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PubMedDate not supplied

A bipartite NLS at the SHP-1 C-terminus mediates cytokine-induced SHP-1 nuclear localization in cell growth control.

SHP-1 protein tyrosine phosphatase is a critical regulator of signaling in hematopoietic cells as illustrated by the lethal hematopoietic disorders in SHP-1-deficient mice. We and others have shown in previous studies that SHP-1 regulates membrane receptor signaling: it binds via its N-terminal region SH2 domains to tyrosine phosphorylated membrane receptors to dephosphorylate key substrates in the receptor complexes. Here we demonstrate that the SHP-1 C-terminal region contains a bipartite NLS that mediates SHP-1 nuclear localization in response to cytokines. This NLS was located within amino acids 576-595 of the PTPase and, when fused by itself to EGFP, targeted the fluorescent protein into the nuclei of transiently transfected NIH3T3 fibroblasts and Bac1.2f5 macrophage cells. When positioned within SHP-1, the activity of the NSL was under tight regulation as indicated by the predominant cytoplasmic distribution of the EGFP/SHP-1 fusion protein in NIH3T3 transfectants and the exclusive cytoplasmic localization of the endogenous SHP-1 in hematopoietic cell line PBLC-1. Activation of the NLS in SHP-1 by IL-4 was demonstrated by increased nuclear localization of the EGFP/SHP-1 fusion protein in NIH3T3 transfectants and of the endogenous SHP-1 protein in PBCL-1 cells at 4, 6 and 8 h post-IL-4 stimulation. SHP-1 nuclear localization in PBCL-1 cells was also induced by IL-7 in a similar manner, suggesting it as a common event in cytokine signaling. In comparison to that of the wild-type phosphatase, an SHP-1 mutant lacking the NLS showed only approximately half of the activity in inhibiting proliferation of NIH3T3 transfectants. These results provide evidence of cytokine-regulated SHP-1 nuclear localization mediated by a bipartite NLS and suggest that SHP-1 regulates nuclear signaling in cell growth control.

3T3 Cells↗
PubMed2006

Nuclear import of Epstein-Barr virus nuclear antigen 1 mediated by NPI-1 (Importin alpha5) is up- and down-regulated by phosphorylation of the nuclear localization signal for which Lys379 and Arg380 are essential.

Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA-1) is essential for replication of episomal EBV DNAs and maintenance of latency. Multifunctional EBNA-1 is phosphorylated, but the significance of EBNA-1 phosphorylation is not known. Here, we examined the effects on nuclear translocation of Ser phosphorylation of the EBNA-1 nuclear localization signal (NLS) sequence, 379Lys-Arg-Pro-Arg-Ser-Pro-Ser-Ser386. We found that Lys379Ala and Arg380Ala substitutions greatly reduced nuclear transport and steady-state levels of green fluorescent protein (GFP)-EBNA1, whereas Pro381Ala, Arg382Ala, Pro384Ala, and Glu378Ala substitutions did not. Microinjection of modified EBNA-1 NLS peptide-inserted proteins and NLS peptides cross-linked to bovine serum albumin (BSA) showed that Ala substitution for three NLS Ser residues reduced the efficiency of nuclear import. Similar microinjection analyses demonstrated that phosphorylation of Ser385 accelerated the rate of nuclear import, but phosphorylation of Ser383 and Ser386 reduced it. However, transfection analyses of GFP-EBNA1 mutants with the Ser-to-Ala substitution causing reduced nuclear import efficiency did not result in a decrease in the nuclear accumulation level of EBNA-1. The results suggest dynamic nuclear transport control of phosphorylated EBNA-1 proteins, although the nuclear localization level of EBNA-1 that binds to cellular chromosomes and chromatin seems unchanged. The karyopherin alpha NPI-1 (importin alpha5), a nuclear import adaptor, bound more strongly to Ser385-phosphorylated NLS than to any other phosphorylated or nonphosphorylated forms. Rch1 (importin alpha1) bound only weakly and Qip1 (importin alpha3) did not bind to the Ser385-phosphorylated NLS. These findings suggest that the amino-terminal 379Lys-Arg380 is essential for the EBNA-1 NLS and that Ser385 phosphorylation up-regulates nuclear transport efficiency of EBNA-1 by increasing its binding affinity to NPI-1, while phosphorylation of Ser386 and Ser383 down-regulates it.

