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.     

Arf-induced turnover of the nucleolar nucleophosmin-associated SUMO-2/3 protease Senp3

The stabilization and subcellular localization of the p19Arf tumor suppressor protein and the SUMO-2/3 deconjugating protease Senp3 each depend upon their binding to the abundant nucleolar protein nucleophosmin (Npm/B23). Senp3 and p19Arf antagonize each otherâ€?s functions in regulating the SUMOylation of target proteins including Npm itself. The p19Arf protein triggers the sequential phosphorylation, polyubiquitination, and rapid proteasomal degradation of Senp3, and this ability of p19Arf to accelerate Senp3 turnover also depends on the presence of Npm. In turn, endogenous p19Arf and Senp3 are both destabilized in viable Npm-null mouse embryo fibroblasts (that also lack p53), and reintroduction of the human NPM protein into these cells reverses this phenotype. NPM mutants that retain their acidic and oligomerization domains can re-stabilize both p19Arf and Senp3 in this setting, but the nucleolar localization of NPM is not strictly required for these effects. Knockdown of Senp3 with shRNAs mimics the anti-proliferative functions of p19Arf in cells that lack p53 alone or in triple knock-out cells that lack the Arf, Mdm2 and p53 genes. These findings reinforce the hypothesis that the p53-independent tumor suppressive functions of p19Arf may be mediated by its ability to antagonize Senp3, thereby inducing cell cycle arrest by abnormally elevating the cellular levels of SUMOylated proteins.     

Cocompartmentalization of p53 and Mdm2 is a major determinant for Mdm2-mediated degradation of p53

The product of the Mdm2 oncogene directly interacts with p53 and promotes its ubiquitination and proteasomal degradation. Initial biological studies identified nuclear export sequences (NES), similar to that of the Rev protein from the human immunodeficiency virus, both in Mdm2 and p53. The reported phenotypes resulting from mutation of these NESs, together with results obtained using the nuclear export inhibitor leptomycin B (LMB), have led to a model according to which nuclear export of p53 (via either the NES of Mdm2 or its own NES) is required for efficient p53 degradation. In this study we demonstrate that Mdm2 can promote degradation of p53 in the nucleus or in the cytoplasm, provided both proteins are colocalized. We also investigated if nuclear export is an obligate step on the p53 degradation pathway. We find that (1) when proteasome activity is inhibited, ubiquitinated p53 accumulates in the nucleus and not in the cytoplasm; (2) Mdm2 with a mutated NES can efficiently mediate degradation of wild type p53 or p53 with a mutated NES; (3) the nuclear export inhibitor LMB can increase the steady-state level of p53 by inhibiting Mdm2-mediated ubiquitination of p53; and (4) LMB fails to inhibit Mdm2-mediated degradation of the p53NES mutant, demonstrating that Mdm2-dependent proteolysis of p53 is feasible in the nucleus in the absence of any nuclear export. Therefore, given cocompartmentalization, Mdm2 can promote ubiquitination and proteasomal degradation of p53 with no absolute requirement for nuclear to cytoplasmic transport.     

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.     

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.     

Leukemia-Associated Mutations in Nucleophosmin Alter Recognition by CRM1: Molecular Basis of Aberrant Transport

Nucleophosmin (NPM) is a nucleocytoplasmic shuttling protein, normally enriched in nucleoli, that performs several activities related to cell growth. NPM mutations are characteristic of a subtype of acute myeloid leukemia (AML), where mutant NPM seems to play an oncogenic role. AML-associated NPM mutants exhibit altered subcellular traffic, being aberrantly located in the cytoplasm of leukoblasts. Exacerbated export of AML variants of NPM is mediated by the nuclear export receptor CRM1, and due, in part, to a mutationally acquired novel nuclear export signal (NES). To gain insight on the molecular basis of NPM transport in physiological and pathological conditions, we have evaluated the export efficiency of NPM in cells, and present new data indicating that, in normal conditions, wild type NPM is weakly exported by CRM1. On the other hand, we have found that AML-associated NPM mutants efficiently form complexes with CRM1^HA (a mutant CRM1 with higher affinity for NESs), and we have quantitatively analyzed CRM1^HA interaction with the NES motifs of these mutants, using fluorescence anisotropy and isothermal titration calorimetry. We have observed that the affinity of CRM1^HA for these NESs is similar, which may help to explain the transport properties of the mutants. We also describe NPM recognition by the import machinery. Our combined cellular and biophysical studies shed further light on the determinants of NPM traffic, and how it is dramatically altered by AML-related mutations.     

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.     

