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Results for “Nuclear Localization Signals”

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PubMed2004

Regulation of Chk1 kinase by autoinhibition and ATR-mediated phosphorylation.

The checkpoint kinase Chk1 undergoes ATR-mediated phosphorylation and activation in response to unreplicated DNA, but the precise mechanism of Chk1 activation is not known. In this study, we have analyzed the domain structure of Xenopus Chk1 and explored the mechanism of its activation by ATR-mediated phosphorylation. We show that the C-terminal region of Xenopus Chk1 contains an autoinhibitory region (AIR), which largely overlaps with a bipartite, unusually long ( approximately 85-amino acid) nuclear localization signal. When coexpressed in oocytes or embryos, the AIR can interact with and inhibit the kinase domain of Chk1, but not full-length Chk1, suggesting an autoinhibitory intramolecular interaction in the Chk1 molecule. If linked with the preceding ATR phosphorylation domain that has either phospho-mimic mutation or genuine phosphorylation, however, the AIR can no longer interact with or inhibit the kinase domain, suggesting a conformational change of the AIR by ATR-mediated phosphorylation. Even in full-length Chk1, such phospho-mimic mutation can interfere with the autoinhibitory intramolecular interaction, but only if this interaction is somewhat weakened by an additional mutation in the AIR. These results provide significant insights into the mechanism of Chk1 activation at the DNA replication checkpoint.

Amino Acid Motifs↗

PubMed2002

Flp recombinase transgenic mice of C57BL/6 strain for conditional gene targeting.

We constructed an expression vector of Flp recombinase modified by adding a nuclear localization signal. Injection of the expression vector into fertilized eggs of the C57BL/6 strain yielded transgenic mouse lines expressing the Flp recombinase transgene in the testis. We crossed the transgenic mice to reporter mice carrying the neomycin phosphotransferase gene flanked by target sites of Flp recombinase. Examination of the deletion of the neomycin phosphotransferase gene in the progeny showed that Flp-mediated recombination took place efficiently in vivo in FLP66 transgenic mouse line. These results suggest that the Flp recombinase system is effective in mice and in combination with the Cre recombinase system extends the potentials of gene manipulation in mice. One of the useful applications of FLP66 transgenic mouse line is the removal of marker genes from mice manipulated for the conditional gene targeting with the Cre/loxP system in the pure C57BL/6 genetic background.

Animals↗

PubMed2000

Transgenic mice expressing a mutant form of loricrin reveal the molecular basis of the skin diseases, Vohwinkel syndrome and progressive symmetric erythrokeratoderma.

Mutations in the cornified cell envelope protein loricrin have been reported recently in some patients with Vohwinkel syndrome (VS) and progressive symmetric erythrokeratoderma (PSEK). To establish a causative relationship between loricrin mutations and these diseases, we have generated transgenic mice expressing a COOH-terminal truncated form of loricrin that is similar to the protein expressed in VS and PSEK patients. At birth, transgenic mice (ML.VS) exhibited erythrokeratoderma with an epidermal barrier dysfunction. 4 d after birth, high-expressing transgenic animals showed a generalized scaling of the skin, as well as a constricting band encircling the tail and, by day 7, a thickening of the footpads. Histologically, ML. VS transgenic mice also showed retention of nuclei in the stratum corneum, a characteristic feature of VS and PSEK. Immunofluorescence and immunoelectron microscopy showed the mutant loricrin protein in the nucleus and cytoplasm of epidermal keratinocytes, but did not detect the protein in the cornified cell envelope. Transfection experiments indicated that the COOH-terminal domain of the mutant loricrin contains a nuclear localization signal. To determine whether the ML.VS phenotype resulted from dominant-negative interference of the transgene with endogenous loricrin, we mated the ML.VS transgenics with loricrin knockout mice. A severe phenotype was observed in mice that lacked expression of wild-type loricrin. Since loricrin knockout mice are largely asymptomatic (Koch, P.K., P. A. de Viragh, E. Scharer, D. Bundman, M.A. Longley, J. Bickenbach, Y. Kawachi, Y. Suga, Z. Zhou, M. Huber, et al., J. Cell Biol. 151:389-400, this issue), this phenotype may be attributed to expression of the mutant form of loricrin. Thus, deposition of the mutant protein in the nucleus appears to interfere with late stages of epidermal differentiation, resulting in a VS-like phenotype.

