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Results for “Nuclear Localization Signals”

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PubMed2001

Nonviral gene therapy and its delivery systems.

Nonviral gene therapy has significant clinical potential, yet its therapeutic utility has been hindered by low transfection efficiency due to a combination of extracellular and intracellular barriers. Recent developments in formulation and delivery methodology have allowed a number of advances toward high efficiency gene delivery to various cell types and organs. In particular, the extracellular and intracellular pharmacokinetics of plasmid DNA trafficking are better understood in a number of cell systems. Using cationic lipid or polymers (often with receptor targeting), more than 10(5) plasmids can be delivered to a single cell. Endosomolytic agents promote endosome disruption, and include: weak bases, proton-sponge polymers, fusogenic peptides, viral particles, and photosensitizing compounds. Both classical and nonclassical nuclear localization signal (NLS) peptides have also been tested for enhancement of the probability of nuclear import events, a major rate-limiting step in DNA delivery to nondividing cells. For example, the M9 sequence from heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) protein, a nonclassical NLS, has been found to increase gene expression level by more than 10 to 150-fold in a variety of cell types. This review will concentrate on the current understandings of the basic mechanisms of nonviral gene delivery and new approaches in the field.

Animals↗

PubMedDate not supplied

Identification of URIM, a novel gene up-regulated in metastasis.

URIM (Up-Regulated In Metastasis) was identified as a new gene by Differential Display Technique during investigation of the transcriptional pattern of a metastasizing (NMCL-1) and a non-metastasizing (530) human melanoma cell line. The protein is encoded by 206 amino acids with an isoelectric point of 10.4. In addition, URIM displays a putative nuclear localization signal and a putative leucine zipper suggesting that URIM may function as a nuclear protein. Expression of URIM in several normal tissues and tumor cell lines was studied by Northern blotting. Surprisingly, 17 fold increased steady-state mRNA levels for URIM were detected in three cell lines derived from bone marrow micrometastasis of mammary carcinoma and one mammary carcinoma cell line derived from ascites fluid compared to normal epithelial cells from mammary gland and two cell lines derived from primary mammary carcinoma. These findings indicate that expression of URIM might be deregulated in metastases of different types of tumors.

Amino Acid Sequence↗

PubMed1996

Regulation of protein transport to the nucleus: central role of phosphorylation.

Nuclear protein transport is integral to eukaryotic cell processes such as differentiation, transformation, and the control of gene expression. Although the targeting role of nuclear localization signals (NLSs) has been known for some time, more recent results indicate that NLS-dependent nuclear protein import is precisely regulated. Phosphorylation appears to be the main mechanism controlling the nuclear transport of a number of proteins, including transcription factors such as NFkappaB, c-rel, dorsal, and SWI5 from yeast. Cytoplasmic retention factors, intra- and intermolecular NLS masking, and NLS masking by phosphorylation are some of the mechanisms by which phosphorylation specifically regulates nuclear transport. Even nuclear localization of the archetypal NLS-containing simian virus 40 large tumor antigen (T-ag) is regulated, namely by the "CcN motif," which comprises the T-ag NLS ("N") determining ultimate subcellular destination, a casein kinase II site ("C") 13 amino acids NH2-terminal to the NLS modulating the rate of nuclear import, and a cyclin-dependent kinase site ("c") adjacent to the NLS regulating the maximal level of nuclear accumulation. The CcN motif appears to be a special form of phosphorylation-regulated NLS (prNLS), where phosphorylation at site(s) close to the NLS specifically regulates NLS function. The regulation of nuclear transport through phosphorylation and prNLSs appears to be common in eukaryotic cells from yeast and plants to higher mammals.

Amino Acid Sequence↗

PubMed1997

Nuclear localization of the NS3 protein of hepatitis C virus and factors affecting the localization.

