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Results for “Nuclear Localization Signals”

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PubMed1997

Oligomerization of HIV-1 Rev mutants in the cytoplasm and during nuclear import.

Oligomerization of Rev molecules has been shown to be required for Rev function. In addition to a Western blot assay monitoring dimer formation, a new in vivo assay analyzing formation of Rev heteromers in the cytoplasm and during nuclear import is presented here. The oligomerization assay is based upon the ability of Rev mutants with an intact nuclear localization signal (NLS) to interact specifically with mutants with a defective NLS and translocate such mutants to the nuclear compartments. Several of the mutants previously reported to be oligomerization defective were found to mediate nuclear and nucleolar localization of the NLS mutant. The Rev mutant previously named M4 was the only mutant tested that did not translocate the mutant with a defective NLS to the nucleus. Furthermore, the predominantly cytoplasmic localization of the M4 mutant suggests that oligomerization is important for effective nuclear import of Rev.

Animals↗

PubMed1994

Nuclear transport of the U2 snRNP-specific U2B'' protein is mediated by both direct and indirect signalling mechanisms.

Experiments investigating the nuclear import of the U2 snRNP-specific B'' protein (U2B'') are presented. U2B'' nuclear transport is shown to be able to occur independently of binding to U2 snRNA. The central segment of the protein (amino acids 90-146) encodes an unusual nuclear localization signal (NLS) that is related to that of the U1 snRNP-specific A protein. However, nuclear import of U2B'' does not depend on this NLS. Sequences in the N-terminal RNP motif of the protein are sufficient to direct nuclear transport, and evidence is presented that the interaction of U2B'' with the U2A' protein mediates this effect. This suggests that U2B'' can 'piggy-back' to the nucleus in association with U2A', and thus be imported to the nucleus by two different mechanisms. U2A' nuclear transport, on the other hand, can occur independently of both U2B'' binding and of U2 snRNA.

Amino Acid Sequence↗

PubMed2000

Human ribosomal protein L5 contains defined nuclear localization and export signals.

Ribosomal protein L5 is part of the 60 S ribosomal subunit and localizes in both the cytoplasm and the nucleus of eukaryotic cells, accumulating particularly in the nucleoli. L5 is known to bind specifically to 5 S rRNA and is involved in nucleocytoplasmic transport of this rRNA. Here, we report a detailed analysis of the domain organization of the human ribosomal protein L5. We show that a signal that mediates nuclear import and nucleolar localization maps to amino acids 21-37 within the 297-amino acid L5 protein. Furthermore, carboxyl-terminal residues at positions 255-297 serve as an additional nuclear/nucleolar targeting signal. Domains involved in 5 S rRNA binding are located at both the amino terminus and the carboxyl terminus of L5. Microinjection studies in somatic cells demonstrate that a nuclear export signal (NES) that maps to amino acids 101-111 resides in the central region of L5. This NES is characterized by a pronounced clustering of critical leucine residues, which creates a peptide motif not previously observed in other leucine-rich NESs. Finally, we present a refined model of the multidomain structure of human ribosomal protein L5.

Amino Acid Sequence↗

PubMed2002

Human brain acyl-CoA hydrolase isoforms encoded by a single gene.

Acyl-CoA hydrolases are a group of enzymes that catalyze the hydrolysis of acyl-CoA thioesters to free fatty acids and CoA-SH. The human brain acyl-CoA hydrolase (BACH) gene comprises 13 exons, generating several isoforms through the alternative use of exons. Four first exons (1a-1d) can be used, and three patterns of splicing occur at exon X located between exons 7 and 8 that contains an internal 3(')-splice acceptor site and creates premature stop codons. When examined with green fluorescent protein-fusion constructs expressed in Neuro-2a cells, the nuclear localization signal encoded by exon 9 was functional by itself, whereas the whole structure was cytosolic, suggesting nuclear translocation of the enzyme. This was consistent with dual staining of the cytosol and nucleus in certain neurons by immunohistochemistry using anti-BACH antibody. The mitochondrial targeting signals encoded by exons 1b and 1c were also functional and directed mitochondrial localization of BACH isoforms with the signals. Although BACH mRNA containing the sequence derived from exon 1a, but not exon X, was exclusively expressed in human brain, these results suggest that the human BACH gene can express long-chain acyl-CoA hydrolase activity in multiple intracellular compartments by generating BACH isoforms with differential localization signals to affect various cellular functions that involve acyl-CoAs.

