Tumor Suppressor ARF Degrades B23, a Nucleolar Protein Involved in Ribosome Biogenesis and Cell Proliferation

Molecular Cell, Vol. 12, 1151–1164, November, 2003, Copyright 2003 by Cell Press

Tumor Suppressor ARF Degrades B23, a Nucleolar Protein Involved in Ribosome Biogenesis and Cell Proliferation Koji Itahana,1 Krishna P. Bhat,1 Aiwen Jin,1 Yoko Itahana,1 David Hawke,2 Ryuji Kobayashi,2 and Yanping Zhang1,* 1 Department of Molecular and Cellular Oncology 2 Department of Molecular Pathology University of Texas M.D. Anderson Cancer Center Houston, Texas 77030

Summary The tumor suppressor ARF induces a p53-dependent and -independent cell cycle arrest. Unlike the nucleoplasmic MDM2 and p53, ARF localizes in the nucleolus. The role of ARF in the nucleolus, the molecular target, and the mechanism of its p53-independent function remains unclear. Here we show that ARF interacts with B23, a multifunctional nucleolar protein involved in ribosome biogenesis, and promotes its polyubiquitination and degradation. Overexpression of B23 induces a cell cycle arrest in normal fibroblasts, whereas in cells lacking p53 it promotes S phase entry. Conversely, knocking down B23 inhibits the processing of preribosomal RNA and induces cell death. Further, oncogenic Ras induces B23 only in ARF null cells, but not in cells that retain wild-type ARF. Together, our results reveal a molecular mechanism of ARF in regulating ribosome biogenesis and cell proliferation via inhibiting B23, and suggest a nucleolar role of ARF in surveillance of oncogenic insults. Introduction The genomic locus at p16INK4a is frequently mutated at an overall frequency of approximately 40% in human cancer, second only to p53 mutations (Kamb et al., 1994; Nobori et al., 1994; Ruas and Peters, 1998). This extraordinarily high incidence of mutation at this locus is now believed to be the result of its unusual organization. Three exons at the p16INK4a locus, designated 1␣, 2, and 3, encode the CDK inhibitor p16INK4a. About 20 kb upstream from exon 1␣ is an alternative exon termed 1␤. Transcripts initiated from exon 1␤ pass over exon 1␣ and extend into the common exon 2 (and exon 3 in the mouse) and encode ARF. Because the two AUG start codons of the transcripts for p16INK4a and ARF fall into alternative reading frames at the shared exon 2, the two proteins have no similarity in their primary amino acid sequences (Quelle et al., 1995). Early studies have shown that enforced expression of ARF causes cell cycle arrest at both the G1 and G2 phases in a p53-dependent fashion (Quelle et al., 1995). Mice with a homozygous deletion of the ARF-specific exon 1␤ but retaining normal p16INK4a expression are highly tumor prone, indicating ARF is a bona fide tumor suppressor (Kamijo et al., 1997). Early observations that ARF expression is elevated by oncogenic signals such as Myc, E2F1, oncogenic Ras, adenovirus E1A, and v-Abl (Bates et al., 1998; *Correspondence: [email protected]

de Stanchina et al., 1998; Palmero et al., 1998; Radfar et al., 1998; Zindy et al., 1998) led to the proposal that ARF mediates a p53-dependent checkpoint that responds to oncogenic, hyperproliferative signals (Sherr, 1998). Functionally, ARF binds to MDM2 (Pomerantz et al., 1998; Zhang et al., 1998), inhibits its E3 ligase activity (Honda and Yasuda, 1999), and stabilizes and activates p53. This is achieved, at least in part, by blocking the nuclear export of both p53 and MDM2 (Tao and Levine, 1999; Zhang and Xiong, 1999). Normally, ARF protein localizes in the nucleolus (Pomerantz et al., 1998; Weber et al., 1999; Zhang and Xiong, 1999), a subcellular compartment recognized primarily as the site of ribosome biogenesis (Olson et al., 2002), whereas p53 and MDM2 localize in the nucleoplasm and shuttle between the nucleus and the cytoplasm (Freedman and Levine, 1998; Roth et al., 1998; Stommel et al., 1999; Zhang and Xiong, 2001b). The mechanism by which ARF inhibits MDM2 and induces p53 has been a subject of debate (Zhang and Xiong, 2001a). It has been proposed that ARF sequesters MDM2 into the nucleolus, thereby freeing up and activating p53 in the nucleoplasm (Tao and Levine, 1999; Weber et al., 1999). This model, however, cannot justify the readily observed formation of ARF-MDM2-p53 ternary complex (Kamijo et al., 1998; Pomerantz et al., 1998; Stott et al., 1998; Zhang et al., 1998), and how p53 can be stabilized and activated even though still-abundant MDM2 is in the nucleoplasm when ARF is overexpressed (Tao and Levine, 1999; Weber et al., 1999, 2000b). Another model proposes that ARF forms a ternary complex with MDM2 and p53 in the nucleoplasm, thereby blocking p53 nuclear export and stabilizing and activating p53 (Zhang and Xiong, 1999). This model implies that the MDM2bound, thus inactivated p53 can be reactivated by ARF without dissociation from the binding of MDM2, an assumption that has not yet been experimentally verified. Recent studies have demonstrated that ARF can inhibit MDM2 and activate p53 without localizing MDM2 into the nucleolus (Korgaonkar et al., 2002; Llanos et al., 2001), and proposed, for the first time, that ARF may have other functions in the nucleolus (Llanos et al., 2001). Indeed, it has been shown that ARF has p53- and MDM2-independent functions (Hemmati et al., 2002; Korgaonkar et al., 2002; Matsuoka et al., 2003; Tsuji et al., 2002; Weber et al., 2000a; Yarbrough et al., 2002). The nature of such p53- and MDM2-independent function of ARF and its actual role in the nucleolus if it is not to sequester MDM2 remains unclear. It is well established that the most prominent function of nucleolus is to act as a site for ribosomal subunit assembly (Olson et al., 2002). As a major consumer of the cell’s resources, ribosome biogenesis consumes a major part of the cell’s energy and resources and plays a key role in the cell’s life cycle (Conlon and Raff, 1999; Neufeld and Edgar, 1998; Warner, 1999). The entire ribosome, composed of four rRNA species and approximately 75 ribosomal proteins (r-proteins) distributed between two subunits of 40S and 60S, is assembled in the nucleolus. The 18S, 5.8S, and 28S rRNA species are

