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At least 127 records · Page 7Linked to original sources
PubMed2001

Cell surface-localized nucleolin is a eukaryotic receptor for the adhesin intimin-gamma of enterohemorrhagic Escherichia coli O157:H7.

Intimin-gamma is an outer membrane protein of enterohemorrhagic Escherichia coli (EHEC) O157:H7 that is required for the organism to adhere tightly to HEp-2 cells and to colonize experimental animals. Another EHEC O157:H7 protein, the Transferred intimin receptor (Tir), is considered the primary receptor for intimin-gamma. Nevertheless, Tir-independent binding of intimin-gamma to HEp-2 cells has been reported. This observation suggests the existence of a eukaryotic receptor(s) for intimin-gamma. In this study, we sought to identify that receptor(s). First, we determined by equilibrium binding titration that the association of purified intimin-gamma with HEp-2 cells was specific and consistent with a single host cell receptor. Second, we isolated a protein from lysates of HEp-2 cells that bound intimin-gamma and subsequently identified this molecule as nucleolin, a protein involved in cell growth regulation that can be cell surface-expressed. Third, we established that purified intimin-gamma and nucleolin were co-localized on the surface of HEp-2 cells and that the site of EHEC O157:H7 attachment was associated with regions of nucleolin expression. Finally, we demonstrated that mouse anti-nucleolin sera significantly decreased the adherence of EHEC O157:H7 to HEp-2 cells. From this, we conclude that nucleolin is the HEp-2 cell receptor for intimin-gamma expressed by EHEC O157:H7.

Adhesins, Bacterial↗
PubMed2003

Nucleolin expressed at the cell surface is a marker of endothelial cells in angiogenic blood vessels.

A tumor-homing peptide, F3, selectively binds to endothelial cells in tumor blood vessels and to tumor cells. Here, we show that the cell surface molecule recognized by F3 is nucleolin. Nucleolin specifically bound to an F3 peptide affinity matrix from extracts of cultured breast carcinoma cells. Antibodies and cell surface biotin labeling revealed nucleolin at the surface of actively growing cells, and these cells bound and internalized fluorescein-conjugated F3 peptide, transporting it into the nucleus. In contrast, nucleolin was exclusively nuclear in serum-starved cells, and F3 did not bind to these cells. The binding and subsequent internalization of F3 were blocked by an antinucleolin antibody. Like the F3 peptide, intravenously injected antinucleolin antibodies selectively accumulated in tumor vessels and in angiogenic vessels of implanted "matrigel" plugs. These results show that cell surface nucleolin is a specific marker of angiogenic endothelial cells within the vasculature. It may be a useful target molecule for diagnostic tests and drug delivery applications.

Active Transport, Cell Nucleus↗
PubMed1993

The Gly/Arg-rich (GAR) domain of Xenopus nucleolin facilitates in vitro nucleic acid binding and in vivo nucleolar localization.

Epitope-tagged Xenopus nucleolin was expressed in Escherichia coli cells and in Xenopus oocytes either as a full-length wild-type protein or as a truncation that lacked the distinctive carboxy glycine/arginine-rich (GAR) domain. Both full-length and truncated versions of nucleolin were tagged at their amino termini with five tandem human c-myc epitopes. Whether produced in E. coli or in Xenopus, epitope-tagged full-length nucleolin bound nucleic acid probes in in vitro filter binding assays. Conversely, the E. coli-expressed GAR truncation failed to bind the nucleic acid probes, whereas the Xenopus-expressed truncation maintained slight binding activity. Indirect immunofluorescence staining showed that myc-tagged full-length nucleolin properly localized to the dense fibrillar regions within the multiple nucleoli of Xenopus oocyte nuclei. The epitope-tagged GAR truncation also translocated to the oocyte nuclei, but it failed to efficiently localize to the nucleoli. Our results show that the carboxy GAR domain must be present for nucleolin to efficiently bind nucleic acids in vitro and to associate with nucleoli in vivo.

Amino Acid Sequence↗
PubMed2000

Molecular basis of sequence-specific recognition of pre-ribosomal RNA by nucleolin.

