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Results for “Nuclear Localization Signals”

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PubMed1999

Apoptosis inhibitory activity of cytoplasmic p21(Cip1/WAF1) in monocytic differentiation.

p21(Cip1/WAF1) inhibits cell-cycle progression by binding to G1 cyclin/CDK complexes and proliferating cell nuclear antigen (PCNA) through its N- and C-terminal domains, respectively. The cell-cycle inhibitory activity of p21(Cip1/WAF1) is correlated with its nuclear localization. Here, we report a novel cytoplasmic localization of p21(Cip1/WAF1) in peripheral blood monocytes (PBMs) and in U937 cells undergoing monocytic differentiation by in vitro treatment with vitamin D3 or ectopic expression of p21(Cip1/WAF1), and analyze the biological consequences of this cytoplasmic expression. U937 cells which exhibit nuclear p21(Cip1/WAF1) demonstrated G1 cell-cycle arrest and subsequently differentiated into monocytes. The latter event was associated with a cytoplasmic expression of nuclear p21(Cip1/WAF1), concomitantly with a resistance to various apoptogenic stimuli. Biochemical analysis showed that cytoplasmic p21(Cip1/WAF1) forms a complex with the apoptosis signal-regulating kinase 1 (ASK1) and inhibits stress-activated MAP kinase cascade. Expression of a deletion mutant of p21(Cip1/WAF1) lacking the nuclear localization signal (DeltaNLS-p21) did not induce cell cycle arrest nor monocytic differentiation, but led to an apoptosis-resistant phenotype, mediated by binding to and inhibition of the stress-activated ASK1 activity. Thus, cytoplasmic p21(Cip1/WAF1) itself acted as an inhibitor of apoptosis. Our findings highlight the different functional roles of p21(Cip1/WAF1), which are determined by its intracellular distribution and are dependent on the stage of differentiation.

Amino Acid Sequence↗

PubMed1999

Critical residues of the homeodomain involved in contacting DNA bases also specify the nuclear accumulation of thyroid transcription factor-1.

The N-terminal end of thyroid transcription factor-1 (TTF-1) homeodomain is composed of a stretch of five basic amino-acids that is conserved in both POU- and NK2-class homeodomains and constitutes a functional nuclear localization signal. By analyzing the cellular distribution of fusion proteins, composed of a jellyfish green fluorescent variant and different parts of TTF-1, we show here that the presence of this basic sequence is not sufficient by itself to confer complete nuclear accumulation. By mutagenesis, we identified a second region located in the center of the DNA recognition helix of the homeodomain that is also able to specify a predominantly nuclear localization of the chimeric proteins, independently of the presence of the basic NLS. The destruction, by mutagenesis, of both the basic stretch and the motif in the DNA recognition helix led to the total loss of nuclear accumulation, indicating that complete nuclear accumulation of TTF-1 results from the concerted action of these two proteic signals. Both of the regions of the homeodomain that are involved in nuclear targeting also encompass critical amino-acids responsible for DNA binding site recognition, as evidenced by the loss of DNA binding activity in vitro upon mutagenesis. Specifically, residues in the central part of the DNA recognition helix are involved in contacting bases in the major groove of DNA and are the most conserved in homeodomain proteins, suggesting that this part of the homeodomain could play a general role in the nuclear localization of members of this family of proteins.

Animals↗

PubMed2001

Signaling through the Smad pathway by insulin-like growth factor-binding protein-3 in breast cancer cells. Relationship to transforming growth factor-beta 1 signaling.