Amino Acid Substitution↗
PubMed1998

Nuclear localization and export signals of the human aryl hydrocarbon receptor.

The aryl hydrocarbon receptor (Ahr) is a ligand-activated transcription factor that binds DNA in the form of a heterodimer with the Ahr nuclear translocator (hypoxia-inducible factor 1beta). We found in this study that Ahr contains both nuclear localization and export signals in the NH2-terminal region. A fusion protein composed of beta-galactosidase and full-length Ahr translocates from the cytoplasm to the nucleus in a ligand-dependent manner. However, a fusion protein lacking the PAS (Per-Ahr nuclear translocator-Sim homology) domain of the Ahr showed strong nuclear localization activity irrespective of the presence or absence of ligand. A minimum bipartite Ahr nuclear localization signal (NLS) consisting of amino acid residues 13-39 was identified by microinjection of fused proteins with glutathione S-transferase-green fluorescent protein. A NLS having mutations in bipartite basic amino acids lost nuclear translocation activity completely, which may explain the reduced binding activity to the NLS receptor, PTAC58. A 21-amino acid peptide (residues 55-75) containing the Ahr nuclear export signal is sufficient to direct nuclear export of a microinjected complex of glutathione S-transferase-Ahr-green fluorescent protein. These findings strongly suggest that Ahr act as a ligand- and signal-dependent nucleocytoplasmic shuttling protein.

Biological Transport↗
PubMed2000

Cyclin F regulates the nuclear localization of cyclin B1 through a cyclin-cyclin interaction.

The key regulator of G(2)-M transition of the cell cycle is M-phase promoting factor (MPF), a complex composed of cdc2 and a B-type cyclin. Cyclin B1 nuclear localization involves phosphorylation within a region called the cytoplasmic retention signal, which also contains a nuclear export signal. The mechanism of MPF nuclear localization remains unclear since it contains no functional nuclear localization signal (NLS). We exploited the yeast two-hybrid screen to find protein(s) potentially mediating localization of cyclin B1 and identified a novel interaction between cyclin B1 and cyclin F. We found that cdc2, cyclin B1 and cyclin F form a complex that exhibits histone H1 kinase activity. Cyclin B1 and cyclin F also colocalize through immunofluorescence studies. Additionally, deletion analysis revealed that each putative NLS of cyclin F is functional. Taken together, the data suggest that the NLS regions of cyclin F regulate cyclin B1 localization to the nucleus. The interaction between cyclin B1 and cyclin F represents the first example of direct cyclin-cyclin binding, and elucidates a novel mechanism that regulates MPF localization and function.

Animals↗
PubMed2001

The C-terminal nuclear localization signal of the sex-determining region Y (SRY) high mobility group domain mediates nuclear import through importin beta 1.

The sex-determining factor SRY is a DNA-binding protein that diverts primordial gonads from the ovarian pathway toward male differentiation to form testes. It gains access to the nucleus through two distinct nuclear localization signals (NLSs) that flank the high mobility group (HMG) DNA-binding domain, but the mechanisms through which these NLSs operate have not been studied. In this study, we reconstitute the nuclear import of SRY in vitro, demonstrating a lack of requirement for exogenous factors for nuclear accumulation and a significant reduction in nuclear transport in the presence of antibodies to importin beta but not importin alpha. Using a range of quantitative binding assays including enzyme-linked immunosorbent assay, fluorescence polarization, and native gel mobility electrophoresis, we assess the binding of importins to SRY, demonstrating a high affinity recognition (in the low nm range) by Imp beta independent of Imp alpha. In assessing the contribution of each NLS, we found that the N-terminal NLS was recognized poorly by importins, whereas the C-terminal NLS was bound by importin beta with similar affinity to SRY. We also found that RanGTP, but not RanGDP, could dissociate the SRY-importin beta complex in solution using FP. We describe a novel double-fluorescent label DNA binding assay to demonstrate mutual exclusivity between importin beta recognition and DNA binding on the part of SRY, which may represent an alternative release mechanism upon nuclear entry. This study represents the first characterization of the nuclear import pathway for a HMG domain-containing protein. Importantly, it demonstrates for the first time that recognition of SRY by Imp beta is of comparable affinity to that with which Imp alpha/beta recognizes conventional NLS-containing substrates.

