Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Nucleolin”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 109 records · Page 6Linked to original sources
PubMed2005

Novel checkpoint response to genotoxic stress mediated by nucleolin-replication protein a complex formation.

Human replication protein A (RPA), the primary single-stranded DNA-binding protein, was previously found to be inhibited after heat shock by complex formation with nucleolin. Here we show that nucleolin-RPA complex formation is stimulated after genotoxic stresses such as treatment with camptothecin or exposure to ionizing radiation. Complex formation in vitro and in vivo requires a 63-residue glycine-arginine-rich (GAR) domain located at the extreme C terminus of nucleolin, with this domain sufficient to inhibit DNA replication in vitro. Fluorescence resonance energy transfer studies demonstrate that the nucleolin-RPA interaction after stress occurs both in the nucleoplasm and in the nucleolus. Expression of the GAR domain or a nucleolin mutant (TM) with a constitutive interaction with RPA is sufficient to inhibit entry into S phase. Increasing cellular RPA levels by overexpression of the RPA2 subunit minimizes the inhibitory effects of nucleolin GAR or TM expression on chromosomal DNA replication. The arrest is independent of p53 activation by ATM or ATR and does not involve heightened expression of p21. Our data reveal a novel cellular mechanism that represses genomic replication in response to genotoxic stress by inhibition of an essential DNA replication factor.

Ataxia Telangiectasia Mutated Proteins↗
PubMed2007

Overexpression of nucleolin in chronic lymphocytic leukemia cells induces stabilization of bcl2 mRNA.

B-cell chronic lymphocytic leukemia (CLL) is characterized by the accumulation of clonal B cells that are resistant to apoptosis as a result of bcl2 oncogene overexpression. Studies were done to determine the mechanism for the up-regulation of bcl-2 protein observed in CD19+ CLL cells compared with CD19+ B cells from healthy volunteers. The 11-fold higher level of bcl-2 protein in CLL cells was positively correlated with a 26-fold elevation in the cytosolic level of nucleolin, a bcl2 mRNA-stabilizing protein. Measurements of the bcl2 heterogeneous nuclear/bcl2 mRNA (hnRNA)/mRNA ratios and the rates of bcl2 mRNA decay in cell extracts indicated that the 3-fold higher steady-state level of bcl2 mRNA in CLL cells was the result of increased bcl2 mRNA stability. Nucleolin was present throughout the nucleus and cytoplasm of CLL cells, whereas in normal B cells nucleolin was only detected in the nucleus. The addition of recombinant human nucleolin to extracts of normal B cells markedly slowed the rate of bcl2 mRNA decay. SiRNA knockdown of nucleolin in MCF-7 cells resulted in decreased levels of bcl2mRNA and protein but no change in beta-actin. These results indicate that bcl-2 overexpression in CLL cells is related to stabilization of bcl2 mRNA by nucleolin.

B-Lymphocytes↗
PubMed2001

Activation of the EBV/C3d receptor (CR2, CD21) on human B lymphocyte surface triggers tyrosine phosphorylation of the 95-kDa nucleolin and its interaction with phosphatidylinositol 3 kinase.

We previously demonstrated that CR2 activation on human B lymphocyte surface triggered tyrosine phosphorylation of a p95 component and its interaction with p85 subunit of phosphatidylinositol 3' (PI 3) kinase. Despite identical molecular mass of 95 kDa, this tyrosine phosphorylated p95 molecule was not CD19, the proto-oncogene Vav, or the adaptator Gab1. To identify this tyrosine phosphorylated p95 component, we first purified it by affinity chromatography on anti-phosphotyrosine mAb covalently linked to Sepharose 4B, followed by polyacrylamide gel electrophoresis. Then, the isolated 95-kDa tyrosine phosphorylated band was submitted to amino acid analysis by mass spectrometry; the two different isolated peptides were characterized by amino acid sequences 100% identical with two different domains of nucleolin, localized between aa 411--420 and 611--624. Anti-nucleolin mAb was used to confirm the antigenic properties of this p95 component. Functional studies demonstrated that CR2 activation induced, within a brief span of 2 min, tyrosine phosphorylation of nucleolin and its interaction with Src homology 2 domains of the p85 subunit of PI 3 kinase and of 3BP2 and Grb2, but not with Src homology 2 domains of Fyn and Gap. These properties of nucleolin were identical with those of the p95 previously described and induced by CR2 activation. Furthermore, tyrosine phosphorylation of nucleolin was also induced in normal B lymphocytes by CR2 activation but neither by CD19 nor BCR activation. These data support that tyrosine phosphorylation of nucleolin and its interaction with PI 3 kinase p85 subunit constitute one of the earlier steps in the specific intracellular signaling pathway of CR2.

