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Previous Article | Next Article 
Molecular and Cellular Biology, November 2004, p. 9327-9338, Vol. 24, No. 21
0270-7306/04/$08.00+0 DOI: 10.1128/MCB.24.21.9327-9338.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Yue Yu,
Leonard B. Maggi Jr., and
Jason D. Weber*
Department of Medicine, Division of Molecular Oncology, Siteman Cancer Center, Washington University School of Medicine, St. Louis, Missouri
Received 28 April 2004/ Returned for modification 1 June 2004/ Accepted 6 August 2004
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ABSTRACT |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mounting evidence suggests that the ARF-p53-Mdm2 pathway is not strictly linear. Mice engineered to overexpress a Myc transgene under the control of the immunoglobulin heavy chain enhancer (Eµ) develop B-cell lymphomas that exhibit biallelic ARF deletion, mutation of p53, or Mdm2 overexpression (11). Additional molecular analysis revealed that several tumors which lacked functional p53 also displayed Mdm2 overexpression, arguing against a simple epistatic relationship among ARF, p53, and Mdm2 (11). Additionally, Carnero and colleagues showed that diminished ARF expression resulted in bypassing of replicative senescence, whereas induction of ARF restored ARF's tumor-suppressive properties, even in the presence of a dominant-negative p53 mutant (7). In support of these initial findings, two other groups have demonstrated the physiological significance of the p53-independent ARF pathway through examination of relevant mouse model systems. Mice lacking ARF primarily develop lymphomas and fibrosarcomas (18, 19); in contrast, p53-null animals consistently display lymphomas and osteosarcomas (16). Surprisingly, ARF/p53 double-null mice exhibit multiple tumors with distinct origins, namely, the simultaneous presentation of carcinomas with lymphomas (45). In a corroborative study, wild-type and p53-null mice displayed normal development of the hyaloid vascular system in the eye, yet ARF-null and ARF/p53 double-null animals failed to show proper regression of this structure (27). Collectively, these data reinforce the fact that ARF and p53 play shared, as well as unique roles, as sensors of inappropriate cell growth.
Clearly, genetic studies have confirmed the existence of an alternative, p53-independent ARF tumor surveillance pathway. In an effort to uncover novel players in this pathway, we isolated ARF-containing protein complexes from cells devoid of p53 and Mdm2. Here, we report the identification of nucleophosmin (NPM), a nucleolar phosphoprotein and clinical marker of highly proliferative cells, as a bona fide ARF-interacting protein. Recently, two studies have also described the formation of ARF-NPM complexes in mouse fibroblasts but drew two differing conclusions: ARF targets NPM/B23 for degradation (15) and/or ARF prevents rRNA processing (4). Our data offer a contrasting view. Specifically, ARF binds and retains NPM in the cell nucleolus, effectively impeding the nucleocytoplasmic shuttling of NPM, resulting in subsequent growth arrest. Importantly, Mdm2 antagonizes these effects, thereby preventing ARF-induced withdrawal from the cell cycle. Two intriguing implications of our findings are that (i) ARF, through its interaction with NPM, may directly access and inhibit additional cytoplasmic growth-promoting events and (ii) Mdm2, a well-established mediator of oncogenesis, dictates ARF's tumor-suppressive capacity in the absence of functional p53.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Plasmid constructs.
Vectors encoding p19ARF, p19ARF 
1-14, and Mdm2(210-304) were used as previously described (44) or were subcloned into the EcoRI sites of pEGFP-C1 plasmids (Clontech). A retroviral plasmid encoding full-length Mdm2 was a generous gift from Martine Roussel (St. Jude's Children's Research Hospital). Full-length NPM was cloned from wild-type MEF reverse-transcribed RNAs using the following PCR primers: 5'-GCGCATATGGAAGACTCGATGGATATGGAC-3' (sense) and 5'-GCGGGATCCTTAAAGAGATTTCCTCCACTGCCAGAG-3' (antisense). The NPM PCR product was digested with the NdeI and BamHI restriction enzymes and subcloned into the NdeI and BamHI sites of the pET28a vector (Novagen, Madison, Wis.). The pET28a six-histidine-tagged NPM insert was PCR subcloned using the following primers: 5'-GCGGAATTCATGGGCAGCAGCCATCATCAT-3' (sense) and 5'-GCGGAATTCTTAAAGAGATTTCCTCCACTG-3' (antisense). The resultant PCR product was subcloned into the EcoRI site of the pSR
-MSV-tkneo retroviral vector. A retroviral vector encoding DMP1 was a generous gift from Charles J. Sherr (St. Jude's Children's Research Hospital).