Amino Acid Sequence↗

PubMed2006

Knockdown of nuclear Ca2+/calmodulin-dependent protein kinase phosphatase causes developmental abnormalities in zebrafish.

Nuclear Ca2+/calmodulin-dependent protein kinase phosphatase (CaMKP-N) is an enzyme that dephosphorylates and concomitantly downregulates multifunctional Ca2+/calmodulin-dependent protein kinases (CaMKs) in vitro. However, the functional roles of this enzyme in vivo are not well understood. To investigate the biological significance of CaMKP-N during zebrafish embryogenesis, we cloned and characterized zebrafish CaMKP-N (zCaMKP-N). Based on the nucleotide sequences in the zebrafish whole genome shotgun database, we isolated a cDNA clone for zCaMKP-N, which encoded a protein of 633 amino acid residues. Transiently expressed full-length zCaMKP-N in mouse neuroblastoma, Neuro2a cells, was found to be localized in the nucleus. In contrast, the C-terminal truncated mutant lacking RKKRRLDVLPLRR (residues 575-587) had cytoplasmic staining, suggesting that the nuclear localization signal of zCaMKP-N exists in the C-terminal region. Ionomycin treatment of CaMKIV-transfected Neuro2a cells resulted in a marked increase in the phosphorylated form of CaMKIV. However, cotransfection with zCaMKP-N significantly decreased phospho-CaMKIV in ionomycin-stimulated cells. Whole mount in situ hybridization analysis of zebrafish embryos showed that zCaMKP-N is exclusively expressed in the head and neural tube regions. Gene knockdown of zCaMKP-N using morpholino-based antisense oligonucleotides induced significant morphological abnormalities in zebrafish embryos. A number of apoptotic cells were observed in brain and spinal cord of the abnormal embryos. These results suggest that zCaMKP-N plays a crucial role in the early development of zebrafish.

Amino Acid Sequence↗

PubMed2006

Evaluation of transfection protocols for unmodified and modified peptide nucleic acid (PNA) oligomers.

We have compared the efficacy of different transfection protocols reported for peptide nucleic acid (PNA) oligomers. A precise evaluation of uptake efficacy was achieved by using a positive readout assay based on the ability of a PNA oligomer to correct aberrant splicing of a recombinant luciferase gene. The study comprised transfection of PNA conjugated to acridine, adamantyl, decanoic acid, and porphyrine (acr-PNA, ada-PNA, deca-PNA, and por-RNA, respectively) and unmodified PNA partially hybridized to a DNA oligomer (PNA/DNA cotransfection). Furthermore, the effect of conjugation to a nuclear localization signal (NLS) was evaluated as part of the PNA/DNA cotransfection protocol. Transfection of the tested PNAs was systematically optimized. PNA/DNA cotransfection was found to produce the highest luciferase activity, but only after careful selection of the DNA oligonucleotide. Both a cationic lipid, Lipofectamine, and a nonliposomal cationic polymer, polyethylenimine (PEI, ExGen 500), were efficient transfection reagents for the PNA/DNA complex. However, Lipofectamine, in contrast to PEI, showed severe side effects, such as cytotoxicity. acr-PNA, ada-PNA, and por-PNA were transfectable with efficacies between 5 and 10 times lower than that seen with PNA/DNA cotransfection. Conjugation of PNA to NLS had no effect on PNA/DNA cotransfection efficacy. An important lesson from the study was the finding that because of uncontrollable biologic variations, even optimal transfection conditions differed to a certain extend from experiment to experiment in an unpredictable way.

Cell Count↗

PubMed2006

Noncovalently linked nuclear localization peptides for enhanced calcium phosphate transfection.