The in vivo role of the RP-Mdm2-p53 pathway in signaling oncogenic stress induced by pRb inactivation and Ras overexpression

The Mdm2-p53 tumor suppression pathway plays a vital role in regulating cellular homeostasis by integrating a variety of stressors and eliciting effects on cell growth and proliferation. Recent studies have demonstrated an in vivo signaling pathway mediated by ribosomal protein (RP)-Mdm2 interaction that responds to ribosome biogenesis stress and evokes a protective p53 reaction. It has been shown that mice harboring a Cys-to-Phe mutation in the zinc finger of Mdm2 that specifically disrupts RP L11-Mdm2 binding are prone to accelerated lymphomagenesis in an oncogenic c-Myc driven mouse model of Burkitt's lymphoma. Because most oncogenes when upregulated simultaneously promote both cellular growth and proliferation, it therefore stands to reason that the RP-Mdm2-p53 pathway might also be essential in response to oncogenes other than c-Myc. Using genetically engineered mice, we now show that disruption of the RP-Mdm2-p53 pathway by an Mdm2(C305F) mutation does not accelerate prostatic tumorigenesis induced by inactivation of the pRb family proteins (pRb/p107/p130). In contrast, loss of p19Arf greatly accelerates the progression of prostate cancer induced by inhibition of pRb family proteins. Moreover, using ectopically expressed oncogenic H-Ras we demonstrate that p53 response remains intact in the Mdm2(C305F) mutant MEF cells. Thus, unlike the p19Arf-Mdm2-p53 pathway, which is considered a general oncogenic response pathway, the RP-Mdm2-p53 pathway appears to specifically suppress tumorigenesis induced by oncogenic c-Myc.     

Cytoplasmically "sequestered" wild type p53 protein is resistant to Mdm2-mediated degradation.

The Mdm2 oncoprotein mediates p53 degradation at cytoplasmic proteasomes and is the principal regulator for maintaining low, often undetectable levels of p53 in unstressed cells. However, a subset of human tumors including neuroblastoma constitutively harbor high levels of wild type p53 protein localized to the cytoplasm. Here we show that the abnormal p53 accumulation in such cells is due to a profound resistance to Mdm2-mediated degradation. Overexpression of Mdm2 in neuroblastoma (NB)(1) cell lines failed to decrease the high steady state levels of endogenous p53. Moreover, exogenous p53, when introduced into these cells, was also resistant to Mdm2-directed degradation. This resistance is not due to a lack of Mdm2 expression in NB cells or a lack of p53-Mdm2 interaction, nor is it due to a deficiency in the ubiquitination state of p53 or proteasome dysfunction. Instead, Mdm2-resistant p53 from NB cells is associated with covalent modification of p53 and masking of the modification-sensitive PAb 421 epitope. This system provides evidence for an important level of regulation of Mdm2-directed p53 destruction in vivo that is linked to p53 modification.     

Nucleolar Arf sequesters Mdm2 and activates p53

The Ink4/Arf locus encodes two tumour-suppressor proteins, p16Ink4a and p19Arf, that govern the antiproliferative functions of the retinoblastoma and p53 proteins, respectively. Here we show that Arf binds to the product of the Mdm2 gene and sequesters it into the nucleolus, thereby preventing negative-feedback regulation of p53 by Mdm2 and leading to the activation of p53 in the nucleoplasm. Arf and Mdm2 co-localize in the nucleolus in response to activation of the oncoprotein Myc and as mouse fibroblasts undergo replicative senescence. These topological interactions of Arf and Mdm2 point towards a new mechanism for p53 activation.     

Reactivating the ARF-p53 axis in AML cells by targeting ULF

The tumor suppressor ARF plays an essential role in the cellular response to oncogenic stress mainly through activation of p53. Nucleophosmin (NPM), a multifunctional protein, forms a stable protein complex with ARF in the nucleolus and protects ARF from the proteasome-mediated degradation. Notably, NPM is mutated in about one third of acute myeloid leukaemia (AML) patients and these mutations lead to aberrant cytoplasmic dislocation of nucleophosmin (NPM-c). Cytoplasmic NPM mutants lose their abilities to retain ARF in the nucleolus and fail to stabilize ARF. Thus, activation of the ARF-p53 axis is significantly compromised in these AML cells. We have recently identified the ubiquitin ligase of ARF (ULF) as a key factor that controls ARF turnover in human cells. Here, we found that the steady levels of both ARF and p53 are very low in human acute myeloid leukaemia OCI-AML3 cells expressing cytoplamsic dislocated nucleophosmin (NPM-c). As expected, ARF is very unstable and rapidly degraded by proteasome. Nevertheless, ULF knockdown stabilizes ARF and reactivates p53 responses in these AML cells. These results further demonstrate that ULF is a bona fide E3 ligase for ARF and also suggest that ULF is an important target for activating the ARF-p53 axis in human AML cells.Key words: ARF, ubiquitination, ULF, p53, NPM, B23, NPM-c     

