Alterations in immunolocalization of the phosphoprotein B23 in HeLa cells during serum starvation

Bright nucleolar immunofluorescence was observed in HeLa S3 cells by immunostaining with a monoclonal antibody to the nucleolar phosphoprotein B23 (MW 37 kD/pI 5.1). After 48 h of incubation in a serum-free medium, the nucleolar fluorescence was diminished and a general nuclear immunofluorescence was observed. This change in localization of fluorescence indicated that protein B23 had migrated out of the nucleoli. No gross morphological change in nucleoli was observed by light microscopy and the immunolocalization of another nucleolar phosphoprotein, C23, was unaffected by serum deprivation. Relocation of protein B23 in nucleoli was observed after refeeding with serum-containing medium. This re-entry process was not observed after treatment with actinomycin D (50 ng/ml-5 micrograms/ml), but the process was unaffected by cycloheximide (0.2 mM). Quantitation of protein B23 in the nucleoli of the control (fed) or starved HeLa cells was done by ELISA immunoassay. A marked decrease in the amount of protein B23 occurred in the nucleoli of the starved cells (11.8 micrograms B23/mgDNA) as compared with the control nucleoli (20.8 micrograms B23/mgDNA). The amount of protein B23 in the nucleoplasm (excluding nucleoli) was 70% higher in the starved cells. Protein B23 was analysed by one- and two-dimensional PAGE. Three components of protein B23 with slightly different molecular weights and pIs (37 kD/5.1, 35 kD/5.1 and 35 kD/5.3) were observed in nucleoli. The lower molecular weight components were predominantly found in the nucleoplasm.     

Further evidence that phosphoprotein C23 (110 kD/pI 5.1) is the nucleolar silver staining protein

Previously we demonstrated a similar distribution between nucleolar organizing region-(NOR)-specific silver staining and localization of nucleolar phosphoprotein C23 (MW 110 kD/pI 5.1) [1, 2]. We now report that under fixation conditions which allow for antibody binding and subsequent silver staining, monoclonal antibody against protein C23 blocks NOR silver staining as well as silver staining in interphase nucleoli. Monoclonal antibody against nucleolar phosphoprotein B23 (MW 37 kD/pI 5.1) did not block silver staining in either NORs or interphase nucleoli. These, along with earlier observations, provide evidence that nucleolar phosphoprotein C23 is the major silver staining protein of the nucleolus and that it is directly or indirectly associated with rDNA.     

Epitope distribution and immunochemical characterization of nucleolar phosphoprotein C23 using ten monoclonal antibodies

Summary Extractable nucleolar proteins from HeLa cells were used as a source of antigen to immunize mice for monoclonal antibody (MAb) production. Ten of the resulting MAbs shown to identify nucleolar phosphoprotein (110 kD/pI 5.5) were purified and used in immunochemical studies to further characterize protein C23. All ten MAbs showed nucleolar localization by indirect immunofluorescence; one antibody (FR2) also showed some nucleoplasmic localization that was attributed to a shared epitope between protein C23 and a 72 kD nuclear/nucleolar antigen. Reciprocal antibody cross blocking studies indicated that the ten MAbs identified nine distinct epitopes on protein C23. Interestingly, seven of the nine epitopes were shown by immunofluorescence and competitive ELISA studies to be species related. Immunostained patterns of exponentially growing HeLa cells suggest that protein C23 exists in vivo solely as a 110 kD peptide. However, protein C23 was subject to rapid degradation into a number of proteolytic fragments upon extraction or storage of isolated nucleoli. The failure to find protein C23 related peptides with molecular sizes less than 110 kD in exponentially growing cells and the lack of cytoplasmic localization of any of the ten MAbs suggests that protein C23 is not a prepro-protein processed in vivo to form ribosomal proteins as previously suggested (1).     

Translocation of nucleolar phosphoprotein B23 (37 kDa/pI 5.1) induced by selective inhibitors of ribosome synthesis

To elucidate the possible role of nucleolar phosphoprotein B23 in ribosome synthesis, drugs which inhibit the processing of ribosomal RNA were employed. After treatment with actinomycin D, toyocamycin or high doses of alpha-amanitin, a uniform nucleoplasmic fluorescence was observed. Low doses of alpha-amanitin and the protein synthesis inhibitor puromycin and cycloheximide had no effect on protein B23 translocation. By ELISA immunoassay, there was a 60% decrease in the amount of protein B23 in the nucleoli of the actinomycin D-treated cells as compared with the control nucleoli. Conversely, the amount of protein B23 in the nucleoplasm (excluding nucleoli) was 3-fold higher in the actinomycin D-treated cells. Preribosomal ribonucleoprotein particles (pre-rRNPs) were extracted from isolated nucleoli of Novikoff hepatoma ascites cells and fractionated on sucrose density gradients. Protein B23 was found co-localized with the pre-rRNPs as determined by ELISA assays which agrees with previous studies. The proteins in these 80 S and 55 S pre-ribosomal ribonucleoprotein particles were fractionated by 10% gel electrophoresis. Immunoblots showed protein B23 was present in both pre-rRNPs.     