The generation of cell lines stably expressing recombinant material is a lengthy process and there has thus been much interest in the use of transient expression systems to rapidly produce recombinant material. To achieve this, the DNA of interest must be delivered into the nucleus of the target cell. The mechanisms by which this process occurs are poorly understood and the efficiency of various methods differs widely. Recently, nuclear localization signals (NLSs) have been investigated to target entry of DNA into the nucleus of mammalian cells. We have used NLSs from the SV40 and Tat antigens mixed with our model luciferase reporter gene plasmid for the transfection of Chinese hamster ovary (CHO) cells using calcium phosphate and FuGENE 6 transfection technology. The noncovalent complexation of NLSs with plasmid DNA before calcium phosphate-mediated transfection resulted in enhanced reporter gene expression with increasing ratios of NLS to plasmid until reaching a maximum. At higher ratios than maximum expression, the expression levels decreased. On the other hand, when using FuGENE 6 reagent NLSs did not enhance reporter gene expression. Cell cycle arrest in G(2)/M phase obliterated the effect of the NLS on reporter gene expression when using the calcium phosphate transfection method.

Animals↗

PubMed2006

Domain analysis of a groundnut calcium-dependent protein kinase: nuclear localization sequence in the junction domain is coupled with nonconsensus calcium binding domains.

The signature of calcium-dependent protein kinases (CDPKs) is a C-terminal calmodulin-like domain (CaMLD) with four consensus calcium-binding sites. A junction domain (JD) joins the kinase with CaMLD and interacts with them through its autoinhibitory and CaMLD binding subdomains, respectively. We noted several CDPKs additionally have a bipartite nuclear localization signal (NLS) sequence as a subdomain in their JD, and this feature is obligatorily coupled with the absence of consensus calcium-binding sites in their respective CaMLDs. These predicted features are substantiated by undertaking investigations on a CDPK (gi:67479988) isolated from cultured groundnut (Arachis hypogea) cells. This kinase can bind 3.1 mol of Ca(2+) under saturating conditions with a considerably high K(d) of 392 mum as compared with its canonical counterparts. CD spectroscopic analysis, however, indicates the intramolecular structural changes accompanied with calcium binding to be similar to canonical CDPKs. Attesting to the presence of NLS in the JD, the endogenous kinase is localized in the nucleus of osmotically stressed Arachis cells, and in vitro binding assays indicate the NLS in the JD to interact with nuclear transport factors of the importin family. Homology modeling also indicates the feasibility of interaction of importins with the NLS present in the JD of such CDPKs in their activated form. The possible significance of obligatory coupling between the presence of NLS in the junction domain and atypical calcium binding properties of these CDPKs is discussed in the light of the known mechanisms of activation of these kinases.

Allergy and Immunology↗

PubMed2006

High-throughput, library-based selection of a murine leukemia virus variant to infect nondividing cells.

Gammaretroviruses, such as murine leukemia virus (MLV), are functionally distinguished from lentiviruses, such as human immunodeficiency virus, by their inability to infect nondividing cells. Attempts to engineer this property into MLV have been hindered by an incomplete understanding of early events in the viral life cycle. We utilized a transposon-based method to generate saturated peptide insertion libraries of MLV gag-pol variants with nuclear localization signals randomly incorporated throughout these overlapping genes. High-throughput selection of the libraries via iterative retroviral infection of nondividing cells led to the identification of a novel variant that successfully transduced growth-arrested cells. Vector packaging by cotransfection of the gag-pol.NLS variant with wild-type gag-pol produced high-titer virions capable of infecting neurons in vitro and in vivo. The capacity of mutant virions to transduce nondividing cells could help to elucidate incompletely understood mechanisms of the viral life cycle and greatly broaden the gene therapy applications of retroviral vectors. Furthermore, the ability to engineer key intracellular viral infection steps has potential implications for the understanding, design, and control of other post-entry events. Finally, this method of library generation and selection for a desired phenotype directly in a mammalian system can be readily expanded to address other challenges in protein engineering.

Active Transport, Cell Nucleus↗

PubMed2004

LOCnet and LOCtarget: sub-cellular localization for structural genomics targets.