We previously demonstrated in T47D cells transfected to express the transforming growth factor-beta receptor type II (TGF-betaRII) that insulin-like growth factor binding protein-3 (IGFBP-3) could stimulate Smad2 and Smad3 phosphorylation, potentiate TGF-beta1-stimulated Smad phosphorylation, and cooperate with exogenous TGF-beta1 in cell growth inhibition (Fanayan, S., Firth, S. M., Butt, A. J., and Baxter, R. C. (2000) J. Biol. Chem. 275, 39146-39151). This study further explores IGFBP-3 signaling through the Smad pathway. Like TGF-beta1, natural and recombinant IGFBP-3 stimulated the time- and dose-dependent phosphorylation of TGF-betaR1 as well as Smad2 and Smad3. This effect required the presence of TGF-betaRII. IGFBP-3 mutated in carboxyl-terminal nuclear localization signal residues retained activity in TGF-betaR1 and Smad phosphorylation, whereas IGFBP-5 was inactive. Immunoneutralization of endogenous TGF-beta1 suggested that TGF-beta1 was not essential for IGFBP-3 stimulation of this pathway, but it increased the effect of IGFBP-3. IGFBP-3, like TGF-beta1, elicited a rapid decline in immunodetectable Smad4 and Smad4.Smad2 complexes. IGFBP-3 and nuclear localization signal mutant IGFBP-3 stimulated the activation of the plasminogen activator inhibitor-1 promoter but was not additive with TGF-beta, suggesting that this end point is not a direct marker of the IGFBP-3 effect on cell proliferation. This study defines a signaling pathway for IGFBP-3 from a cell surface receptor to nuclear transcriptional activity, requiring TGF-betaRII but not dependent on the nuclear translocation of IGFBP-3. The precise mechanism by which IGFBP-3 interacts with the TGF-beta receptor system remains to be established.

Activin Receptors, Type I↗

PubMed2006

Involvement of a Xenopus nuclear GTP-binding protein in optic primordia formation.

Using a subtracted Xenopus cDNA library based on the differential sensitivity of anterior and posterior genes to retinoic acid, we isolated a novel Xenopus nuclear GTP-binding protein (XGB). XGB is expressed prominently in the optic primordia at the tailbud stage. The N-terminal region of XGB contains a set of GTP-binding protein motifs, and the C-terminal region contains two putative nuclear localization signals and two coiled regions. A GFP-XGB fusion protein was expressed in the nucleus of NIH3T3 cells where it bound to subnuclear structures. Truncated C-terminal constructs of XGB containing both nuclear localization signal(s) and coiled region(s) suppressed eye formation, whereas neither the N-terminal construct nor constructs with a mutated GTP-binding protein motif affected eye formation. Expression of Pax6 and Rx1 genes, which are crucial for eye development, was reduced in embryos overexpressing the C-terminal constructs of XGB. Suppression of Pax6 and Rx1 at earlier developmental stages as well as perturbation of eye formation at later stages was counteracted by co-expression of wild-type XGB. We conclude that XGB plays a role in the formation of optic primordia through activation of at least two eye field transcription factors.

Amino Acid Sequence↗

PubMed2005

A nuclear targeting determinant for SATB1, a genome organizer in the T cell lineage.

SATB1 is a nuclear protein, which acts as a cell-type specific genome organizer and gene regulator essential for T cell differentiation and activation. Several functional domains of SATB1 have been identified. However, the region required for nuclear localization remains unknown. To delineate this region, we employed sequence analysis to identify phylogenetically diverse members of the SATB1 protein family, and used hidden Markov model (HMM)-based analysis to define conserved regions and motifs in this family. One of the regions conserved in SATB1- and SATB2-like proteins in mammals, fish, frog and bird, is located near the N-terminus of family members. We found that the N-terminus of human SATB1 was essential for the nuclear localization of the protein. Furthermore, fusing residues 20-40 to a cytoplasmic green fluorescence protein (GFP) fused to pyruvate kinase (PK) was sufficient to quantitatively translocate the pyruvate kinase into the nucleus. The nuclear targeting sequence of human SATB1 (residues 20-40) is novel and does not contain clusters of basic residues, typically found in 'classical' nuclear localization signals (NLSs). We investigated the importance of four well-conserved residues (Lys29, Arg32, Glu34, and Asn36) in this nuclear targeting sequences. Remarkably, full-length SATB1 harboring a single point mutation at either Lys29 or Arg32, but not Glu34 or Asn36, did not enter the nucleus. Our results indicate that SATB1 N-terminal residues 20-40 represent a novel determinant of nuclear targeting.

Amino Acid Sequence↗

PubMed1995

Identification and characterization of a novel cytokine-inducible nuclear protein from human endothelial cells.

