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Results for “Nucleolin”

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At least 19 recordsLinked to original sources

PubMed2002

Stress-dependent nucleolin mobilization mediated by p53-nucleolin complex formation.

We recently discovered that heat shock causes nucleolin to relocalize from the nucleolus to the nucleoplasm, whereupon it binds replication protein A and inhibits DNA replication initiation. We report that nucleolin mobilization also occurs following exposure to ionizing radiation (IR) and treatment with camptothecin. Mobilization was selective in that another nucleolar marker, upstream binding factor, did not relocalize in response to IR. Nucleolin relocalization was dependent on p53 and stress, the latter initially stimulating nucleolin-p53 complex formation. Nucleolin relocalization and complex formation in vivo were independent of p53 transactivation but required the p53 C-terminal regulatory domain. Nucleolin and p53 also interact directly in vitro, with a similar requirement for p53 domains. These data indicate a novel p53-dependent mechanism in which cell stress mobilizes nucleolin for transient replication inhibition and DNA repair.

Active Transport, Cell Nucleus↗

PubMed2003

Stability of Nucleolin protein as the basis for the differential expression of Nucleolin mRNA and protein during serum starvation.

Nucleolin is a nucleolar phosphoprotein that plays a direct role in ribosome biogenesis. Our aim was to determine how its activity as a growth-promoting factor is coordinated with, if not regulated by, the cell cycle machinery. In serum starting and then rescuing these cells with serum, we found that the protein level did not drop in the same way that the mRNA level did. In addition, although the mRNA level rises during the immediate period during serum rescue, the protein level remained the same. We found that the protein level was maintained after serum starvation as a result of high stability. There was no selective enhanced translation of the remaining amount of Nucleolin mRNA. With regard to the constancy in protein level despite the rise in mRNA level during serum rescue, there is no concomitant degradation of newly synthesized or old protein and synthesis of new protein. Because Nucleolin has been documented to bind mRNA, APP mRNA being one among them, we propose a autoregulatory model in which Nucleolin regulates the translation of Nucleolin mRNA, such that during a period of excess protein, translation is inhibited through direct binding of Nucleolin protein to its mRNA.

Culture Media, Serum-Free↗

PubMed2003

[Protein phosphatases and nucleolin in osteoblastic cells: cleavage of nucleolin in apoptotic cells].

Protein phosphorylation and dephosphorylation has been recognized as a key mechanism in cell proliferation, differentiation, and apoptosis in various tissues. Okadaic acid is a potent inhibitor of protein phosphatase type 1 (PP1) and type 2A and induces apoptosis in human osteoblastic Saos-2 and MG63 cells. Nucleolin is an abundantly expressed nucleolar phosphoprotein and is located mainly in the nucleolus. The staining pattern of nucleolin in Saos-2 and MG63 cells is similar to that of PP1 delta. Nucleolin was demonstrated to bind to PP1 delta in nucleolus by using immunocytochemical and immunoprecipitation methods. AgNORs and nucleolin, visible as dots in the nucleus of the control cells, disappeared from the apoptotic nuclei. A major band, 110 kDa, was detected in the proteins obtained from the control cells. The level of the 110 kDa protein decreased in the apoptotic cells, whereas an additional band, 80 kDa, appeared and the level of this protein increased in the proteins prepared from okadaic acid-induced apoptotic cells. Our results indicate that PP1 delta directly binds to nucleolin in the nucleolus and that nucleolin is cleaved during apoptosis.

Apoptosis↗

PubMed1998

Inhibition of nucleolar protein nucleolin by electroporation with anti-nucleolin antibodies results in an increase of the nucleolar size.

