Physical and functional interactions of the Arf tumor suppressor protein with nucleophosmin/B23

The Arf tumor suppressor inhibits cell cycle progression through both p53-dependent and p53-independent mechanisms, including interference with rRNA processing. Using tandem-affinity-tagged p19(Arf), we purified Arf-associated proteins from mouse NIH 3T3 fibroblasts undergoing cell cycle arrest. Tagged p19(Arf) associated with nucleolar and ribosomal proteins, including nucleophosmin/B23 (NPM), a protein thought to foster the maturation of preribosomal particles. NPM is an abundant protein, only a minor fraction of which binds to p19(Arf); however, a significant proportion of p19(Arf) associates with NPM. The interaction between p19(Arf) and NPM requires amino acid sequences at the Arf amino terminus, which are also required for Mdm2 binding, as well as the central acidic domain of NPM and an adjacent segment that regulates NPM oligomerization. The interaction between p19(Arf) and NPM occurs in primary mouse embryonic fibroblasts, including those lacking both Mdm2 and p53. In an NIH 3T3 derivative cell line (MT-Arf) engineered to conditionally express an Arf transgene, induced p19(Arf) associates with NPM and colocalizes with it in high-molecular-weight complexes (2 to 5 MDa). An NPM mutant lacking its carboxyl-terminal nucleic acid-binding domain oligomerizes with endogenous NPM, inhibits p19(Arf) from entering into 2- to 5-MDa particles, and overrides the ability of p19(Arf) to retard rRNA processing.     

Nucleophosmin and its AML-associated mutant regulate c-Myc turnover through Fbw7 gamma

Mutations leading to aberrant cytoplasmic localization of nucleophosmin (NPM) are the most frequent genetic alteration in acute myelogenous leukemia (AML). NPM binds the Arf tumor suppressor and protects it from degradation. The AML-associated NPM mutant (NPMmut) also binds p19Arf but is unable to protect it from degradation, which suggests that inactivation of p19Arf contributes to leukemogenesis in AMLs. We report here that NPM regulates turnover of the c-Myc oncoprotein by acting on the F-box protein Fbw7gamma, a component of the E3 ligase complex involved in the ubiquitination and proteasome degradation of c-Myc. NPM was required for nucleolar localization and stabilization of Fbw7gamma. As a consequence, c-Myc was stabilized in cells lacking NPM. Expression of NPMmut also led to c-Myc stabilization because of its ability to interact with Fbw7gamma and delocalize it to the cytoplasm, where it is degraded. Because Fbw7 induces degradation of other growth-promoting proteins, the NPM-Fbw7 interaction emerges as a central tumor suppressor mechanism in human cancer.     

Nucleophosmin is required for DNA integrity and p19Arf protein stability

Nucleophosmin (NPM) is a nucleolar phosphoprotein that binds the tumor suppressors p53 and p19(Arf) and is thought to be indispensable for ribogenesis, cell proliferation, and survival after DNA damage. The NPM gene is the most frequent target of genetic alterations in leukemias and lymphomas, though its role in tumorigenesis is unknown. We report here the first characterization of a mouse NPM knockout strain. Lack of NPM expression results in accumulation of DNA damage, activation of p53, widespread apoptosis, and mid-stage embryonic lethality. Fibroblasts explanted from null embryos fail to grow and rapidly acquire a senescent phenotype. Transfer of the NPM mutation into a p53-null background rescued apoptosis in vivo and fibroblast proliferation in vitro. Cells null for both p53 and NPM grow faster than control cells and are more susceptible to transformation by activated oncogenes, such as mutated Ras or overexpressed Myc. In the absence of NPM, Arf protein is excluded from nucleoli and is markedly less stable. Our data demonstrate that NPM regulates DNA integrity and, through Arf, inhibits cell proliferation and are consistent with a putative tumor-suppressive function of NPM.     

Nucleophosmin (B23) targets ARF to nucleoli and inhibits its function

The ARF tumor suppressor is a nucleolar protein that activates p53-dependent checkpoints by binding Mdm2, a p53 antagonist. Despite persuasive evidence that ARF can bind and inactivate Mdm2 in the nucleoplasm, the prevailing view is that ARF exerts its growth-inhibitory activities from within the nucleolus. We suggest ARF primarily functions outside the nucleolus and provide evidence that it is sequestered and held inactive in that compartment by a nucleolar phosphoprotein, nucleophosmin (NPM). Most cellular ARF is bound to NPM regardless of whether cells are proliferating or growth arrested, indicating that ARF-NPM association does not correlate with growth suppression. Notably, ARF binds NPM through the same domains that mediate nucleolar localization and Mdm2 binding, suggesting that NPM could control ARF localization and compete with Mdm2 for ARF association. Indeed, NPM knockdown markedly enhanced ARF-Mdm2 association and diminished ARF nucleolar localization. Those events correlated with greater ARF-mediated growth suppression and p53 activation. Conversely, NPM overexpression antagonized ARF function while increasing its nucleolar localization. These data suggest that NPM inhibits ARF's p53-dependent activity by targeting it to nucleoli and impairing ARF-Mdm2 association.     