Reactivating the ARF-p53 axis in AML cells by targeting ULF

The tumor suppressor ARF plays an essential role in the cellular response to oncogenic stress mainly through activation of p53. Nucleophosmin (NPM), a multifunctional protein, forms a stable protein complex with ARF in the nucleolus and protects ARF from the proteasome-mediated degradation. Notably, NPM is mutated in about one third of acute myeloid leukaemia (AML) patients and these mutations lead to aberrant cytoplasmic dislocation of nucleophosmin (NPM-c). Cytoplasmic NPM mutants lose their abilities to retain ARF in the nucleolus and fail to stabilize ARF. Thus, activation of the ARF-p53 axis is significantly compromised in these AML cells. We have recently identified the ubiquitin ligase of ARF (ULF) as a key factor that controls ARF turnover in human cells. Here, we found that the steady levels of both ARF and p53 are very low in human acute myeloid leukaemia OCI-AML3 cells expressing cytoplamsic dislocated nucleophosmin (NPM-c). As expected, ARF is very unstable and rapidly degraded by proteasome. Nevertheless, ULF knockdown stabilizes ARF and reactivates p53 responses in these AML cells. These results further demonstrate that ULF is a bona fide E3 ligase for ARF and also suggest that ULF is an important target for activating the ARF-p53 axis in human AML cells.     

Decreased Mdm2 Expression Inhibits Tumor Development and Extends Survival Independent of Arf and Dependent on p53

Inactivation of the Arf-Mdm2-p53 tumor suppressor pathway is a necessary event for tumorigenesis. Arf controls Mdm2, which in turn regulates p53, but Arf and Mdm2 also have p53-independent functions that affect tumor development. Moreover, inhibition of oncogene-induced tumorigenesis relies on Arf and p53, but the requirements of Arf and p53 in tumor development initiated in the absence of overt oncogene overexpression and the role of Mdm2 in this process remain unclear. In a series of genetic experiments in mice with defined deficiencies in Arf, Mdm2 and/or p53, we show Mdm2 haploinsufficiency significantly delayed tumorigenesis in mice deficient in Arf and p53. Mdm2 heterozygosity significantly inhibited tumor development in the absence of Arf, and in contrast to Myc oncogene-driven cancer, this delay in tumorigenesis could not be rescued with the presence of one allele of Arf. Notably, Mdm2 haploinsufficieny blocked the accelerated tumor development in Arf deficient mice caused by p53 heterozygosity. However, tumorigenesis was not inhibited in Mdm2 heterozygous mice lacking both alleles of p53 regardless of Arf status. Surprisingly, loss of Arf accelerated tumor development in p53-null mice. Tumor spectrum was largely dictated by Arf and p53 status with Mdm2 haploinsufficiency only modestly altering the tumor type in some of the genotypes and not the number of primary tumors that arose. Therefore, the significant effects of Mdm2 haploinsufficiency on tumor latency were independent of Arf and required at least one allele of p53, and an Mdm2 deficiency had minor effects on the types of tumors that developed. These data also demonstrate that decreased levels of Mdm2 are protective in the presence of multiple genetic events in Arf and p53 genes that normally accelerate tumorigenesis.     

hGTSE-1 expression stimulates cytoplasmic localization of p53

hGTSE-1 (human G(2) and S phase-expressed-1) is a cell cycle-regulated protein mainly localized in the cytoplasm and apparently associated with the microtubules. hGTSE-1 is able to down-regulate levels and activity of the p53 tumor suppressor protein: it binds the C-terminal region of p53 and represses its ability to induce apoptosis after DNA damage. Here we report that, after DNA damage, hGTSE-1 becomes stabilized in a p53-independent way and accumulated in the nucleus. Further characterization of hGTSE-1 localization revealed increased nuclear staining in unstressed cells after treatment with the nuclear export inhibitor leptomycin B, or when a nuclear export signal (NES) located in its C-terminal region was mutated. Finally, we provide evidence that hGTSE-1 ectopic expression, in addition to p53 protein levels down-regulation, is able to enhance cytoplasmic localization of p53. Interestingly, NES-mutated hGTSE-1 accumulates in the nucleus, binds p53 but looses its ability to enhance cytoplasmic redistribution of p53 and to regulate p53 protein levels. Similarly, when wild type hGTSE-1 functions on p53 were analyzed in cells lacking Mdm2, it failed in regulating both p53 localization and protein levels, thus indicating that hGTSE-1 requires an intact NES and functional Mdm2 for the regulation of p53. Our results provide new insights into the mechanism of hGTSE-1 function, whereby its characterized nucleo-cytoplasmic shuttling ability is required to regulate p53.     

