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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The yeast transcription factor Gcn4p contains two stretches of amino acid residues, NLS1 and NLS2, which are independently able to relocate the cytoplasmic protein chorismate mutase into the nucleus. Only NLS2 is conserved among fungi. A truncated version of CPCA (the counterpart of Gcn4p in Aspergillus nidulans), which lacks the conserved NLS, accumulates in the cytoplasm instead of the nucleus. Nuclear uptake mediated by the NLS1 of Gcn4p is impaired by defects in genes for several different karyopherins, whereas NLS2-dependent nuclear import specifically requires the alpha-importin Srp1p and the beta-importin Kap95p. Yeast strains that are defective in either of these two karyopherins are unable to respond to amino acid starvation. We have thus identified Gcn4p as a substrate for the Srp1p/Kap95p transport complex. Our data suggest that NLS2 is the essential and specific nuclear transport signal; NLS1 may play only an unspecific or accessory role.
Mol Genet Genomics 2004 Apr
PMID:Nuclear import of yeast Gcn4p requires karyopherins Srp1p and Kap95p. 1464

In spite of recent efforts to elucidate the nuclear import pathway of the human immunodeficiency virus type 1 (HIV-1) integrase protein (IN), its exact route as well as the domains that mediate its import are still unknown. Here, we show that a synthetic peptide bearing the amino acid residues 161-173 of the HIV-1 IN is able to mediate active import of covalently attached bovine serum albumin molecules into nuclei of permeabilized cells and therefore was designated as nuclear localization signal-IN (NLS(IN)). A peptide bearing residues 161-173 in the reversed order showed low karyophilic properties. Active nuclear import was demonstrated by using fluorescence microscopy and a quantitative ELISA-based assay system. Nuclear import was blocked by addition of the NLS(IN) peptide, as well as by a peptide bearing the NLS of the simian virus 40 T-antigen (NLS-SV40). The NLS(IN) peptide partially inhibited nuclear import mediated by the full-length recombinant HIV-1 IN protein, indicating that the sequence of the NLS(IN) is involved in mediating nuclear import of the IN protein. The NLS(IN) as well as the full-length IN protein interacted specifically with importin alpha, binding of which was blocked by the NLS(IN) peptide itself as well as by the NLS-SV40.
J Mol Biol 2004 Mar 05
PMID:A synthetic peptide bearing the HIV-1 integrase 161-173 amino acid residues mediates active nuclear import and binding to importin alpha: characterization of a functional nuclear localization signal. 1503 73

Antisense and antigene oligonucleotides (ONs) are attractive drugs for gene therapy, but major limiting factors for their routine use are inefficient cellular uptake and low accessibility to the target sites. Adding various lipophilic conjugates to the ON improves intracellular delivery as has been previously reported. We studied the cellular delivery of various ON modifications, as well as their cytosolic and nuclear distribution in mammalian Hep2-EGFP-NLS cell line. We compared uptake efficacy of ON and LNA, both conjugated with cholesterol at the 5' end. All ONs were 3' labeled with fluorescent Cy 5 dye. We made a comparison of the ONs uptake efficacy and the kinetics, both adding ONs to the culture medium, and using streptolysin-O (SL-O) permeabilization. The cellular uptake of each ON used in this study was visualized by fluorescent microscopy. We confirmed the results by FACS analysis. We determined the ratio between initial ON-chol concentration (0.4 microM) and the final amount in nucleus.SL-O can highly improve kinetics of ON delivery; not only into the cytoplasm but also to the nucleus, the presumed site of antigene ON action. The most effective nuclear uptake was observed when ON conjugated with cholesterol (ON-chol) and SL-O was used. Nuclear distribution of ON was reached within few minutes. In contrast, ON simply added to the medium reached cytoplasm only and the process of delivery took several hours.
Mol Cell Biochem 2005 Aug
PMID:Cholesterol conjugated oligonucleotide and LNA: a comparison of cellular and nuclear uptake by Hep2 cells enhanced by streptolysin-O. 1613 86