Amino Acid Motifs↗

PubMed2000

The karyophilic properties of human immunodeficiency virus type 1 integrase are not required for nuclear import of proviral DNA.

Integrase (IN) is a key component of the preintegration nucleoprotein complex (PIC), which transports the retroviral genome from the cytoplasm to the nucleus of newly infected cells. Retroviral IN proteins have intrinsic karyophilic properties, which for human immunodeficiency virus type 1 (HIV-1) are currently attributed to regions that display sequence homology to previously characterized nuclear localization signals. We asked here whether the karyophilic properties of HIV-1 IN are involved in the nuclear import of PIC. We mutated three conserved basic regions in the C-terminal domain of IN and analyzed the effects of mutations on subcellular localization of the protein, viral particle composition, IN dimerization within virions, and infectivity. Alteration of two sequences caused the loss of nuclear accumulation of IN and drastically reduced the capacity of the protein to multimerize. Mutation of the most C-terminal sequence had no effect on the subcellular localization and dimerization of IN. Nevertheless, conservation of all three sequences was required for viral infectivity. Despite the perturbation of IN subcellular localization, all mutant viruses displayed normal reverse transcription and nuclear transport of PICs in newly infected cells. The replicative defect was instead at the level of integration, for which all mutants were markedly affected in vivo. Besides reinforcing the association between dimerization of IN and nuclear accumulation of the enzyme, our data demonstrate that subcellular localization of IN alone cannot predict the fate of the PICs.

Amino Acid Sequence↗

PubMed2000

Localization of BAI-associated protein1/membrane-associated guanylate kinase-1 at adherens junctions in normal rat kidney cells: polarized targeting mediated by the carboxyl-terminal PDZ domains.

Brain-specific angiogenesis inhibitor (BAI)-associated protein (BAP)1 (also called membrane-associated guanylate kinase [MAGI]-1) is composed of six PSD-95/Dlg-A/ZO-1 (PDZ) domains, two WW domains, and one guanylate kinase (GK) domain. We previously reported that BAP1 is localized at tight junctions in Madine Darby canine kidney (MDCK) cells and intestinal epithelial cells. Here, we have determined the localization of BAP1 in normal rat kidney (NRK) cells that do not form tight junctions. BAP1 was colocalized with E-cadherin along the lateral membrane, suggesting its localization at adherens junctions. Green fluorescent protein (GFP)-BAP1 was distributed in the cytosol in separate NRK cells, and accumulated to the cell-cell contacts when NRK cells have contact with each other. The GFP-BAP1 mutant containing either the first PDZ and GK domains or the WW and second PDZ domains was localized in the cytosol and the nucleus. The GFP-BAP1 mutant containing the second to fourth PDZ domains was distributed in the cytosol. The construct containing the fifth and sixth PDZ domains was localized at the cell-cell contacts along the lateral membrane and slightly in the nucleus, whereas the construct lacking the fifth and sixth PDZ domains was localized in the cytosol and in the nucleus. BAP1 was tyrosine-phosphorylated in vivo, but the tyrosine phosphorylation of BAP1 was not correlated with its localization. These results suggest that the signal in the carboxyl-terminal PDZ domains functions dominantly in vivo to target BAP1 to the lateral membrane, although potential nuclear localization signals exist in the N-terminal region of BAP1.

Adherens Junctions↗

PubMed2002

The biology of the opioid growth factor receptor (OGFr).