Antigens, CD19↗
PubMed1990

Genomic organization and chromosomal localization of the human nucleolin gene.

Nucleolin, a eukaryotic nucleolar phosphoprotein, is involved in the synthesis and maturation of ribosomes. To characterize the genomic organization and regulatory sequences of this gene, two overlapping lambda clones containing the human nucleolin gene plus flanking regions were isolated from a genomic library using human nucleolin cDNA. Southern blots of genomic DNA from human, several mammals, chicken, and yeast revealed that the nucleolin gene is well conserved across these species. The gene consists of 14 exons with 13 intervening sequences and spans approximately 11 kilobases of DNA. Analysis of the splice junctions indicated that the amino-terminal domain and the four RNA binding domains plus the nuclear localization signal are split into adjacent exons. Sequences from the 5'-flanking and the first intron contain a high content of GC residues which is consistent with nucleolin being a "housekeeping" gene. Promoter elements include an atypical TATA box (GTTA), one CCAAT box much further from the initiation site, three reverse compliments of CCAAT (ATTGG), and two pyrimidine-rich nucleotide stretches. In addition, this region and the first intron contain numerous potential Sp1, GCF, CRE-fos, GCN, AP-1, AP-2, UCE, and sequences similar to the glucocorticoid receptor binding site. The transcription start site was determined by primer extension and S1 nuclease mapping of RNA from human liver. One Kpn and three Alu repeats were found within two of the middle introns. The 3'-untranslated portion of the gene contains five homology blocks in a 100-base pair region that are highly conserved among human, mouse, and hamster genomes. Finally, we have determined that the human nucleolin gene is located on chromosome 2q12-qter and is present at one copy per haploid genome. A restriction fragment length polymorphism with EcoRI has been detected in the gene.

Amino Acid Sequence↗
PubMed2000

Recognition and binding of the human selenocysteine insertion sequence by nucleolin.

Prokaryotic and eukaryotic cells cotranslationally incorporate the unusual amino acid selenocysteine at a UGA codon, which conventionally serves as a termination signal. Translation of selenoprotein gene transcripts in eukaryotes depends upon a "selenocysteine insertion sequence" in the 3'-untranslated region. We have previously shown that DNA-binding protein B specifically binds this sequence element. We now report the identification of nucleolin as a partner in the selenoprotein translation complex. In RNA electromobility shift assays, nucleolin binds the selenocysteine insertion sequence from the human cellular glutathione peroxidase gene, competes with binding activity from COS cells, and shows diminished affinity for probes with mutations in functionally important, conserved sequence elements. Antibody to nucleolin interferes with the gel shift activity of COS cell extract. Antibody to DNA-binding protein B co-extracts nucleolin from HeLa cell cytosol, and the two proteins co-sediment in glycerol gradient fractions of ribosomal high salt extracts. Thus, nucleolin appears to join DNA-binding protein B and possibly other partners to form a large complex that links the selenocysteine insertion sequence in the 3'-untranslated region to other elements in the coding region and ribosome to translate the UGA "stop" codon as selenocysteine.

Amino Acid Sequence↗
PubMed1988

Hyperphosphorylation of N-60, a protein structurally and immunologically related to nucleolin after tumour-promoter treatment.

Okadaic acid, a non-TPA-type tumour promoter, induces hyperphosphorylation of a 60-kd protein in primary human fibroblasts. Treatment with TPA-type tumour promoters (e.g. TPA and teleocidin) did not cause this hyperphosphorylation. Phosphorylation of this protein was not seen at times earlier than 90 min after the addition of 75 ng/ml okadaic acid to the proliferating cell cultures. The presence of inhibitors such as actinomycin D and cycloheximide, did not significantly influence the level of hyperphosphorylation induced by okadaic acid treatment. By immunoblotting using an antibody anti-nucleolin, the 60-kd protein was identified as a fragment of nucleolar protein, nucleolin. Similarly, antibodies against the 60-kd protein cross-reacted with nucleolin. Furthermore peptide mapping, using staphylococcal V8 protease, showed that the 60-kd protein phosphorylated by casein kinase II in vitro and the okadaic-acid-induced hyperphosphorylated 60-kd protein exhibited identical phosphopeptide maps, indicating that there is also structural relatedness between N-60 and nucleolin. Hyperphosphorylation of the nucleolin fragment (N-60) was suppressed by anti-tumour promoter retinoic acid.