Progesterone receptors (PR) are ligand-activated transcription factors that modulate transcription by activating genes. There are two isoforms of PR, PRA and PRB. In most cell contexts, the PRA isoform is a repressor of the PRB isoform. Without hormone induction, PRA is mostly located in the nucleus whereas PRB distributes both in the nucleus and in the cytoplasm. In this paper, a new model system has been used to study the impact of initial subcellular localization, and import rate of progesterone receptor on transcriptional activity. This new model system involves using a mutant version of PRB which is found only in the cytoplasmic compartment of cells in the unliganded state, making the distribution of the receptor more homogeneous to start with compared with the previous model, wild type (wt) PRB, which has a more heterogeneous distribution (nuclear and cytoplasmic even without ligand). Import kinetics has been shown to be one of the major means by which to regulate PR transcriptional activity. Fluorescence microscopy was used to measure green fluorescent protein tagged PRB import rate into the nucleus. Luciferase reporter gene assay was used to measure transcriptional activity of PRB. In addition, a two-hybrid assay was performed to measure the interaction between PRB and importin alpha. Mutant versions of PRA and PRB with the constitutively active nuclear localization signal removed were created (PRA-NLSc mutant and PRB-NLSc mutant). These PR mutants were found to localize mainly in the cytoplasm in the absence of hormone. With addition of hormone, PR mutants translocated to the nucleus, although at a slower rate compared to wt PRB. Our results show that the activation of reporter gene transcription is proportional to the nuclear import rate of PRB-NLSc mutant, and the difference in import kinetics between wt PRB and the PRB-NLSc mutant is due to a stronger interaction of wt PRB with importin alpha. We also show that the hormone inducible NLS in PR, NLSh, is a weak nuclear localization signal even without hormone and can act as a weak hormone dependent nuclear localization signal when combined with the ligand binding domain of PR. In addition, by changing the initial subcellular localization of PRA from the nucleus to the cytoplasm, this diminished PRA's ability to act as an inhibitor of PRB.
Mol Pharm
PMID:Effect of initial subcellular localization of progesterone receptor on import kinetics and transcriptional activity. 1632 58

Subcellular compartmentalization and dynamic movements of steroid receptors are major steps in executing their transcription regulatory function. Though significant progress has been made in understanding the mechanisms underlying nuclear import of NLS-bearing proteins, our general and mechanistic understanding about the nuclear export processes has begun to emerge only recently. The discovery of most commonly utilized CRM1/exportin1 dependent nuclear export pathway is attributed to a potent nuclear export inhibitor leptomycin B that helped dissecting this and other nuclear export pathways. Simultaneously, utilization of green fluorescent protein (GFP)-tagged intracellular steroid receptors has contributed to not only resolving controversial issue of subcellular localization of unliganded hormone receptors but also provided further insight into finer details of receptor dynamics in living cells. With judicious use of leptomycin B and expression of GFP-tagged receptors in living cells, existence of exportin1/CRM1 independent pathway(s), nuclear export signals and receptors for bi-directional translocation that are unique to steroid receptor trafficking have been specified. Currently, we appear to be arriving at a consensus that steroid/nuclear receptors follow dynamic nucleocytoplasmic processes that deviate from the ones commonly utilized by majority of other proteins.
Mol Cell Endocrinol 2006 Feb 26
PMID:Intracellular localization and nucleocytoplasmic trafficking of steroid receptors: an overview. 1638 93