Electroporation of exponentially growing human larynx epidermoid carcinoma cells (HEp-2) with a serum against nucleolin, one of the most abundant non-histone nuclear proteins, has shown, 24 h after electroporation, a significant increase in the size of the nucleolus of these cells compared with normal HEp-2 cells (non-electroporated) and electroporated HEp-2 cells in the absence of anti-nucleolin serum (P < 0.01). Image analysis evaluation of the different nucleolar components proved a major contribution of the dense fibrillar component to the total nucleolar size in cells electroporated with anti-nucleolin antibodies, more than that corresponding to the dense fibrillar component in cells from any of the control groups (P < 0.01), indicating that the reported increase in nucleolar size was due to a marked enlargement of the dense fibrillar regions. These results, in agreement with previous biochemical and molecular biology studies, suggest a pivotal role for nucleolin in pre-rRNA processing and constitute morphological evidence supporting this role. Following nucleolin inhibition, impaired pre-rRNA processing might result in an accumulation of this molecular species in the dense fibrillar component of the nucleolus, where pre-rRNA is first present.

Antibodies↗

PubMed1991

Increased stability of nucleolin in proliferating cells by inhibition of its self-cleaving activity.

Nucleolin is the major nucleolar phosphoprotein of exponentially growing eukaryotic cells and is presumably involved in pre-rRNA transcription and ribosome biogenesis. Monoclonal antibodies against nucleolin were selected by a differential dot-immunobinding assay. Nucleolin expression during T lymphocyte activation was monitored by the specific antibody. Results showed that nucleolin fluctuated in parallel to DNA synthesis. The intact 105-kDa nucleolin molecule was the major species in actively dividing cells, whereas the degraded forms were relatively abundant in nondividing cells. These results imply that stability of nucleolin molecule is cell proliferation-dependent. When affinity purified nucleolin containing undetectable contaminants was incubated at 37 degrees C, the majority of 105-kDa nucleolin was cleaved by 6 h and completely degraded within 24 h. This purified nucleolin was further separated from possible copurified protease, if any, on a reducing sodium dodecyl sulfate-polyacrylamide gel. After renaturation, the 105-kDa nucleolin immobilized in the gel was also cleaved at 37 degrees C. These data have confirmed that nucleolin protein autocatalyzes its own degradation. The self-cleaving activity of nucleolin was inhibited by nuclear extracts prepared from proliferating cells. Apparently, a proteolytic inhibitor(s) in the nuclei of proliferating cells stabilized the nucleolin molecule. It provides an unique regulatory mechanism for nucleolin expression.

Antibodies, Monoclonal↗

PubMed1993

The self-cleaving activity of nucleolin determines its molecular dynamics in relation to cell proliferation.

Nucleolin (105 kDa) is the most abundant nucleolar phosphoprotein in exponentially growing cells. We have demonstrated that nucleolin has an intrinsic protease activity for autodegradation. This self-cleaving activity is markedly decreased when cells enter the proliferative stage, resulting in significant stabilization of the nucleolin molecule. The fusion protein derived from a partial nucleolin cDNA clone, which encodes the C-terminal two-thirds of nucleolin, maintained the self-cleaving activity similar to that of the nucleolin purified from nonproliferating cells. The enzymatic domain is therefore located within this region. An exogenous peptide, highly negatively charged, exerted an inhibitory effect, indicating that the enzymatic activity can be regulated. The cleavage kinetics of nucleolin isolated from proliferating cells was apparently concentration independent, suggesting that the autoproteolytic reaction of nucleolin in cells at dividing stage is an intramolecular event. There were many cleavage fragments, which were clustered at four major sites, with apparent molecular weights of about 100, 70, 60, and 50 kDa. These results imply that the nucleolin molecule has repeated motifs that serve as the cleavage sites. Since the detailed electrophoresis patterns, including location and intensity, of the in vitro self-cleaved products of nucleolin derived from nonproliferating and proliferating cells were not quite the same, preferential usage of the particular cutting sites according to cell stage was suggested. It is possible that post-translational modification of nucleolin in proliferating cells may cause these variations.

Amino Acid Sequence↗

PubMed2000

The cell-surface-expressed nucleolin is associated with the actin cytoskeleton.