Carcinogens↗
PubMed2002

Nucleolin is a calcium-binding protein.

We have purified a prominent 110-kDa protein (p110) from 1.6 M NaCl extracts of rat liver nuclei that appears to bind Ca2+. p110 was originally identified by prominent blue staining with 'Stains-All' in sodium dodecyl sulfate-polyacrylamide gels and was observed to specifically bind ruthenium red and 45Ca2+ in nitrocellulose blot overlays. In spin-dialysis studies, purified p110 saturably bound approximately 75 nmol Ca2+/mg protein at a concentration of 1 mM total Ca2+ with half-maximal binding observed at 105 microM Ca2+. With purification, p110 became increasingly susceptible to proteolytic (likely autolytic) fragmentation, although most intermediary peptides between 40 and 90 kDa retained "Stains-All", ruthenium red, and 45Ca2+ binding. N-terminal sequencing of intact p110 and a 70-kDa autolytic peptide fragment revealed a strong homology to nucleolin. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)/IEF revealed autolysis produced increasingly acidic peptide fragments ranging in apparent pI's from 5.5 for intact p110 to 3.5 for a 40 kDa peptide fragment. Intact p110 and several peptide fragments were immunostained with a highly specific anti-nucleolin antibody, R2D2, thus confirming the identity of this protein with nucleolin. These annexin-like Ca2+-binding characteristics of nucleolin are likely contributed by its highly acidic argyrophilic N-terminus with autolysis apparently resulting in largely selective removal of its basic C-terminal domain. Although the Ca2+-dependent functions of nucleolin are unknown, we discuss the possibility that like the structurally analogous HMG-1, its Ca2+-dependent actions may regulate chromatin structure, possibly during apoptosis.

Animals↗
PubMed1995

Human myeloid cell nuclear differentiation antigen binds specifically to nucleolin.

The human myeloid cell nuclear differentiation antigen (MNDA) is a nuclear protein expressed specifically in cells of the myelomonocytic lineage and regulated by interferon alpha in a cell-specific fashion. MNDA is also a member of a family of interferon-regulated genes of unknown function. In an effort to elucidate the function of MNDA, three techniques (affinity purification, coimmunoprecipitation, and protein blot assay) were used to characterize its specific protein binding activities. Microsequence analysis showed that MNDA bound the 100 kDa nucleolin protein. The identification of nucleolin was confirmed by immunoreaction with specific antibodies. MNDA contains motifs which could account for specific binding to nucleolin. Nucleolin binds other macromolecules and exhibits features consistent with roles in signal transduction, production of ribosomes, nuclear matrix structure, and regulation of transcription. The present results indicate that the function of MNDA is most likely related to interactions with other proteins. Through these associations, MNDA could contribute cell/lineage- and differentiation-specific limits to the function of ubiquitous proteins such as nucleolin. Further analysis of MNDA protein binding could be critical to elucidating the function of MNDA and could contribute to understanding the function of the products of other members of this interferon-inducible family of genes.

Amino Acid Sequence↗
PubMed2006

Effect of laminin-1 on intestinal cell differentiation involves inhibition of nuclear nucleolin.

Intestinal epithelial cells are characterized by continuous renewal and differentiation events, which may be influenced by the basement membrane, and in particular laminins, which are major components of this specialized extracellular matrix. The function and signaling pathways of laminins in these processes are still poorly documented. In this study, we investigated the possible role and the subcellular localization of nucleolin, a nuclear shuttling protein, in relation to differentiation of human intestinal epithelial Caco2/TC7 cells triggered by exogenous laminin-1. Immunofluorescence and Western blot analysis indicated that laminin-1 induced early differentiation of the cells concomitantly to a decrease in nuclear nucleolin and its a cell surface location. We also showed that both effects of laminin-1 on Caco2/TC7 cells--induction of the differentiation marker sucrase-isomaltase and redistribution of nucleolin--could be mediated by a beta1-integrin dependent cascade that implicated activation of the p38 MAPK pathway. In addition, knock-down of nucleolin expression by the small interfering RNA strategy mimicked the effect of laminin-1 as it resulted in the induction of cell polarization and differentiation. Thus, our study suggests that changes in the subcellular distribution and expression level of nucleolin play an important role in intestinal cell differentiation and relay the signaling pathway induced by laminin-1.