The Atm protein kinase is central to the DNA double-strand break response in mammalian cells. After irradiation, dimeric Atm undergoes autophosphorylation at Ser 1981 and dissociates into active monomers. Atm activation is stimulated by expression of the Mre11/Rad50/nibrin complex. Previously, we showed that a C-terminal fragment of nibrin, containing binding sites for both Mre11 and Atm, was sufficient to provide this stimulatory effect in Nijmegen breakage syndrome (NBS) cells. To discriminate whether nibrin's role in Atm activation is to bind and translocate Mre11/Rad50 to the nucleus or to interact directly with Atm, we expressed an Mre11 transgene with a C-terminal NLS sequence in NBS fibroblasts. The Mre11-NLS protein complexed with Rad50, localized to the nucleus in NBS fibroblasts, and associated with chromatin. However, Atm autophosphorylation was not stimulated in cells expressing Mre11-NLS, nor were downstream Atm targets phosphorylated. To determine whether nibrin-Atm interaction is necessary to stimulate Atm activation, we expressed nibrin transgenes lacking the Atm binding domain in NBS fibroblasts. The nibrin DeltaAtm protein interacted with Mre11/Rad50; however, Atm autophosphorylation was dramatically reduced after irradiation in NBS cells expressing the nibrin DeltaAtm transgenes relative to wild-type nibrin. These results indicate that nibrin plays an active role in Atm activation beyond translocating Mre11/Rad50 to the nucleus and that this function requires nibrin-Atm interaction.
Mol Cell Biol 2006 Mar
PMID:Active role for nibrin in the kinetics of atm activation. 1647 90

The complexation of the new protein vector PGEk--a carrier of nucleic acids into proliferating cells with phosphodiester d(TTAGGG)4 (TMO) and phosphorothioate Sd(TTAGGG)4 (TMS) telomerase inhibitor oligonucleotides was studied. PGEk molecule, consisting of 64 amino acids, is comprising the sequence of the human epidermal growth factor EGFh which is hydrophobic cell targeting moiety serving for receptor-mediated endocytosis and an NLS (nuclear localization signal) which is hydrophilic serving as a DNA-binding and selective nuclear import moiety. Experiments were carried out in 0.01 M Na-phosphate buffer contained 0.1 M NaCl, pH 7.8 at 37 degrees C. CD spectral analysis revealed that TMO molecules folded back into intramolecular antiparallel G-quadruplex while TMS molecules were represented as unstructured thread. The number of adsorbed PGEk molecules were estimated using PGEk intrinsic fluorescence decrease and fluorescence polarization increase of PGEk under oligonucleotide titration. Adsorption isotherms were plotted in Scatchard coordinates. We have shown that adsorption of the first two PGEk molecules on TMO and TMS occurs noncooperatively with the high association constants K1(TMO) = (7 +/- 1) x 10(7) M(-1) and K1(TMS) = (3 +/- 0.5) x 10(7) M(-1), respectively. Further adsorption up to 5-6 PGEk molecules on TMO occurrs cooperatively with still high association constant K2(TMO) = (4.0 +/- 1.5) x 10(6) M(-1). TMS oligonucleotide binds the third PGEk molecule rather weakly, K2(TMS) = (8 +/- 2) x 10(5) M(-1). CD spectral analysis revealed that G-quadruplex structure formed by TMO have undergone a partial unfolding by binding of PGEk molecules while single-stranded structure formed by TMS was not affected by binding PGEk. Thus, the tertiary structure of DNA and the number of adsorbed PGEk molecules formed biologically active compounds PGEk: TMO and PGEk: TMS were defined, which are able to penetrate through the membrane of proliferating cells and to suppress their growth.
Mol Biol (Mosk)
PMID:[Complexes of telomeric oligonucleotide d(TTAGGG)4 with the new recombinant protein PGEk--nucleic acid carrier into proliferating cells]. 1681 68

ICK1 is the first member of a family of plant cyclin-dependent kinase (CDK) inhibitors. It has been shown that ICK1 is localized in the nuclei of transgenic Arabidopsis plants. Since cellular localization is important for the functions of cell cycle regulators, a comprehensive analysis was undertaken to identify specific sequences regulating the cellular localization of ICK1. Deletion and site-specific mutants fused to the green fluorescent protein (GFP) were used in transgenic Arabidopsis plants and transfected tobacco cells. Surprisingly, three separate sequences in the N-terminal, central and C-terminal regions of ICK1 could independently confer nuclear localization of the GFP fusion proteins. The central nuclear localization signal NLS(ICK1) could transport the much larger GUS (beta-glucuronidase)-GFP fusion protein into nuclei, while the other two sequences were unable to. These results suggest that NLS(ICK1) is a strong NLS that actively transports the fusion protein into nuclei, while the other two sequences are either a weaker NLS or confer the nuclear localization of GFP indirectly. It was further observed that the N-terminal sequence specifies a punctate pattern of subnuclear localization, while the C-terminal sequence suppresses it. Furthermore, co-expression of ICK1 and Arabidopsis CDKA, tagged with different GFP variants, showed that ICK1 could mediate the transport of CDKA into nuclei while a mutant ICK1(1-162) that does not interact with CDKA lost this ability. These results illustrate how the nuclear localization of ICK1 is regulated and also suggest a possible role of ICK1 in regulating the cellular distribution of CDKA.
Plant Mol Biol 2006 Sep
PMID:Molecular control of nuclear and subnuclear targeting of the plant CDK inhibitor ICK1 and ICK1-mediated nuclear transport of CDKA. 1684 78