Nucleolin is a RNA- and protein-binding multifunctional protein. Mainly characterized as a nucleolar protein, nucleolin is continuously expressed on the surface of different types of cells along with its intracellular pool within the nucleus and cytoplasm. By confocal and electron microscopy using specific antibodies against nucleolin, we show that cytoplasmic nucleolin is found in small vesicles that appear to translocate nucleolin to the cell surface. Translocation of nucleolin is markedly reduced at low temperature or in serum-free medium, whereas conventional inhibitors of intracellular glycoprotein transport have no effect. Thus, translocation of nucleolin is the consequence of an active transport by a pathway which is independent of the endoplasmic reticulum-Golgi complex. The cell-surface-expressed nucleolin becomes clustered at the external side of the plasma membrane when cross-linked by the nucleolin-specific monoclonal antibody mAb D3. This clustering, occurring at 20 degrees C and in a well-organized pattern, is dependent on the existence of an intact actin cytoskeleton. At 37 degrees C, mAb D3 becomes internalized, thus illustrating that surface nucleolin can mediate intracellular import of specific ligands. Our results point out that nucleolin should also be considered a component of the cell surface where it could be functional as a cell surface receptor for various ligands reported before.

Actins↗

PubMed2001

Up-regulation of nucleolin mRNA and protein in peripheral blood mononuclear cells by extracellular-regulated kinase.

The signal transduction pathways regulating nucleolin mRNA and protein production have yet to be elucidated. Peripheral blood mononuclear cells treated with phorbol 12-myristate 13-acetate showed steady state levels of nucleolin mRNA that were 2-2.5-fold greater than untreated control cells. The up-regulation of nucleolin mRNA was substantially repressed by U0126, a specific inhibitor that blocks phosphorylation of extracellular-regulated kinase (ERK). Calcium ionophores and ionomycin also activated ERK and substantially elevated nucleolin mRNA levels, demonstrating phorbol 12-myristate 13-acetate and calcium signaling converge on ERK. Drugs that affected protein kinase C, protein kinase A, and phospholipase C signal transduction pathways did not alter nucleolin mRNA levels significantly. The half-life of nucleolin mRNA increased from 1.8 h in resting cells to 3.2 h with phorbol ester activation, suggesting ERK-mediated posttranscriptional regulation. Concomitantly, full-length nucleolin protein was increased. The higher levels of nucleolin protein were accompanied by increased binding of a 70-kDa nucleolin fragment to the 29-base instability element in the 3'-untranslated region of amyloid precursor protein (APP) mRNA in gel mobility shift assays. Supplementation of rabbit reticulocyte lysate with nucleolin decreased APP mRNA stability and protein production. These data suggest ERK up-regulates nucleolin posttranscriptionally thereby controlling APP production.

3' Untranslated Regions↗

PubMed2001

Repression of RNA polymerase I transcription by nucleolin is independent of the RNA sequence that is transcribed.

Nucleolin is one of the most abundant non-ribosomal proteins of the nucleolus. Several studies in vitro have shown that nucleolin is involved in several steps of ribosome biogenesis, including the regulation of rDNA transcription, rRNA processing, and ribosome assembly. However, the different steps of ribosome biogenesis are highly coordinated, and therefore it is not clear to what extent nucleolin is involved in each of these steps. It has been proposed that the interaction of nucleolin with the rDNA sequence and with nascent pre-rRNA leads to the blocking of RNA polymerase I (RNA pol I) transcription. To test this model and to get molecular insights into the role of nucleolin in RNA pol I transcription, we studied the function of nucleolin in Xenopus oocytes. We show that injection of a 2-4-fold excess of Xenopus or hamster nucleolin in stage VI Xenopus oocytes reduces the accumulation of 40 S pre-rRNA 3-fold, whereas transcription by RNA polymerase II and III is not affected. Direct analysis of rDNA transcription units by electron microscopy reveals that the number of polymerase complexes/rDNA unit is drastically reduced in the presence of increased amounts of nucleolin and corresponds to the level of reduction of 40 S pre-rRNA. Transcription from DNA templates containing various combinations of RNA polymerase I or II promoters in fusion with rDNA or CAT sequences was analyzed in the presence of elevated amounts of nucleolin. It was shown that nucleolin leads to transcription repression from a minimal polymerase I promoter, independently of the nature of the RNA sequence that is transcribed. Therefore, we propose that nucleolin affects RNA pol I transcription by acting directly on the transcription machinery or on the rDNA promoter sequences and not, as previously thought, through interaction with the nascent pre-rRNA.