Translocation of nucleolar phosphoprotein B23 ( ) induced by selective inhibitors of ribosome synthesis

To elucidate the possible role of nucleolar phosphoprotein B23 in ribosome synthesis, drugs which inhibit the processing of ribosomal RNA were employed. After treatment with actinomycin D, toyocamycin or high doses of α-amanitin, a uniform nucleoplasmic fluorescence was observed. Low doses of α-amanitin and the protein synthesis inhibitor puromycin and cycloheximide had no effect on protein B23 translocation. By ELISA immunoassay, there was a 60% decrease in the amount of protein B23 in the nucleoli of the actinomycin D-treated cells as compared with the control nucleoli. Conversely, the amount of protein B23 in the nucleoplasm (excluding nucleoli) was 3-fold higher in the actinomycin D-treated cells. Preribosomal ribunucleoprotein particles (pre-rRNPs) were extracted from isolated nucleoli of Novikoff hepatoma ascites cells and fractionated on sucrose density gradients. Protein B23 was found co-localized with the pre-rRNPs as determined by ELISA assays which agrees with previous studies. The proteins in these 80 S and 55 S pre-ribosomal ribonucleoprotein particles were fractionated by 10% gel electrophoresis. Immunoblots showed protein B23 was present in both pre-rRNPs.     

Translocation of nucleolar phosphoprotein B23 (37kDapI 5.1) induced by selective inhibitors of ribosome synthesis

To elucidate the possible role of nucleolar phosphoprotein B23 in ribosome synthesis, drugs which inhibit the processing of ribosomal RNA were employed. After treatment with actinomycin D, toyocamycin or high doses of α-amanitin, a uniform nucleoplasmic fluorescence was observed. Low doses of α-amanitin and the protein synthesis inhibitor puromycin and cycloheximide had no effect on protein B23 translocation. By ELISA immunoassay, there was a 60% decrease in the amount of protein B23 in the nucleoli of the actinomycin D-treated cells as compared with the control nucleoli. Conversely, the amount of protein B23 in the nucleoplasm (excluding nucleoli) was 3-fold higher in the actinomycin D-treated cells. Preribosomal ribunucleoprotein particles (pre-rRNPs) were extracted from isolated nucleoli of Novikoff hepatoma ascites cells and fractionated on sucrose density gradients. Protein B23 was found co-localized with the pre-rRNPs as determined by ELISA assays which agrees with previous studies. The proteins in these 80 S and 55 S pre-ribosomal ribonucleoprotein particles were fractionated by 10% gel electrophoresis. Immunoblots showed protein B23 was present in both pre-rRNPs.     

The N-terminal half of NPM dissociates from nucleoli of HeLa cells after anticancer drug treatments.

NPM (nucleophosmin/B23) is a nucleolar phosphoprotein abundant in tumor cells. It dissociates from nucleoli of cells after treatments with various anticancer drugs. To determine the domain of NPM responsible for nucleolar binding, the N- and C-terminal halves of NPM were fused to GFP (green fluorescent protein) and introduced into HeLa cells. The N-terminal half (aa 1-150) of NPM (GFP-NPM(N)) was found localized in the nucleoli. A stable transformant of GFP-NPM(N) in HeLa cells was prepared and tested for association to nucleoli after anticancer drug treatments. GFP-NPM(N) dissociates from nucleoli after treatments with daunomycin, actinomycin D, camptothecin, and toyocamycin. The dissociation is time- and dose-dependent, and correlates with the cytotoxicity induced by the drugs. These results indicate that a stable transformant of GFP-NPM(N) in HeLa cells may be useful for the screening of anticancer drugs.     

Double immunolocalization of major nucleolar proteins, fibrillarin and B23, in dividing mammalian cultured cells.