Opioid peptides act as growth factors in neural and non-neural cells and tissues, in addition to serving for neurotransmission/neuromodulation in the nervous system. The native opioid growth factor (OGF), [Met(5)]-enkephalin, is a tonic inhibitory peptide that plays a role in cell proliferation and tissue organization during development, cancer, cellular renewal, wound healing, and angiogenesis. OGF action is mediated by a receptor mechanism. Assays with radiolabeled OGF have detected specific and saturable binding, with a one-site model of kinetics. Subcellular fractionation studies show that the receptor for OGF (OGFr) is an integral membrane protein associated with the nucleus. Using antibodies generated to a binding fragment of OGFr, this receptor has been cloned and sequenced in human, rat, and mouse. OGFr is distinguished by containing a series of imperfect repeats. The molecular and protein structure of OGFr have no resemblance to that of classical opioid receptors, and have no significant homologies to known domains or functional motifs with the exception of a bipartite nuclear localization signal. Immunoelectron microscopy and immunocytochemistry investigations, including co-localization studies, have detected OGFr on the outer nuclear envelope where it interfaces with OGF. The peptide-receptor complex associates with karyopherin, translocates through the nuclear pore, and can be observed in the inner nuclear matrix and at the periphery of heterochromatin of the nucleus. Signal transduction for modulation of DNA activity is dependent on the presence of an appropriate confirmation of peptide and receptor. This report reviews the history of OGF-OGFr, examines emerging insights into the mechanisms of action of opioid peptide-receptor interfacing, and discusses the clinical significance of these observations.

Amino Acid Sequence↗

PubMed2003

Caspase-dependent cleavage of c-Abl contributes to apoptosis.

The nonreceptor tyrosine kinase c-Abl may contribute to the regulation of apoptosis. c-Abl activity is induced in the nucleus upon DNA damage, and its activation is required for execution of the apoptotic program. Recently, activation of nuclear c-Abl during death receptor-induced apoptosis has been reported; however, the mechanism remains largely obscure. Here we show that c-Abl is cleaved by caspases during tumor necrosis factor- and Fas receptor-induced apoptosis. Cleavage at the very C-terminal region of c-Abl occurs mainly in the cytoplasmic compartment and generates a 120-kDa fragment that lacks the nuclear export signal and the actin-binding region but retains the intact kinase domain, the three nuclear localization signals, and the DNA-binding domain. Upon caspase cleavage, the 120-kDa fragment accumulates in the nucleus. Transient-transfection experiments show that cleavage of c-Abl may affect the efficiency of Fas-induced cell death. These data reveal a novel mechanism by which caspases can recruit c-Abl to the nuclear compartment and to the mammalian apoptotic program.

Active Transport, Cell Nucleus↗

PubMed2000

Cloning of the cDNA and localization of the gene encoding human NRBP, a ubiquitously expressed, multidomain putative adapter protein.

Adapter proteins modulate multiple signaling pathways by regulating the aggregation of other factors into signaling complexes. Here we have identified a novel human cDNA encoding NRBP, a multidomain putative adapter protein containing (i) two putative nuclear receptor binding motifs (LXXLL), (ii) a putative binding domain for Src homology-2 (SH2) domain containing proteins, (iii) a kinase-like domain, (iv) a bipartite nuclear localization signal, and (v) three sequences rich in glutamic acid, serine, proline, and threonine (PEST) residues. The NRBP mRNA transcript, of approximately 2.4 kb, was ubiquitously expressed in a wide range of normal human tissues and 15 human tumor cell lines. The NRBP cDNA is predicted to encode a polypeptide of 535 amino acids with a molecular mass of 59.8 kDa. Translation of NRBP mRNA in vitro reveals three translation products of 60, 51, and 43 kDa, suggesting that translation of NRBP may initiate at multiple sites. The NRBP gene was localized to human chromosome 2p23, near the location of the NCOA1 gene encoding the nuclear receptor coactivator, steroid receptor coactivator-1 (SRC-1). The features of NRBP predict a function as an adapter protein potentially linking signaling pathways involving nuclear receptors and SH2 domain containing proteins.