Animals↗

PubMed2001

Light differentially regulates cell division and the mRNA abundance of pea nucleolin during de-etiolation.

The abundance of plant nucleolin mRNA is regulated during de-etiolation by phytochrome. A close correlation between the mRNA abundance of nucleolin and mitosis has also been previously reported. These results raised the question of whether the effects of light on nucleolin mRNA expression were a consequence of light effects on mitosis. To test this we compared the kinetics of light-mediated increases in cell proliferation with that of light-mediated changes in the abundance of nucleolin mRNA using plumules of dark-grown pea (Pisum sativum) seedlings. These experiments show that S-phase increases 9 h after a red light pulse, followed by M-phase increases in the plumule leaves at 12 h post-irradiation, a time course consistent with separately measured kinetics of red light-induced increases in the expression of cell cycle-regulated genes. These increases in cell cycle-regulated genes are photoreversible, implying that the light-induced increases in cell proliferation are, like nucleolin mRNA expression, regulated via phytochrome. Red light stimulates increases in the mRNA for nucleolin at 6 h post-irradiation, prior to any cell proliferation changes and concurrent with the reported timing of phytochrome-mediated increases of rRNA abundance. After a green light pulse, nucleolin mRNA levels increase without increasing S-phase or M-phase. Studies in animals and yeast indicate that nucleolin plays a significant role in ribosome biosynthesis. Consistent with this function, pea nucleolin can rescue nucleolin deletion mutants of yeast that are defective in rRNA synthesis. Our data show that during de-etiolation, the increased expression of nucleolin mRNA is more directly regulated by light than by mitosis.

Cell Cycle↗

PubMedDate not supplied

Differential expression of protein phosphatase type 1 isotypes and nucleolin during cell cycle arrest.

In the present study, we examined the expression and cytolocalization of protein phosphatase type 1 (PP1) isoforms and nucleolin in human osteoblastic cell line MG63 cells at two boundaries in the cell cycle. We treated MG63 cells with hydroxyurea and nocodazole to arrest the cells at the G(1)/S and G(2)/M boundaries, respectively. As judged from the results of Western blot analysis, PP1 isoforms were expressed differently at each boundary of the cell cycle. Nucleolin was also shown to have a different expression pattern at each boundary. In the hydroxyurea-treated cells, nucleolus-like bodies were bigger in size and decreased in number compared with those in asynchronized cells. However, the subcellular localization of PP1s and nucleolin was not changed. Anti-nucleolin antibody interacted with 110-kDa and 95-kDa proteins present in asynchronized cells and in the cells treated with hydroxyurea. Treatment of the cells with nocodazole decreased the level of the 95-kDa form of nucleolin. In the nocodazole-treated cells, it was impossible to distinguish the distribution of each protein. The phosphorylation status of nucleolin in the cell cycle arrested samples was examined by 2D-IEF-PAGE followed by Western blot analysis. In the case of asynchronized cells or hydroxyurea-treated ones, nucleolin was located at a basic isoelectric point (dephosphorylated status); whereas in the G(2)/M arrest cells, the isoelectric point of nucleolin shifted to an acidic status, indicating that nucleolin was phosphorylated. The present results indicate that PP1 and nucleolin were differently expressed at G(1)/S and G(2)/M boundaries of the cell cycle and acted in a different fashion during cell-cycle progression.

Cell Cycle↗

PubMed2006

Up-regulation of the KLF2 transcription factor by fluid shear stress requires nucleolin.