Molecular Cell 1152

Inhibition of Nucleolar B23 by ARF 1153

derived from a single 47S rRNA precursor that is synthesized by RNA polymerase I from multiple copies of the genes for preribosomal RNA (rDNA) and then processed by a series of endonucleolytic and exonulceolytic cleavages. The 5S rRNA is synthesized separately by RNA polymerase III and is associated with the 60S preribosomal subunit early in assembly. The mature 40S ribosomal subunit contains the 18S rRNA and approximately 32 r-proteins, whereas the 60S subunit is composed of the 5S, 5.8S, and 25/28S rRNAs and approximately 45 r-proteins (Kressler et al., 1999; Venema and Tollervey, 1999). Proper assembly of each ribosomal subunit requires the coordination of several events, including the synthesis and import of r-proteins, synthesis and processing of rRNA, and the concomitant assembly of r-proteins into the preribosomal subunits. Conceivably, perturbation of ribosome biogenesis will have a profound effect on the cell, and a tight control system to coordinate cell division with growth must be involved in all multicellular organisms. Nonetheless, little is known about how cells coordinate ribosome biogenesis and cell cycle regulation. Here, we report that ARF interacts with and inhibits the function of B23, a nucleolar endoribonuclease involved in maturation of 28S rRNA (Savkur and Olson, 1998), through promoting B23 polyubiquitination and proteosomal degradation. Our data are consistent with a recent report showing that ARF inhibits the processing of preribosomal RNA (Sugimoto et al., 2003), and provide a molecular mechanism by which ARF inhibits nucleolar function and suggests an additional activity of ARF in promoting polyubiquitination and degradation of the nucleolar protein B23. Results ARF Interacts with B23 Utilizing adenovirus-mediated overexpression of human ARF and coimmunoprecipitation (Co-IP) with an antibody specific for the human ARF protein, a number of polypeptides were detected, specifically in the ARF immunocomplex but not in the control sample (Figure 1A). One of the most prominent bands on the silverstained gel, with an apparent molecular mass of 33,000, was subjected to mass spectrometry. Three peptides were obtained from the band and all matched perfectly

with the human nucleolar protein B23 (also known as nucleophosmin, numatrin, or NO38). Full-length B23 (B23 cDNA was purchased from Invitrogen, accession number BC008495) contains 294 amino acid residues with a calculated molecular mass of 32,575, a size corresponding to the apparent mass of the band on the silverstained gel. B23 is an abundant nucleolar phosphoprotein implicated in multiple cellular functions, including ribosomal protein assembly and transport (Olson et al., 1986), centrosome duplication (Okuda et al., 2000), molecular chaperone activity in preventing protein aggregation (Szebeni and Olson, 1999), and regulating the stability and activity of p53 (Colombo et al., 2002). B23 is also shown to possess an endoribonuclease activity (Savkur and Olson, 1998). The in vivo interaction of ARF and B23 was confirmed by Co-IP of ectopically overexpressed ARF and B23 proteins (Figure 1B, left), and by Co-IP of ectopic ARF and endogenous B23 (Figure 1B, right). The ARF-B23 interaction is not a nonspecific consequence of their colocalization in the nucleolus because another abundant nucleolar protein C23, also involved in pre-rRNA processing (Lischwe et al., 1981; Olson et al., 2002), did not interact with ARF (Figure 1B, right). The ARF-B23 interaction was further confirmed at physiological conditions with endogenous proteins in HeLa cells (Hela cells contain relatively high levels of ARF) (Figure 1C), and with E2F1-induced ARF in WI-38 normal human fibroblasts (Figure 1D). The possibility of ribosomal RNA mediating the binding was ruled out since the binding was not altered when RNase A was included throughout the cell lysis and IP (Figure 1E). B23 did not interact with MDM2 (Figure 1F) and, contrary to a recent report (Colombo et al., 2002), it did not interact with p53. We failed to detect p53-B23 interaction under conditions where p53 was either ectopically overexpressed or induced by UV in WI-38 cells (Figure 1F). To determine the domains involved in ARF-B23 interaction, we constructed deletion mutants of each protein, expressed them in U2OS cells, and tested their binding activity by Co-IP. The exon 1␤-encoded N-terminal domain of ARF (amino acids 1-64) was sufficient to bind with B23 (Figure 2A, lane 1). In contrast, the exon 2-encoded C-terminal domain of ARF (amino acids 65-132) did not interact with B23, even though it was expressed at a very high level (Figure 2A, lane 5). On the other hand, the N-terminal half of B23 (amino acids 1-113), which en-