Base Sequence↗
PubMed1995

A 110-kD nuclear shuttling protein, nucleolin, binds to the neurite-promoting IKVAV site of laminin-1.

The basement membrane protein laminin and the IKVAV-containing sequence from the laminin alpha 1 chain have been found to promote the differentiation of primary neurons and a variety of neural cell lines. We previously reported that a 110-kd IKVAV-binding protein (LBP110) isolated from brain appears to be a member of the beta-amyloid precursor protein (APP) family by immunologic and functional studies, which showed that LBP110/APP is also important in neurite outgrowth (Kibbey et al.: Proc Natl Acad Sci USA 90:10150-10153, 1993). In the preparation of this binding protein, a contaminating IKVAV-binding protein of identical molecular weight, nucleolin, was also identified. Here we have studied the relationship between these binding proteins. We find that nucleolin binds specifically to the IKVAV sequence independently of LBP110/ApP. We have also demonstrated significant levels of nucleolin in mature brain and in differentiating neural cells, suggesting that nucleolin functions not only in cell proliferation and in ribosome biogenesis as was previously reported, but also in the differentiation and maintenance of neural tissue. Our identification of cytoplasmic and cell-surface nucleolin, an IKVAV-binding protein, suggests that this protein may function in signalling by extra-cellular matrix.

Amino Acid Sequence↗
PubMed1998

Effect of laminin on the nuclear localization of nucleolin in rat intestinal epithelial IEC-6 cells.

Laminin is a major component of extracellular matrix. The mechanism of action of laminin on cell proliferation, differentiation, and migration is not fully understood. In this study, we investigated the role of extracellular matrix, especially laminin, on the cellular localization of the nuclear protein, nucleolin, and on cell proliferation. Immunofluorescent and western blot analysis indicated that nucleolin was translocated most efficiently to the nucleus in the small intestinal rat epithelial cell line (IEC-6) when cultured on laminin-coated plates. Specifically, nucleolin was observed predominantly in cytoplasm in the cells cultured without laminin. In contrast, nuclear localization was observed in the cells cultured on laminin. This effect of laminin on nucleolin translocation was time-dependent. Laminin was also observed to stimulate proliferation of IEC-6 cells in serum free medium. Our results suggest that laminin alters the distribution of nucleolin which may be an early signal for cell proliferation.

Animals↗
PubMed1995

The murine nucleolin protein is an inducible DNA and ATP binding protein which is readily detected in nuclear extracts of lipopolysaccharide-treated splenocytes.

A 100-kDa DNA binding protein was found to be dramatically up-regulated upon the mitogenic stimulation of murine splenocytes with bacterial lipopolysaccharide (LPS). The induced DNA binding protein was also found to exhibit moderate binding specificity for the immunoglobulin isotype switch DNA repeats. Furthermore, the induction of the 100-kDa protein by LPS was found to be mediated by both an increase in the protein's stability and an increase in the synthesis of the protein. In vitro phosphorylation experiments revealed that the 100-kDa DNA binding protein was one of the most heavily phosphorylated proteins in both lymphoid and nonlymphoid nuclear extracts. Although this in vitro phosphorylation initially appeared to be mediated by a potent nuclear kinase activity, it was later determined that a significant part of the detected labeling was due to the direct binding of ATP by the 100-kDa protein. Antibodies raised to the 100-kDa DNA binding protein were used to isolate cDNA clones from a lymphocyte cDNA lambda gt11 expression library. Nucleotide sequence analysis revealed that the cloned cDNAs were identical to the mouse nucleolin gene. The beta-galactosidase fusion proteins (encoded by exons 3-14 of nucleolin) and a more severely truncated 45-kDa protein (encoded by exons 5-14 of nucleolin) were both found to bind strongly to DNA and ATP. Furthermore, the strength of DNA binding was found to be highly dependent on the overall dG content of the DNA probes. Our experiments also revealed that apart from binding ATP and G-rich DNA, nucleolin directly bound GTP, dATP, and dGTP, but not dCTP, dTTP, or dUTP. Computer analysis revealed that the putative ATP binding domains appear to fall within two of the phylogenetically conserved RNA binding domains of nucleolin.