We have previously characterized the regulation of the KLF2 transcription factor gene by describing an induction complex that binds to and regulates its promoter. In the present study, by using DNA affinity chromatography and mass spectrometry, we have identified nucleolin as an additional protein that binds to a palindromic response region in the KLF2 promoter. The presence of nucleolin on the KLF2 promoter in macrophages was verified by electrophoretic mobility shift assays. Interestingly, in mouse and human endothelial cell lines, electrophoretic mobility shift assays and chromatin immunoprecipitation analyses indicated that nucleolin binds the KLF2 promoter only upon application of fluid shear stress. Pretreatment of the endothelial cells with LY294002, a specific inhibitor of phosphatidylinositol 3-kinase (PI3K), blocked the shear stress-induced binding of nucleolin to the promoter, demonstrating its PI3K-dependent regulation. Additionally, nucleolin exhibited dynamic flow-specific, PI3K-dependent alterations in size. Anti-nucleolin antibodies interacted with a 110-kDa form in static endothelial cells and with several catalytic forms that changed in abundance after the application of shear stress. Immunoprecipitation experiments demonstrated that fluid flow induced the interaction of nucleolin with the p85 regulatory subunit of PI3K. Finally, introduction of small interfering RNAs targeting the nucleolin genetic sequence selectively reduced nucleolin expression and was sufficient to block the induction of KLF2 by shear stress. These data support a general role for nucleolin in gene regulation and identify it as a novel factor involved in regulation of KLF2 expression.

Amino Acid Sequence↗

PubMed1999

Molecular dissection of nucleolin's role in growth and cell proliferation: new insights.

Cells require optimum protein synthetic activity in order to support cell proliferation, maintain homeostatic and metabolic integrity, and repair damage. Since growth depends on protein synthesis through ribosome biogenesis, the control of biosynthesis of ribosomes is necessarily a key element for control of growth. Nucleolin is a major nucleolar protein of exponentially growing eukaryotic cells, which is directly involved in the regulation of ribosome biogenesis and maturation. The highly conserved nucleolin contains three major domains through which it controls the organization of nucleolar chromatin, packaging of pre-RNA, rDNA transcription, and ribosome assembly. Numerous reports have implicated the involvement of nucleolin either directly or indirectly in the regulation of cell proliferation and growth, cytokinesis, replication, embryogenesis, and nucleogenesis. Nucleolin, an RNA binding protein, is also an autoantigen, a transcriptional repressor, and a switch region targeting factor. In addition, nucleolin exhibits autodegradation, DNA and RNA helicase activities, and DNA-dependent ATPase activity. An interesting aspect of nucleolin action is that it is a target for regulation by proteolysis, methylation, ADP-ribosylation, and phosphorylation by CKII, cdc2, PKC-xi, cyclic AMP-dependent protein kinase, and ecto-protein kinase. For these and other reasons, nucleolin is fundamental to the survival and proliferation of cells. Considerable progress has been made in recent years with the identification of new nucleolin binding proteins that may mediate these many nucleolin-dependent functions. Nucleolin also functions as a cell surface receptor, where it acts as a shuttling protein between cytoplasm and nucleus, and thus can even provide a mechanism for extracellular regulation of nuclear events. Exploration of the regulation of this multifaceted protein in a remarkable number of diverse functions is challenging.

Animals↗

PubMed1995

The quantity of nucleolar proteins nucleolin and protein B23 is related to cell doubling time in human cancer cells.

