-independent nuclear export signal.

Morbilliviruses, which belong to the Mononegavirales, replicate its RNA genome in the cytoplasm of the host cell. However, they also form characteristic intranuclear inclusion bodies, consisting of nucleoprotein (N), in infected cells. To analyze the mechanisms of nucleocytoplasmic transport of N protein, we characterized the nuclear localization (NLS) and nuclear export (NES) signals of canine distemper virus (CDV) N protein by deletion mutation and alanine substitution of the protein. The NLS has a novel leucine/isoleucine-rich motif (TGILISIL) at positions 70-77, whereas the NES is composed of a leucine-rich motif (LLRSLTLF) at positions 4-11. The NLS and NES of the N proteins of other morbilliviruses, that is, measles virus (MV) and rinderpest virus (RPV), were also analyzed. The NLS of CDV-N protein is conserved at the same position in MV-N protein, whereas the NES of MV-N protein is located in the C-terminal region. The NES of RPV-N protein is also located at the same position as CDV-N protein, whereas the NLS motif is present not only at the same locus as CDV-N protein but also at other sites. Interestingly, the nuclear export of all these N proteins appears to proceed via a CRM1-independent pathway.

Amino Acid Sequence↗

PubMed1998

Nuclear import of protein kinase C occurs by a mechanism distinct from the mechanism used by proteins with a classical nuclear localization signal.

Protein kinase C does not have any known nuclear localization signal but, nevertheless, is redistributed from the cytoplasm to the nucleus upon various stimuli. In NIH 3T3 fibroblasts stimulation with phorbol ester leads to a translocation of protein kinase C alpha to the plasma membrane and into the cell nucleus. We compared the mechanism of protein kinase C alpha's transport into the nucleus with the transport mechanism of a protein with a classical nuclear localization signal at several steps. To this end, we co-microinjected fluorescently labeled bovine serum albumin to which a nuclear localization signal peptide was coupled, together with substances interfering with conventional nuclear protein import. Thereafter, the distribution of both the nuclear localization signal-bearing reporter protein and protein kinase C alpha was analyzed in the same cells. We can show that, in contrast to the nuclear localization signal-dependent transport, the phorbol ester-induced transport of protein kinase C alpha is not affected by microinjection of antibodies against the nuclear import factor p97/importin/karyopherin beta or microinjection of non-hydrolyzable GTP-analogs. This suggests that nuclear import of protein kinase C alpha is independent of p97/importin/karyopherin beta and independent of GTP. At the nuclear pore there are differences between the mechanisms too, since nuclear transport of protein kinase C alpha cannot be inhibited by wheat germ agglutinin or an antibody against nuclear pore complex proteins. Together these findings demonstrate that the nuclear import of protein kinase C alpha occurs by a mechanism distinct from the one used by classical nuclear localization signal-bearing proteins at several stages.

3T3 Cells↗

PubMed2005

Regulation of Gli1 localization by the cAMP/protein kinase A signaling axis through a site near the nuclear localization signal.

The hedgehog (Hh) pathway plays a critical role during development of embryos and cancer. Although the molecular basis by which protein kinase A (PKA) regulates the stability of hedgehog downstream transcription factor cubitus interruptus, the Drosophila homologue of vertebrate Gli molecules, is well documented, the mechanism by which PKA inhibits the functions of Gli molecules in vertebrates remains elusive. Here, we report that activation of PKA retains Gli1 in the cytoplasm. Conversely, inhibition of PKA activity promotes nuclear accumulation of Gli1. Mutation analysis identifies Thr374 as a major PKA site determining Gli1 protein localization. In the three-dimensional structure, Thr374 resides adjacent to the basic residue cluster of the nuclear localization signal (NLS). Phosphorylation of this Thr residue is predicted to alter the local charge and consequently the NLS function. Indeed, mutation of this residue to Asp (Gli1/T374D) results in more cytoplasmic Gli1 whereas a mutation to Lys (Gli1/T374K) leads to more nuclear Gli1. Disruption of the NLS causes Gli1/T374K to be more cytoplasmic. We find that the change of Gli1 localization is correlated with the change of its transcriptional activity. These data provide evidence to support a model that PKA regulates Gli1 localization and its transcriptional activity, in part, through modulating the NLS function.