Figure 1. ARF Interacts with B23 (A) Mass spectrometry detection ARF binding proteins. Extracts of U2OS cells infected with indicated adenoviruses were immunoprecipitated with ARF antibodies and were resolved on a silver-stained gel. Peptide bands unique in ad-ARF-infected samples are subjected for mass spectrometry. (B) The binding between ectopically expressed ARF and B23. U2OS cells were transfected with plasmids encoding myc-B23 and/or infected with indicated viruses. Cell extracts were immunoprecipitated with an anti-ARF antibody and Western blotting was performed with indicated antibodies. (C) The binding of endogenous B23 and endogenous ARF in HeLa cells. (D) The binding between endogenous ARF induced by E2F1 with endogenous B23 in normal fibroblasts. WI-38 cells and U2OS cells were infected with indicated viruses. Cell extracts were immunoprecipitated with either ARF (left panel) or B23 (right panel) antibodies. (E) No effect for RNase A treatment on ARF-B23 complex formation. Extracts from U2OS cells transfected with plasmids encoding ARF and myc-B23 were immunoprecipitated with ARF antibodies. RNase A (200 ␮g/ml) was added throughout immunoprecipitation and IP. (F) MDM2 or p53 do not interact with B23. Extracts from U2OS cells infected with indicated viruses were immunoprecipitated with indicated antibodies (upper panel). WI-38 cells were exposed to UV (25J/m2) for 24 hr and cell extracts were immunoprecipitated with indicated p53 antibodies. Untreated WI-38 cells and p53 negative Saos2 and H1299 cells served as a control. Note that a nonspecific band at the position of B23 was observed in samples immunoprecipitated with DO1 antibody.

Molecular Cell 1154

Figure 2. Binding Domains for ARF and B23 (A) Requirement of N-terminal of ARF for binding with B23. Extracts from U2OS cells transfected with plasmids encoding deletion mutants of myc-ARF were immunoprecipitated with myc antibodies and blotted as indicated. (B) Requirement of N-terminal of B23 for binding with ARF. Extracts from U2OS cells transfected with plasmids encoding ARF and deletion mutants of myc-B23 were immunoprecipitated with ARF antibodies and blotted as indicated.

compasses the entire homodimerization domain (Hingorani et al., 2000), was sufficient for binding with ARF (Figure 2B, lane 2), whereas the C-terminal half of B23 (amino acids 117-294) did not interact with ARF (Figure 2B, lane 3). Notably, even though ARF is an extremely basic protein with a calculated pI of 12.9 (13.0 for the N-terminal 64 amino acids), it does not interact with the central acidic region of B23 (amino acid 119-188, pI ⫽ 3.5), indicating the interaction is sequence specific and not simply due to electrostatic force. A previous study has shown that B23 binds both DNA and RNA with its C-terminal end (Wang et al., 1994); mapping of the ARF binding site on B23’s N-terminal domain supports the notion that the ARF-B23 interaction is not mediated by DNA or RNA.

ARF Induces B23 Rapid Degradation We have noticed in our study that the level of endogenous B23 appeared to be lower in cells infected with ad-ARF than in cells infected with control virus (see right panel in Figure 1B). To investigate the functional consequence of the ARF-B23 interaction and to verify if there is a potential ARF-induced B23 reduction, we examined the protein levels of B23 under the conditions of ARF overexpression. We infected U2OS cells with ad-ARF and examined the levels of endogenous B23 by Western blotting. Confirming our early observation, ARF induced decrease of endogenous B23, and the reduction of B23 by ARF was seen within 24 hr postinfection, and the level of B23 in ARF-infected cells remained low for at least 4 days (Figure 3A). A similar level of B23