The structure of the 28 kDa complex of the first two RNA binding domains (RBDs) of nucleolin (RBD12) with an RNA stem-loop that includes the nucleolin recognition element UCCCGA in the loop was determined by NMR spectroscopy. The structure of nucleolin RBD12 with the nucleolin recognition element (NRE) reveals that the two RBDs bind on opposite sides of the RNA loop, forming a molecular clamp that brings the 5' and 3' ends of the recognition sequence close together and stabilizing the stem-loop. The specific interactions observed in the structure explain the sequence specificity for the NRE sequence. Binding studies of mutant proteins and analysis of conserved residues support the proposed interactions. The mode of interaction of the protein with the RNA and the location of the putative NRE sites suggest that nucleolin may function as an RNA chaperone to prevent improper folding of the nascent pre-rRNA.

Amino Acid Sequence↗
PubMed1992

Nucleolin forms a specific complex with a fragment of the viral (minus) strand of minute virus of mice DNA.

Nucleolin, a major nucleolar protein, forms a specific complex with the genome (a single-stranded DNA molecule of minus polarity) of parvovirus MVMp in vitro. By means of South-western blotting experiments, we mapped the binding site to a 222-nucleotide motif within the non-structural transcription unit, referred to as NUBE (nucleolin-binding element). The specificity of the interaction was confirmed by competitive gel retardation assays. DNaseI and nuclease S1 probing showed that NUBE folds into a secondary structure, in agreement with a computer-assisted conformational prediction. The whole NUBE may be necessary for the interaction with nucleolin, as suggested by the failure of NUBE subfragments to bind the protein and by the nuclease footprinting experiments. The present work extends the previously reported ability of nucleolin to form a specific complex with ribosomal RNA, to a defined DNA substrate. Considering the tropism of MVMp DNA replication for host cell nucleoli, these data raise the possibility that nucleolin may contribute to the regulation of the parvoviral life-cycle.

Base Sequence↗
PubMed2002

Identification of nucleolin and nucleophosmin as genotoxic stress-responsive RNA-binding proteins.

Genotoxic stress (DNA damage) can elicit multiple responses in mammalian cells, including the activation of numerous cascades of signal transduction that result in the activation of cellular genes involved in growth control, DNA repair and apoptosis. In an earlier report, we have shown that DNA-damaging agents can also induce the RNA-binding activity of several specific proteins that favor a double stem-loop RNA structure. Here we report the purification and identification of nucleophosmin (NPM) and nucleolin as two genotoxic stress-responsive RNA-binding proteins. UV radiation induces the protein expression levels and RNA-binding activity of NPM while nucleolin RNA-binding activity increases after UV or ionizing radiation exposure. Moreover, we have identified 40 mRNA ligands that are potentially regulated by nucleolin, several of which are stress-responsive transcripts. In addition, our data indicate that activation of nucleolin RNA-binding activity by genotoxic stress is mediated by stress-activated protein kinase p38. Our findings suggest that activation of the RNA-binding properties of nucleolin and NPM is part of the cellular response to genotoxic stress.

Amino Acid Sequence↗
PubMed1994

Identification of nucleolin as a binding protein for midkine (MK) and heparin-binding growth associated molecule (HB-GAM).

Midkine (MK) is a heparin-binding growth/differentiation factor with a molecular weight of 13 kDa, and is structurally unrelated to fibroblast growth factors (FGF). We studied MK-binding proteins in order to clarify the action mechanism of MK. A 100-kDa protein was identified in PYS-2, 3T3, and L cells as an MK-binding protein by a ligand blot experiment. This MK-binding protein was purified by affinity chromatography on an MK-agarose column followed by SDS polyacrylamide gel electrophoresis. Sequence determination of N-terminal 23 amino acid residues revealed that the MK-binding protein was nucleolin, a major nucleolar protein, which functions as a shuttle protein between the nucleus and cytoplasm and is located also on the cell surface. Heparin-binding growth associated molecule (HB-GAM), which has 50% sequence identity with MK, fused to maltose-binding protein also bound to nucleolin. On the other hand, basic FGF (bFGF) scarcely bound to nucleolin in the absence of heparin, while both MK and bFGF bound weakly to nucleolin in the presence of heparin. Nuclear localization of MK was shown in hemangioma cells by immunohistochemical staining. These findings supported the hypothesis that parts of the MK and HB-GAM are translocated to the nucleus after binding with nucleolin.

Amino Acid Sequence↗
PubMed1995

Induction of heat shock protein 70 and nucleolin and their intracellular distribution during early stage of liver regeneration.