3' Untranslated Regions↗

PubMed2002

A developmentally regulated ARF-like 5 protein (ARL5), localized to nuclei and nucleoli, interacts with heterochromatin protein 1.

ARF-like proteins (ARLs) are distinct group of members of the ARF family of Ras-related GTPases. Although ARLs are very similar in primary structure to ARFs, their functions remain unclear. We cloned mouse (m) and human (h) ARL5 cDNAs to characterize the protein products and their molecular properties. mARL5 mRNA was more abundant in liver than in other adult tissues tested. mARL5, similar to mARL4, was developmentally regulated and localized to nuclei. hARL5 interacted with importin-alpha through its C-terminal bipartite nuclear localization signal. When expressed in COS-7 cells, mutant hARL5(T35N), which is predicted to be GDP bound, was concentrated in nucleoli. The N-terminus of hARL5, like that of ARF, was myristoylated. Yeast two-hybrid screening and in vitro protein-interaction assays showed that hARL5(Q80L), predicted to be GTP bound, interacted with heterochromatin protein 1alpha (HP1alpha), which is known to be associated with telomeres as well as with heterochromatin, and acted as a transcriptional suppressor in mammalian cells. The interaction was reproduced in COS cells, where hARL5(Q80L) was co-immunoprecipitated with HP1alpha. hARL5 interaction with HP1alpha was dependent on the nucleotide bound, and required the MIR-like motif. Moreover, hARL5(Q80L), but not hARL5 lacking the MIR-like motif, was partly co-localized with overexpressed HP1alpha. Our findings suggest that developmentally regulated ARL5, with its distinctive nuclear/nucleolar localization and interaction with HP1alpha, may play a role(s) in nuclear dynamics and/or signaling cascades during embryonic development.

ADP-Ribosylation Factors↗

PubMed1991

The role of phosphorylation and the CDC28 protein kinase in cell cycle-regulated nuclear import of the S. cerevisiae transcription factor SWI5.

The intracellular localization of the S. cerevisiae transcription factor SWI5 is cell cycle dependent. The protein is nuclear in G1 cells but cytoplasmic in S, G2, and M phase cells. We have identified SWI5's nuclear localization signal (NLS) and show that it can confer cell cycle-dependent nuclear entry to a heterologous protein. Located within or close to the NLS are three serine residues, mutation of which results in constitutive nuclear entry. These residues are phosphorylated in a cell cycle-dependent manner in vivo, being phosphorylated when SWI5 is in the cytoplasm and dephosphorylated when it is in the nucleus. As all three serines are phosphorylated by purified CDC28-dependent H1 kinase activity in vitro, we propose a model in which the CDC28 kinase acts directly to control nuclear entry of SWI5.

Amino Acid Sequence↗

PubMed2005

Developmentally regulated cytoplasmic retention of the transcription factor XMI-ER1 requires sequence in the acidic activation domain.

Xmi-er1 is a fibroblast growth factor regulated immediate-early gene that is activated during mesoderm induction in Xenopus embryonic explants. This gene encodes a nuclear protein with potent transcriptional regulator activity and overexpression of XMI-ER1 in Xenopus embryos inhibits mesoderm induction and leads to truncations along the anteroposterior axis. We showed previously that XMI-ER1 is retained in the cytoplasm during cleavage stages and only begins to appear in the nucleus at mid-blastula. Such developmentally regulated nuclear translocation may represent an important mechanism for regulating XMI-ER1 activity in the early embryo. Here, we investigate different mechanisms that might control nuclear translocation of XMI-ER1. Using alpha-amanitin to inhibit transcription, we show that nuclear localization is not dependent on zygotic transcription. Nor is it the result of a developmentally regulated import pathway, as the XMI-ER1 nuclear localization signal (NLS) fused to beta-galactosidase (betagal) was able to direct nuclear translocation prior to mid-blastula. Fusion of an additional, heterologous NLS to the N-terminus of XMI-ER1 was not sufficient to overcome cytoplasmic retention, indicating that retention does not involve NLS masking, but rather binding to a cytoplasmic anchor. The anchoring molecule is not an RNA, as microinjection of RNase A did not affect the timing of nuclear translocation. Western blot analysis using antibodies that recognize phosphorylated residues revealed that, while XMI-ER1 is not itself phosphorylated, it is associated with two differentially phosphorylated proteins, suggesting that the anchoring mechanism may involve interaction with a cytoplasmic protein(s). A series of XMI-ER1 deletion mutants was utilized to map the putative retention domain. Our analysis revealed that amino acids 144-175, containing the fourth acidic stretch of the acidic activation domain, are required for retention. These results suggest that XMI-ER1 is retained in the cytoplasm of the early embryo by interaction of the region containing amino acids 144-175 with a cytoplasmic anchor.