Nucleolar isolation and MALDI-TOF. TKO MEFs labeled with [35S]methionine and infected for 96 h with retroviruses encoding hemagglutinin-tagged ARF (HA-ARF) were harvested in ice-cold phosphate-buffered saline (PBS) and homogenized with a glass douncer. Nucleoli were isolated as previously described (2). Nucleoli were verified by staining them in 0.1% azure C dye (Sigma, St. Louis, Mo.). The purified nucleoli were lysed in 25 mM Tris-HCl, pH 7.4-150 mM NaCl-1 mM MgCl2-1 mM EDTA-1% Tween-20 and sonicated with three 10-s bursts at 20% output. HA-ARF complexes were immunoprecipitated with antibodies against the HA epitope conjugated to protein A-Sepharose (Amersham, Piscataway, N.J.). Proteins were eluted with 100 mM glycine (pH 3.0), neutralized in 1 M Tris-HCl (pH 7.4), and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The separated proteins were transferred to polyvinylidine difluoride (PVDF) membranes (Millipore, Bedford, Mass.) and exposed to film. Identified bands were excised and digested with 0.1 µg of trypsin per ml (Promega, Rockford, Ill.) overnight at 30°C. Tryptic peptide fragments were extracted with 60% acetonitrile and 0.1% trifluoroacetic acid using an automated Multiprobe II system (Packard Biosciences, Meriden, Conn.). The extracted peptides were dried under vacuum, purified with Zip Plates (Millipore), resuspended in matrix, and subjected to matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) analysis using a Voyager DE Pro spectrometer (Applied Biosystems, Foster City, Calif.).
Immunoprecipitation and Western blot analysis.
Wild-type, DKO (p53/Mdm2 null), and TKO (ARF/p53/Mdm2 null) MEFs were infected with retroviruses encoding MycER (a gift from J. Bishop), DMP1, ARF, or ARF 1-14 and lysed in binding buffer (25 mM Tris-HCl, pH 8, 150 mM NaCl, 1 mM EDTA, 0.1% NP-40, 1 mM phenylmethylsulfonyl fluoride, and 0.4 U of aprotinin) either 48 (wild type and DKO) or 96 (TKO) h after infection. For MycER infections, cells were lysed 48 h after the addition of 4-hydroxytamoxifen (4-HT) (1 µM). Antibody to the ARF C terminus (a gift from Charles Sherr), antibody to NPM (Zymed, San Francisco, Calif.), or nonimmune rabbit serum (NRS) or nonimmune mouse serum was added to the binding reaction mixtures for 1 h at 4°C. Immune complexes were precipitated with protein A-Sepharose and washed with binding buffer. The precipitated proteins, as well as direct protein lysates, were separated by SDS-PAGE and transferred to PVDF membranes preactivated in methanol. Mdm2, ARF, NPM, and 
-tubulin proteins were visualized by direct immunoblotting with 2A10, ab80 (abcam, Cambridge, United Kingdom), NPM (Zymed), and -tubulin (Santa Cruz, Santa Cruz, Calif.) antibodies, respectively.