Our previous work demonstrated a simultaneous induction of Hsp70 and nucleolin mRNA during the prereplicative stage of hepatocytes after partial hepatectomy [Ohmori, H. et al. (1990) Exp. Cell Res. 189, 227-232]. In the present study, changes of intracellular localization of these proteins were examined. Nucleolin, which mainly localized in the nucleus, increased with time and accumulated in the nucleolus around 12-18 h after partial hepatectomy. Hsp70 protein also increased slightly around 6-12 h after the operation. In accordance with this increase, immunohistochemical staining revealed that almost all nuclei of hepatocytes became Hsp70-positive, although Hsp70 was seen to be dispersed throughout the nucleoplasm and nucleolus at all times examined. Next, we isolated a cDNA clone of ribosomal protein (S-17) and examined its behavior. Induction of S-17 mRNA was observed to be essentially similar to that of nucleolin mRNA in regenerating rat liver, although inductions of histone H2A and H4 occurred at a later time, that is, in parallel with DNA synthesis. Furthermore, we observed a simultaneous induction of Hsp70 and nucleolin mRNA by serum-stimulation after serum-depletion in HeLa and IAR-20 (rat) culture cells. These results suggest that the induction of Hsp70, in addition to nucleolin, was not fortuitous but may be involved in the early events of liver regeneration.

Animals↗
PubMed1995

Nucleolin and fibrillarin expression in stimulated lymphocytes and differentiating HL-60 cells. A flow cytometric assay.

Nucleolin and fibrillarin are two histone-like major proteins in the nucleolus that were found to be overexpressed in proliferating cells. Using specific antibodies to either nucleolin or fibrillarin flow cytometric, measurements were carried out to demonstrate quantitative changes of these proteins during lymphocyte mitogenic activation and differentiation of HL-60 promyelocytic leukaemia cells. The expression of nucleolin increased during lymphocyte stimulation and decreased slowly but constantly in the course of differentiation of HL-60 cells. Expression of fibrillarin reached a maximum in the first cell cycle and then dropped to a basic level in stimulated lymphocytes. Compared to nucleolin, the level of fibrillarin decreased more rapidly and more extensively in differentiating HL-60 cells. The data support other observations that nucleolin is a stabile structural protein at the ribosomal genes while fibrillarin may have a more specific functional role in nucleologenesis and ribosome production.

Cell Differentiation↗
PubMed1989

Characterization of a 48-kDa nucleic-acid-binding fragment of nucleolin.

Nucleolin (C23 or 100 kDa) is an abundant single-stranded-nucleic-acid-binding nucleolar protein proposed to be involved in the early stages of ribosome assembly. A stable 48-kDa fragment of the protein was produced either by proteolytic activity present in nucleolar extracts or by added trypsin. The hydrodynamic and DNA-binding properties of the 48-kDa fragment were compared with the parent molecule. Protein sequencing indicated that the fragment begins at residue 282; amino acid composition of the fragment including 10-12 methylated arginine residues suggested that the fragment contains the entire COOH-terminal two-thirds of the protein. The 48-kDa fragment was more globular than nucleolin, as indicated by a lower frictional coefficient (1.3 vs. 2.0 for nucleolin) and a similar sedimentation coefficient (4.1-4.3S) in spite of the reduction in molecular mass. Although the 48-kDa fragment retained single-stranded-DNA-binding activity, the binding capacity and the ability to reassociate DNA were about fivefold and sixfold lower, respectively, than nucleolin. Similarly, tenfold higher concentrations of the 48-kDa fragment were required to form nucleoprotein aggregates. These results suggest that nucleolin contains a globular COOH-terminal domain for nucleic-acid binding and a NH2-terminal region which is involved in protein-protein interactions and modulating nucleic-acid-binding activity.

Amino Acid Sequence↗
PubMed2005

Cleavage of nucleolin and AgNOR proteins during apoptosis induced by anticancer drugs in human salivary gland cells.

BACKGROUND: To investigate the behavior of nuclear proteins in apoptosis induced by anticancer drugs in cultured human salivary gland (HSG) cells. METHODS: Dynamic alternations of nucleolin and argyrophilic nucleolar organizer region (AgNOR) proteins in anticancer drug-induced apoptosis of HSG cells and in a cell-free apoptotic system were examined using Western blot analysis and immunocytochemical method. RESULTS: The 110-kDa form of nucleolin and AgNOR protein decreased and the 80- and 95-kDa forms appeared during apoptosis in HSG cells and in a cell-free apoptotic system. In addition, the induction of DNA ladder formation coincided with the appearance of alternation of nucleolin and AgNOR proteins in a cell-free apoptosis. Nucleolin diffusely spread out into the nuclear material in the apoptotic body of HSG cells. CONCLUSIONS: The present results indicate that alternations of nucleolin and AgNOR proteins are associated with the induction of DNA fragmentation and the final active phase of apoptosis induced by anticancer drugs in malignant salivary gland cells.