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↗
PubMed1995

Cytoplasmic localization of a mutant M(r) 160,000 topoisomerase II alpha is associated with the loss of putative bipartite nuclear localization signals in a drug-resistant human lung cancer cell line.

Many clinically important antineoplastic agents exert their cytotoxicity through interaction with the M(r) 170,000 topoisomerase II alpha, an essential nuclear enzyme. Resistance to these agents has been associated frequently with either a decrease in the levels of topoisomerase II alpha or a qualitative change that alters the interaction of this enzyme with a drug or DNA. Using a VP-16-selected lung cancer cell line, H209/V6, we have identified a third resistance mechanism which involves an aberrant subcellular location of the topoisomerase II alpha isoenzyme. We have shown previously that H209/V6 cells express two topoisomerase II alpha mRNAs (6.1 and 4.8 kilobases) but only a single catalytically active protein which has a M(r) of 160,000 and is located primarily in the cytoplasm (Mirski et al., Cancer Res., 53: 4866-4873, 1993; Feldhoff et al., Cancer Res., 54: 756-762, 1994). In the present study we have determined that this mutant M(r) 160,000 topoisomerase II alpha is encoded by the shorter 4.8-kilobase mRNA. The sequencing of reverse transcriptase-PCR products from H209/V6 cells and subsequent Northern blot analyses showed that a sequence of 988 nucleotides from the 3'-coding and 3'-noncoding region of the normal topoisomerase II alpha is absent from the 4.8-kilobase mRNA. This shorter mRNA is predicted to encode a topoisomerase II alpha protein that no longer contains the 109 COOH-terminal amino acids of the normal enzyme but instead contains 34 new amino acids encoded by a sequence that was previously in the 3'-noncoding region of the mRNA. Confirmation that the COOH terminus of topoisomerase II alpha is no longer present in the M(r) 160,000 protein in H209/V6 cells was obtained by immunoblot analysis. Sequence analyses indicate that 3 putative bipartite nuclear localization signals in the M(r) 160,000 protein are disrupted or lost. Our results suggest that sequences within the COOH-proximal domain of human topoisomerase II alpha serve an important nuclear localization function.

Base Sequence↗
PubMed2002

The hsp90 Co-chaperone XAP2 alters importin beta recognition of the bipartite nuclear localization signal of the Ah receptor and represses transcriptional activity.

The mouse aryl hydrocarbon receptor (mAhR) is a ligand-activated transcription factor that exists in a tetrameric, core complex with a dimer of the 90-kDa heat shock protein, and the hepatitis B virus X-associated protein 2 (XAP2). Transiently expressed mAhR-YFP (yellow fluorescent protein fused with the mAhR) localizes throughout cells, with a majority occupying nuclei. Co-expression of XAP2 with mAhR-YFP results in a distinct redistribution to the cytoplasm. We have utilized several approaches to attempt to identify the mechanism by which XAP2 modulates the sub-cellular localization of the mAhR. The nuclear export inhibitor, leptomycin B, was used to demonstrate that XAP2 inhibits ligand-independent nucleocytoplasmic shuttling of the receptor. Results from cytoskeletal disruption and the addition of an alternate nuclear localization sequence (NLS) to mAhR-YFP suggest that XAP2 does not physically tether the complex in the cytoplasm. The use of a rabbit polyclonal antibody raised against a portion of the bipartite NLS of the mAhR revealed that XAP2 does not appear to block access to the NLS. However, XAP2 hinders importin beta binding to the mAhR complex, suggesting that XAP2 alters the conformation of the bipartite NLS of mAhR. XAP2 also represses the transactivation potential of the AhR, in contrast to previously published reports, perhaps by stabilizing the receptor complex and/or blocking nucleocytoplasmic shuttling of the AhR complex.