Ewing sarcoma (EWS) protein, a member of a large family of RNA-binding proteins, contains an N-terminal transcriptional activation domain (EAD) and a C-terminal RNA-binding domain (RBD). Due to its multifunctional properties EWS protein is involved in processes such as gene expression, RNA processing and transport, and cell signaling. Chimeric EWS proteins generated by chromosomal translocations cause malignant tumors. EWS protein is located predominantly in the nucleus, but was found also in the cytosol and associated with the cell membrane. The determinants responsible for the nuclear localization of the protein were as yet unknown. We identified the nuclear localization signal of EWS protein at its C terminus (C-NLS), which is required for the nuclear import and retention of the protein. The C-NLS sequence is conserved in related proto-oncoproteins suggesting an NLS function also in these proteins. Two arginine residues, due to their positive charge, a proline residue and a tyrosine residue are essential for C-NLS function. The nuclear localization of EWS protein is independent of the regions in RBD containing numerous arginine methylation sites, RNA-recognition and zinc finger motifs. Regions in EAD guide the subnuclear partition of EWS protein and contain another but different NLS that allows nucleocytoplasmic shuttling of the N-terminal domain.
J Mol Biol 2006 Oct 13
PMID:Identification and characterization of the nuclear localization/retention signal in the EWS proto-oncoprotein. 1696 92

Cardiac Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) in heart has been implicated in Ca(2+) current (I(Ca)) facilitation, enhanced sarcoplasmic reticulum (SR) Ca(2+) release and frequency-dependent acceleration of relaxation (FDAR) via enhanced SR Ca(2+) uptake. However, questions remain about how CaMKII may work in these three processes. Here we tested the role of CaMKII in these processes using transgenic mice (SR-AIP) that express four concatenated repeats of the CaMKII inhibitory peptide AIP selectively in the SR membrane. Wild type mice (WT) and mice expressing AIP exclusively in the nucleus (NLS-AIP) served as controls. Increasing stimulation frequency produced typical FDAR in WT and NLS-AIP, but FDAR was markedly inhibited in SR-AIP. Quantitative analysis of cytosolic Ca(2+) removal during [Ca(2+)](i) decline revealed that FDAR is due to an increased apparent V(max) of SERCA. CaMKII-dependent RyR phosphorylation at Ser2815 and SR Ca(2+) leak was both decreased in SR-AIP vs. WT. This decrease in SR Ca(2+) leak may partly balance the reduced SERCA activity leading to relatively unaltered SR-Ca(2+) load in SR-AIP vs. WT myocytes. Surprisingly, CaMKII regulation of the L-type Ca(2+) channel (I(Ca) facilitation and recovery from inactivation) was abolished by the SR-targeted CaMKII inhibition in SR-AIP mice. Inhibition of CaMKII effects on I(Ca) and RyR function by the SR-localized AIP places physical constraints on the localization of these proteins at the junctional microdomain. Thus SR-targeted CaMKII inhibition can directly inhibit the activation of SR Ca(2+) uptake, SR Ca(2+) release and I(Ca) by CaMKII, effects which have all been implicated in triggered arrhythmias.
J Mol Cell Cardiol 2007 Jan
PMID:CaMKII inhibition targeted to the sarcoplasmic reticulum inhibits frequency-dependent acceleration of relaxation and Ca2+ current facilitation. 1705 27


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