BACKGROUND: The quantity of the silver-stained nucleolar proteins (AgNOR proteins) measured in situ in cytohistologic preparations is related to the rapidity of cell proliferation. The term "AgNOR proteins" comprises several proteins. The relationship between the individual AgNOR protein amount and cell proliferating activity is not yet known. We studied the quantitative distribution of the individual AgNOR proteins, with specific attention to the two major AgNOR proteins, nucleolin and protein B23, in seven human cancer cell lines characterized by different cell doubling times. DESIGN: The doubling time of cancer cells was measured by counting the asynchronously growing cells at regular time intervals. The AgNOR proteins were quantified in situ, after a specific one-step staining procedure, by computerized image analysis. For the quantitative evaluation of nucleolin and protein B23, two methods were followed. Nuclear proteins after separation by SDS-PAGE were transferred onto nitrocellulose membranes and were either: 1) stained by the silver staining procedure for AgNOR proteins or 2) treated with anti-nucleolin and anti-protein B23 mAb followed by reaction with secondary Ab linked to peroxidase and revealed by chemiluminescence and autoradiography. In both cases, measurement of individual AgNOR protein and nucleolin and protein B23 amount was carried out using computerized densitometric analysis. RESULTS: Integrated density values of the silver-stained bands at 105 kDa (nucleolin) and 38 to 39 kDa (protein B23) represented, in all cell lines, more than 60% of the total silver-stained band value. A relationship was found between the densitometric values of silver-stained nucleolin and protein B23 and rapidity of cell proliferation (r = 0.85 and r = 0.86, respectively, p < 0.05). The values of nucleolin and protein B23 obtained using the Western blots were strictly related to the rapidity of cell proliferation (r = 0.93 and 0.96, respectively, p < 0.001). Finally, a good correlation was observed between the mean AgNOR protein area value, as defined in cytologic preparations in situ, and nucleolin and protein B23 amounts as evaluated in silver-stained nitrocellulose membranes (r = 0.92 and r = 0.90, respectively, p < 0.01) and in Western blots (r = 0.95 and r = 0.94, respectively, p < 0.001). CONCLUSIONS: These data indicate that the quantitative changes of AgNOR proteins observed in cytohistologic preparations in situ mainly reflect the quantitative changes of nucleolin and protein B23 and demonstrate that nucleolin and protein B23 amounts are inversely related to cell doubling time in human cancer cells.

Adenocarcinoma↗

PubMed1996

Nucleolin is a sequence-specific RNA-binding protein: characterization of targets on pre-ribosomal RNA.

Nucleolin is an abundant nucleolar protein, which plays an essential, but largely unknown role in ribosome biogenesis. Nucleolin contains four consensus RNA-binding domains (CS-RBD), the presence of which suggests that the molecular function of this protein is likely reflected by its RNA-binding properties. Indeed, by immunocytological analysis performed on ribosomal transcription units, we have found several nucleolin molecules associated with nascent pre-rRNA. In mouse, two high-affinity binding sites with an apparent dissociation constant (Kd) of 50 to 100 nM have been mapped in the 5' ETS upstream from the early pre-rRNA processing site. Interestingly, nucleolin of mouse origin has recognized analogous sequences in the 5' ETS of human pre-rRNA. In parallel, selection-amplification (SELEX) experiments have identified an 18-nucleotide long RNA sequence that binds nucleolin with high affinity (Kd 5 to 20 nM) and shares a common UCCCGA motif with the characterized pre-rRNA binding sites. By mutagenesis and a structural analysis, we have characterized the nucleolin RNA binding site and found that it is constituted by a minimal 18-nucleotide long stem-loop structure. The sequence UCCCGA that is found within the hairpin loop is necessary for the specific interaction. Mutation of any of the C or G residues within this motif abolishes nucleolin interaction. Furthermore, point mutation in the stem that completely disrupt the hairpin structure also prevents nucleolin binding. By determining the minimal 5' and 3' ends of the RNA that is bound to the protein we concluded that nucleolin binding site is constituted by a short four to five-base-pair stem and an eight-nucleotide loop. This structural motif is very similar to hairpins recognized by two other CS-RBD-containing proteins (U1 snRNP A and U2 snRNP B"). Possible functional implications of our findings are discussed.

Animals↗

PubMed1992

Identification and localization of a nucleolin homologue in onion nucleoli.