Inhibition of Nucleolar B23 by ARF 1155

reduction was also seen in cells infected with ad-ARF in other cell types including Saos2 and WI-38, albeit prolonged ARF infection eventually induced cell death in p53-positive cells after 3 days (data not shown). To examine whether the reduction of B23 by ARF was due to a decrease of transcription of B23, total RNA was isolated from ad-ARF-infected cells and the level of B23 mRNA was probed. Under the same condition of adARF infection, in which B23 protein was significantly reduced (Figure 3A), B23 mRNA levels remained constant (Figure 3B), indicating that ARF does not affect the transcription of B23. To examine whether the reduction of B23 by ARF was due to a decrease of B23 protein stability, we performed B23 half-life assay in ad-ARFinfected Saos2 cells that do not have p53. In the absence of ad-ARF infection, endogenous B23 appeared to be very stable and its half-life was without discernible change after 24 hr of chasing (Figure 3C, lanes 5–8). In the presence of ad-ARF infection, however, B23 became unstable and its half-life was reduced to about 12 hr, indicating that ARF promotes a rapid degradation of B23 protein (Figure 3C, lanes 1–4). The extent of this ARF-induced B23 degradation was likely an underestimate, because the infection of Saos2 cells was less than 100% and the expression level of ARF in the infected cells varied. To further show that the ARF-induced B23 degradation is transcription independent, a U2OS cell line stably expressing pcDNA3-myc-B23 was established and the cells were infected with ad-ARF. As shown in Figure 3D, ARF promoted the degradation of ectopic myc-B23, demonstrating that this degradation is transcription independent. ARF did not induce degradation of C23, which, like B23, is a nucleolar protein but does not bind with ARF, demonstrating that only colocalizing but not interacting with ARF in the nucleolus is not sufficient for ARF-induced degradation (Figure 3E). To investigate whether oncogene-induced physiological ARF promotes B23 degradation, we examined B23 level in WI-38 cells in which ARF was elevated by infecting cells with ad-E2F1. As shown in Figure 3F, B23 was evidently reduced in WI-38 cells infected with adE2F1, but remained unchanged in ad-E2F1-infected U2OS cells, which lack functional ARF, strongly suggesting a direct role of ARF in mediating B23 degradation. MDM2 and p53 Do Not Induce B23 Degradation The results that ARF induces B23 degradation in different cell types, including those cells with or without functional p53, suggest that this may be a p53- and MDM2independent function. To determine whether MDM2 and p53 can also affect B23 stability, we compared the level of endogenous B23 in U2OS and Saos2 cells infected with viruses expressing MDM2, p53, and ARF. U2OS cells contain a functional p53 and a relatively high level of MDM2, whereas Saos2 cells lack p53. When infected with an equal amount of ARF virus, B23 was reduced to a comparatively low level in U2OS and Saos2 cells, confirming that the ARF-induced B23 degradation was not cell type specific and was not dependent on the presence of p53 or MDM2 (Figure 4A). On the other hand, overexpression of neither MDM2 nor p53 in U2OS and Saos2 cells induced B23 degradation (Figure 4A),

indicating MDM2 and p53 do not directly affect the protein stability of B23. The level of B23 in cells appears to be cell cycle independent, since it remained unchanged when cells were promoted into S phase by MDM2 or arrested at the G1 phase by p53 after cells were infected with each virus for 24 hr. However, whether cell cycle alterations by prolonged existence of high levels of MDM2 or p53 will eventually affect the level of B23 remain to be determined. We further examined the reduction of B23 by immunofluorescence staining of Saos2 cells infected with adARF. Consistent with a previous study (Spector et al., 1984), endogenous B23 was seen concentrated in the nucleolus with evident nucleoplasmic accumulation, but not in the cytoplasm (Figure 4B, panel 1). In agreement with the Western blotting results, where the level of B23 remained unchanged in cells expressing a high level of p53 (panel 2), it was clearly reduced in cells expressing ARF (panel 3). We have noticed that the level of B23 reduction in individual cells closely correlated with the level of ARF expression, in that the cells expressing higher levels of ARF had lower levels of B23. Together, our data demonstrate that ARF promotes an MDM2and p53-independent degradation of B23 protein.

Evidence of ARF-Induced B23 Polyubiquitination and Proteosomal Degradation In search of a mechanism for ARF-induced B23 degradation, we thought to determine whether B23 is degraded by the 26S proteosome complex by treating ad-ARFinfected cells with the 26S proteosome inhibitor MG132. Treating U2OS cells with 20 ␮M MG132 for 12 hr stabilized endogenous p53 to a level similar to that of the p53 level stabilized by ARF, showing an efficient blocking of the 26S proteosomal degradation pathway. Under the same condition of MG132 treatment, the majority of B23 degradation induced by ARF was blocked demonstrating that ARF-induced B23 degradation requires the function of the 26S proteosomal pathway (Figure 5A). The dependency of 26S proteosomal degradation suggests that B23 might be polyubiquitinated. To decide whether B23 can be polyubiquitinated and if the polyubiquitination can be promoted by ARF, we carried out in vivo ubiquitination assay of B23. We transfected H1299 cells with a plasmid expressing His-tagged B23 with or without plasmids expressing ARF and HA-ubiquitin. Total His-B23 species were pulled down by Ni-beads, separated by SDS-PAGE, and HA-Ub-conjugated HisB23 was detected by an anti-HA antibody. As shown in Figure 5B, in a sample coexpressing His-B23, ARF, and HA-Ub, the level of high-molecular weight, HA-Ub-conjugated His-B23 species was evidently increased, demonstrating that ARF promotes B23 polyubiquitination (Figure 5B, lane 5). In addition, we found that B23 degradation was not dependent on CRM1-mediated nuclear export since treating the cells with the CRM1 inhibitor LMB did not affect ARF-induced B23 degradation (Figure 5C). Together, these data support that B23 is polyubiquitinated in vivo and the polyubiquitination can be enhanced by ARF. The ARF-induced B23 reduction is, therefore, likely through ARF promoting B23 polyubiquitination and the 26S proteosomal degradation.

Molecular Cell 1156

Inhibition of Nucleolar B23 by ARF 1157

Figure 4. ARF Degrades B23 in a p53- and MDM2-Independent Manner (A) p53 or MDM2 do not induce B23 degradation. U2OS and Saos2 cells were infected with indicated viruses for 1 day. Protein extracts were analyzed by Western blotting. (B) ARF but not p53 decreases nucleolar and nucleoplasmic B23. Saos2 cells were infected with indicated viruses for 2 days (panels 2 and 3) and double-immunostained with indicated antibodies. Nuclei were visualized by DAPI staining. ARF or p53 infected cells were indicated by arrows.