Actins↗
PubMed1994

Studies on FGF-2: nuclear localization and function of high molecular weight forms and receptor binding in the absence of heparin.

Multiple forms of FGF-2 have been shown to exist in many cell types. These different species of molecular masses of 18, 21.5, 22, and 24 kDa are all translated via the use of alternate initiation codons. The three forms of HMW FGF-2 initiate at CUGs codons, whereas the 18 kDa form initiates at an AUG codon. The entire 18 kDa sequence is contained within the larger forms of HMW FGF-2 as the AUG codon is 3' to the CUG codons. Although the 18 kDa form FGF-2 is localized primarily in the cytosol, a significant fraction of the HMW FGF-2 has a nuclear location. The nuclear localization of HMW FGF-2 is determined by amino acid residues in the amino-terminal extended sequence. The residues required for nuclear localization appear to be RG repeats that are found at multiple sites within the amino-terminal extension of HMW FGF-2. The nuclear localization of HMW FGF-2 suggested that these species may have unique properties. By selecting permanent transfectants of 3T3 cells expressing HMW, 18 kDa FGF-2, or all forms of FGF-2, we have found that HMW FGF-2 can endow cells with a phenotype different from that of cells expressing 18 kDa FGF-2. These cells are transformed by what appears to be the intracellular action of HMW FGF-2. The interaction of FGF-2 with heparin has also been examined.(ABSTRACT TRUNCATED AT 250 WORDS)

3T3 Cells↗
PubMed1994

The nuclear localization signal of NGFI-A is located within the zinc finger DNA binding domain.

NGFI-A is an immediate-early gene that encodes a transcription factor whose DNA binding domain is composed of three C2H2 zinc fingers. To identify its nuclear localization signal (NLS), wild type NGFI-A and various mutants were transfected into COS cells and their cellular location assayed by indirect immunofluorescence. Although wild type NGFI-A was located exclusively within the nucleus, deletions lacking the highly basic zinc finger region were not efficiently translocated to the nucleus. However, DNA binding per se is not required for nuclear localization, as an NGFI-A mutant (A Y339G), which does not bind DNA, is still faithfully directed to the nucleus. To determine the minimal region(s) of NGFI-A sufficient to direct nuclear localization, the cellular location of various NGFI-A/beta-galactosidase fusion proteins was examined. Fusion proteins containing all three zinc fingers were found in the nucleus, but those containing only two zinc fingers were predominantly cytoplasmic. When the zinc finger structure was altered by mutating a zinc-chelating cysteine residue in any one of the three zinc fingers, the resulting domain was no longer capable of directing beta-galactosidase to the nucleus. Furthermore, the mutation of an arginine residue in the third zinc finger of NGFI-A, a position which is occupied by a leucine residue in most C2H2 zinc fingers, abolished nuclear localization, but had no effect on DNA binding. These studies suggest that NGFI-A contains a novel NLS which is dependent on the overall structure of the DNA binding domain and not solely upon its highly basic nature.

Amino Acid Sequence↗
PubMed2001

Analysis of murine Brca2 reveals conservation of protein-protein interactions but differences in nuclear localization signals.

In this report, we have analyzed the protein encoded by the murine Brca2 locus. We find that murine Brca2 shares multiple properties with human BRCA2 including its regulation during the cell cycle, localization to nuclear foci, and interaction with Brca1 and Rad51. Murine Brca2 stably interacts with human BRCA1, and the amino terminus of Brca2 is sufficient for this interaction. Exon 11 of murine Brca2 is required for its stable association with RAD51, whereas the carboxyl terminus of Brca2 is dispensable for this interaction. Finally, in contrast to human BRCA2, we demonstrate that carboxyl-terminal truncations of murine Brca2 localize to the nucleus. This finding may explain the apparent inconsistency between the cytoplasmic localization of carboxyl-terminal truncations of human BRCA2 and the hypomorphic phenotype of mice homozygous for similar carboxyl-terminal truncating mutations.