Immunofluorescence and confocal microscopy. Wild-type or TKO MEFs (3 x 104) were seeded onto glass coverslips and infected with retroviruses encoding MycER, tkNeo (vector), ARF, or ARF in combination with Mdm2 or NPM. The cells were fixed 96 h after infection (TKO MEFs) or 48 h after the addition of 4-hydroxytamoxifen (MycER-infected wild-type MEFs) with ice-cold methanol-acetone (1:1 [vol/vol]) and stained for 1 h with either a rabbit ARF C-terminal antibody (4 µg per ml), human fibrillarin (Sigma), or mouse NPM antibody (4 µg per ml), followed by fluorescein isothiocyanate (FITC)-conjugated anti-mouse or anti-rabbit immunoglobulin (Pierce) or tetramethyl rhodamine isothiocyanate-conjugated anti-rabbit or anti-human immunoglobulin (Pierce). For measurement of DNA replication, 5-bromodeoxyuridine (BrdU) (Sigma) was added to the culture medium 72 h after infection at a final concentration of 10 µM. Twenty-four hours after the addition of BrdU, the cells were fixed in ice-cold methanol-acetone as described above, treated for 10 min with 1.5 N HCl, and stained for 1 h with a mouse monoclonal anti-BrdU antibody (Amersham), followed by FITC-conjugated anti-mouse immunoglobulin (Pierce). Nuclei were visualized with DAPI (4',6-diamidino-2-phenylindole) (Sigma). At least 100 cells were counted on each of three coverslips for all experimental conditions. Fluorescence signals were detected using a Nikon epifluorescent compound microscope (100x) fitted with a Nikon FDX-35 charge-coupled device camera.
FPLC. ARF synthetic peptides were coupled to cyanogen bromide-activated Sepharose (Amersham) and equilibrated as previously described (44). TKO lysates (200 µg) were injected at a flow rate of 1.0 ml per min, washed with 20 ml of 25 mM Tris-HCl (pH 7.4) at 1.0 ml per min, and eluted with a 25 ml of NaCl gradient (0 to 1.5 M) at 1.0 ml per min, followed by 20 ml of 100 mM glycine (pH 3.0) at 1.0 ml per min, using BioLogic fluid phase liquid affinity chromatography (FPLC) and BioLogic HR software (Bio-Rad, Hercules, Calif.). The 1.0-ml collected fractions were precipitated with trichloroacetic acid, resuspended in 1 M Tris-HCl (pH 8.0), and electrophoretically separated on denaturing polyacrylamide gels containing SDS. The separated proteins were transferred to PVDF membranes, and individual proteins were detected using antibodies to cyclin A (Santa Cruz), fibrillarin (Sigma), NPM (Zymed), and Mdm2 (2A10).
Heterokaryon assay.
HeLa cells (2 x 105) were seeded onto glass coverslips in six-well plates and transfected with His-NPM alone or in combination with green fluorescent protein (GFP)-ARF, GFP-ARF N62, or GFP-ARF 1-14. As a positive shuttling control, Myc-tagged NPC-M9 (a gift from J. Alan Diehl, University of Pennsylvania) was transfected in combination with GFP-ARF N62. NIH 3T3 cells (6 x 105) were seeded onto the HeLa cells 24 h posttransfection and cocultured for an additional 16 h at 37°C. The cocultures were then incubated for 30 min with DMEM containing cycloheximide (100 µg per ml). For fusion of mouse and human plasma membranes, the medium was removed and the cells were incubated with 50% polyethylene glycol in PBS (prewarmed to 37°C) for 105 s, followed by three PBS washes (prewarmed to 37°C). The cocultures were incubated with DMEM containing cycloheximide (100 µg per ml) for an additional 4 h to permit protein shuttling. Heterokaryons were fixed with formalin-methanol (10% [vol/vol] in H2O) for 15 min and permeabilized with 1% NP-40 in PBS for 5 min at room temperature, followed by three PBS washes. The cells were then blocked for 1 h with 5% fetal bovine serum in PBS and stained for 1 h with a rabbit anti-His antibody (1:25; Santa Cruz) or mouse anti-myc antibody (1:10; Santa Cruz), followed by either FITC-conjugated anti-rabbit or -mouse immunoglobulin (Pierce) or rhodamine X-conjugated anti-rabbit or -mouse immunoglobulin (Pierce) for 30 min. Nuclei were stained with DAPI. Fluorescent signals were detected using a Nikon epifluorescent compound microscope (100x) fitted with a Nikon FDX-35 charge-coupled device camera.