Figure 3. ARF Promotes Rapid Degradation of B23 (A and B) Downregulation of endogenous B23 by ectopically expressed ARF. U2OS cells were infected with indicated viruses. Protein or RNA was harvested at indicated times post infections and analyzed by Western blotting or Northern blotting, respectively. (C) Half-life assay of endogenous B23. Saos2 cells were infected with adenoviruses expressing indicated proteins. Twenty-four hours after infection, cells were pulsed with [35S]-methionine for 2 hr and then chased for the indicated length of time. Cell lysates were immunoprecipitated with an antibody specific to B23. The resulting B23 immunoprecipitates were separated electrophoretically by SDS-PAGE and visualized by autoradiography. The amount of labeled B23 protein at each time point was quantified on a PhosphoImager and normalized relative to the amount of radio labeled B23 present in cells following the 0 hr chase and the results are plotted. (D) Downregulation of stably overexpressed B23 by ARF. Stable U2OS clones expressing myc-B23 were infected with indicated adenoviruses for 2 days and extracts were analyzed by Western blotting. (E) Downregulation of endogenous B23 but not C23 by ARF. U2OS cells were infected with indicated viruses for 2 days and cell extracts were analyzed by Western blotting. (F) Downregulation of endogenous B23 by endogenous ARF induced by E2F1 in WI-38 cells. WI-38 and U2OS cells were infected with indicated viruses for 2 days and cell extracts were analyzed by Western blotting.

Molecular Cell 1158

Downregulation of B23 Inhibits rRNA Processing We next examined alterations of B23 biological function by ARF. Among several measurable functions of B23, its endoribonuclease activity is well documented (Herrera et al., 1995; Savkur and Olson, 1998). Utilizing transcripts synthesized in vitro, it has been demonstrated that B23 cleaves the second internal transcribed spacer (ITS2) in rRNA precursor. This cleavage prepares rRNA for digestion by exonucleases and subsequent maturation into 28S rRNA, a major RNA component in the 60S ribosomal subunit (Figure 6A) (Hadjiolova et al., 1994). It has been recently reported that ARF inhibits the processing of pre-ribosomal RNA, linking ARF with control of ribosomal biogenesis (Sugimoto et al., 2003). To determine the dynamic processing of rRNA precursors and intermediates, we carried out pulse-chase labeling analysis of newly synthesized rRNA under conditions in which B23 was reduced by either ARF overexpression or siRNA B23 inhibition in Saos2 cells (Saos2 cells were chosen to avoid p53-dependent effect after ad-ARF infection). We adjusted the experimental conditions so that both ad-ARF infection and B23 siRNA transfection achieved a comparable downregulation (approximately 50%) of endogenous B23 (Figure 6B). As shown in Figure 6C, the pulse-labeled 47S rRNA precursor was readily detected in all treatments at the beginning of chasing (0 min), indicating the transcription of the rRNA precursor was not blocked by any of the treatments. At the 60 min timepoint, the amount of mature 28S rRNA was approximately equal to the 32S intermediate in cells transfected with scrambled siRNA or infected with adGFP, with a 28S/32S ratio of 1.16 and 1.01, respectively (Figure 6C, lanes 3 and 9; Figure 6D, columns 1 and 3), indicating an active processing of the 32S precursor. In contrast, when cells were transfected with B23 siRNA or infected with ad-ARF, the level of 28S rRNA was markedly decreased and the ratio of 28S/32S was reduced to 0.45 and 0.33, respectively (Figure 6C, lanes 6 and 12; Figure 6D, columns 2 and 4), demonstrating that downregulation of B23 inhibited processing of the 32S intermediate for subsequent maturation of the 28S rRNA. Notably, while the maturation of 28S rRNA was markedly reduced, the maturation of 18S rRNA was not significantly affected by B23 siRNA. This is consistent with the role of B23 as an ITS2-specific endoribonuclease that cleaves 32S intermediate into 28S rRNA (Savkur and Olson, 1998). Our data also showed that overexpression of ARF inhibited not only the processing of the 32S precursor but also the processing of the 47S rRNA precursor, suggesting that ARF has a broader spectrum of targets than just B23 in its inhibition of rRNA processing (Figure 6C, lanes 10 to 12).

Figure 5. ARF-Induced B23 Degradation Involves B23 Polyubiquitination and the 26S Proteasome Pathway but Not CRM1-Mediated Nuclear Export (A) The 26S proteasome inhibitor MG132 prevents ARF-induced B23 degradation. U2OS cells were infected with indicated viruses for 2 days. Cells were treated with 20 ␮M of MG132 for 12 hr before harvesting. Protein extracts were analyzed by Western blotting. (B) ARF promotes polyubiquitination of B23. H1299 cells were trans-

fected with indicated plasmids for 3 days. In vivo ubiquitination assay was carried out as described in Experimental Procedures. His-tagged B23 was precipitated by nickel-charged resin beads. Polyubiquitinated bands of His-B23 modified by HA-polyubiqutine were revealed using the HA antibody by Western blotting (upper panel). The membrane was reprobed with His antibody to reveal precipitated, nonubiquitinated His-B23 (middle panel). Cell extracts were analyzed by Western blotting using ARF antibody (lower panel) (C) No effect of LMB on B23 degradation by ARF. U2OS cells were infected with indicated viruses for 2 days. Cells were treated with 10 ␮M of LMB for 12 hr before harvesting. Protein extracts were analyzed by Western blotting.