Amino Acid Sequence↗

PubMed1997

Nuclear localization signal binding protein from Arabidopsis mediates nuclear import of Agrobacterium VirD2 protein.

T-DNA nuclear import is a central event in genetic transformation of plant cells by Agrobacterium. Presumably, the T-DNA transport intermediate is a single-stranded DNA molecule associated with two bacterial proteins, VirD2 and VirE2, which most likely mediate the transport process. While VirE2 cooperatively coats the transported single-stranded DNA, VirD2 is covalently attached to its 5' end. To better understand the mechanism of VirD2 action, a cellular receptor for VirD2 was identified and its encoding gene cloned from Arabidopsis. The identified protein, designated AtKAPalpha, specifically bound VirD2 in vivo and in vitro. VirD2-AtKAPalpha interaction was absolutely dependent on the carboxyl-terminal bipartite nuclear localization signal sequence of VirD2. The deduced amino acid sequence of AtKAPalpha was homologous to yeast and animal nuclear localization signal-binding proteins belonging to the karyopherin alpha family. Indeed, AtKAPalpha efficiently rescued a yeast mutant defective for nuclear import. Furthermore, AtKAPalpha specifically mediated transport of VirD2 into the nuclei of permeabilized yeast cells.

Amino Acid Sequence↗

PubMed1999

Appropriately spaced nuclear localizing signals are necessary for efficient nuclear import of nonnuclear proteins.

To deliver nonnuclear proteins into the nucleus, we have examined the locations and number of nuclear localizing signals by use of simian virus 40 large T-antigen (SV40Ta) and yeast enhanced green fluorescent protein (yEGFP) in Saccharomyces cerevisiae as a model system. When only one SV40Ta was added to either the N- or C-terminus of yEGFP, the fluorescence of yEGFP was detected in both the nucleus and the cytoplasm. When two SV40Ta signals were added, one to the N-terminus and one to the C-terminus of yEGFP (SV40Ta-yEGFP-SV40Ta), the fluorescence of yEGFP was localized in only the nucleus. When the presequence of cytochrome oxidase subunit IV (pCOXIV) was inserted between the SV40Ta and the N-terminus of yEGFP (SV40Ta-pCOXIV-yEGFP-SV40Ta) in this construct, the fluorescence was located in both the nucleus and the cytoplasm, suggesting that the increased distance between the two SV40Ta signals decreased the efficiency of transport into the nucleus. When an additional SV40Ta signal was inserted between pCOXIV and yEGFP (SV40Ta-pCOXIV-SV40Ta-yEGFP), the fluorescence was localized only in the nucleus, indicating that two SV40Ta signals spaced by pCOXIV of 28 amino acid residues forming an alpha-helix are potent in transporting yEGFP into the nucleus. These results indicate that two SV40Ta signals spaced appropriately are essential for the efficient transport of the nonnuclear protein into the nucleus.

Amino Acid Sequence↗

PubMed1992

Hormonal regulation of the nuclear localization signals of the human glucocorticosteroid receptor.