rRNA processing. HeLa cells (106) were seeded onto 60-mm-diameter dishes and transfected with plasmids encoding GFP or GFP-ARF. The cells were pulse-labeled for 45 min with [5,6-3H]uridine (Amersham) and chased with complete medium for 1 h as previously described (39). Total RNA was harvested from cells using Trizol (GIBCO), and labeled rRNAs (2,000 cpm/lane) were separated on 1% formaldehyde-agarose gels. The gels were denatured for 20 min (0.05 N NaOH, 0.15 M NaCl) and neutralized for 30 min (0.1 M Tris, pH 7.5, 0.15 M NaCl) before being transferred to Hybond N+ membranes. Labeled rRNAs were visualized on EN3HANCE (Perkin-Elmer)-sprayed membranes using standard autoradiography.
RNA interference. The following 19-nucleotide duplex, corresponding to nucleotides 133 to 151 downstream of the murine NPM transcriptional start site, was synthesized and cloned into the pSUPER.retro plasmid vector (OligoEngine, Seattle, Wash.) according to the manufacturer's instructions: 5'-AGAACGGTCAGTTTAGGAG-3' (sense) and 5'-CTCCTAAACTGACCGTTCT-3' (antisense). Retroviruses encoding NPM-targeting small interfering RNAs (siRNAs) were generated via cotransfection of pSUPER.retro-NPM RNA interference (RNAi) and helper plasmids into packaging cells as described above. TKO MEFs seeded onto 100-mm-diameter dishes were infected with pSUPER-NPM RNAi retroviruses and subjected to Western blotting and indirect immunofluorescent analyses 96 h after retroviral transduction, using antibodies described above. For indirect immunofluorescent analysis of NPM expression and BrdU incorporation, cells infected on 100-mm-diameter dishes were seeded onto glass coverslips (105 per coverslip) 24 h after infection, followed by addition of BrdU (10 µM), fixation, and staining for NPM and BrdU at 96 h posttransduction, as described above. Nuclei were demarcated with DAPI, and fluorescence was visualized as described above. As a control, TKO MEFs were infected with empty siRNA vector and scrambled siRNAs (gifts from Helen Piwnica-Worms) (5'-AGGGATGTGTCCCCTTGTG-3' [sense] and 5'-CTCTTGGGGTCTCTTCCC-3' [antisense]) and were assayed in parallel with those infected with pSUPER-NPM RNAi retroviruses.
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RESULTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To establish the biochemical composition of an Mdm2-free ARF complex, we infected MEFs derived from mice lacking ARF, p53, and Mdm2 (TKO) with retroviruses encoding HA-ARF and labeled cellular proteins with [35S]methionine. To reduce nonspecific binding of abundant cytoplasmic and nuclear proteins during cell lysis, HA-ARF complexes were isolated from purified nucleoli. Intact nucleoli (Fig. 1A, inset) were lysed, and ARF-containing protein complexes were precipitated using antibodies against the HA moiety (Fig. 1A). HA-ARF is difficult to detect at 
20 kDa due to its extreme lack of methionine and cysteine residues. Proteins within ARF complexes were separated via SDS-PAGE, digested with trypsin to obtain individual peptide fragments, and combined with matrices. Dried matrix-peptide mixtures were subjected to MALDI-TOF analysis. Peptide masses obtained from a protein band at 38 kDa matched the peptide fingerprint for murine NPM (Fig. 1A). An abundant nucleolar phosphoprotein, NPM is upregulated in response to mitogenic signals (12, 20) and is necessary for entry into S phase of the cell cycle in established fibroblast cell lines (17). NPM's reported role as a marker of cell proliferation prompted us to further investigate its association with the ARF tumor suppressor.