Using specific autoimmune sera to the nucleolar protein fibrillarin and monoclonal antibodies to B23/nucleophosmin, we localized early and late nucleolar rRNA-processing factors in cycling human HeLa and pig PK cells. It was shown that, at the electron microscopic level, fibrillarin was located over the nucleolar fibrillar compartment, but was absent in the fibrillar centres. During mitosis, fibrillarin was located within the same domains as B23, namely, the cytoplasm, the perichromosomal layer, prenucleolar bodies, and the nucleolar cytoplasmic derivatives, but the kinetics of the two proteins during mitosis was essentially different. Thus, fibrillarin dissociated from the nucleolar remnant at prophase of mitosis or following actinomycin D treatments after B23, but was found to be more prominent within the perichromosomal layer at metaphase, and earlier migrated to the reassembled nucleoli at telophase. In contrast to B23, fibrillarin was found to be resistant to the treatment with 2 M NaCl.     

Nucleolar translocalization of GRA10 of Toxoplasma gondii transfectionally expressed in HeLa cells

Toxoplasma gondii GRA10 expressed as a GFP-GRA10 fusion protein in HeLa cells moved to the nucleoli within the nucleus rapidly and entirely. GRA10 was concentrated specifically in the dense fibrillar component of the nucleolus morphologically by the overlap of GFP-GRA10 transfection image with IFA images by monoclonal antibodies against GRA10 (Tg378), B23 (nucleophosmin) and C23 (nucleolin). The nucleolar translocalization of GRA10 was caused by a putative nucleolar localizing sequence (NoLS) of GRA10. Interaction of GRA10 with TATA-binding protein associated factor 1B (TAF1B) in the yeast two-hybrid technique was confirmed by GST pull-down assay and immunoprecipitation assay. GRA10 and TAF1B were also co-localized in the nucleolus after co-transfection. The nucleolar condensation of GRA10 was affected by actinomycin D. Expressed GFP-GRA10 was evenly distributed over the nucleoplasm and the nucleolar locations remained as hollows in the nucleoplasm under a low dose of actinomycin D. Nucleolar localizing and interacting of GRA10 with TAF1B suggested the participation of GRA10 in rRNA synthesis of host cells to favor the parasitism of T. gondii.     

Analysis of the proliferative activity of a cell using new monoclonal antibodies to nucleolar protein B23/nucleophosmin

Nowadays, antinucleolar antibodies are widely used for exploration of the nucleolar organization and molecular mechanisms of ribosome production. Here we have described a new monoclonal antibody against the major nucleolar phosphoprotein B23/nucleophosmin (3C9) that is involved in the terminal stages of ribosome production. It is used to examine immunocytochemical peculiarities of the nucleolus in terms of the cell proliferative status and also during mitosis. In human peripheral blood lymphocytes, activated for proliferation with phytohaemagglutinin (PHA), PHA stimulation of lymphocytes was shown to result in accumulation of protein B23 in augmentative nucleoli. A comparative study of 3C9 and two other anti-B23 antibodies 20B2 and anti-B23 by Western blots and indirect immunofluorescence favored the idea that 3C9 cross-reacted with the major isoform of B23, B23.1, that have an apparent molecular weight of 40 kDa.     

Characterization and cellular localization of nucleophosmin/B23 in HeLa cells treated with selected cytotoxic agents (studies of B23-translocation mechanism).

Previous studies indicated that nucleophosmin/B23, an abundant nucleolar phosphoprotein, accumulated in the nucleoplasm (B23-translocation) of cells after exposure to selected cytotoxic drugs. Attempts were made to understand the B23-translocation mechanism. This paper reports that: (1) B23-translocation is a reversible process. Upon removal of camptothecin, which induced B23-translocation in HeLa cells, nucleophosmin/B23 relocalized into nucleoli within 2 h. Relocation occurs in the presence of cycloheximide which inhibits new protein synthesis. There is no reduction or degradation of nucleophosmin/B23 detected during drug treatments. Nucleophosmin/B23 has a half-life of 18-20 h. Taken together, these results indicate that B23-translocation is a reversible process. Drug treatment causes redistribution of nucleophosmin/B23 in nucleoplasm. (2) Inhibition of RNA synthesis does not cause the B23-translocation. Over 80% of RNA synthesis was inhibited in HeLa cells by treatment with actinomycin D, camptothecin, and methotrexate. While actinomycin D and camptothecin cause B23-translocation in all cells, 40% of methotrexate-treated cells remain untranslocated. (3) There is no significant change of phosphorylation in nucleophosmin/B23 during drug treatment. An identical oligomeric cross-linkage pattern was obtained in drug-treated cells. (4) HeLa cells treated with B23-translocation effective drugs have small and round nucleoli while control cells have large and irregular-shaped nucleoli.     