Vascular endothelial cells undergo profound changes upon cellular activation including expression of a spectrum of cell activation-associated genes. These changes play important roles in many physiological and pathological events. By differential screening of a cDNA library prepared from interleukin-1 alpha and tumor necrosis factor-alpha-stimulated human dermal microvascular endothelial cells, we have identified a novel cytokine-inducible gene, designated as C-193. The compiled cDNA sequence of C-193 is 1901 base pairs long and shows no significant homology with any known gene sequence. Genomic DNA analysis revealed that C-193 is encoded by a single gene, which is conserved in different mammalian species. The C-193 gene was localized to human chromosome 10 by Southern blot analysis of somatic cell hybrids. Multiple AT-rich mRNA decay elements were identified in the 3'-untranslated region. C-193 mRNA expression was rapidly and transiently induced by treatment with interleukin-1 alpha or tumor necrosis factor-alpha, reached a peak of expression about 16 h post tumor necrosis factor-alpha stimulation, and the induction of C-193 was protein synthesis independent. Lipopolysaccharide and cycloheximide were also potent inducers of C-193 mRNA. Therefore, C-193 represents a new addition to the primary response gene family. In vitro translation of C-193 yielded a 36-kDa protein product, consistent with the predicted open reading frame of 318 amino acids and a calculated molecular mass of 36 kDa for C-193 protein. The predicted protein sequence contains a basic amino acid cluster similar to a nuclear localization signal, four tandem repeats of ankyrin-like sequence, and multiple consensus protein phosphorylation sites. C-193 was engineered with a FLAG tag at its carboxyl terminus and transiently expressed in COS cells. Consistent with the presence of a putative nuclear localization signal, the C-193-FLAG protein was localized to the nucleus of transfected COS cells by indirect immunofluorescence microscopy. C-193-FLAG prepared in vitro was capable of binding DNA cellulose. These results indicate that C-193 protein may play an important role in endothelial cell activation.

Amino Acid Sequence↗

PubMed2004

Bidirectional increase in permeability of nuclear envelope upon poliovirus infection and accompanying alterations of nuclear pores.

Poliovirus and some other picornaviruses trigger relocation of certain nuclear proteins into the cytoplasm. Here, by using a protein changing its fluorescence color with time and containing a nuclear localization signal (NLS), we demonstrate that the poliovirus-triggered relocation is largely due to the exit of presynthesized nuclear protein into the cytoplasm. The leakiness of the nuclear envelope was also documented by the inability of nuclei from digitonin-permeabilized, virus-infected (but not mock-infected) cells to retain an NLS-containing derivative of green fluorescent protein (GFP). The cytoplasm-to-nucleus traffic was also facilitated during infection, as evidenced by experiments with GAPDH (glyceraldehyde-3-phosphate dehydrogenase), cyclin B1, and an NLS-lacking derivative of GFP, which are predominantly cytoplasmic in uninfected cells. Electron microscopy demonstrated that a bar-like barrier structure in the channel of the nuclear pores, seen in uninfected cells, was missing in the infected cells, giving the impression of fully open pores. Transient expression of poliovirus 2A protease also resulted in relocation of the nuclear proteins. Lysates from poliovirus-infected or 2A-expressing cells induced efflux of 3xEGFP-NLS from the nuclei of permeabilized uninfected cells. This activity was inhibited by the elastase inhibitors elastatinal and N-(methoxysuccinyl)-L-alanyl-L-alanyl-L-prolyl-L-valine chloromethylketone (drugs known also to be inhibitors of poliovirus protease 2A), a caspase inhibitor zVAD(OMe), fmk, and some other protease inhibitors. These data suggest that 2A elicited nuclear efflux, possibly in cooperation with a zVAD(OMe).fmk-sensitive protease. However, poliovirus infection facilitated nuclear protein efflux also in cells deficient in caspase-3 and caspase-9, suggesting that the efflux may occur without the involvement of these enzymes. The biological relevance of nucleocytoplasmic traffic alterations in infected cells is discussed.

Active Transport, Cell Nucleus↗

PubMed2000

The RING finger protein SNURF modulates nuclear trafficking of the androgen receptor.