Nuclear localization of the rat glucocorticosteroid receptor (rGR) transiently expressed in COS-7 cells appears to be mediated by two nuclear localization signals, NL1 and NL2, in a hormone-dependent mechanism. We investigated the intracellular distribution of the human GR (hGR) expressed in COS-7 cells, by a different immunohistochemical technique involving immunostaining of cell pellet sections, thus avoiding the use of cell permeabilizing agents and allowing rigorous comparison between successive experiments. With a large set of hGR mutants, we could define determinants of the hGR nuclear localization and compare them with those previously reported for rGR. Our study demonstrated two hormone-dependent nuclear localization signals. NL1 activity, overlapping the DNA-binding domain (DBD)-hinge boundary, was repressed by the unliganded ligand-binding domain (LBD), even if the repressed NL1 retained a residual potency to target hGR in the nucleus. Structure/function analysis suggested a bipartite structure of NL1, analogous to that of other nuclear targeting signals (the carboxy-terminal part of DBD between amino acids 478 and 487 and the beginning of the hinge region which includes a basic amino acid stretch between 491 and 498). Upon hormone binding, NL2, located in the LBD, was activated, but was unable by itself to sustain full nuclear localization, which required the derepressed NL1 activity. Only two sequences in the LBD, localized between amino acids 600 and 626 and from amino acid 696 up to the carboxyl-terminal amino acid 777, respectively, were found to inhibit NL1 activity. As previously reported, efficient nuclear retention, mandatory for gene expression, did not required DNA-binding activity. The controversial intracellular localization of the unliganded form of hGR and the role of hsp90 in cytoplasmic localization are further discussed.

Cell Line↗

PubMed1995

Identification of the nuclear localization signal of rat liver CTP:phosphocholine cytidylyltransferase.

CTP:phosphocholine cytidylyltransferase (CT) is a major regulatory enzyme in phosphatidylcholine synthesis in mammalian cells. CT is found in both soluble and particulate forms, both of which are nuclear. We report here the identification of a 21-residue sequence at the amino terminus of CT, 8KVNSRKRRKEVPGPNGATEED28, which was sufficient to direct beta-galactosidase into the cell nucleus. Further deletions from either end of this sequence greatly reduced the nuclear localization of beta-galactosidase. Deletions of amino acids within the nuclear localization signal or of the entire signal disrupted CT nuclear localization, but CT was not completely excluded from the nucleus. Clones of stable transfectants of the nuclear localization signal-deficient CT expressed in Chinese hamster ovary (CHO) 58 cells, which is temperature-sensitive for growth and CT activity, were isolated and characterized. The deletion mutants were active under the same conditions as the wild-type enzyme. Despite the difference in subcellular location from wild-type CT, the nuclear localization mutants were fully able to complement the CT-deficient cell line CHO 58 for both growth and choline incorporation into phosphatidylcholine at the nonpermissive temperature. The mobility of the mutant enzymes on SDS gels was altered relative to the mobility of wild-type CT; however, the extent of phosphorylation of the mutant enzymes was decreased only slightly. Thus, the distribution of CT in both cytoplasm and nucleus, rather than exclusively nucleus, has little effect on the ability of CT to function in growing CHO cells.

Amino Acid Sequence↗

PubMed2001

The C-terminal region of PTHrP, in addition to the nuclear localization signal, is essential for the intracrine stimulation of proliferation in vascular smooth muscle cells.

PTHrP is secreted by most cell types. In addition to a paracrine/autocrine role, PTHrP has "intracrine" actions, entering the nuclear compartment under the direction of a classic bipartite nuclear localization signal. In vascular smooth muscle cells, nuclear entry stimulates mitogenesis. In the current study, we sought to more precisely define the regions of PTHrP required for the activation of mitogenesis in vascular smooth muscle cells. PTHrP deletion mutants missing large regions [i.e. the signal peptide, N terminus (1--36), mid region (38--86), nuclear localization signal, C terminus (108--139), or combinations of the above] were expressed in A-10 vascular smooth muscle cells. The consequences on nuclear localization and proliferation were examined. Deletion of the nuclear localization signal prevented nuclear entry and slowed proliferation. Deletion of the highly conserved N terminus or mid region had no impact on nuclear localization or on proliferation. Deletion of the C terminus had no deleterious effect on nuclear localization but dramatically reduced proliferation. Thus, the nuclear localization signal is both necessary and sufficient for nuclear localization of PTHrP. In contrast, activation of proliferation in vascular smooth muscle cells requires both an intact nuclear localization signal and an intact C terminus. Whereas the nuclear localization signal is required for nuclear entry, the C terminus may serve a trans-activating function to stimulate mitogenesis once inside the nucleus of vascular smooth muscle cells.