Mechanism of cholesteryl ester transfer protein inhibition by a neutralizing monoclonal antibody and mapping of the monoclonal antibody epitope

The plasma cholesteryl ester transfer protein (CETP, Mr 74,000) has a binding site for neutral lipid which can readily equilibrate with lipoprotein cholesteryl esters or triglycerides. Recently, a monoclonal antibody (TP2) was obtained which neutralizes the cholesteryl ester (CE) and triglyceride (TG) transfer activities of the CETP. In this report, the epitope of the inhibitory monoclonal antibody has been localized to a hydrophobic 26-amino acid sequence at the COOH terminus of CETP. The Fab fragments of TP2 caused partial (50%) inhibition of CE transfer and complete inhibition of TG transfer by the CETP. Similarly, the Fab fragments inhibited (37%) the binding of CE to the CETP and abolished the binding of TG to the CETP. Surprisingly, the TP2 Fab was also found to enhance the binding of CETP to plasma lipoproteins and to phospholipid vesicles. In conclusion, the TP2 monoclonal antibody inhibits lipid transfer by blocking the uptake of lipid by CETP. The COOH-terminal epitope may be in or near the neutral lipid binding site. Occupancy of this site by TP2 Fab fragments or by neutral lipid may result in a conformational change of CETP causing enhanced binding to lipoproteins or vesicles.     

Overexpression of the nucleolar protein nucleophosmin/B23 in collagen lattice-cultured fibroblasts: Potential role in the control of protein synthesis

Fibroblasts cultivated in tridimensional collagen lattices exhibit a downregulation of protein synthesis, related to decreased ribosomal RNA (rRNA) content and half life, when compared to monolayer cultivated cells. The involvement in this process of nucleophosmin/B23, a nucleolar phosphoprotein with ribonuclease properties, was checked. We compared production of nucleophosmin/B23 in monolayer and collagen lattice cultured fibroblasts. A significant increase of nucleophosmin/B23 mRNA levels was noticed in lattice-cultured fibroblasts vs monolayers (+154%, p < 0.05). A concomitant enhancement of nucleolar nucleophosmin/B23 content was found (+112%, p < 0.001). Simultaneously, ribonuclease activity contained in nucleolar extracts from collagen lattice-cultured fibroblasts was significantly increased (+54%, p < 0.01). These data demonstrate that extracellular collagen matrix induces the overexpression of nucleophosmin/B23, and suggest that the regulation of protein syntheses in collagen lattice cultures may be explained, at least partly, by an increased degradation of neosynthesized rRNAs dependent on nucleophosmin.     

The RNA binding activity of a ribosome biogenesis factor,nucleophosmin/B23, is modulated by phosphorylation with a cell cycle-dependent kinase and by association with its subtype

Nucleophosmin/B23 is a nucleolar phosphoprotein. It has been shown that B23 binds to nucleic acids, digests RNA, and is localized in nucleolar granular components from which preribosomal particles are transported to cytoplasm. The intracellular localization of B23 is significantly changed during the cell cycle. Here, we have examined the cellular localization of B23 proteins and the effect of mitotic phosphorylation of B23.1 on its RNA binding activity. Two splicing variants of B23 proteins, termed B23.1 and B23.2, were complexed both in vivo and in vitro. The RNA binding activity of B23.1 was impaired by hetero-oligomer formation with B23.2. Both subtypes of B23 proteins were phosphorylated during mitosis by cyclin B/cdc2. The RNA binding activity of B23.1 was repressed through cyclin B/cdc2-mediated phosphorylation at specific sites in B23. Thus, the RNA binding activity of B23.1 is stringently modulated by its phosphorylation and subtype association. Interphase B23.1 was mainly localized in nucleoli, whereas B23.2 and mitotic B23.1, those of which were incapable of binding to RNA, were dispersed throughout the nucleoplasm and cytoplasm, respectively. These results suggest that nucleolar localization of B23.1 is mediated by its ability to associate with RNA.     

Nucleolar protein B23 interacts with Japanese encephalitis virus core protein and participates in viral replication

Japanese encephalitis virus (JEV) core protein is detected not only in the cytoplasm but also in the nucleoli of infected cells. We previously showed that a mutant JEV lacking the nucleolar localization of the core protein impaired viral replication in mammalian cells. In this study, we identified a nucleolar phosphoprotein B23 as a protein binding with the core protein of JEV but not with that of dengue virus. The region binding with JEV core protein was mapped to amino acid residues 38 to 77 of B23. Upon JEV infection, some fraction of B23 was translocated from the nucleoli to the cytoplasm, and cytoplasmic B23 was colocalized with the core protein of wild-type JEV but not with that of the mutant JEV. Furthermore, overexpression of dominant negatives of B23 reduced JEV replication. These results suggest that B23 plays an important role in the intracellular localization of the core protein and replication of JEV.     

