DNA damage, p14ARF, Nucleophosmin (NPM/B23), and cancer

The p53/p14ARF/mdm2 stress response pathway plays a central role in mediating cellular responses to oncogene activation, genome instability, and therapy-induced DNA damage. Abrogation of the pathway occurs in most if not all cancers, and may be essential for tumor development. The high frequency with which the pathway is disabled in cancer and the fact that the pathway appears to be incompatible with tumor cell growth, has made it an important point of focus in cancer research and therapeutics development. Recently, Nucleophosmin (NPM, B23, NO38 and numatrin), a multifunctional nucleolar protein, has emerged as a p14ARF binding protein and regulator of p53. While complex formation between ARF and NPM retains ARF in the nucleolus and prevents ARF from activating p53, DNA damaging treatments promote a transient subnuclear redistribution of ARF to the nucleoplasm, where it interacts with mdm2 and promotes p53 activation. The results add support to a recently proposed model in which the nucleolus serves as a p53-uspstream sensor of stress, and where ARF links nucleolar stress signals to nucleoplasmic effectors of the stress response. A better understanding of ARF’s nucleolar interactions could further elucidate the regulation of the p53 pathway and suggest new therapeutic approaches to restore p53 function.     

B23 and ARF

B23 (nucleophosmin/NPM) is a multifunctional protein that recently has been directly implicated in the p53 network by its documented interaction with the p14(ARF)/p19(Arf) tumor suppressor, a major upstream activator of p53. Here we provide an overview of the functional interactions of B23 and ARF. We also integrate the current models into a unified picture, showing that B23 is essential for stabilizing and maintaining a basal level of ARF in the nucleolus, whereas increasing levels of ARF after oncogenic stress promotes B23 degradation and interferes with B23 nucleocytoplasmic shuttling. In this way, ARF can be regarded as a parasitic peptide on the B23 molecule, because ARF uses this chaperone for its own survival but also antagonizes normal activities of B23. Finally, the functional significance of the ARF-B23 interaction for tumor development and the prospects for novel cancer therapies are evaluated.     

Immunofluorescence studies on proteins B23 and C23 in nucleoli of human lymphocytes

Nucleoli of normal and leukemic lymphocytes were studied by cytochemical and immunofluorescence methods to provide more information on the nucleolar presence and distribution of proteins B23 and C23. Annular nucleoli of human lymphocytes represent a very convenient subject for such studies, since they consist of one centrally located large fibrillar center surrounded by RNP components. In such nucleoli, protein C23 was present mainly in the central nucleolar region and protein B23 was found mostly in the periphery. The nucleolar area immunostained for protein B23 was usually larger than that stained for protein C23. The distribution of protein C23 appeared to be similar to that of intensely stained nucleolar argyrophilic components. No substantial differences were found between the distribution of proteins B23 and C23 in nucleoli of normal and leukemic lymphocytes. In lymphocytes of patients treated with chemotherapy, the immunofluorescence was diminished for protein B23 and particularly so for protein C23.     

Essential role of the B23/NPM core domain in regulating ARF binding and B23 stability

How cells coordinate inhibition of growth and division during genotoxic events is fundamental to our understanding of the origin of cancer. Despite increasing interest and extensive study, the mechanisms that link regulation of DNA synthesis and ribosomal biogenesis remain elusive. Recently, the tumor suppressor p14(ARF) (ARF) has been shown to interact functionally with the nucleolar protein B23/NPM (B23) and inhibit rRNA biogenesis. However, the molecular basis of the ARF-B23 interaction is hitherto unclear. Here we show that a highly conserved motif in the B23 oligomerization domain is essential for mediating ARF binding in vivo. Mutagenesis of conserved B23 core residues (L102A, G105A, G107A) prevented B23 from interacting with ARF. Modeling of the B23 core indicated that substitutions in the GSGP loop motif could trigger conformational changes in B23 thereby obstructing ARF binding. Interestingly, the GSGP loop mutants were unstable, defective for oligomerization, and delocalized from the nucleolus to the nucleoplasm. B23 core mutants displayed increased ubiquitination and proteasomal degradation. We conclude that the functional integrity of the B23 core motif is required for stability, efficient nucleolar localization as well as ARF binding.     