LOCtarget is a web server and database that predicts and annotates sub-cellular localization for structural genomics targets; LOCnet is one of the methods used in LOCtarget that can predict sub-cellular localization for all eukaryotic and prokaryotic proteins. Targets are taken from the central registration database for structural genomics, namely, TargetDB. LOCtarget predicts localization through a combination of four different methods: known nuclear localization signals (PredictNLS), homology-based transfer of experimental annotations (LOChom), inference through automatic text analysis of SWISS-PROT keywords (LOCkey) and de novo prediction through a system of neural networks (LOCnet). Additionally, we report predictions from SignalP. The final prediction is based on the method with the highest confidence. The web server can be used to predict sub-cellular localization of proteins from their amino acid sequence. The LOCtarget database currently contains localization predictions for all eukaryotic proteins from TargetDB and is updated every week. The server is available at http://www.rostlab.org/services/LOCtarget/.

Databases, Genetic↗

PubMed2002

Model system to study classical nuclear export signals.

Signal-mediated protein transport through the nuclear pore complex is of considerable interest in the field of molecular pharmaceutics. Nuclear localization signals can be used to target genes/antisense delivery systems to the nucleus. Studying nuclear export is useful in enhancing the expression and the efficiency of action of these therapeutic agents. The mechanism of nuclear import has been well studied and most of the proteins participating in this mechanism have been identified. The subject of nuclear export is still in the initial stages, and there is a considerable amount of uncertainty in this area. Two main export receptors identified so far are Exportin 1 (Crm1) and Calreticulin. Crm1 recognizes certain leucine-rich amino acid sequences in the proteins it exports called classical nuclear export signals. This paper describes a model system to study, identify, and establish these classical nuclear export signals using green fluorescent protein (GFP). Two putative export signals in the human progesterone receptor (PR) and the strongest nuclear export signal known (from mitogen activated protein kinase kinase [MAPKK]) were studied using this model system.

Active Transport, Cell Nucleus↗

PubMed2003

LOC3D: annotate sub-cellular localization for protein structures.

LOC3D (http://cubic.bioc.columbia.edu/db/LOC3d/) is both a weekly-updated database and a web server for predictions of sub-cellular localization for eukaryotic proteins of known three-dimensional (3D) structure. Localization is predicted using four different methods: (i) PredictNLS, prediction of nuclear proteins through nuclear localization signals; (ii) LOChom, inferring localization through sequence homology; (iii) LOCkey, inferring localization through automatic text analysis of SWISS-PROT keywords; and (iv) LOC3Dini, ab initio prediction through a system of neural networks and vector support machines. The final prediction is based on the method that predicts localization with the highest confidence. The LOC3D database currently contains predictions for >8700 eukaryotic protein chains taken from the Protein Data Bank (PDB). The web server can be used to predict sub-cellular localization for proteins for which only a predicted structure is available from threading servers. This makes the resource of particular interest to structural genomics initiatives.

Animals↗

PubMed2003

The GA octodinucleotide repeat binding factor BBR participates in the transcriptional regulation of the homeobox gene Bkn3.

In the dominant mutant Hooded (K), the barley gene BKn3 is overexpressed as a result of a duplication of 305 bp in intron IV. When fused to a cauliflower mosaic virus 35S minimal promoter, the 305 bp element activates gene expression in tobacco, as does a 655 bp BKn3 promoter sequence. Both DNA fragments contain a (GA)8 repeat (GA/TC)8. A one-hybrid screen using the 305 bp element as the DNA target led to the cloning of the barley b recombinant (BBR) protein, which binds specifically to the (GA/TC)8 repeat. BBR is nuclear targeted and is a characterized nuclear localization signal (NLS) sequence, a DNA-binding domain extended up to 90 aa at the C-terminus and a putative N-terminal activation domain. The corresponding gene has no introns and is ubiquitously expressed in barley tissues. In co-transfection experiments, BBR activates (GA/TC)8-containing promoters, and its overexpression in tobacco leads to a pronounced leaf shape modification. BBR has properties of a GAGA-binding factor, but the corresponding gene has no sequence homology to Trl and Psq of Drosophila, which encode functionally analogous proteins. In Arabidopsis, (GA/TC)8 repeats occur particularly within 1500 bp upstream of gene start codons included in some homeodomain genes of different classes. The data presented suggest that expression of the barley BKn3 is regulated, at least in part, by the binding of the transcription factor BBR to GA/TC repeats.