Nucleolar Arf tumor suppressor inhibits ribosomal RNA processing

The p19(Arf) tumor suppressor, a nucleolar protein, binds to Mdm2 to induce p53-dependent cell cycle arrest. Arf also prevents the proliferation of cells lacking Mdm2 and p53, albeit less efficiently. We show that p19(Arf) inhibits production of ribosomal RNA, retarding processing of 47/45S and 32S precursors. These effects correlate with but do not strictly depend upon inhibition of rRNA biosynthesis or cell cycle arrest, are not mimicked by p53, and require neither p53 nor Mdm2. Arf mutants lacking conserved amino acid residues 2-14 do not block rRNA synthesis and processing or inhibit cell proliferation. Evolution may have linked a primordial nucleolar Arf function to Mdm2 and p53, creating a more efficient checkpoint-signaling pathway for coordinating ribosomal biogenesis and cell cycle progression.     

p19Arf-p53 tumor suppressor pathway regulates cell motility by suppression of phosphoinositide 3-kinase and Rac1 GTPase activities

The p19(Arf)-p53 tumor suppressor pathway plays a critical role in cell-cycle checkpoint control and apoptosis, whereas Rho family small GTPases are key regulators of actin structure and cell motility. By using primary mouse embryonic fibroblasts that lack Arf, p53, or both, we studied the involvement of the p19(Arf)-p53 pathway in the regulation of cell motility and its relationship with Rho GTPases. Deletion of Arf and/or p53 led to actin cytoskeleton reorganization and a significant increase in cell motility. The endogenous phosphoinositide (PI) 3- kinase and Rac1 activities were elevated in Arf(-/-) and p53(-/-) cells, and these activities are required for p19(Arf)- and p53-regulated migration. Reintroduction of the wild type Arf or p53 genes into Arf(-/-) or p53(-/-) cells reversed the PI 3-kinase and Rho GTPase activities as well as the migration phenotype. These results suggest a functional relationship between an established tumor suppressor pathway and a signaling module that controls actin structure and cell motility and show that p19(Arf) and p53 negatively regulate cell migration by suppression of PI 3-kinase and Rac1 activities.     

TRIM28 Is an E3 Ligase for ARF-Mediated NPM1/B23 SUMOylation That Represses Centrosome Amplification

The tumor suppressor ARF enhances the SUMOylation of target proteins; however, the physiological function of ARF-mediated SUMOylation has been unclear due to the lack of a known, associated E3 SUMO ligase. Here we uncover TRIM28/KAP1 as a novel ARF-binding protein and SUMO E3 ligase for NPM1/B23. ARF and TRIM28 cooperate to SUMOylate NPM1, a nucleolar protein that regulates centrosome duplication and genomic stability. ARF-mediated SUMOylation of NPM1 was attenuated by TRIM28 depletion and enhanced by TRIM28 overexpression. Coexpression of ARF and TRIM28 promoted NPM1 centrosomal localization by enhancing its SUMOylation and suppressed centrosome amplification; these functions required the E3 ligase activity of TRIM28. Conversely, depletion of ARF or TRIM28 increased centrosome amplification. ARF also counteracted oncogenic Ras-induced centrosome amplification. Centrosome amplification is often induced by oncogenic insults, leading to genomic instability. However, the mechanisms employed by tumor suppressors to protect the genome are poorly understood. Our findings suggest a novel role for ARF in maintaining genome integrity by facilitating TRIM28-mediated SUMOylation of NPM1, thus preventing centrosome amplification.     

Nucleolar stress characterized by downregulation of nucleophosmin: a novel cause of neuronal degeneration

Nucleophosmin (NPM) is a multifunctional nucleolar protein that has been linked with nucleolar stress. In non-neuronal cell lines, NPM may enhance or inhibit the activity of tumor suppressor p53, a major apoptotic protein. The relationship between NPM and p53 in the central nervous system (CNS) remains unknown. Here, we assessed the role of NPM in the CNS using a model of seizure-induced neurodegeneration. We show that NPM overexpression is neuroprotective against kainic acid-induced excitotoxicity, and that downregulation of NPM is pro-apoptotic in a p53-independent manner. These results suggest a key role for NPM in promoting neuronal survival and a novel mechanism of neuronal degeneration triggered by nucleolar stress.     