Identification of numatrin, the nuclear matrix protein associated with induction of mitogenesis, as the nucleolar protein B23. Implication for the role of the nucleolus in early transduction of mitogenic signals

We have previously described and characterized a nuclear protein at 40 kDa/pI 5 termed "numatrin" which is tightly bound to the nuclear matrix. We demonstrated that a rapid increase in the synthesis of numatrin at early G1 phase is closely correlated with receptor-mediated induction of cellular proliferation by various mitogens and that elevated amounts of numatrin are found in tumor cells, suggesting that numatrin may have an important role in regulation of cellular growth in normal and malignant cells. Further experiments were undertaken to compare the biochemical characteristics of numatrin to those of other known proteins that are associated with cellular mitogenesis. Comparison of the electrophoretic mobility of numatrin with the proliferation cell nuclear antigen/cyclin showed that these proteins are not identical. However, numatrin had an identical electrophoretic migration on two-dimensional gel electrophoresis to that of a previously described nucleolar protein B23. The tryptic digest peptide map of 125I-labeled B23 was identical to that of numatrin on two-dimensional thin layer electrophoresis/chromatography. Labeling of cells with 32P further showed that numatrin is a major phosphoprotein as previously reported for protein B23. Using the protocol for purification of B23, we purified numatrin from nucleoli of HL-60 cells and produced two polyclonal antibodies (303 and 339) to this protein. We further show that numatrin is recognized by anti-B23 monoclonal antibody as well as by polyclonal antibodies 303 and 339 in enzyme-linked immunosorbent assay. Conversely, these anti-numatrin polyclonal antibodies cross-react with protein B23 as shown in immunoblot analysis. These results, taken collectively, prove that numatrin is identical to the nucleolar protein B23 and thus suggest that protein B23 and events which occur at the nucleolus might have an important role in early transduction of mitogenic signals at the G1 phase of the cell cycle.     

Nucleolar localization of protein B23 (375.1) by immunocytochemical techniques

Highly acidic phosphoprotein B23 ($\text{37}\text{5.1}\text{;}\text{M.W. x}\text{10}{}^3\text{/}\text{pI}$) which is in preribosomal RNP particles in nucleoli of Novikoff hepatoma cells (1) was found to be one of the two major silver staining nucleolar proteins (2). An improved isolation method was developed for protein B23 which included 4 M urea/3 M LiCl extraction of nucleoli, dialysis of the extract against 4 M urea/20 mM Tris-malate/pH 5.5 and DEAE-cellulose chromatography. For studies on cellular localization of this protein, highly purified protein B23 was used to produce anti- B23 antibodies in rabbits. The specificity of the anti- B23 antibodies was demonstrated by formation of immunoprecipitin bands with the purified antigen and crude nucleolar extracts from Novikoff hepatoma cells. With the indirect peroxidase immunostaining method, a specific localization of protein B23 was demonstrated in the nucleoli of normal rat liver, thioacetamide-treated rat liver and Novikoff hepatoma cells.     

Protein B23 is an important human factor for the nucleolar localization of the human immunodeficiency virus protein Tat.

Nucleolar shuttle protein B23 was found to bind to human immunodeficiency virus protein Tat, and this binding required the nucleolar localization motif of Tat. A fusion protein containing the B23 binding domain and beta-galactosidase caused mislocalization of Tat to the cytoplasm and inhibited the transactivation activity of Tat. These data suggest that B23 is a human factor necessary for the nucleolar localization of Tat.     

Cytoplasmic microinjection of immunoglobulin Gs recognizing RNA helices inhibits human cell growth

We report here that nucleolar and cytoplasmic RNA in mammalian cells is recognized specifically by both experimentally induced monoclonal IgG unique for left-handed Z-RNA and by autoimmune mouse monoclonal IgG specific for ribosomal RNA. Nucleolar Z-RNA synthesis, like nucleolar ribosomal RNA synthesis, is inhibited by actinomycin D treatment and dimethylsulfoxide-induced differentiation. Immune anti-Z-RNA IgGs microinjected into living nuclei bind nucleolar RNA, and these complexes appear to be removed from the nucleus within minutes. Cytoplasmically microinjected monoclonal or polyclonal anti-Z-RNA IgGs specifically bind cytoplasmic RNA and inhibit cell multiplication. Microinjection of antibodies directed against double-stranded RNAs. Elevated ionic conditions, which in energy-minimized models can cause the walls of the groove in Z-RNA (but not Z-DNA) to approach each other and close, also prevent antibody binding to specific synthetic or cellular Z-RNA determinants. Our antibodies binding unique Z-RNA structures probably recognize antigens determined by the exposed 2'-OH ribose sugar-phosphate groups.