Amino Acid Sequence↗

PubMed2005

[Antigenic analysis of the recombinant capsid protein of porcine circovirus type 2].

The nuclear localization signal (NLS)-defected capsid protein gene (dCap) of porcine circovirus type 2 (PCV2) was expressed firstly in Escherichia coli as a fusion protein with glutathione S-transferase (rGST-dCap protein). The purified rGST-dCap protein and NLS-defected Cap protein of PCV2 (rdCap protein) from the purified rGST-dCap protein reacted specifically with swine antiserum to PCV2. Furthermore, the obtained monoclonal antibodies (mAbs) to rdCap protein were shown to bind to PCV2 particles replicated in PK15 cell. MAbs to rdCap protein also revealed the neutralizing ability to PCV2 particles. These results demonstrate that rGST-dCap protein expressed in E. coli is folded correctly or at least partly, and all mAbs to rdCap protein possess the binding epitopes of PCV2 particle whereas mAbs 4C4,3F6 and 2G7 to rdCap protein keep the neutralization epitopes of PCV2 particle, showing a potential of rGST-dCap protein as a vaccine antigen or serodiagnostic reagent.

Animals↗

PubMed1998

Characterization of photolyase/blue-light receptor homologs in mouse and human cells.

We isolated and characterized mouse photolyase-like genes, mCRY1 (mPHLL1) and mCRY2 (mPHLL2), which belong to the photolyase family including plant blue-light receptors. The mCRY1 and mCRY2 genes are located on chromosome 10C and 2E, respectively, and are expressed in all mouse organs examined. We raised antibodies specific against each gene product using its C-terminal sequence, which differs completely between the genes. Immunofluorescent staining of cultured mouse cells revealed that mCRY1 is localized in mitochondria whereas mCRY2 was found mainly in the nucleus. The subcellular distribution of CRY proteins was confirmed by immunoblot analysis of fractionated mouse liver cell extracts. Using green fluorescent protein fused peptides we showed that the C-terminal region of the mouse CRY2 protein contains a unique nuclear localization signal, which is absent in the CRY1 protein. The N-terminal region of CRY1 was shown to contain the mitochondrial transport signal. Recombinant as well as native CRY1 proteins from mouse and human cells showed a tight binding activity to DNA Sepharose, while CRY2 protein did not bind to DNA Sepharose at all under the same condition as CRY1. The different cellular localization and DNA binding properties of the mammalian photolyase homologs suggest that despite the similarity in the sequence the two proteins have distinct function(s).

Amino Acid Sequence↗

PubMed2003

Basic residues of the helix six domain of influenza virus M1 involved in nuclear translocation of M1 can be replaced by PTAP and YPDL late assembly domain motifs.

Influenza type A virus matrix (M1) protein possesses multiple functional motifs in the helix 6 (H6) domain (amino acids 91 to 105), including nuclear localization signal (NLS) (101-RKLKR-105) involved in translocating M1 from the cytoplasm into the nucleus. To determine the role of the NLS motif in the influenza virus life cycle, we mutated these and the neighboring sequences by site-directed mutagenesis, and influenza virus mutants were generated by reverse genetics. Our results show that infectious viruses were rescued by reverse genetics from all single alanine mutations of amino acids in the H6 domain and the neighboring region except in three positions (K104A and R105A within the NLS motif and E106A in loop 6 outside the NLS motif). Among the rescued mutant viruses, R101A and R105K exhibited reduced growth and small-plaque morphology, and all other mutant viruses showed the wild-type phenotype. On the other hand, three single mutations (K104A, K105A, and E106A) and three double mutations (R101A/K102A, K104A/K105A, and K102A/R105A) failed to generate infectious virus. Deletion (Delta YRKL) or mutation (4A) of YRKL also abolished generation of infectious virus. However, replacement of the YRKL motif with PTAP or YPDL as well as insertion of PTAP after 4A mutation yielded infectious viruses with the wild-type phenotype. Furthermore, mutant M1 proteins (R101A/K102A, Delta YRKL, 4A, PTAP, 4A+PTAP, and YPDL) when expressed alone from cloned cDNAs were only cytoplasmic, whereas the wild-type M1 expressed alone was both nuclear and cytoplasmic as expected. These results show that the nuclear translocation function provided by the positively charged residues within the NLS motif does not play a critical role in influenza virus replication. Furthermore, these sequences of H6 domain can be replaced by late (L) domain motifs and therefore may provide a function similar to that of the L domains of other negative-strand RNA and retroviruses.