Animals↗

PubMed2001

Intracellular trafficking and transgene expression of viral and non-viral gene vectors.

The objective of this review is to summarize the critical steps in intracellular trafficking and the principal factors involved in transgene expression by a comparison of viral and non-viral gene vectors. Intracellular trafficking of viral and non-viral gene vectors are reviewed kinetically as well as from the mechanistic point of view. The peptide-dependent specific mechanism of endosomal escape by viral vectors is compared with the non-specific mechanism of non-viral vectors. Regarding the nuclear transport of DNA, a number of recently developed strategies in non-viral vectors such as the application of nuclear localization signals or cell specific transcription factors are summarized in comparison with viral nuclear gene delivery. The molecular mechanisms of transcription and the translation of delivered genes to nucleus are also summarized in view of drug delivery systems. This information is intended to serve as a basis for developing a new gene delivery system for both viral and non-viral gene vectors. Optimizing the gene delivery system by integrating this intracellular trafficking as well as transgene expression will be required in order to develop an efficient and an safe gene delivery system.

Animals↗

PubMed2000

Modulation of retinoid signalling through NGF-induced nuclear export of NGFI-B.

The retinoid-X receptor (RXR) regulates multiple hormonal pathways through heterodimerization with nuclear receptors such as the all-trans retinoic acid receptor (RAR). The orphan nuclear receptor NGFI-B (also called Nur77) can heterodimerize with RXR. Here we show that nerve growth factor (NGF) induces the phosphorylation of Ser 105 of NGFI-B in PC12 phaeochromocytoma cells, resulting in translocation of the NGFI-B-RXR heterodimer complex out of the nucleus using nuclear export signals within NGFI-B. As a consequence of the redistribution of RXR, the transcriptional activity of RXR-RAR is reduced. NGFI-B-mediated nuclear export of receptors may serve as a mechanism for crosstalk between NGF and retinoid pathways.

Amino Acid Sequence↗

PubMed2005

Nuclear proteins as gene-transfer vectors.

An interest in nuclear proteins as possible gene vectors arose as early as 1988. Nuclear proteins possess important features, such as DNA condensing capacity and the presence of NLSs (nuclear localization signals), which are thought to be perfect tools to combine cellular and nuclear uptake of DNA. The NLS of the proteins might support the intracellular trafficking of the complexes, whereas their condensing capacity enables DNase stability. Nuclear proteins, histones and also non-histones, such as the high-mobility-group proteins, have been successfully employed in in vitro gene transfer by a variety of workers over the last decade. In the pertinent published data, almost all nuclear proteins tested showed gene-transfer activity. The degree of activity of the individual nuclear proteins tested varied according to sequential and structural differences of the proteins, discriminating their DNA binding behaviour, but reported transfection efficiency differences are inconsistent. They do not reveal a logical pattern, such as a correlation with their DNA affinity. The differences in transfection efficiency of certain nuclear proteins reported by different authors could be based on a lot of factors, including the source or method of protein preparation, the cells used, the media or the transfection additives, such as lysosomotropic agents. The variances are quite random, which suggests an involvement of physicochemical parameters of the nuclear protein-DNA complexes in the efficiency of gene expression. Physicochemical features of the complexes are also determined by factors such as transfection additives (e.g. Ca(2+)). The present review therefore includes, in addition to an overview about the nuclear proteins used in gene-transfer experiments, some assumptions about the mechanism of the cellular uptake of nuclear protein-DNA complexes and their translocation to the nucleus.