Shuttling imbalance of MLF1 results in p53 instability and increases susceptibility to oncogenic transformation

Myeloid leukemia factor 1 (MLF1) stabilizes the activity of the tumor suppressor p53 by suppressing its E3 ubiquitin ligase, COP1, through a third component of the COP9 signalosome (CSN3). However, little is known about how MLF1 functions upstream of the CSN3-COP1-p53 pathway and how its deregulation by the formation of the fusion protein nucleophosmin (NPM)-MLF1, generated by t(3;5)(q25.1;q34) chromosomal translocation, leads to leukemogenesis. Here we show that MLF1 is a cytoplasmic-nuclear-shuttling protein and that its nucleolar localization on fusing with NPM prevents the full induction of p53 by both genotoxic and oncogenic cellular stress. The majority of MLF1 was located in the cytoplasm, but the treatment of cells with leptomycin B rapidly induced a nuclear accumulation of MLF1. A mutation of the nuclear export signal (NES) motif identified in the MLF1 sequence enhanced the antiproliferative activity of MLF1. The fusion of MLF1 with NPM translocated MLF1 to the nucleolus and abolished the growth-suppressing activity. The introduction of NPM-MLF1 into early-passage murine embryonic fibroblasts allowed the cells to escape from cellular senescence at a markedly earlier stage and induced neoplastic transformation in collaboration with the oncogenic form of Ras. Interestingly, disruption of the MLF1-derived NES sequence completely abolished the growth-promoting activity of NPM-MLF1 in murine fibroblasts and hematopoietic cells. Thus, our results provide important evidence that the shuttling of MLF1 is critical for the regulation of cell proliferation and a disturbance in the shuttling balance increases the cell's susceptibility to oncogenic transformation.     

ARF impedes NPM/B23 shuttling in an Mdm2-sensitive tumor suppressor pathway

The ARF tumor suppressor is widely regarded as an upstream activator of p53-dependent growth arrest and apoptosis. However, recent findings indicate that ARF can also regulate the cell cycle in the absence of p53. In search of p53-independent ARF targets, we isolated nucleophosmin (NPM/B23), a protein we show is required for proliferation, as a novel ARF binding protein. In response to hyperproliferative signals, ARF is upregulated, resulting in the nucleolar retention of NPM and concomitant cell cycle arrest. The Mdm2 oncogene outcompetes NPM/B23 for ARF binding, and introduction of Mdm2 reverses ARF's p53-independent properties: in vitro, NPM is released from ARF-containing protein complexes, and in vivo S phase progression ensues. ARF induction by oncogenes or replicative senescence does not alter NPM/B23 protein levels but rather prevents its nucleocytoplasmic shuttling without inhibiting rRNA processing. By actively sequestering NPM in the nucleolus, ARF utilizes an additional mechanism of tumor suppression, one that is readily antagonized by Mdm2.     

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 phosphoprotein modifications as a marker of apoptosis induced by RITA treatment

Reactivating p53 and Inducing Tumor Apoptosis (RITA) has been reported to increase the p53 activity and to trigger p53-dependent apoptosis in cancer cells with wild-type p53. Tumor suppressor p53 interacts with nucleolar phosphoproteins nucleophosmin (NPM) and nucleolin (NCL), which have crucial role in many cellular processes. Specific NPM mutations associated with acute myeloid leukemia (AML) cause aberrant localization of NPM and p53 in the cytoplasm with possible impact on the p53 function. We tested an effect of RITA on primary cells, and we found significant RITA-induced changes in NPM and NCL phosphorylation associated with apoptosis in cells of AML patients, but not that of healthy donors. Subsequent screening of several AML cell lines revealed heterogeneous response to RITA, and confirmed an association of the specific phosphorylation with apoptosis. While decreased NCL phosphorylation at Threonines T76 and T84 could be attributed to RITA-induced cell cycle arrest, enhanced NPM phosphorylation at Threonine T199 was not accompanied by the cell cycle changes and it correlated with sensitivity to RITA. Simultaneously, inverse changes occurred at Serine S4 of the NPM. These new findings of RITA mechanism of action could establish the NPM pT199/pS4 ratio as a marker for suitability of RITA treatment of AML cells.