The androgen receptor (AR) is a transcription factor that mediates androgen action. We have used the green fluorescent protein (GFP) technique to investigate dynamics of nuclear trafficking of human AR in living cells. In the absence of ligand, the GFP-AR fusion protein is distributed between cytoplasm and nuclei. Androgen exposure leads to a rapid and complete import of GFP-AR to nuclei of CV-1 cells (>=90% nuclear in 30 minutes), whereas a pure antiandrogen, Casodex, elicits a slower (<40% nuclear in 30 minutes) and incomplete transfer. Unliganded ARs with mutations in the basic amino acids of the bipartite nuclear localization signal (NLS) within the second zinc finger and the hinge region are predominantly cytoplasmic and their androgen-dependent nuclear import is severely compromised ((3/4)20% nuclear in 30 minutes). Interestingly, substitutions of the Leu residues flanking the bipartite NLS lead to inefficient nuclear transfer in response to androgen ((3/4)20% nuclear in 30 minutes). The ligand-binding domain of AR, which represses bipartite NLS activity, contains an agonist-specific NLS. The small nuclear RING finger protein SNURF, which interacts with AR through a region overlapping with the bipartite NLS, facilitates AR import to nuclei and retards its export on hormone withdrawal. More AR is associated with the nuclear matrix in the presence than absence of coexpressed SNURF. We suggest that the SNURF-mediated tethering of AR in nuclei represents a novel mechanism for activating steroid receptor functions.

Amino Acid Sequence↗

PubMed2001

A high-efficiency protein transduction system demonstrating the role of PKA in long-lasting long-term potentiation.

Proteins and peptides have been demonstrated to penetrate across the plasma membrane of eukaryotic cells by protein transduction domains. We show that protein transduction by 11 arginine (11R) is an efficient method of delivering proteins into the neurons of brain slices. Here, we demonstrate that PKA inhibitory peptide, fused with 11R and nuclear localization signal, delivers the peptide exclusively into the nuclear compartment of neurons in brain slices. This inhibitory peptide blocked both cAMP responsive element-binding protein phosphorylation and long-lasting long-term potentiation (LTP) induction, but not early LTP. These results highlight transduction of proteins and peptides into specific neuronal subcellular compartments in brain slices as a powerful tool for studying neuronal plasticity.

Active Transport, Cell Nucleus↗

PubMed2006

Mapping nucleolar localization sequences of 1A6/DRIM.

Human 1A6/downregulated in metastasis (DRIM) is a nucleolar protein with multiple HEAT-repeat motifs (Huntington, elongation factor 3, a subunit of protein phosphatase 2A, target of rapamycin). The yeast homologue to 1A6/DRIM, Utp20, is part of the small subunit processome and functions in 18S RNA processing. In the present study, we utilized the green fluorescent protein as the fusion protein marker to investigate the sequence responsible for 1A6/DRIM accumulation in nucleolus. Deletion sequence analysis demonstrated that a single region located between amino acids 2744 and 2761 at the C-terminus of 1A6/DRIM is capable of nucleolar accumulation. Two basic amino acid clusters within this region are essential for nucleolar accumulation. The sequences required for nucleolar accumulation overlaps the putative nuclear localization signal of 1A6/DRIM.

3T3 Cells↗

PubMed2001

Nuclear entry mechanism of rat PER2 (rPER2): role of rPER2 in nuclear localization of CRY protein.

Mammalian PERIOD2 protein (PER2) is the product of a clock gene that controls circadian rhythms, because PER2-deficient mice have an arrhythmic phenotype. The nuclear entry regulation of clock gene products is a key step in proper circadian rhythm formation in both Drosophila and mammals, because the periodic transcription of clock genes is controlled by an intracellular, oscillating, negative feedback loop. The present study used deletion mutants of rat PER2 (rPER2) to identify the functional nuclear localization signal (NLS) in rPER2. The elimination of putative NLS (residues 778 to 794) from the rPER2 fragment resulted in the loss of nuclear entry activity. Adding the NLS to the cytosolic protein (bacterial alkaline phosphatase) translocates the fusion protein to the nuclei. The data indicate the presence of a functional NLS in rPER2. Furthermore, intact rPER2 was preferentially translocated from the cytoplasm to the nucleus when coexpressed with human CRY1 (hCRY1). However, rPER2 mutants lacking a carboxyl-terminal domain could not enter the nucleus even in the presence of hCRY1. In addition, coexpression of the nuclear localization domain (residues 512 to 794) lacking rPER2 and CRY1 changed the subcellular localization of CRY1 from the nucleus to the cytoplasm. In vitro protein interaction studies demonstrated that the carboxyl-terminal domain of rPER2 is essential for binding to CRY1. The data suggested that both the rPER2 NLS and carboxyl-terminal CRY binding domain are essential for nuclear entry of the rPER2-CRY1 complex.