Adenosine Triphosphate↗
PubMed2005

Nucleolin is regulated both at the level of transcription and translation.

Nucleolin is a multi-functional protein that is located to the nucleolus. In tissue culture cells, the stability of nucleolin is related to the proliferation status of the cell. During development, rat cardiomyocytes proliferate actively with increases in the mass of the heart being due to both hyperplasia and hypertrophy. The timing of this shift in the phenotype of the myocyte from one capable of undergoing hyperplasia to one that can grow only by hypertrophy occurs within 4 days of post-natal development. Thus, cardiomyocytes are an ideal model system in which to study the regulation of nucleolin during growth in vivo. Using Western blot and quantitative RT-PCR (TaqMan) we found that the amount of nucleolin is regulated both at the level of transcription and translation during the development of the cardiomyocyte. However, in cells which had exited the cell cycle and were subsequently given a hypertrophic stimulus, nucleolin was regulated post-transcriptionally.

Animals↗
PubMed2006

K-Ras4B proteins are expressed in the nucleolus: Interaction with nucleolin.

Kirsten Ras4B (K-Ras4B) is a potent onco-protein that is expressed in the majority of human cell types and is frequently mutated in carcinomas. K-Ras4B, like other members of the Ras family of proteins, is considered to be a cytoplasmic protein that must be localized to the plasma membrane for activation. Here, using confocal microscopy and biochemical analysis, we show that K-Ras4B, but not H-Ras or the closely related K-Ras4A, is also present in the nucleoli of normal and transformed cells. Subcellular fractionation and immunostaining show that K-Ras4B is located not only in the cytoplasm, but also in the nucleolar compartment. Modification of a C-terminal hexa-lysine motif unique to K-Ras4B results in exclusively cytoplasmic forms of the protein. Nucleolin, a pleiotropic regulator of cellular processes, including transcriptional regulation, is also characterized by a nucleolar-like nuclear appearance. We show that K-Ras4B and nucleolin co-localize within the nucleus and that nucleolin physically associates with K-Ras4B. Inhibition of K-Ras4B/nucleolin association blocked nucleolar localization of K-Ras4B. Using siRNA to knockdown the expression of nucleolin eliminated the nucleolar localization of K-Ras4B and significantly repressed the activation of the well-characterized K-Ras4B transcriptional target Ap-1, but stimulated Elk1. These data provide evidence of a nucleolar localization of K-Ras4B and describe a functional association between K-Ras4B and nucleolin.

Cell Nucleolus↗
PubMed2004

Solution structure of the complex formed by the two N-terminal RNA-binding domains of nucleolin and a pre-rRNA target.

Nucleolin is a 70 kDa multidomain protein involved in several steps of eukaryotic ribosome biogenesis. In vitro selection in combination with mutagenesis and structural analysis identified binding sites in pre-rRNA with the consensus (U/G)CCCG(A/G) in the context of a hairpin structure, the nucleolin recognition element (NRE). The central region of the protein contains four tandem RNA-binding domains (RBDs), of which the first two are responsible for the RNA-binding specificity and affinity for NREs. Here, we present the solution structure of the 28 kDa complex formed by the two N-terminal RNA-binding domains of nucleolin (RBD12) and a natural pre-rRNA target, b2NRE. The structure demonstrates that the sequence-specific recognition of the pre-rRNA NRE is achieved by intermolecular hydrogen bonds and stacking interactions involving mainly the beta-sheet surfaces of the two RBDs and the linker residues. A comparison with our previously determined NMR structure of RBD12 in complex with an in vitro selected RNA target, sNRE, shows that although the sequence-specific recognition of the loop consensus nucleotides is the same in the two complexes, they differ in several aspects. While the protein makes numerous specific contacts to the non-consensus nucleotides in the loop E motif (S-turn) in the upper part of the sNRE stem, nucleolin RBD12 contacts only consensus nucleotides in b2NRE. The absence of these upper stem contacts from the RBD12/b2NRE complex results in a much less stable complex, as demonstrated by kinetic analyses. The role of the loop E motif in high-affinity binding is supported by gel-shift analyses with a series of sNRE mutants. The less stable interaction of RBD12 with the natural RNA target is consistent with the proposed role of nucleolin as a chaperone that interacts transiently with pre-rRNA to prevent misfolding.

Animals↗
PubMed1991

Phosphorylation and proteolytic degradation of nucleolin from 3T3-F442A cells.