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ARF colocalizes with NPM in the granular region of the nucleolus. In mouse and primary diploid human cells, ARF is expressed at low basal levels and localizes to the granular region of the nucleolus (21, 25, 32, 43), a subnuclear organelle that is readily detected upon immunostaining for fibrillarin. NPM, a highly abundant nucleolar protein, localizes primarily to fibrillarin-positive nucleoli, but additional staining throughout the nucleus is observed (Fig. 2A and C). To corroborate our finding that ARF and NPM interact and reside within the same protein complex, we next wanted to verify that NPM colocalizes with ARF in the granular region of the nucleolus under conditions of ARF induction in vivo. As expected, myc-mediated upregulation of endogenous ARF in wild-type MEFs resulted in the accumulation of ARF in nucleoli, as indicated by the colocalization of ARF with fibrillarin (Fig. 2A). Similarly, retroviral transduction of exogenous ARF into TKO MEFs produced a robust nucleolar pattern of ARF expression (Fig. 2B). Coimmunostaining for NPM and ARF in myc-transduced wild-type MEFs and ARF-transduced TKO MEFs revealed that NPM and ARF colocalize to the granular region of nucleoli, as confirmed by indirect immunofluorescent (Fig. 2B, overlay) and confocal (Fig. 2C, overlay) microscopy.
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1-14) could interact with NPM in vivo. TKO MEFs were infected with retroviruses encoding full-length ARF or ARF 1-14, followed by coimmunoprecipitation against ARF and NPM to assess formation of the ARF-NPM complex. Whereas full-length murine ARF readily bound NPM (Fig. 3A, upper left) ARF 1-14 failed to exhibit a significant in vivo interaction with NPM (Fig. 3A, upper right), consistent with its inability to induce complete cell cycle arrest in TKO MEFs (45). However, we did observe some residual binding of this mutant (1-14) with NPM, leaving open the possibility that other regions within ARF may be important in mediating ARF-NPM interactions in vivo. Contiguous deletion of ARF residues 26 to 37 (which define the low-affinity Mdm2 binding site) or 38 to 52 did not alter ARF-NPM complex formation (Fig. 3A, bottom), and unlike ARF 1-14, these mutants are fully capable of inducing cell cycle arrest (43).
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Mdm2 releases NPM from ARF-containing protein complexes. Previous work established ARF's reasonably strong affinity for a central region of Mdm2 (residues 210 to 304) (6, 10), and interestingly, both Mdm2 and NPM interact with the first 14 amino acids of ARF (Fig. 3B) (44). To compare the relative binding strength of the ARF-NPM association with that of ARF-Mdm2, we injected purified full-length NPM onto a peptide column composed of ARF residues 1 to 14. NPM did not interact with the Sepharose used to make the column (Fig. 4A, top, lanes 1 to 3) and was not nonspecifically outcompeted once it was bound to the ARF 1 to 14 peptide column (Fig. 4A, bottom, lanes 4 to 8). Once NPM had bound to the ARF column (Fig. 4B, diagram), the column was washed with buffer containing an excess molar amount of purified Mdm2 residues 210 to 304, which corresponds to the ARF binding domain (6, 44). Bound proteins were eluted with a salt gradient followed by an acid wash, and the resultant fractions were subjected to Western blot analysis for detection of NPM and Mdm2. Initially, NPM was retained on the ARF column yet was exchanged for Mdm2 in subsequent wash steps and eluted in the flowthrough (Fig. 4B, lanes 1 to 3). Newly formed ARF-Mdm2 complexes were stable but eventually eluted under high-salt or acidic conditions (Fig. 4B, lanes 6 to 9), demonstrating ARF's preference for Mdm2 over NPM as a binding partner. In agreement with this finding, Mdm2 remained bound to the ARF peptide column when the column was washed with buffer containing an excess molar amount of purified full-length NPM (Fig. 4C, lanes 6 to 9).
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ARF prevents NPM's nucleocytoplasmic shuttling. As an upstream activator of p53-dependent pathways of growth arrest and apoptosis, ARF stabilizes p53 protein levels through binding and sequestration of Mdm2 in the nucleolus, away from nucleoplasmic pools of p53, suggesting that regulation of protein topology may be an underlying feature of ARF-mediated tumor suppression (25, 41, 43). The notion that subcellular compartmentalization can either enable or disable a protein's function is further supported by our present understanding of NPM dynamics within the cell. Previous studies have established NPM's participation in nucleocytoplasmic shuttling (5, 47), while more recent work indicates that a relatively small fraction of NPM appears to transit from the nucleolus to the cytosol in a cell cycle-dependent manner (29, 42). Given that nucleolar ARF limits Mdm2's oncogenic potential by preventing its access to, and export from, the nucleus (41, 43), we hypothesized that ARF may utilize a similar strategy to inhibit NPM function, thereby achieving p53-independent command of the cell cycle.