Subcellular localization of the NS2 and NS3 proteins of hepatitis C virus was analyzed. In stable Ltk transfectants inducibly expressing an NS2-NS3 polyprotein (amino acids [aa] 810 to 1463), processed full-size NS2 (aa 810 to 1026) was detected exclusively in a cytoplasmic membrane fraction. On the other hand, the other processed product, carboxy-truncated NS3 (NS3 deltaC1463; aa 1027 to 1463), was present in both cytoplasmic and nuclear fractions. To further analyze subcellular localization of NS3, NS3 deltaC1459 (aa 1027 to 1459), full-size NS3 (NS3F; aa 1027 to 1657), and both amino- and carboxy-truncated NS3 (NS3 deltaNdeltaC; aa 1201 to 1459) were expressed in HeLa cells by using a vaccinia virus-T7 hybrid expression system. NS3 deltaC1459 and NS3F accumulated in the nucleus as well as in the cytoplasm, exhibiting a dot-like staining pattern. On the other hand, NS3 deltaNdeltaC was localized predominantly in the cytoplasm, suggesting the presence of a nuclear localization signal(s) in the amino-terminal sequence of NS3. NS4A, a viral cofactor for the NS3 protease, inhibited nuclear transport of NS3 deltaC1459 and NS3F, with the latter inhibited to a lesser extent than was the former. Interestingly, wild-type p53 tumor suppressor augmented nuclear localization of NS3 deltaC1459 and NS3F, whereas mutant-type p53 inhibited nuclear localization and augmented cytoplasmic localization of NS3 deltaC1459. However, subcellular localization of NS3 deltaNdeltaC was not affected by either type of p53. Wild-type p53-mediated nuclear accumulation of NS3 deltaC1459 and NS3F was inhibited partially, but not completely, by coexpressed NS4A, with NS3F again affected less prominently than was NS3 deltaC1459.

Animals↗

PubMed2003

Transport signals and transcription-dependent nuclear localization of the putative DEAD-box helicase MDDX28.

The human protein MDDX28 is a putative RNA helicase and a nucleocytoplasmic shuttling protein also localized to the mitochondria. Its localization is novel among RNA helicases. We have studied its intracellular targeting signals and show that the first 20 amino acids of MDDX28 are necessary and sufficient for both mitochondrial import and nuclear export of the protein. Mutation of the five leucines in the sequence to alanines abolished the mitochondrial targeting signal as well as greatly reducing the nuclear export signal, indicating that these signal sequences are highly overlapping. Two short stretches of basic amino acids separated by 44 residues were both necessary and sufficient for full nuclear localization. However, they were not absolutely essential, because the protein was present in 7% of the nuclei when both signals were mutated. This indicates that MDDX28 contains another unidentified weak nuclear localization signal(s). Three basic domains in the N-terminal half of the protein and its RNA binding ability were essential for nucleolar localization as well as transcription-inhibition-dependent localization to nuclear subcompartments. Two of these basic domains were the same as those constituting the nuclear localization signal, suggesting that they are responsible for bringing the protein into the nucleus to the sites of RNA binding. Our results indicate that MDDX28 nucleo-cytoplasmic shuttling is dependent on the availability of nascent RNA.

Active Transport, Cell Nucleus↗

PubMed1996

Nuclear localization of the Arabidopsis APETALA3 and PISTILLATA homeotic gene products depends on their simultaneous expression.

The Arabidopsis APETALA3 (AP3) and PISTILLATA (PI) proteins are thought to act as transcription factors and are required for specifying floral organ identities. To define the nuclear localization signals within these proteins, we generated translational fusions of the coding regions of AP3 and PI to the bacterial uidA gene that encodes beta-glucuronidase (GUS). Transient transformation assays of either the AP3-GUS or PI-GUS fusion protein alone resulted in cytoplasmic localization of GUS activity. However, coexpression of AP3-GUS with PI, or PI-GUS with AP3, resulted in nuclear localization of GUS activity. Stable transformation with these fusion proteins in Arabidopsis showed similar results. The nuclear colocalization signals in AP3 and PI were mapped to the amino-terminal regions of each protein. These observations suggest that the interaction of the AP3 and PI gene products results in the formation of a protein complex that generates or exposes a colocalization signal required to translocate the resulting complex into the nucleus. The colocalization phenomenon that we have described represents a novel mechanism to coordinate the functions of transcription factors within the nucleus.