Active Transport, Cell Nucleus↗

PubMed2001

Nuclear exclusion of Cdc25 is not required for the DNA damage checkpoint in fission yeast.

Maintenance of genome integrity requires a checkpoint that restrains mitosis in response to DNA damage [1]. This checkpoint is enforced by Chk1, a protein kinase that targets Cdc25 [2--7]. Phosphorylated Cdc25 associates with 14-3-3 proteins, which appear to occlude a nuclear localization signal (NLS) and thereby inhibit Cdc25 nuclear import [6, 8--14]. Proficient checkpoint arrest is thought to require Cdc25 nuclear exclusion, although definitive evidence for this model is lacking. We have tested this hypothesis in fission yeast. We show that elimination of an NLS in Cdc25 causes Cdc25 nuclear exclusion and a mitotic delay, as predicted by the model. Attachment of an exogenous NLS forces nuclear inclusion of Cdc25 in damaged cells. However, forced nuclear localization of Cdc25 fails to override the damage checkpoint. Thus, nuclear exclusion of Cdc25 is unnecessary for checkpoint enforcement. We propose that direct inhibition of Cdc25 phosphatase activity by Chk1, as demonstrated in vitro with fission yeast and human Chk1 [15, 16], is sufficient for proficient checkpoint regulation of Cdc25 and may be the primary mechanism of checkpoint enforcement in fission yeast.

Cell Cycle Proteins↗

PubMed2003

Novel mechanism of the co-regulation of nuclear transport of SmgGDS and Rac1.

The armadillo protein SmgGDS promotes guanine nucleotide exchange by small GTPases containing a C-terminal polybasic region (PBR), such as Rac1 and RhoA. Because the PBR resembles a nuclear localization signal (NLS) sequence, we investigated the nuclear transport of SmgGDS with Rac1 or RhoA. We show that the Rac1 PBR has significant NLS activity when it is fused to green fluorescent protein (GFP) or in the context of full-length Rac1. In contrast, the RhoA PBR has very poor NLS activity when it is fused to GFP or in the context of full-length RhoA. The nuclear accumulation of both Rac1 and SmgGDS is enhanced by Rac1 activation and diminished by mutation of the Rac1 PBR. Conversely, SmgGDS nuclear accumulation is diminished by interactions with RhoA. An SmgGDS nuclear export signal sequence that we identified promotes SmgGDS nuclear export. These results suggest that SmgGDS. Rac1 complexes accumulate in the nucleus because the Rac1 PBR has NLS activity and because Rac1 supplies the appropriate GTP-dependent signal. In contrast, SmgGDS.RhoA complexes accumulate in the cytoplasm because the RhoA PBR does not have NLS activity. This model may be applicable to other armadillo proteins in addition to SmgGDS, because we demonstrate that activated Rac1 and RhoA also provide stimulatory and inhibitory signals, respectively, for the nuclear accumulation of p120 catenin. These results indicate that small GTPases with a PBR can regulate the nuclear transport of armadillo proteins.

Active Transport, Cell Nucleus↗

PubMed2000

Interdependence between a 55 kDa protein (p55) and a 12 kDa protein (p12) in facilitating the nuclear entry of goat uterine estrogen receptor under cell-free conditions.

A 55 kDa nuclear localization signal binding protein (p55) is involved in the transport of the goat uterine estrogen receptor from the cytoplasm to the nuclear pore complex (NPC). p55 forms a complex with a 12 kDa protein (p12) which in turn becomes 'docked' at the NPC. The present study reports on the purification and functional characterization of p12. Both p55 and p12 are Mg2+-dependent ATPases. The protein-protein interactions that take place between these two molecules at the NPC cause an enhancement in the net ATPase activity associated with the protein complex. Presumably, this enhanced ATPase function helps in the final nuclear entry of the estrogen receptor; p55 remains associated with p12 at the nuclear entry site under these conditions.

Adenosine Triphosphate↗

PubMedDate not supplied

Nuclear import: a tale of two sites.