Inhibition of Nucleolar B23 by ARF 1159

Figure 6. Downregulation of B23 Inhibits Ribosome Biogenesis (A) A diagram of RNA processing (Ruggero and Pandolfi, 2003). (B) Downregulation of B23 by siRNA and ARF. Saos2 cells were transfected with either a control scrambled RNA duplex (Scr) or B23 siRNA (B23) for 3 days, or infected with indicated viruses for 2 days. Cell extracts were analyzed by Western blotting. (C and D) Blocking of 28S rRNA maturation by ARF overexpression and by B23 inhibition. Saos2 cells were either transfected with siRNA for 3 days or infected with indicated viruses for 2 days, pulse labeled with L-[methyl-3H]-methionine, and chased for the indicated times. An equal amount of radioactivity was loaded into each lane. Ethidium bromide staining of 28S rRNA is shown at the bottom. The ratio of densitometry signals of 28S/32S for lanes 3, 6, 9, and 12 calculated on a PhosphoImager are shown.

Effect of B23 Level on Cell Proliferation and Survival Because the tumor suppressor ARF induces B23 degradation, and because B23 is an important enzyme in rRNA processing, a probable role of altering the level of B23 in cell proliferation and survival was examined. We first determined the effect of high level B23 on cell cycle progression. Consistent with a previous report showing that high level B23 induces growth arrest (Colombo et al., 2002), ad-B23 infection in WI-38 cells reduced S phase population (Figure 7A). However, we found that in the absence of p53, B23 promoted S phase entry, which was observed in both the Saos2 tumor cells and the p53 null MEF cells (Figure 7A), indicating that a high level B23 may have an oncogenic potential to promote cell growth that may provoke a p53 response. We next examined the effect of low level B23 on cells. B23 was reduced by siRNA transfection in WI-38 and U2OS cells to about half of amount compared to control transfections (Figure 7B, right). Under these conditions, the distribution of cells in G1, S, and G2 phases were not significantly altered (Figure 7B). However, we observed a large increase of sub-G1 population, indicative of cell death, in

siB23 transfected cells but not in control transfections, indicating that downregulation of B23 induces cell death. After sub-G1 cells were gated out, a slight increase of S phase in siB23-transfected cells was observed (see Supplemental Figure S1 at http://www.molecule.org/ cgi/content/full/12/5/1151/DC1), which is consistent with a previous report (Colombo et al., 2002). Together, our data show that in absence of p53, an abnormally high level of B23 promotes S phase entry, while in the presence of p53 it induces cell cycle arrest. Conversely, an abnormally low level of B23 induces cell death. Whether or not B23 has an oncogenic potential to induce ARF expression when deregulated remains to be investigated. A Role of ARF in Preventing Oncogenic Insult-Induced B23 Overexpression If ARF acts as an antagonizer in nucleolus to counterbalance B23, one would expect to see an inverse correlation of the level of ARF and B23, especially in normal cells in which the signaling pathways controlling the expression of ARF and B23 are intact. To test this hy-

Molecular Cell 1160

Figure 7. The Effect of B23 Level on Cell Proliferation and Survival and a Role of ARF in Preventing Induction of B23 by Oncogenic Insult (A) B23 induces cell cycle arrest in WI-38 cells but promotes S phase entry in Saos2 and p53⫺/⫺ MEFs. WI-38, Saos2, and p53⫺/⫺ MEF cells were infected with indicated viruses. Cells were harvested after 2 days of infection, stained with propidium iodide (PI), and the cell cycle distribution was determined by flow cytometry. Percentage of cells in the S phase is indicated. (B) Cell death caused by B23 downregulation. WI-38 and U2OS cells were transfected with indicated siRNA for 3days and analyzed by flow