Arabidopsis↗

PubMed1994

Determinants for intracellular sorting of cytoplasmic and nuclear intermediate filaments.

The mechanism by which nuclear and cytoplasmic filaments are sorted in vivo was studied by examining which lamin sequences are required to target an otherwise cytoplasmic IF protein, the small neurofilament subunit (NF-L), to the nuclear lamina. By swapping corresponding domains between NF-L and lamin A, nuclear envelope targeting of NF-L was shown to require the presence of the "head" domain, a 42-amino acid sequence unique to lamin rod domains, a nuclear localization signal and the CAAX motif. Replacement of the entire COOH-terminal tail of lamin A with that of NF-L had no discernible effect on nuclear localization of lamin A, provided the substituted NF-L tail contained a NLS and a CAAX motif. This chimeric protein exhibited characteristics more typical of lamin B than that of the parental lamin A. With regard to cytoplasmic assembly properties, substitution of the head domain of lamin A for that of NF-L did not substantially affect the ability of NF-L to coassemble with vimentin in the cytoplasm. In contrast, insertion of a 42-amino acid sequence unique to lamin rod domains into NF-L profoundly affected NF-L coassembly with vimentin indicating that the 42-amino acid insertion in lamins may be important for sorting lamins from cytoplasmic IF proteins.

Amino Acid Sequence↗

PubMed1995

A nuclear localization domain in the hnRNP A1 protein.

The heterogeneous nuclear RNP (hnRNP) A1 protein is one of the major pre-mRNA/mRNA binding proteins in eukaryotic cells and one of the most abundant proteins in the nucleus. It is localized to the nucleoplasm and it also shuttles between the nucleus and the cytoplasm. The amino acid sequence of A1 contains two RNP motif RNA-binding domains (RBDs) at the amino terminus and a glycine-rich domain at the carboxyl terminus. This configuration, designated 2x RBD-Gly, is representative of perhaps the largest family of hnRNP proteins. Unlike most nuclear proteins characterized so far, A1 (and most 2x RBD-Gly proteins) does not contain a recognizable nuclear localization signal (NLS). We have found that a segment of ca. 40 amino acids near the carboxyl end of the protein (designated M9) is necessary and sufficient for nuclear localization; attaching this segment to the bacterial protein beta-galactosidase or to pyruvate kinase completely localized these otherwise cytoplasmic proteins to the nucleus. The RBDs and another RNA binding motif found in the glycine-rich domain, the RGG box, are not required for A1 nuclear localization. M9 is a novel type of nuclear localization domain as it does not contain sequences similar to classical basic-type NLS. Interestingly, sequences similar to M9 are found in other nuclear RNA-binding proteins including hnRNP A2.

Amino Acid Sequence↗

PubMed1993

Functional analysis of phosphorylation sites in human lamin A controlling lamin disassembly, nuclear transport and assembly.