Nucleophosmin/B23 is a proliferate shuttle protein associated with nuclear matrix

It has become obvious that a better understanding and potential elucidation of the nucleolar phosphoprotein B23 involving in functional interrelationship between nuclear organization and gene expression. In present study, protein B23 expression were investigated in the regenerative hepatocytes at different periods (at days 0, 1, 2, 3, 4, 7) during liver regeneration after partial hepatectomy on the rats with immunohistochemistry and Western blot analysis. Another experiment was done with immunolabeling methods and two-dimensional (2-D) gel electrophoresis for identification of B23 in the regenerating hepatocytes and HepG2 cells (hepatoblastoma cell line) after sequential extraction with detergents, nuclease, and salt. The results showed that its expression in the hepatocytes had a locative move and quantitative change during the process of liver regeneration post-operation. Its immunochemical localization in the hepatocytes during the process showed that it moved from nucleoli of the hepatocytes in the stationary stage to nucleoplasm, cytoplasm, mitotic spindles, and mitotic chromosomes of the hepatocytes in the regenerating livers. It was quantitatively increased progressively to peak level at day 3 post-operation and declined gradually to normal level at day 7. It was detected in nuclear matrix protein (NMP) composition extracted from the regenerating hepatocytes and HepG2 cells and identified with isoelectric point (pI) value of 5.1 and molecular weight of 40 kDa. These results indicated that B23 was a proliferate shuttle protein involving in cell cycle and cell proliferation associated with nuclear matrix.     

B23/Nucleophosmin promotes reconstitution of synaptic path in hippocampus after injury

B23, also known as nucleophosmin (NPM), is multifunctional protein directly implicated in cell proliferation, cell cycle progression, and cell survival. In the current study, in addition to confirming its anti-apoptotic function in neuronal survival, we demonstrated that the spatial-temporal expression profile of B23 during development of hippocampal neurons is high in the embryonic stage, down-regulated after birth, and preferentially localized at the tips of growing neuritis and branching points. Overexpression of B23 promotes axon growth with abundant branching points in growing hippocampal neurons, but depletion of B23 impairs axon growth, leading to neuronal death. Following injury to the trisynaptic path in hippocampal slice, overexpression of B23 remarkably increased the number and length of regenerative fibers in the mossy fiber path. Our study suggests that B23 expression in developing neurons is essential for neuritogenesis and axon growth and that up-regulation of B23 may be a strategy for enhancing the reconstitution of synaptic paths after injury to hippocampal synapses.     

Nucleophosmin/B23, a nuclear PI(3,4,5)P(3) receptor, mediates the antiapoptotic actions of NGF by inhibiting CAD

Phosphatidylinositol 3,4,5-triphosphate [PI(3,4,5)P(3)] is an essential second messenger implicated in various cellular processes. Cytoplasmic PI(3,4,5)P(3) has been well characterized, but little is known about the physiological role of nuclear PI(3,4,5)P(3). Here, we describe a nuclear PI(3,4,5)P(3) receptor, nucleophosmin (NPM)/B23, that mediates the antiapoptotic effects of NGF by inhibiting DNA fragmentation activity of caspase-activated DNase (CAD). Employing PI(3,4,5)P(3) column and NGF-treated PC12 nuclear extracts, we identified B23 as a nuclear PI(3,4,5)P(3) binding protein. Purification from nuclear extract demonstrates that B23 contributes to DNA fragmentation inhibitory activity. Depletion of B23 from nuclear extracts or knockdown B23 in PC12 cells abolishes NGF-provoked protective effect, whereas overexpression of B23 in PC12 cells prevents apoptosis. Further, hydrolyzing PI(3,4,5)P(3) with PTEN or SHIP abrogates its antiapoptotic activity. Moreover, B23 mutants that can not associate with PI(3,4,5)P(3) fail to prevent DNA fragmentation. Thus, the nuclear B23-PI(3,4,5)P(3) complex regulates the antiapoptotic activity of NGF in the nucleus.     

Nucleophosmin interacts with and inhibits the catalytic function of eukaryotic initiation factor 2 kinase PKR

In normal cells the protein kinase PKR effects apoptosis in response to various extra and intracellular cues and can also function to suppress the neoplastic phenotype. Because most neoplastic cells are resistant to certain apoptotic cues, we reasoned that an early molecular event in carcinogenesis or leukemogenesis might be the inactivation of PKR by expression or activation of intracellular PKR inhibitors. Seeking novel PKR-modulating proteins we report here that nucleophosmin (NPM), a protein frequently overexpressed in a variety of human malignancies, binds to PKR, and inhibits its activation. Co-immunoprecipitation and in vitro binding experiments showed that NPM associated with PKR. Kinase assays demonstrated that recombinant NPM inhibited PKR activation in a dose-dependent manner. In addition, purified recombinant NPM was phosphorylated by activated PKR. Most importantly, overexpression of NPM suppressed PKR activity, enhanced protein synthesis, and inhibited apoptosis. Lymphoblasts from patients with Fanconi anemia (FA) expressed low levels of NPM, which correlated with high ground-state activation of PKR and cellular hypersensitivity to apoptotic cues, but enforced expression of NPM in these mutant cells reduced aberrant apoptotic responses. Inhibition of PKR by NPM may be one mechanism by which neoplastic clones evolve in sporadic malignancies and in neoplastic cells arising in the context of the cancer predisposition syndrome, Fanconi anemia.     