Inhibition of Nucleolar B23 by ARF 1161

pothesis, we examined endogenous levels of ARF and B23 in early- and late-passage wild-type MEF cells. As shown in Figure 7C, late-passage MEFs express high levels of ARF, a characteristic feature of MEF cells approaching senescence. Notably, these late-passage MEF cells express much less B23 than those early-passage MEFs, which express a high level of B23 and a low level of ARF. This inverse correlation of the levels of ARF and B23 was also seen between the wild-type and ARF null MEFs. As shown in Figure 7D, ARF null MEF cells express much higher levels of B23 than those wild-type MEFs, even though both cell types are from early passages and have similar doubling times, suggesting that both the existence and the level of ARF counterbalance B23. Finally, we tested the idea that cells sustaining oncogenic insults might induce B23 and that the induction of B23 might also be counteracted by ARF. We used retroviruses expressing Ha-Ras (V12) to infect MEF cells to first test whether overexpression of an oncogene can induce B23. ARF null, p53 null, and p53/MDM2 double null MEFs were chosen for the experiment to avoid Rasmediated premature senescence. ARF null MEF cells were infected with control or retro-Ras. After selection, the level of B23 was determined by Western blotting. In both early- and late-passage ARF null MEFs, Ras induced expression of B23 (Figure 7E). Next, we examined whether induction of ARF by RAS may prevent the induction/accumulation of B23. As shown in Figure 7F, in both p53 null and p53/MDM2 double null MEFs Ras induced expression of ARF. Significantly however, Ras did not induce B23 in these cells, strongly suggesting that the presence of ARF might have prevented the induction/ accumulation of B23. Discussion The nucleolus has long been recognized as the site of ribosome biogenesis. Major steps of ribosome biogenesis, such as preribosomal RNA transcription, rRNA modification and processing, and ribosomal subunit assembly, all occur in the nucleolus (Olson et al., 2002). Interestingly, the tumor suppressor ARF, whose currently known major function is to interact with MDM2 and to stabilize and activate p53, is also localized in the nucleolus. However, in contrast to the nucleolar ARF, MDM2 and p53 are both nuclear proteins normally localized in the nucleoplasm and excluded from the nucleolus. The distinct cellular compartmentalization of ARF from its binding target, MDM2, led to a proposal that ARF inhibits MDM2 by sequestering it in the nucleolus, away from the nucleoplasmic p53. Although evidence

indicates this is happening in cells (Tao and Levine, 1999; Weber et al., 1999), recent studies have demonstrated that ARF can efficiently inhibit MDM2 without sequestering it into the nucleolus (Korgaonkar et al., 2002; Llanos et al., 2001). Then, why does ARF predominantly localize to the nucleolus, especially when it is activated by oncogenic insults and its function becomes essential? Whether ARF has other binding partners, particularly those in the nucleolus where ARF normally resides remains unclear, and is also a critical question to our understanding of physiological role of ARF in the nucleolus. Recently, Sugimoto et al. (2003) showed that ARF inhibits the processing of preribosomal RNA, and this function of ARF is probably through its binding with 5.8S rRNA. We report here that ARF interacts with the nucleolar protein B23. Through this interaction, ARF inhibits the endoribinuclease activity of B23, which is due, at least in part, to an accelerated protein degradation of B23 by ARF. Although the exact mechanism of B23 degradation by ARF is not clear, our data indicate that B23 can be polyubiquitinated in vivo and be degraded through the 26S proteasomal pathway, and that ARF promotes the polyubiquitination of B23. Whether there exists a specific E3 ubiquitin ligase complex for B23 and whether ARF acts as a linker to bring together B23 and the E3 complex remain to be determined. Alternatively, ARF may reduce B23 stability by other mechanisms. For example, B23 has a tendency to oligomerize in vivo and exists as a hexamer (Yung and Chan, 1987) via its N-terminal domain (Hingorani et al., 2000). In the absence of ARF, B23 most likely exists as a hexamer. This hexamer B23 may not be accessible to E3 ligase activity and is, therefore, very stable. The fact that we did not detect an obvious decrease of 35S-labeled B23 after 24 hr of chasing in the absence of ARF infection indicates the high stability of the B23 protein (Figure 3C). ARF, by interacting with the N-terminal, oligomerization domain of B23, may disrupt the hexamer form of B23, leaving a nonhexamer form of B23 that becomes accessible to the polyubiquitnation degradation pathway. B23 is an abundant protein localized in both the nucleolus and the nucleoplasm (Figure 4B). We have noticed that the fraction of B23 localized in the nucleoplasm is preferentially degraded by ARF, whereas the nucleolar localized B23 is more resistant to ARF-mediated degradation (our unpublished data). Even though ARF may not completely degrade B23, it may still be able to block its activity by forming a complex with it in the nucleolus. An ARF-bound B23 may lose its endoribonuclease activity initially and subsequently be tagged for polyubiquitination and degradation. Such a mechanism of B23 inhi-

cytometry as in (A). Percentage of cells in the sub-G1-phase is indicated. Cell extracts from cells transfected with scrambled siRNA (siScr) and B23 siRNA (siB23) were analyzed by Western blotting and the results are shown in the right panels. (C) Inverse correlation between the level of B23 and ARF in MEFs during passage. Wild-type MEFs were serially passaged and cell extracts were isolated and analyzed by Western blotting. (D) Levels of B23 in MEFs. Cell extracts from wild-type (P2) and ARF⫺/⫺ MEFs (P4) were analyzed by Western blotting. (E) H-Ras induce B23 in ARF⫺/⫺ MEFs. Early (P4) and late (⬎P20) passage ARF⫺/⫺ MEFs were infected with retroviruses carrying pBabe vector (vector) or pBabe-Ha-RAS (V12) (Ras). After 2 days of infection, cells were selected by puromycine (2 ␮g/ml) for 3 days and cell extracts were harvested and analyzed by Western blotting. (F) H-Ras does not induce B23 in p53⫺/⫺ and p53⫺/⫺/MDM2⫺/⫺ MEFs. Indicated cells were infected and selected as in (E). Cell extracts were harvested and analyzed by Western blotting.