Amino Acid Sequence↗

PubMed2006

The monopartite nuclear localization signal of autoimmune regulator mediates its nuclear import and interaction with multiple importin alpha molecules.

Autoimmune regulator (AIRE) is a transcriptional regulator involved in establishing immunological self-tolerance. Mutations in the AIRE gene lead to the development of the autosomal, recessively inherited, organ-specific autoimmune disease, autoimmune polyendocrinopathy candidiasis ectodermal dystrophy (APECED). The AIRE protein is mainly localized in the cell nucleus where it is associated with nuclear bodies. The N-terminal part of AIRE has been previously shown to mediate nuclear localization of the protein. However, the functional nuclear localization signal (NLS) and nuclear import mechanisms of AIRE have not been identified. We show that, although the amino-acid sequence of AIRE contains a potential bipartite NLS consisting of amino acids 110-114 and 131-133, only the latter part constitutes a functional NLS. Furthermore, we show by in vitro binding assays that AIRE interacts with multiple members of the nuclear transport receptor importin alpha family, mainly alpha1, alpha3, and alpha5, and that these interactions depend on the intactness of the Arg-Lys-rich NLS of AIRE. In addition, we found that AIRE binds to the 'minor' NLS-binding site of importin alpha3 and alpha5 proteins consisting of the C-terminal armadillo repeats 7-9. Our findings strongly suggest that the nuclear import of AIRE is mediated by the classical importin alpha/beta pathway through binding to several importin alpha family members.

Amino Acid Sequence↗

PubMed2005

Characterization of nuclear localization signals and cytoplasmic retention region in the nuclear receptor CAR.

The constitutive androstane receptor (CAR) is a ligand/activator-dependent transactivation factor that resides in the cytoplasm and forms part of an as yet unidentified protein complex. Upon stimulation, CAR translocates into the nucleus where it modulates the transactivation of target genes. However, CAR exogenously expressed in rat liver RL-34 cells is located in the nucleus even in the absence of activators. By transiently transfecting RL-34 cells with various mutated rat CAR segments, we identified two nuclear localization signals: a basic amino acid-rich sequence (RRARQARRR) between amino acids 100 and 108; and an assembly of noncontiguous residues widely spread over amino acid residues 111 to 320 within the ligand binding domain. A C-terminal leucine-rich segment corresponding to a previously reported murine xenochemical response signal was not found to exhibit nuclear import activity in cultured cells. Using rat primary hepatocytes transfected with various CAR segments, we identified the region required for the cytoplasmic retention of CAR. Based on these results, the intracellular localization of CAR would be determined by the combined effects of nuclear localization signals, the xenochemical response signal, and the cytoplasmic retention region.

Animals↗

PubMed1998

Search for a nuclear localization signal in the prion protein.

Spongiform transmissible encephalopathies are neurodegenerative diseases characterized by the accumulation, in infected brains, of a pathological form of a normal host-encoded protein called PrP. Previous data have shown that PrP could interact with cytosolic factors, including nuclear molecules, emphasizing the possible function of such interactions. Moreover, in infected cells, PrP is observed not only at the plasma membrane but also in the nuclear compartment. The N-terminal extremity of the mature PrP has been thought to harbor a nuclear localization signal reminiscent of the nuclear localization signal of the simian virus 40 large T antigen. By designing a fusion protein between the putative nuclear localization signal of PrP and the green fluorescent protein, we have shown that the N-terminal sequence of PrP is not efficient in targeting the protein in the nuclear compartment. This implies new insights regarding the way by which PrP could, however, reach the nuclear compartment.

Amino Acid Sequence↗

PubMed1998

Cytomegalovirus assembly protein precursor and proteinase precursor contain two nuclear localization signals that mediate their own nuclear translocation and that of the major capsid protein.