The recently determined crystal structure of a nuclear localization sequence receptor has revealed an exquisitely specific interaction between ligand and receptor, and explains how simple and complex nuclear localization signals can both be recognized specifically by the same molecule.

Animals↗

PubMed1996

Transportin: nuclear transport receptor of a novel nuclear protein import pathway.

Many nuclear proteins are imported into the cell nucleus by the "classical" nuclear localization signal (NLS)-mediated import pathway. In this pathway, a sequence rich in basic residues in the protein interacts with a heterodimeric complex termed importin and this, along with the GTPase Ran, mediates nuclear import of the NLS-bearing protein. The heterogeneous nuclear ribonucleoprotein (hnRNP) A1 protein contains a novel nuclear localization sequence, termed M9, that does not contain any clusters of basic residues. Very recently, we showed that M9 directs import into the nucleus by a novel protein import pathway distinct from the classical NLS pathway. A 90-kilodalton protein termed transportin was identified as a protein that specifically interacts with wild-type M9 but not transport-defective M9 mutants. Transportin and an ATP-regenerating system were found to be necessary and sufficient for import of M9-containing proteins in an in vitro import assay. In this report, we provide additional evidence that transportin can interact directly with M9-containing proteins and also show that it can mediate import of full-length hnRNP A1. In addition, Ran, or a Ran-binding protein, is identified as a second protein component of this novel nuclear import pathway. Transportin relatives from Saccharomyces cerevisiae which likely serve as additional nuclear transport receptors are described.

Amino Acid Sequence↗

PubMed2001

Nucleocytoplasmic shuttling by human immunodeficiency virus type 1 Vpr.

Human immunodeficiency virus type 1 (HIV-1) is capable of infecting nondividing cells such as macrophages because the viral preintegration complex is able to actively traverse the limiting nuclear pore due to the redundant and possibly overlapping nuclear import signals present in Vpr, matrix, and integrase. We have previously recognized the presence of at least two distinct and novel nuclear import signals residing within Vpr that, unlike matrix and integrase, bypass the classical importin alpha/beta-dependent signals and do not require energy or a RanGTP gradient. We now report that the carboxy-terminal region of Vpr (amino acids 73 to 96) contains a bipartite nuclear localization signal (NLS) composed of multiple arginine residues. Surprisingly, when the leucine-rich Vpr(1-71) fragment, previously shown to harbor an NLS, or full-length Vpr is fused to the C terminus of a green fluorescent protein-pyruvate kinase (GFP-PK) chimera, the resultant protein is almost exclusively detected in the cytoplasm. However, the addition of leptomycin B (LMB), a potent inhibitor of CRM1-dependent nuclear export, produces a shift from a cytoplasmic localization to a nuclear pattern, suggesting that these Vpr fusion proteins shuttle into and out of the nucleus. Studies of nuclear import with GFP-PK-Vpr fusion proteins in the presence of LMB reveals that both of the leucine-rich alpha-helices are required for effective nuclear uptake and thus define a unique NLS. Using a modified heterokaryon analysis, we have localized the Vpr nuclear export signal to the second leucine-rich helix, overlapping a portion of the amino-terminal nuclear import signal. These studies thus define HIV-1 Vpr as a nucleocytoplasmic shuttling protein.

Amino Acid Sequence↗

PubMed1997

Sequence determinants for hnRNP I protein nuclear localization.

hnRNP I, also referred to as polypyrimidine tract binding protein, is one of the proteins associated with nascent pre-mRNA in the heterogeneous nuclear ribonucleoprotein (hnRNP) complexes. As for all karyophilic proteins, the nuclear import of hnRNP proteins requires specific sequence determinants that in many instances differ from the canonical import signal. In order to identify the sequences responsible for the nuclear localization, various hnRNP I portions were fused to a reporter protein (bacterial chloramphenicol acetyl transferase) and used in transient transfection assay. By this approach we identified a 60-amino-acid sequence located at the amino terminus of hnRNP I (designated NLD-I) that is both necessary and sufficient for nuclear localization. NLD-I represents a novel bipartite type of nuclear localization signal that bears no resemblance to other nuclear localization determinants so far identified in hnRNP proteins.

3T3 Cells↗