Antineoplastic Agents↗
PubMed1991

Autoantibodies to nucleolin in systemic lupus erythematosus and other diseases.

The 110-kDa intracellular phosphoprotein (110K) described previously by this laboratory as a common IgM autoantigen in SLE and certain other systemic autoimmune disorders and viral infections is identified as nucleolin in the present investigation. Using rabbit antiserum to rat nucleolin as a probe, IgM autoantibody-reactive 110K co-migrated with human lymphocyte nucleolin in one- and two-dimensional immunoblots. Rabbit anti-nucleolin also specifically depleted autoreactive 110K from detergent lysates of human cells. Because nucleolin shares amino acid sequence similarity and/or forms dynamic particles with other prominent autoantigens, the present observation raises the possibility that the nucleolin/anti-nucleolin system may be of special significance for the development of humoral autoreactivity to nuclear Ag.

Antibody Specificity↗
PubMed1993

Insulin induces the phosphorylation of nucleolin. A possible mechanism of insulin-induced RNA efflux from nuclei.

Insulin induces the serine phosphorylation of the nucleolar protein nucleolin at subnanomolar concentrations in differentiated 3T3-442A cells. The stimulation is biphasic with phosphorylation reaching a maximum at 10 pM insulin and then declining to only 40% of basal levels at insulin concentrations of 1 microM. These changes are rapid, reaching half-maximal after 4 min and maximal after 15 min of incubation. The cell-permeable casein kinase II inhibitor 5,6-dichlorobenzimidazole-riboside prevents the insulin-stimulated phosphorylation of nucleolin suggesting that casein kinase II may mediate this effect of the hormone. Insulin-like growth factor 1 mimics the action of insulin on dephosphorylation of nucleolin at nanomolar concentrations suggesting that the latter effect may be mediated by insulin-like growth factor 1 receptors. Insulin treatment of 3T3-442A cells also results in a stimulation of RNA efflux from isolated, intact cell nuclei. The dose dependence of insulin-induced nucleolin phosphorylation and insulin-stimulated RNA efflux from intact cell nuclei are almost identical. Insulin induces an increase in the RNA efflux at subnanomolar concentrations in 3T3-442A adipocytes, while high (micromolar) concentrations of insulin inhibited the efflux of RNA. These data indicate that insulin regulates the phosphorylation/dephosphorylation of nucleolin, possibly via stimulation of casein kinase II, and this may play a role in regulation of the RNA efflux from nuclei.

3T3 Cells↗
PubMed1993

Requirements for nuclear translocation and nucleolar accumulation of nucleolin of Xenopus laevis.

The intracellular localization of germinal vesicle proteins is regulated during early Xenopus development. Here we have analyzed the determinants that control the localization of nucleolin, a nucleolar protein of vertebrates. Our immunological analyses and isolation of cDNAs have revealed the presence of a second nucleolin gene in addition to that previously identified. The two nucleolin polypeptides of 95 kDa and 90 kDa molecular mass are both expressed in oocytes and are found predominantly in the nucleoli. During oocyte maturation, both polypeptides are hyperphosphorylated and found distributed throughout the cytoplasm. Hyperphosphorylated forms and cytoplasmic localization of nucleolin both prevail in early embryos up to the midblastula transition. Subsequently, the proteins are found in the nuclei. Accumulation in nucleoli is only detected at later stages, beginning with gastrulation. Thus nucleolar localization is temporally uncoupled from nuclear translocation. Consistent with these observations, our molecular analyses have revealed that the nuclear location signal which is present in nucleolin and which is sufficient for nuclear location, is not sufficient for nucleolar accumulation. Nucleolar accumulation requires, in addition to the nuclear location signal, the presence of the RNA binding domains and of an RG-rich domain, which is also thought to interact with RNA.

Amino Acid Sequence↗
PubMed1993

The amino terminus of mammalian nucleolin specifically recognizes SV40 T-antigen type nuclear localization sequences.