We have constructed point mutations in human lamin A cDNA at conserved serine and threonine residues, some of which were shown to be phosphorylated in vitro by cdc2-kinase and protein kinase C and in vivo. Using a functional in vivo assay system, we identified three categories of mutant phenotypes. (i) Dominant negative phenotypes in mitosis result from mutation of Thr-19 and Ser-22 within the amino-terminal cdc2-kinase motif of lamin A. An increase of aberrant mitotic phenotypes in the double mutants Thr-19/Ser-392 and Ser-22/Ser-392 suggests that concomitant phosphorylation of the three residues regulates mitotic lamin A disassembly. (ii) Mutation of both Ser-403/Ser-404 within a PKC motif flanking the nuclear localization signal inhibits transport of mutant lamin A to the nucleus in 64% of the cells. It is proposed that phosphorylation of the motif in vivo positively regulates nuclear localization together with the nuclear localization sequence. (iii) The assembly of lamin A into the perinuclear lamina is disturbed by mutation of the carboxy-terminal Ser-525, previously shown to be interphase-specifically phosphorylated (Eggert et al., Eur. J. Biochem. 213, 659-671 (1993)). The phenotype shows discontinuous and patch-like aggregates of the mutant protein in the nucleus. We suggest that phosphorylation of the site either regulates lamina assembly or lamina-chromatin interaction in interphase.

Amino Acid Sequence↗

PubMed2006

Neuronal PAS domain protein 1 is a transcriptional repressor and requires arylhydrocarbon nuclear translocator for its nuclear localization.

Neuronal PAS domain protein 1 (NPAS1), a basic helix-loop-helix-PAS transcription factor expressed in the central nervous system, has been suggested to be involved in neuronal differentiation. However, relatively little is known about the molecular mechanism underlying the role of NPAS1 during development. In this study we set out to characterize the different domains within NPAS1. We showed that the nuclear localization of NPAS1 is dependent on the presence of ARNT. In addition, the transcriptional potential of ARNT is not required for this localization. In the absence of ARNT, NPAS1 is excluded from the nucleus, and this exclusion is due to the presence of a nuclear export signal within the N terminus of NPAS1. The interaction between NPAS1 and ARNT is via their N termini. We found no transactivation domain within NPAS1; instead, we mapped out at least three repression domains within NPAS1, suggesting that NPAS1 acts as a repressor. Furthermore, our experiments showed that NPAS1 is able to repress the transactivation functions of ARNT and ARNT2. We suggest that NPAS1 is guided into the nucleus by ARNT via the ARNT nuclear localization signal, and NPAS1 can override the activation function of adjacent transcription factors, providing a mechanism by which NPAS1 may inhibit transcription.

Animals↗

PubMed2004

Integration of retrograde axonal and nuclear transport mechanisms in neurons: implications for therapeutics.

The elongated morphology of neuronal processes imposes a significant challenge for effective intracellular communication between the neurites and the cell body. This problem is especially acute upon injury, when the cell body must receive accurate and timely information on the site and extent of axonal damage to mount an appropriate response. Recent work has demonstrated that nuclear import factors from the importin (karyopherin) alpha and beta families provide a mechanism for retrograde injury signaling. Importins are found throughout axons and dendrites at significant distances from the cell body, and importin beta protein is increased after nerve lesion by local translation of axonal mRNA. This leads to formation of a high-affinity nuclear localization signal (NLS) binding complex that traffics retrogradely due to an interaction of importin alpha with the motor protein dynein. Disruption of the complex with excess NLS peptides delays regeneration of injured sensory neurons. The dual role of importins in retrograde transport in axons and nuclear import in cell bodies suggests new avenues for manipulating intrinsic regeneration mechanisms in the nervous system and may provide a novel route for drug delivery to the CNS.

Active Transport, Cell Nucleus↗

PubMed1994

Nuclear signaling pathways for polypeptide ligands and their membrane receptors?

Classical signal transduction theory revolves around the premise that the role of membrane receptors is to transfer the signal represented by ligand binding from the external cell surface across the membrane to within the cell. Other components of the signaling cascade such as second-messenger molecules and kinases then convey the signal from the cytoplasm to the nucleus to effect changes in gene expression. Membrane receptor endocytosis is seen as part of the cellular down-regulation and desensitization apparatus rather than as having an active signaling function. Evidence is mounting, however, that polypeptide ligands and their membrane receptors may have an important additional signaling role within the cell including the nucleus. Several ligands such as those of the platelet-derived and fibroblast growth factor classes have been found not only to localize in the nucleus, but also to possess sequences similar to the nuclear localization signal of the simian virus SV40 large T antigen. In most cases where they have been examined, these sequences appear to be both functional in nuclear targeting and essential for full signaling activity. The implication is that subsequent to internalization, polypeptide ligands and/or their receptors may translocate to the nucleus and participate directly in regulating gene expression.