Animals↗
PubMed1994

Molecular and structural analysis of nuclear localizing anti-DNA lupus antibodies.

To determine the structure of three nuclear localizing lupus anti-DNA immunoglobulins (Igs) and to search for clues to mechanisms of cellular and/or nuclear access, their H- and L-chain variable region sequences were determined and subjected to three-dimensional modeling. Although the results indicate heterogeneity in their primary structures, the H chains are encoded by 3 members of the J558 VH gene family with a common tertiary conformation that is not shared by a J558-encoded nonnuclear localizing anti-DNA control Ig. Furthermore, at least two of the Igs share a conformational motif in the H-chain CDR3, and all three Igs contain multiple positively charged amino acids in their CDRs, resembling nuclear localization signals that direct protein nuclear import. Notably, each VH and VK gene is also found recurrently among previously described autoantibodies. Molecular analysis further indicates that both germline-encoded and significantly mutated V genes can generate nuclear localizing anti-DNA Ig.

Amino Acid Sequence↗
PubMed2006

Identification of the nuclear localization motif in the ETV6 (TEL) protein.

ETV6, or Translocation-Ets-Leukemia (TEL), is an ETS family transcriptional repressor that is essential for establishing hematopoiesis in neonatal bone marrow, and is frequently a target of chromosomal translocations in human cancer. ETV6 is predominantly a nuclear phosphoprotein that represses transcription by binding directly to the promoters of target genes. The nuclear localization mechanism of ETV6, however, is not well understood. In this report, we provide evidence that a nuclear localization signal (NLS) exists in the C-terminal region of ETV6. ETV6 proteins with mutations outside of amino acids 332-452 localize to the nucleus, whereas proteins with mutations within amino acids 332-452 remain in the cytoplasm. Furthermore, when a fragment of ETV6 comprised of amino acids 332-452 was fused to cytoplasmic beta-galactosidase protein, the fusion protein was able to enter the nucleus. These results strongly indicate that residues 332-452 mediate nuclear localization of ETV6.

Amino Acid Motifs↗
PubMed2000

Characterization of a zinc finger protein ZAN75: nuclear localization signal, transcriptional activator activity, and expression during neuronal differentiation of P19 cells.

The ZAN75 cDNA was first identified in NIH 3T3 cells and codes for a DNA-binding protein with two zinc finger motifs. In this study, we characterized the nuclear localization signal of ZAN75, tested if ZAN75 regulates transcription, and examined its expression during embryonic development and neuronal differentiation of P19 mouse embryonal carcinoma cells. By examining the cellular localization of deletion mutants of ZAN75 fused to green fluorescence protein, ZAN75 was revealed to have a bipartite nuclear localization signal sequence upstream of the zinc finger domains. The N-terminal region of ZAN75, when fused to the GAL4 DNA-binding domain, strongly activated transcription. The expression of ZAN75 mRNA was found to be developmentally regulated, showing the highest expression in E11.5 embryos. In situ hybridization experiments using E11.5 embryos showed a high expression of the transcripts in neuronal tissues such as brain and neural tube. The expression of ZAN75 was transiently increased at both the mRNA and the protein levels when P19 cells were treated with retinoic acid to induce neuronal differentiation. Taken together, these results indicate that ZAN75 is a transcriptional activator with a bipartite nuclear localization signal and may play a role in neuronal differentiation.

Amino Acid Sequence↗
PubMed1998

A constitutive nuclear localization signal from the second zinc-finger of orphan nuclear receptor TR2.

The orphan nuclear receptor TR2 and its truncated isoform deleted in the ligand binding domain (LBD) were localized exclusively in the nuclei as revealed by two methods of detection. An anti-hemagglutinin (HA) antibody detected specific nuclear localization of HA-tagged receptors and the green fluorescent protein (GFP)-tagged receptors were found to be distributed in the nuclei of living cells. By deletion analyses, the sequence responsible for targeting this receptor into the nucleus was defined. A stretch of 20 amino acid residues (KDCVINKHHRNRCQYCRLQR) within the second zinc-finger of this receptor is required for its nuclear localization and this signal is constitutively active. No nuclear localization signal was found in the N-terminus or the LBD. The GFP-tagged receptor remained biologically active, as evidenced by its repressive activity on the reporter that carried a binding site for this receptor, a direct repeat-5 (DR5). An electrophoretic mobility shift assay was performed to characterize the binding property of TR2 and its truncated isoform. TR2 bound to the DR5 as dimers whereas its truncated isoform bound as monomers.