Nucleophosmin/B23 activates Aurora A at the centrosome through phosphorylation of serine 89

Aurora A (AurA) is a major mitotic protein kinase involved in centrosome maturation and spindle assembly. Nucleophosmin/B23 (NPM) is a pleiotropic nucleolar protein involved in a variety of cellular processes including centrosome maturation. In the present study, we report that NPM is a strong activator of AurA kinase activity. NPM and AurA coimmunoprecipitate and colocalize to centrosomes in G2 phase, where AurA becomes active. In contrast with previously characterized AurA activators, NPM does not trigger autophosphorylation of AurA on threonine 288. NPM induces phosphorylation of AurA on serine 89, and this phosphorylation is necessary for activation of AurA. These data were confirmed in vivo, as depletion of NPM by ribonucleic acid interference eliminated phosphorylation of CDC25B on S353 at the centrosome, indicating a local loss of AurA activity. Our data demonstrate that NPM is a strong activator of AurA kinase activity at the centrosome and support a novel mechanism of activation for AurA.     

Nucleophosmin/B23 Inhibits Eg5-mediated Microtubule Depolymerization by Inactivating Its ATPase Activity

Nucleophosmin/B23, an abundant nucleolar protein, plays multiple roles in cell growth and proliferation, and yet, little has been studied about its function in regulating dynamics of microtubules. Here, we report that B23 directly interacts with Eg5, a member of the kinesin family, in the cytosol. The DNA/RNA binding domain of B23 and the motor domain of Eg5 were found to be involved in their interaction. Both in vivo and in vitro evidences showed that B23 acts as an upstream regulator of Eg5 in promoting microtubule polymerization. Moreover, we further demonstrated that B23 regulates microtubule dynamics by directly inhibiting Eg5 ATPase activity.     

RRP1B targets PP1 to mammalian cell nucleoli and is associated with Pre-60S ribosomal subunits

A pool of protein phosphatase 1 (PP1) accumulates within nucleoli and accounts for a large fraction of the serine/threonine protein phosphatase activity in this subnuclear structure. Using a combination of fluorescence imaging with quantitative proteomics, we mapped the subnuclear localization of the three mammalian PP1 isoforms stably expressed as GFP-fusions in live cells and identified RRP1B as a novel nucleolar targeting subunit that shows a specificity for PP1β and PP1γ. RRP1B, one of two mammalian orthologues of the yeast Rrp1p protein, shows an RNAse-dependent localization to the granular component of the nucleolus and distributes in a similar manner throughout the cell cycle to proteins involved in later steps of rRNA processing. Quantitative proteomic analysis of complexes containing both RRP1B and PP1γ revealed enrichment of an overlapping subset of large (60S) ribosomal subunit proteins and pre-60S nonribosomal proteins involved in mid-late processing. Targeting of PP1 to this complex by RRP1B in mammalian cells is likely to contribute to modulation of ribosome biogenesis by mechanisms involving reversible phosphorylation events, thus playing a role in the rapid transduction of cellular signals that call for regulation of ribosome production in response to cellular stress and/or changes in growth conditions.     

Curcumin inhibits immunostimulatory function of dendritic cells: MAPKs and translocation of NF-kappa B as potential targets

Curcumin has been shown to exhibit anti-inflammatory, antimutagenic, and anticarcinogenic activities. However, the effect of curcumin on the maturation and immunostimulatory function of dendritic cells (DC) largely remains unknown. In this study, we examined whether curcumin can influence surface molecule expression, cytokine production, and their underlying signaling pathways in murine bone marrow-derived DC. DC were derived from murine bone marrow cells and used as immature or LPS-stimulated mature cells. The DC were tested for surface molecule expression, cytokine production, dextran uptake, the capacity to induce T cell differentiation, and their underlying signaling pathways. Curcumin significantly suppressed CD80, CD86, and MHC class II expression, but not MHC class I expression, in the DC. The DC also exhibited impaired IL-12 expression and proinflammatory cytokine production (IL-1beta, IL-6, and TNF-alpha). The curcumin-treated DC were highly efficient at Ag capture, via mannose receptor-mediated endocytosis. Curcumin inhibited LPS-induced MAPK activation and the translocation of NF-kappaB p65. In addition, the curcumin-treated DC showed an impaired induction of Th1 responses and a normal cell-mediated immune response. These novel findings provide new insight into the immunopharmacological role of curcumin in impacting on the DC. These novel findings open perspectives for the understanding of the immunopharmacological role of curcumin and therapeutic adjuvants for DC-related acute and chronic diseases.