Amino Acid Sequence↗

PubMed2004

Role of Ca2+/calmodulin-dependent protein kinase II in cardiac hypertrophy and heart failure.

Ca2+/calmodulin-dependent protein kinase II (CaMKII), a critical transducer of Ca2+ signaling, is a multifunctional protein kinase which can phosphorylate a wide range of substrates and regulate numerous cellular functions. The delta isoforms of CaMKII predominate in the heart and two splice variants of CaMKIIdelta, deltaB and deltaC, have been demonstrated to be present in the adult mammalian myocardium. The deltaB isoform contains a nuclear localization signal (NLS) that is absent from deltaC, and consequently, the two isoforms have different subcellular localization. Recent work from our laboratory and others has implicated CaMKII in the development of cardiac hypertrophy and heart failure. The specific roles of these CaMKII isoforms in regulating cardiac function appear to be determined by their subcellular localization. The nuclear deltaB isoform plays a key role in hypertrophic gene expression, whereas the cytoplasmic deltaC isoform can affect excitation-contraction (E-C) coupling through phosphorylation of Ca2+ regulatory proteins and may also transduce signals leading to apoptosis. In addition, the nuclear deltaB and the cytoplasmic deltaC isoforms of CaMKII are differentially regulated in pressure overload-induced cardiac hypertrophy. This review focuses on evidence that CaMKII plays an essential role in transcriptional activation associated with cardiac hypertrophy, as well as the aberrant Ca2+ handling and apoptosis that may contribute to heart failure. The hypothesis that CaMKII isoform selective activation, localization and substrate phosphorylation lead to specificity in the resultant signaling pathways is discussed.

Animals↗

PubMed2005

Regulating inducible transcription through controlled localization.

Many signaling pathways regulate the activity of effector transcription factors by controlling their subcellular localization. Until recently, the cytoplasmic retention of inactive transcription factors was mainly attributed to binding partners that mask the nuclear localization signals (NLSs) of target proteins. Inactive transcription factors were thought to be exclusively cytoplasmic until their activation, after which the NLSs were unmasked to allow nuclear translocation. There is now a growing body of evidence, however, that challenges this simple model. This review discusses recent reports that suggest that inducible transcription factors can constantly shuttle between the cytoplasm and the nucleus, and that their apparent cytoplasmic retention can be achieved by binding partners that mask the NLSs, tether the transcription factor to cytoplasmic structures, or mark the transcription factor for proteasomal degradation. We also discuss the possibility that this more complex model of cytoplasmic retention might be applicable to a broader range of transcription factors and their associated signaling pathways.

Animals↗

PubMed2001

Investigation of nucleo-cytoplasmic transport using UV-guided microinjection.

Active nucleo-cytoplasmic transport is mediated by dynamic signal-mediated pathways. We investigated the effects of transcription inhibitors or fluorescent lectins on nuclear import mediated by nuclear localization signals (NLSs). Therefore, a novel experimental approach that allows the controlled sequential introduction of fluorescent substances into living cells was established. A microinjection system equipped with an UV-source enabled us to identify fluorescent-labeled cells for the subsequent introduction of additional fluorescent compounds, in order to study their interactions in vivo. Cells were initially labeled either by expression of autofluorescent proteins or by microinjection of fluorescent substances. Transcription inhibitors did not affect nuclear transport mediated by classical NLSs but inhibited import mediated by the M9-domain of hnRNPA1. Comparison of a mono- and bipartite NLS revealed that the bipartite signal was more active in import. Sequential injection of differentially labeled nuclear import and export substrates allowed monitoring of import and export simultaneously in the same living cell. The introduced experimental approach will also be useful to analyze a variety of biological processes in living mammalian cells.