To address ARF's potential role in regulating NPM trafficking throughout the cell, we conducted in vivo heterokaryon shuttling assays (41). An expression construct encoding six-histidine-tagged full-length murine NPM (His-NPM) was transiently transfected alone or in combination with green fluorescent protein-tagged constructs encoding either full-length murine ARF (GFP-ARF) or ARF deletion mutants that either retain (GFP-ARF N62) or lack (GFP-ARF 1-14) the conserved amino terminus into asynchronously growing HeLa cells. Mouse NIH 3T3 fibroblasts were seeded onto transfected HeLa cells, yielding a heterogeneous human-mouse cell population, followed by fusion of plasma membranes and subsequent immunofluorescent detection of protein-shuttling events. As evidenced by staining of DNA with DAPI, the nuclei of NIH 3T3 and HeLa cells are easily distinguished by the greater heterochromatin focus content of mouse cells (speckled pattern) (Fig. 6, DAPI). In the absence of GFP-ARF, NPM readily migrated from human nucleoli to mouse nucleoli, as visualized in interspecies heterokaryons (Fig. 6A). However, in the presence of nucleolar GFP-ARF or GFP-ARF N62, NPM failed to shuttle and was restricted to human nucleoli within heterokaryons (Fig. 6B and C). Conversely, GFP-ARF 1-14, devoid of the NPM binding domain, was unable to restrict NPM shuttling between human and mouse nucleoli (Fig. 6D). As GFP-ARF did not hinder the nucleocytoplasmic trafficking of Myc-NPC-M9, an hnRNP protein that readily shuttles (Fig. 6E) (31), our combined body of data indicates that ARF specifically binds and sequesters NPM into nucleolar ARF complexes to completely impede NPM nucleocytoplasmic shuttling (Fig. 6F). Inhibition of NPM shuttling was not due to reduction of NPM protein, as GFP-ARF had no effect on NPM protein expression in HeLa cells (Fig. 6G). Additionally, GFP-ARF did not inhibit rRNA processing of 28S, 18S, and 5.8S subunits in HeLa cells (Fig. 6H), indicating that NPM's roles in rRNA processing and nuclear export are distinct and separate events.
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-tubulin (Fig. 7A), as well as other nucleolar proteins (data not shown), remained unchanged, demonstrating the specificity of our siRNA targeting sequence. Additionally, a scrambled siRNA did not reduce NPM levels (Fig. 7A), underscoring the fact that NPM protein expression is not influenced by introduction of nonspecific RNAi molecules. Importantly, pSUPER-NPM RNAi-infected TKO MEFs that displayed reduced NPM protein expression were significantly impaired in their ability to enter S phase compared to cells infected with control viruses that exhibited normal levels of NPM (Fig. 7B). Given that the reduction of NPM protein expression and the inhibition of NPM's nucleocytoplasmic shuttling each trigger growth arrest in MEFs, we conclude that NPM is an essential player in the process of cell cycle progression.
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The key to ARF's proficiency in arresting cell proliferation, irrespective of p53 status, resides within its extreme amino terminus, namely, the first 14 residues of the ARF amino acid sequence. In search of additional interacting partners of this domain, we have verified the formation of both endogenous and exogenous ARF-NPM nucleolar complexes in primary mouse fibroblasts. Notably, ARF utilizes these exact residues to establish its high-affinity association with Mdm2, raising the possibility that ARF may affiliate with two distinct nucleolar complexes and/or represent a source of competition between Mdm2 and NPM. Upon its induction by hyperproliferative signals, ARF readily draws both Mdm2 and NPM into seemingly independent, distinct nucleolar complexes, evidenced by the absence of a ternary complex. However, overexpression of Mdm2 results in the release of NPM from ARF-containing protein complexes, suggesting that ARF's ability to dictate growth arrest in the presence or absence of p53 is largely determined by the stoichiometry of its binding partners. In light of this finding, it is appealing to think of Mdm2 as a target of ARF suppression, as well as a dampener of persistent ARF function. Specifically, Mdm2 may negatively regulate the p53 response through a dual mechanism: degradation of its direct transcriptional activator, p53 (38, 40), and inhibition of ARF function via preferential binding over NPM. We show here that Mdm2 can antagonize both the p53-dependent and -independent functions of ARF. By uncovering a pathway through which Mdm2 can promote cell growth independently of its classic upstream activator, p53, our study has exposed an unforeseen capacity of the Mdm2 oncoprotein.