A protein homologous to nucleolin, a major nucleolar protein with multifunctional features involved in pre-rRNA synthesis and early processing, has been identified and localized in situ in onion root meristematic cells by different techniques, which have included the use of an antibody raised against hamster nucleolin. The protein was identified on Western blots of nucleolar proteins as a 64-kDa band, by means of the anti-nucleolin antibody, bismuth staining, and the silver staining-nucleolar organizer (Ag-NOR) method. The experiments also suggested that nucleolin could be a target of these two cytochemical stainings. Although the 64-kDa band corresponds to a major nucleolar protein, it is a minor one among total nuclear proteins. The same techniques were used in situ at the ultrastructural level, and the immunogold detection of the nucleolin homologue was quantitatively evaluated. The protein accumulates in the transition area from nucleolar fibrillar centers to the dense fibrillar component, which is considered to be the structural result of ribosomal gene transcription. Out of this transition area, the dense fibrillar component may be divided into two regions, proximal and distal with respect to fibrillar centers, which show, respectively, the significant and unsignificant presence of nucleolin; we interpret this fact as the expression of the topological arrangement of pre-rRNA processing. Fibrillar centers themselves showed a weak but significant labeling with the anti-nucleolin antibody. However, bismuth staining was absent from the interior of fibrillar centers, indicating that the nucleolin in them is not phosphorylated. Ag-NOR staining uniformly covered fibrillar centers and the dense fibrillar component (at least in its proximal region), but it did not stain condensed chromatin inclusions in heterogeneous fibrillar centers, showing that the binding of nucleolin to chromatin is associated with its decondensation. This work provides additional evidence of the high phylogenetic conservation of molecular motifs which take part in ribosome biogenesis.

Allium↗

PubMed2006

Nucleolin inhibits Hdm2 by multiple pathways leading to p53 stabilization.

Nucleolin is a c-Myc-induced gene product with defined roles in ribosomal RNA processing and the inhibition of chromosomal DNA replication following stress. Here we find that changes in nucleolin protein levels in unstressed cells cause parallel changes in the amount of p53 protein. Alterations in p53 levels arise from nucleolin binding to the p53 antagonist Hdm2, resulting in the inhibition of both p53 ubiquitination and Hdm2 auto-ubiquitination. Nucleolin does not alter p53 ubiquitination by human papillomavirus E6, indicating that the effect is specific for Hdm2. Although the inhibition of ligase activity would be expected to stabilize Hdm2, we instead find that nucleolin also reduces Hdm2 protein levels, demonstrating that nucleolin inhibits Hdm2 using multiple mechanisms. Increases in nucleolin levels in unstressed cells led to higher expression of p21(cip1/waf1), a reduced rate of cellular proliferation, and an increase in apoptosis. Thus, nucleolin has a number of properties in common with the tumor suppressor ARF (alternate reading frame). We propose that nucleolin, like ARF, responds to hyperproliferative signals by upregulation of p53 through Hdm2 inhibition.

Blotting, Western↗

PubMed1993

Limited proteolysis of rat liver nucleolin by endogenous proteases: effects of polyamines and histones.

Nucleolin is a major nucleolar phosphoprotein and is presumably involved in rDNA transcription and ribosome biosynthesis. This protein is known to be very labile and to be cleaved by endogenous proteases into many small peptides. We found that, when rat liver nucleolar suspension (Nu-1) or nucleolin-rich extract (Nu-2) was incubated under conventional conditions, polyamines and histones interacted with the nucleolin to lead to its preferential degradation to 60 kDa phosphopeptide (p60). The peptide p60 was identified as a peptide containing the N-terminal half of the nucleolin molecule, as judged from peptide-map analysis. Whereas spermine binding to the purified nucleolin was decreased by KCl concentrations above 50 mM, histones (H1, H2B and H3) were able to bind to the nucleolin in the presence of up to 300 mM KCl. A distinct difference between H1 and other histones was found in that H1 could produce p60 from nucleolin in both Nu-1 and Nu-2, whereas H2B and H3 stimulated the degradation of nucleolin to p60 only when Nu-2 was used for the source of nucleolin. A possible relationship between p60 formation and rRNA synthesis is discussed, but its exact role remains to be studied.

Animals↗