The cytomegalovirus (CMV) assembly protein precursor (pAP) interacts with the major capsid protein (MCP), and this interaction is required for nuclear translocation of the MCP, which otherwise remains in the cytoplasm of transfected cells (L. J. Wood et al., J. Virol. 71:179-190, 1997). We have interpreted this finding to indicate that the CMV MCP lacks its own nuclear localization signal (NLS) and utilizes the pAP as an NLS-bearing escort into the nucleus. The CMV pAP amino acid sequence has two clusters of basic residues (e.g., KRRRER [NLS1] and KARKRLK [NLS2], for simian CMV) that resemble the simian virus 40 large-T-antigen NLS (D. Kalderon et al., Cell 39:499-509, 1984) and one of these (NLS1) has a counterpart in the pAP homologs of other herpesviruses. The work described here establishes that NLS1 and NLS2 are mutually independent NLS that can act (i) in cis to translocate pAP and the related proteinase precursor (pNP1) into the nucleus and (ii) in trans to transport MCP into the nucleus. By using combinations of NLS mutants and carboxy-terminal deletion constructs, we demonstrated a self-interaction of pAP and cytoplasmic interactions of pAP with pNP1 and of pNP1 with itself. The relevance of these findings to early steps in capsid assembly, the mechanism of MCP nuclear transport, and the possible cytoplasmic formation of protocapsomeric substructures is discussed.

Amino Acid Sequence↗

PubMed2006

Parafibromin is a nuclear protein with a functional monopartite nuclear localization signal.

Parafibromin is a nuclear protein with a tumour suppressor role in the development of non-hereditary and hereditary parathyroid carcinomas, and the hyperparathyroidism-jaw tumour (HPT-JT) syndrome, which is associated with renal and uterine tumours. Nuclear localization signal(s), (NLS(s)), of the 61 kDa parafibromin remain to be defined. Utilization of computer-prediction programmes, identified five NLSs (three bipartite (BP) and two monopartite (MP)). To investigate their functionality, wild-type (WT) and mutant parafibromin constructs tagged with enhanced green fluorescent protein or cMyc were transiently expressed in COS-7 cells, or human embryonic kidney 293 (HEK293) cells, and their subcellular locations determined by confocal fluorescence microscopy. Western blot analyses of nuclear and cytoplasmic fractions from the transfected cells were also performed. WT parafibromin localized to the nucleus and deletions or mutations of the three predicted BP and one of the predicted MP NLSs did not affect this localization. In contrast, deletions or mutations of a MP NLS, at residues 136-139, resulted in loss of nuclear localization. Furthermore, the critical basic residues, KKXR, of this MP NLS were found to be evolutionarily conserved, and over 60% of all parafibromin mutations lead to a loss of this NLS. Thus, an important functional domain of parafibromin, consisting of an evolutionarily conserved MP NLS, has been identified.

Amino Acid Sequence↗

PubMed2006

Epstein-Barr virus nuclear antigen 3A contains six nuclear-localization signals.

The Epstein-Barr nuclear antigen 3A (EBNA3A) is one of only six viral proteins essential for Epstein-Barr virus-induced transformation of primary human B cells in vitro. Viral proteins such as EBNA3A are able to interact with cellular proteins, manipulating various biochemical and signalling pathways to initiate and maintain the transformed state of infected cells. EBNA3A has been reported to have one nuclear-localization signal and is targeted to the nucleus during transformation, where it associates with components of the nuclear matrix. By using enhanced green fluorescent protein-tagged deletion mutants of EBNA3A in combination with site-directed mutagenesis, an additional five functional nuclear-localization signals have been identified in the EBNA3A protein. Two of these (aa 63-66 and 375-381) were computer-predicted, whilst the remaining three (aa 394-398, 573-578 and 598-603) were defined functionally in this study.

Epstein-Barr Virus Nuclear Antigens↗