While NPM is an abundant nucleolar phosphoprotein, data from our laboratory and others indicate that it is distributed throughout the cell in discrete pools, and factors such as NPM's state of posttranslational modification, bound protein partners, and subcellular localization may determine the composition and activity of any given NPM pool (5, 8, 22, 23, 30, 35, 47). As revealed by our data and the results of others, we hypothesize that ARF sequesters a specific pool of NPM in the nucleolus, preventing its transit and intended function(s) elsewhere in the cell. The intrinsic growth-promoting potential of the NPM pool immobilized within the ARF complex remains unclear, but recent studies support several possibilities. ARF transduction into MEFs lacking ARF and p53 (ARF/p53-null) was shown to significantly impair the processing of rRNAs, and this effect was strictly dependent upon the highly conserved first 14 amino acids within ARF's extreme amino terminus (39). Importantly, transduction of p53 failed to reproduce this result (39), indicating that ARF's ability to down-regulate rRNA processing is distinct from ARF's established roles within the classical ARF/p53/Mdm2 epistatic pathway. Additionally, two studies recently reported that NPM was a nucleolar ARF binding partner (4, 15). Both studies indirectly demonstrate that ARF can prevent proper rRNA processing, but how this might affect proper protein translation and how this event signals proliferative arrest remain unanswered. Nonetheless, these findings, in combination with our data, suggest that ARF's interaction with NPM may facilitate contact between ARF and the nucleolar ribosomal processing machinery, given that NPM appears to function as an integral component of ribosome maturation and export (30). We have further shown that NPM shuttling is required for cell proliferation, suggesting that ARF, via nucleolar sequestration of NPM, may not only target rRNA processing but might also prevent the nucleolar or nuclear export of processed rRNAs. This would be analogous to an earlier hypothesis in which protein targets of the ARF tumor suppressor may "ride the ribosome" out of the nucleolus and into the cytoplasm to execute their growth-promoting functions (37).
In an unrelated study, NPM was shown to be phosphorylated by cyclin E/cdk2 at the centrosome, resulting in the initiation of centrosome duplication (29). Hence, it is possible that nucleolar ARF, in response to hyperproliferative signals, binds and immobilizes the NPM pool that is designated for transit to the centrosome, consistent with our findings. However, a more recent report failed to detect NPM in isolated centrosomes (1), raising the possibility that other functional targets of NPM reside within or are transported to the cytoplasm, one of which might be the maturing ribosome itself. The models described above warrant further investigation, but we cannot overlook the possibility that NPM may, in addition to its ascribed roles in ribosome biogenesis and centrosome duplication, transmit additional growth-promoting commands and that Mdm2-mediated release of NPM from ARF complexes potentiates these functions. Nonetheless, ARF, in its capacity as a tumor suppressor, employs redundant mechanisms of protein binding and topological sequestration to inhibit both p53-dependent and -independent targets, thereby achieving control over cell proliferation.
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ACKNOWLEDGMENTS |
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S.N.B. is supported through the Siteman Center Cancer Biology Pathway. Y.Y. is the recipient of a grant-in-aid from the Department of Defense Breast Cancer Research Program (BC030793). L.B.M. is supported by institutional training grant T32-HL07873 from the National Institutes of Health. J.D.W. is a Pew Scholar and the recipient of grants-in-aid from the Edward Mallinckrodt, Jr., Foundation, American Cancer Society (IRG-58-010-46), and National Institutes of Health (GM066032).
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FOOTNOTES |
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S.N.B. and Y.Y. contributed equally to this paper.
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