Animals↗

PubMed1996

Fibroblast growth factor (FGF) homologous factors: new members of the FGF family implicated in nervous system development.

Four new members of the fibroblast growth factor (FGF) family, referred to as fibroblast growth factor homologous factors (FHFs), have been identified by a combination of random cDNA sequencing, data base searches, and degenerate PCR. Pairwise comparisons between the four FHFs show between 58% and 71% amino acid sequence identity, but each FHF shows less than 30% identity when compared with other FGFs. Like FGF-1 (acidic FGF) and FGF-2 (basic FGF), the FHFs lack a classical signal sequence and contain clusters of basic residues that can act as nuclear localization signals. In transiently transfected 293 cells FHF-1 accumulates in the nucleus and is not secreted. Each FHF is expressed in the developing and adult nervous systems, suggesting a role for this branch of the FGF family in nervous system development and function.

Adult↗

PubMed2002

Structure of mitogen-activated protein kinase-activated protein (MAPKAP) kinase 2 suggests a bifunctional switch that couples kinase activation with nuclear export.

MAPK-activated protein kinase 2 (MAPKAPK2), one of several kinases directly phosphorylated and activated by p38 MAPK, plays a central role in the inflammatory response. The activated MAPKAPK2 phosphorylates its nuclear targets CREB/ATF1, serum response factor, and E2A protein E47 and its cytoplasmic targets HSP25/27, LSP-1, 5-lipoxygenase, glycogen synthase, and tyrosine hydroxylase. The crystal structure of unphosphorylated MAPKAPK2, determined at 2.8 A resolution, includes the kinase domain and the C-terminal regulatory domain. Although the protein is inactive, the kinase domain adopts an active conformation with aspartate 366 mimicking the missing phosphorylated threonine 222 in the activation loop. The C-terminal regulatory domain forms a helix-turn-helix plus a long strand. Phosphorylation of threonine 334, which is located between the kinase domain and the C-terminal regulatory domain, may serve as a switch for MAPKAPK2 nuclear import and export. Phosphorylated MAPKAPK2 masks the nuclear localization signal at its C terminus by binding to p38. It unmasks the nuclear export signal, which is part of the second C-terminal helix packed along the surface of kinase domain C-lobe, and thereby carries p38 to the cytoplasm.

Amino Acid Sequence↗

PubMed2002

Inhibitory activity of a heterochromatin-associated serpin (MENT) against papain-like cysteine proteinases affects chromatin structure and blocks cell proliferation.

MENT (Myeloid and Erythroid Nuclear Termination stage-specific protein) is a developmentally regulated chromosomal serpin that condenses chromatin in terminally differentiated avian blood cells. We show that MENT is an effective inhibitor of the papain-like cysteine proteinases cathepsins L and V. In addition, ectopic expression of MENT in mammalian cells is apparently sufficient to inhibit a nuclear papain-like cysteine proteinase and prevent degradation of the retinoblastoma protein, a major regulator of cell proliferation. MENT also accumulates in the nucleus, causes a strong block in proliferation, and promotes condensation of chromatin. Variants of MENT with mutations or deletions within the M-loop, which contains a nuclear localization signal and an AT-hook motif, reveal that this region mediates nuclear transport and morphological changes associated with chromatin condensation. Non-inhibitory mutants of MENT were constructed to determine whether its inhibitory activity has a role in blocking proliferation. These mutations changed the mode of association with chromatin and relieved the block in proliferation, without preventing transport to the nucleus. We conclude that the repressive effect of MENT on chromatin is mediated by its direct interaction with a nuclear protein that has a papain-like cysteine proteinase active site.

Amino Acid Sequence↗