Nucleolin is a major nucleolar protein in mammalian cells that is thought to be involved in ribosome biogenesis. The discovery that nucleolin shuttles between the cytoplasm and the nucleus raises the possibility that it is also involved in transporting ribosomal or nuclear proteins to the nucleus. The three structural domains of nucleolin bear a striking resemblance to the domains of a previously identified yeast protein NSR1, although the two proteins do not share a high degree of sequence similarity. NSR1 specifically recognizes the nuclear localization sequence (NLS) of both the simian virus large T antigen (SV40 T-antigen) and the yeast histone H2B by ligand blot analysis, and is a candidate for a receptor involved in the initial stages of nuclear transport. We report here that nucleolin, either purified from Chinese hamster ovary (CHO) cells or expressed in yeast, also specifically recognizes the wild-type, but not a mutant, histone H2B nuclear localization sequence by ligand blot analysis. The NLS recognition site is located within the N-terminal domain of both proteins. In showing that nucleolin, a protein that moves between the cytoplasm and the nucleus, also has the ability to interact with nuclear localization signals, our data support the idea that shuttling nucleolar proteins play a role in nuclear transport.

Amino Acid Sequence↗
PubMed1997

Amount of the two major Ag-NOR proteins, nucleolin, and protein B23 is cell-cycle dependent.

To know the biological basis allowing the use of Ag-NOR protein expression as proliferation marker in human malignancies, the relationship between cell cycle and amount of Ag-NOR protein was analyzed. The quantification of the two major Ag-NOR proteins, nucleolin and protein B23, was performed in exponentially growing, serum-deprived, and cell-cycle stimulated cells. Expression of nucleolin was low in serum-deprived cells and increased mostly in S phase during cell-cycle stimulation. Conversely, expression of protein B23 was slightly repressed in serum-deprived cells, and increased progressively until G2 phase during cell-cycle stimulation. The accumulation of nucleolin and protein B23 in G2 compared to G1 was demonstrated using sorted phase-specific cells. In G0, cells sorted according to their very low RNA content, and the amount of Ag-NOR proteins was half of that found in G1 cells, nucleolin being only weakly detectable. Therefore, the expression of nucleolin increased between G0-G1 and G1-S phases. These data support the hypothesis that quantification of Ag-NOR proteins is an estimation of the percentage of cells in each cell cycle phase because their amount is high in S-G2 and low in G1 phases.

Animals↗
PubMed1985

Cloning of cDNA encoding a 100 kDa nucleolar protein (nucleoline) of Chinese hamster ovary cells.

Nucleoline (100 kDa) is the major nucleolar protein in exponentially growing cells that behaves like a nucleolar organizer protein and plays a key role in rDNA transcription and prerRNA processing. We reported the isolation of 5 cDNA clones by probing a cDNA library, constructed in the expression vector lambda gt11, with a polyclonal serum raised against nucleoline. A new immunoassay, using hybrid proteins (beta gal-cDNA encoded protein) was developed to establish that the isolated cDNAs encoded parts of nucleoline. A further confirmation resulted from the sequence comparison between the cDNA encoded peptide and a 42 aa peptide isolated from rat nucleoline (1). The 5 cDNAs overlapped extensively and covered more than 90% of a full length cDNA. By probing a Northern blot with the 100 kDa cDNA, a 2650 nucleotide polyA+ RNA was detected that contained just enough information to code for nucleoline.

Amino Acid Sequence↗
PubMed1999

Linkage of a nucleolin-related protein and casein kinase II with the detergent-stable photoreceptor cytoskeleton.

Vertebrate photoreceptors are highly polarized sensory neurons with a complex microtubule and actin-based cytoskeletal organization. In the present study, we have used a detergent-extracted cytokeleton preparation from bovine photoreceptors to test the hypothesis that protein kinases and their substrates co-purify with the photoreceptor cytoskeleton. We incubated the cytoskeletal preparation in the presence of [gamma-32P]ATP. Following SDS-PAGE and autoradiography, we found two principal phosphoproteins with apparent molecular weights of 55 kDa (pp55) and 112 kDa (pp112). We have additionally identified the kinase responsible for phosphorylation of pp112 (and possibly pp55) as a casein kinase II-like enzyme. pp55 was identified as beta-tubulin based on Western blotting and its position on two-dimensional gels. Microsequencing revealed that 16 of the first 17 amino acids of pp112 were identical to human nucleolin, a nuclear protein. Western blotting, mobility in SDS PAGE and in two-dimensional gels, predominant localization within the nucleus, and phosphorylation by a casein kinase II all support the conclusion that pp112 is a nucleolin-related protein. Immunocytochemistry revealed a significant extranuclear pool of nucleolin-immunoreactivity within the cell bodies of photoreceptors. These findings suggest an important extranuclear role for nucleolin or a related protein in photoreceptors.

Amino Acid Sequence↗