Animals↗
PubMed1999

Sequence requirements for the nuclear localization of the murine cytomegalovirus M44 gene product pp50.

The murine cytomegalovirus (MCMV) M44 gene product pp50 is normally present in the nuclei of virus-infected cells. During transient expression of pp50 in COS-1 cells, the phosphoprotein was readily detectable in the nuclei, indicating that it possesses a nuclear localization signal (NLS). Studies on the subcellular locations of N- and C-terminal deletion mutants of pp50 suggested that alterations in both the C terminus and the highly conserved N-terminal domains of pp50 affect nuclear localization. In particular, the C-terminal 11 amino acids of pp50, which includes a "KKQK" motif, were able to mediate the import of a beta-galactosidase fusion protein into the nucleus. The pair of lysine residues in this motif constitutes an essential element of the C-terminal NLS as mutation of this motif to AAQK directly affected the nuclear localization of either pp50 or beta-galactosidase fusion proteins containing the C-terminal portion of pp50. Furthermore our results indicated that the functionality of the C-terminal NLS is dependent on the structural integrity of the highly conserved N-terminal portion of the molecule, as deletion of amino acids 157-201 alone adversely affected nuclear localization. In the absence of a functional C-terminal NLS, the subcellular localization of pp50 is sensitive to potential conformational changes induced by mutations within the N-terminal half of the molecule. Under those circumstances, mutation of the YK residues at position 22-23 or deletion of amino acids 267-283 was sufficient to produce a protein that was impaired in nuclear import or retention.

Amino Acid Sequence↗
PubMed2004

Nuclear localization of Tob is important for regulation of its antiproliferative activity.

TOB: is a member of an antiproliferative gene family that includes btg1, pc3/tis21/btg2, pc3b, ana/btg3, and tob2. Exogenous overexpression of the family proteins suppresses cell proliferation. These proteins participate in transcriptional regulation of several genes. Here, we show that Tob is a nuclear protein that is imported into the nucleus through a nuclear localization signal (NLS)-mediated mechanism. Mutation in the NLS sequence of Tob affects its nuclear localization and impairs antiproliferative activity. Additionally, Tob contains a nuclear export signal (NES). In oncogenic ErbB2-transformed cells, nuclear export of Tob is facilitated by NES-mediated mechanism, resulting in decrease of its antiproliferative activity. These results indicate that regulation of nuclear localization of Tob is important for its antiproliferative activity.

Animals↗
PubMed1996

The yeast nucleoporin Nsp1 binds nuclear localization sequences in vitro.

Facilitated transport of proteins into the nucleus requires nuclear localization sequences (NLSs) be present in the protein destined for the nucleus. The specific binding of NLSs by components of the nuclear transport apparatus is essential for these targeting reactions. We now report that the yeast nucleoporin Nsp1 binds specifically nuclear localization sequences in vitro. This nucleoporin recognizes several NLSs that are functional for nuclear targeting in vivo, including the NLS of SV40 T-antigen and of the yeast transcription factor Ga14. Nsp1 is organized into three domains, and we have located NLS binding sites to the N-terminal portion and the middle repetitive region of the protein. For the interaction between the NLS of SV40 T-antigen and Nsp1, we obtained association constants of 1.2 x 10(7) M-1 and 5 x 10(7) M-1. An association constant of 5 x 10(7) M-1 was determined for NLS binding to the repetitive domain of Nsp1. We analyzed binding of Nsp1 and its domains to a mutant version of the NLS derived from SV40 T-antigen, which poorly functions for nuclear targeting in vivo. The affinity for the mutant signal was about two orders of magnitude lower than for the wild-type NLS.

Binding Sites↗