Nucleophosmin is a binding partner of nucleostemin in human osteosarcoma cells

Nucleostemin (NS) is expressed in the nucleoli of adult and embryonic stem cells and in many tumors and tumor-derived cell lines. In coimmunoprecipitation experiments, nucleostemin is recovered with the tumor suppressor p53, and more recently we have demonstrated that nucleostemin exerts its role in cell cycle progression via a p53-dependent pathway. Here, we report that in human osteosarcoma cells, nucleostemin interacts with nucleophosmin, a nucleolar protein believed to possess oncogenic potential. Nucleostemin (NS) and nucleophosmin (NPM) displayed an extremely high degree of colocalization in the granular component of the nucleolus during interphase, and both proteins associated with prenucleolar bodies in late mitosis before the reformation of nucleoli. Coimmunoprecipitation experiments revealed that NS and NPM co-reside in complexes, and yeast two-hybrid experiments confirmed that they are interactive proteins, revealing the NPM-interactive region to be the 46-amino acid N-terminal domain of NS. In bimolecular fluorescence complementation studies, bright nucleolar signals were observed, indicating that these two proteins directly interact in the nucleolus in vivo. These results support the notion that cell cycle regulatory proteins congress and interact in the nucleolus, adding to the emerging concept that this nuclear domain has functions beyond ribosome production.     

Structural consequences of nucleophosmin mutations in acute myeloid leukemia

Mutations affecting NPM1 (nucleophosmin) are the most common genetic lesions found in acute myeloid leukemia (AML). NPM1 is one of the most abundant proteins found in the nucleolus and has links to the MDM2/p53 tumor suppressor pathway. A distinctive feature of NPM1 mutants in AML is their aberrant localization to the cytoplasm of leukemic cells. This mutant phenotype is the result of the substitution of several C-terminal residues, including one or two conserved tryptophan residues, with a leucine-rich nuclear export signal. The exact molecular mechanism underlying the loss of nucleolar retention, and the role of the tryptophans, remains unknown. In this study we have determined the structure of an independently folded globular domain in the C terminus of NPM1 using NMR spectroscopy, and we report that the conserved tryptophans are critical for structure. This domain is necessary for the nucleolar targeting of NPM1 and is disrupted by mutations in AML with cytoplasmic NPM1. Furthermore, we identify conserved surface-exposed lysine residues that are functionally rather than structurally important for nucleolar localization. This study provides new focus for efforts to understand the pathogenesis of AML with cytoplasmic NPM1 and may be used to aid the design of small molecules that target the C-terminal domain of NPM1 to act as novel anti-proliferative and anti-leukemia therapeutics.     

Regulation of p14ARF Through Subnuclear Compartmentalization

The p53-mediated pathway cell cycle arrest and apoptosis is central to cancer and an important point of focus for therapeutics development. The p14ARF ("ARF") tumor suppressor induces the p53 pathway in response to oncogene activation or DNA damage. However, ARF is predominantly nucleolar in localization and engages in several interactions with nucleolar proteins, whereas p53 is nucleoplasmic. This raises the question as to how ARF initiates its involvement in the p53 pathway. We have found that UV irradiation of cells disrupts the interaction of ARF with two of its nucleolar binding partners, B23(NPM, nucleophosmin, NO38, numatrin) and topoisomerase I, and promotes an immediate and transient subnuclear redistribution of ARF to the nucleoplasm, where it can engage the p53 pathway (Lee et al, Cancer Research 65:9834-42; 2005). The results support a model in which the nucleolus serves as a p53 upstream sensor of cellular stress, and add to a growing body of evidence that nucleolar sequestration of ARF prevents activation of p53. The results also have therapeutic implications for therapies based on exploiting p53 and other cellular stress response pathways to suppress cancer.     

The Wnt Target Protein Peter Pan Defines a Novel p53-independent Nucleolar Stress-Response Pathway

Proper ribosome formation is a prerequisite for cell growth and proliferation. Failure of this process results in nucleolar stress and p53-mediated apoptosis. The Wnt target Peter Pan (PPAN) is required for 45 S rRNA maturation. So far, the role of PPAN in nucleolar stress response has remained elusive. We demonstrate that PPAN localizes to mitochondria in addition to its nucleolar localization and inhibits the mitochondrial apoptosis pathway in a p53-independent manner. Loss of PPAN induces BAX stabilization, depolarization of mitochondria, and release of cytochrome c, demonstrating its important role as an anti-apoptotic factor. Staurosporine-induced nucleolar stress and apoptosis disrupt nucleolar PPAN localization and induce its accumulation in the cytoplasm. This is accompanied by phosphorylation and subsequent cleavage of PPAN by caspases. Moreover, we show that PPAN is a novel interaction partner of the anti-apoptotic protein nucleophosmin (NPM). PPAN depletion induces NPM and upstream-binding factor (UBF) degradation, which is independent of caspases. In summary, we provide evidence for a novel nucleolar stress-response pathway involving PPAN, NPM, and BAX to guarantee cell survival in a p53-independent manner.