Amino Acid Motifs↗

PubMed2003

Bpag1 localization to actin filaments and to the nucleus is regulated by its N-terminus.

Plakins are a family of giant cytoskeleton binding proteins. One member of this group is bullous pemphigoid antigen 1 (Bpag1)/dystonin, which has neuronal and muscle isoforms that consist of actin-binding and microtubule-binding domains at either end separated by a plakin domain and several spectrin repeats. The better-characterized epithelial isoform has only the plakin domain in common with the neuronal and muscle isoforms. Here, we have analyzed the localization of muscle/neuronal (Bpag1a/b) isoforms and the epithelial (Bpag1e) isoform within C2C12 myoblast cells. Although an antibody specific to Bpag1a/b isoform 2 detected protein co-aligning actin stress fibers, this same antibody and two Bpag1e antibodies predominantly detected protein in the nuclei. A Bpag1a/b isoform 2 N-terminal fusion protein containing the plakin domain also localized to actin stress fibers and to nuclei. Within the plakin domain, we characterized a functional nuclear localization signal, which was responsible for localization of the fusion protein to the nucleus. Bpag1a/b isoform 1 N-terminal fusion proteins differed in their interaction with the actin cytoskeleton and with their ability to localize to the nucleus, suggesting that Bpag1 isoforms with different N-termini have differing roles. These results show the importance of N-terminal domains in dictating the localization and function of Bpag1 isoforms. We provide the first indication that Bpag1 is not strictly a cytoplasmic/membrane protein but that it can also localize to the nucleus.

Actin Cytoskeleton↗

PubMed2000

A novel gene family with a developmentally regulated expression in Xenopus laevis.

We have isolated a new maternal gene called 4G2. 4G2 cDNA encodes a predicted protein of 501 amino acids, and its apparent molecular mass of 61 kDa was determined by SDS-PAGE of 4G2 recombinant protein expressed in E. coli or in vitro translated in rabbit reticulocyte lysate. Amino acid analysis of 4G2 revealed the RGD and LDV motif with a potential cell attachment activity. The open reading frames (ORF) also contained a consensus bipartite nuclear localization signal (NLS). There were number of expressed tag sequences (ESTs) from Drosophila, zebrafish, chicken, mouse, and human origin that encode a high degree of identity to the predicted 4G2 protein, thereby suggesting that 4G2 may constitute a novel gene family whose function has not been elucidated. We also present evidence that 4G2 transcript is maternally synthesized in stage IV oocyte, localized to animal hemisphere of egg, and zygotically reactivated in mid-neurula stage.

Amino Acid Motifs↗

PubMed2001

Structure, expression and chromosome mapping of MLZE, a novel gene which is preferentially expressed in metastatic melanoma cells.

We isolated a novel gene, termed MLZE, from a B16-BL6 cDNA library after subtraction of B16-F10 mRNA. Expression levels of mouse MLZE (mMLZE) increased in accordance with metastatic ability of B16 melanoma sublines. Human homolog of mMlze (hMlze) contained one leucine zipper structure and two potential nuclear localizing signals. Northern blot analysis of multiple human tissues showed that hMLZE was expressed primarily in trachea and spleen. We mapped the hMLZE gene (by fluorescence in situ hybridization) to 8q24.1 - 2, which contains the c-myc gene and is often amplified in malignant melanoma. Immunohistochemistry revealed that the number of hMlze-positive cases was significantly larger in Clark levels III, IV and V melanomas (6 / 11 = 55%) than in Clark levels I and II melanomas (2 / 15 = 13%). In two cases of hMlze-positive melanomas, the strength of hMlze staining increased substantially in the deep component of the tumor. Considering that melanomas above Clark level II are more metastatic than those below Clark level III, these findings suggested that MLZE is one of the genes whose expression is upregulated during the course of acquisition of metastatic potential in melanoma cells.

Adult↗