Amino Acid Sequence↗

PubMed2003

Functional inactivation of the conserved Sem1p in yeast by intrabodies.

Intrabody technology was applied to characterize the function and intracellular localization of a highly conserved Saccharomyces cerevisiae Sem1 protein. DSS1, the mammalian homologue of Sem1p, is functionally conserved between yeast and mammalian cells, and in mammalian cells physically interacts with the strong tumour supressor BRCA2. Yeast and the generated intrabodies are thus expected to offer a useful system for studies on Sem1p/DSS1 function. Sem1p-specific antibody isolated from a phage display library was expressed intracellularily and targeted to either the cytosol or the nucleus of yeast cells. Analysis of the applicability of different antibody fragments as intrabodies showed that the Fab intrabody was expressed most efficiently. Expression of nuclear-targeted anti-Sem1p Fab intrabodies inhibited the growth of the sigma1278b yeast strain in a manner similar to deletion of the SEM1 gene. This indicates that the Fab intrabodies interact in vivo with Sem1p and result in inactivation of Sem1p. Localization of the Fab intrabody with or without the nuclear localization signal to the nucleus in Sem1p-dependent manner suggests that Sem1p mediates the nuclear transport of the intrabody without any targeting signal. Our results suggest that Sem1p function in yeast cells is in part manifested in the nucleus.

Amino Acid Sequence↗

PubMed1994

Cloning of a gene encoding a human leukocyte protein characterised by extensive heptad repeats.

Complementary DNA (cDNA) clones, encoding a fusion protein that was recognised by an antiserum raised against a purified polypeptide fragment of a 180-kDa human leukocyte protein, were isolated from a lambda gt11 expressed library. The clones encoded a unique amino acid (aa) sequence interspersed with heptad repeats that typify coiled-coil proteins, and hybridised to a 5-kb transcript universally expressed in a panel of eight human tissues. Comparatively high levels of RNA expression were seen in testis, ovary and mitogen-activated peripheral blood leukocytes (PBLs). The deduced 1300-aa sequence reveals a protein with a typical signal peptide, a hydrophilic domain containing an N-terminal globular head with a nuclear localization signal sequence, a C-terminal region of coiled-coil structure, a candidate transmembrane domain, and a short 10-aa C-terminal domain. Rabbit polyclonal antisera raised against a truncated lambda gt11 fusion protein recognized a 150-170-kDa protein (non-reduced) in mitogen-activated PBLs. The protein designated here as CG-1 may exist as a homodimer destined for translocation to the nucleus, with a role in leukocyte differentiation and/or effector function.

Amino Acid Sequence↗

PubMed2006

Analysis of HIV-2 Vpx by modeling and insertional mutagenesis.

Vpx facilitates HIV-2 nuclear localization by a poorly understood mechanism. We have compared Vpx to an NMR structure HIV-1 Vpr in a central helical domain and probed regions of Vpx by insertional mutagenesis. A predicted loop between helices two and three appears to be unique, overlapping with a known novel nuclear localization signal. Overall, Vpx was found to be surprisingly flexible, tolerating a series of large insertions. We found that insertion within the polyproline-containing C-terminus destabilizes nuclear localization, whereas mutating a second helix in the central domain disrupts viral packaging. Other insertional mutants in the predicted loop and in a linker region between the central domain and the C-terminus may be useful as sites of intramolecular tags as they could be packaged adequately and retained preintegration complex associated integration activity in a serum starvation assay. An unexpected result was found within a previously defined nuclear localization motif near aa 71. This mutant retained robust nuclear localization in a GFP fusion assay and was competent for preintegration complex associated nuclear import. In summary, we have modeled helical content in Vpx and assessed potential sites of intramolecular tags which may prove useful for protein-protein interactions studies.

Amino Acid Sequence↗