The effect of phosphorylation on the proteolysis of nucleolin has been investigated. Nucleolin is readily phosphorylated both in vitro and in vivo. Utilizing phosphorylation assays and immunoblotting with anti-nucleolin serum, we have observed that phosphorylation enhances nucleolin as a substrate for a protease. This protease activity cleaves the protein into a highly phosphorylated 30 kDa peptide and a 72 kDa peptide. The involvement of casein kinase II is suggested since this cleavage is promoted by spermine and inhibited by heparin, which are, respectively, a stimulator and an inhibitor of casein kinase II activity. The molecular identity of the protease and the physiologic significance of the proteolytic cleavage of nucleolin remain to be studied.

3T3 Cells↗
PubMed1991

Synergistic stimulatory effect of glucocorticoid, EGF and insulin on the synthesis of ribosomal RNA and phosphorylation of nucleolin in primary cultured rat hepatocytes.

Effects of dexamethasone, EGF and insulin on the synthesis of rRNA and phosphorylation of nucleolin in primary cultures of adult rat hepatocytes were studied. Hepatocytes were incubated for 8 h with EGF (20 ng/ml) plus insulin (0.1 microM) and/or for 20 h with dexamethasone (1 microM) before the end of incubation. The incorporation of [3H]uridine into acid-insoluble materials and the nuclear activity of RNA polymerase I were stimulated approx. 2-fold with EGF plus insulin and these were further enhanced 2-3-times by dexamethasone, although dexamethasone alone exerted no stimulation. When hepatocytes were incubated with [32P]orthophosphate, similar enhancement by these hormones was also observed in the phosphorylation of a nucleolar protein, nucleolin, which was detected by immunoprecipitation with anti-nucleolin antibodies. The amount of nucleolin was slightly increased by EGF plus insulin in the presence of dexamethasone, but scarcely changed by treatment with EGF plus insulin or dexamethasone alone. Cycloheximide inhibited RNA synthesis to a greater or lesser degree in the case of all hepatocytes which were cultured with or without these hormonal treatments. These results indicate that the in vivo effect of glucocorticoid on rRNA synthesis and nucleolin phosphorylation in liver is primarily a direct action on parenchymal cells and requires other growth factors such as EGF and insulin.

Animals↗
PubMed1997

Nucleolin is a protein kinase C-zeta substrate. Connection between cell surface signaling and nucleus in PC12 cells.

We have previously shown that protein kinase C (PKC)-zeta is activated and required for nerve growth factor (NGF)-induced differentiation of rat pheochromocytoma PC12 cells (Wooten, M. W., Zhou, G., Seibenhener, M. L., and Coleman, E. S. (1994) Cell Growth & Diff. 5, 395-403; Coleman, E. S., and Wooten, M. W. (1994) J. Mol. Neurosci. 5, 39-57). Here we report the characterization and identification of a 106-kDa nuclear protein as a specific substrate of PKC-zeta. NGF treatment of PC12 cells resulted in translocation of PKC-zeta and coincident phosphorylation of a protein that was localized within the nucleoplasm of nuclei isolated from PC12 cells. Addition of PKC-zeta pseudosubstrate peptide in vitro or myristoylated peptide in vivo diminished phosphorylation of pp106 in a dose-dependent fashion. Likewise, addition of purified PKC-zeta, but neither PKC-alpha nor delta, to nuclear extracts resulted in an incremental increase in the phosphorylation of pp106. Expression of dominant-negative PKC-zeta inhibited NGF-induced phosphorylation of pp106, by comparison overexpression of PKC-zeta enhanced basal phosphorylation without a noticeable effect upon NGF-induced effects. Amino acid sequence analysis of four peptides derived from purified pp106 revealed that this protein was homologous to nucleolin. Using an in vitro reconstitution system, purified nucleolin was likewise shown to be phosphorylated by purified PKC-zeta. The staining intensity of both enzyme and substrate in the nucleus increased upon treatment with NGF. In vivo labeling with 32Pi and stimulation of PC12 cells with NGF followed by immunoprecipitation with anti-nucleolin antibody corroborated the in vitro approach documenting enhanced phosphorylation of nucleolin by NGF treatment. Taken together, the findings presented herein document that nucleolin is a target of PKC-zeta that serves to relay NGF signals from cell surface to nucleus in PC12 cells.

Amino Acid Sequence↗