Molecular Cell 1162

bition by ARF could thus ensure a fast and efficient ribosome biogenesis blockade in response to hyperproliferative signals. In addition, ARF may target multiple steps in the ribosome biogenesis pathway. We have noticed that the extent of rRNA processing inhibition by ARF is not identical to that observed by knocking down B23. Our data show that knocking down B23 inhibited only the processing of the 32S rRNA precursor into 28S rRNA, which is consistent with the biochemical activity of B23 as the ITS2-specific endoribonuclease (Savkur and Olson, 1998). However, ARF overexpression not only inhibited the processing of 32S into 28S rRNA to a similar extent as seen in B23 knocking down by siRNA, but also clearly retarded processing of the 47S precursor (Figure 6C). We have identified several other polypeptides in the ARF immunocomplex whose functions involve ribosome biogenesis (our unpublished data). We speculate that a major biological function of ARF is to inhibit ribosome biogenesis in response to environmental cues by directly binding to multiple key components involved in rRNA processing and ribosomal assembly in the nucleolus. The importance of coordinating cell growth (increase of cell size) with proliferation (increase of cell number) has been recognized for a long time (Johnston et al., 1977). However, the molecular basis of this relationship is poorly understood, and the proteins responsible for mediating the crosstalk between ribosome biogenesis and cell cycle progression remain largely unknown (Ruggero and Pandolfi, 2003). Our findings suggest that the ARF-B23 interaction may function in coordinating cell growth with proliferation. For example, when a harmful oncogenic signal triggers a cell cycle checkpoint by activating the ARF-MDM2-p53 pathway, it may simultaneously activate a ribosome biogenesis checkpoint by the ARF-B23-ribosome pathway. In such a situation, ARF serves a dual function to restrain both growth and proliferation. Given the large quantity of B23 versus the relatively small amount of MDM2 in growing cells, it is conceivable that the majority of ARF protein could be participating in inhibiting B23 in the nucleolus, whereas a fraction of ARF participates in inhibiting MDM2 in the nucleoplasm. This notion is consistent with a recent report that a subfraction of ARF in the nucleoplasm is sufficient to block MDM2 function (Llanos et al., 2001). Ample evidences have indicated that B23 may have an oncogenic potential when overexpressed. For example, high levels of B23 have been linked with neoplastic growth in various cell types (Feuerstein and Mond, 1987; Feuerstein et al., 1988; Nozawa et al., 1996). In our study, overexpression of B23 reduced the percentage of G1 phase and increased the S phase population in p53 negative cells, but induced cell cycle arrest in normal cells (Figure 7A). Previously, Colombo et al. (2002) have shown that B23 overexpression induces senescence in normal fibroblasts. B23 can either cause a growth arrest or promote S phase depending on p53. This property is quite similar to the one for oncogenic RAS, which induces senescence in MEFs but induces growth in p53 null MEFs (Serrano et al., 1997). We showed that B23 was induced by oncogenic Ras in an ARF null background and the presence of ARF can attenuate B23 induction. On the other hand, we also observed that virus-mediated B23 overexpression can

overcome ARF-induced cell cycle arrest (data not shown) suggesting that some of the potential oncogenic activity of B23 may reside in its ability to inactivate ARF function. Whether or not a high level of B23 will provoke ARF expression, thus comprising a negative feedback regulatory loop between ARF and B23, remains to be determined. Experimental Procedures Cell Lines and Cell Culture U2OS, Saos2, HeLa, and H1299 cells were obtained from either ATCC or the UNC Lineberger Tissue Culture Facility. WI-38 cells (PD25) were obtained from Dr. Judith Campisi (Lawrence Berkeley National Laboratory, Berkeley, CA). p19ARF⫺/⫺, p53⫺/⫺MEFs, and p53⫺/⫺/MDM2⫺/⫺ MEFs were gift from Dr. Guillermina Lozano. All cells were cultured in a 37⬚C incubator with 5% CO2 in DMEM with 10%FBS. Vectors, Adenoviruses, Retroviruses, Transfections, and FACS Analysis Full-length B23 cDNA was purchased from Invitrogen (accession number BC008495), tagged with myc, and cloned into pcDNA3 (Invitrogen). Deletion constructs were made by PCR-mediated mutagenesis. All mutants were confirmed by direct DNA sequencing. ARF constructs, adenoviruses, adenovirus infections, transfections, and FACS analysis were described elsewhere (Zhang and Xiong, 1999; Zhang et al., 1998). H-RAS retroviruses were obtained from Dr. Sandy Chang. Retroviruses were produced and infected as described (Itahana et al., 2003). Protein Analysis B23 and ␣-actin antibodies were purchased from Zymed and Chemicon, respectively. p53 (FL-393) and C23 (MS-3) antibodies were obtained from Santa Cruz. MDM2 (Ab1) antibodies were purchased from Calbiochem. p53 (DO1), Myc (9E10.3), and ARF (P02) and E2F-1 (Ab-1;SQ41) antibodies were purchased from NeoMarkers. RAS (clone18) was from BD Transduction Laboratories. P53 (PAb421) was obtained from Oncogene Science. HA (16B12) antibody was from SIGMA. Cells were lysed in 0.1% NP-40 buffer for immunoprecipitation and 0.5% NP-40 buffer for straight Western as previously described (Zhang and Xiong, 1999). The indirect immunofluorescence was previously described (Zhang and Xiong, 1999). The affinity purified rabbit polyclonal ␣-human ARF and ␣-p53 antibodies, Western blotting, immunoprecipitations, pulse-chase experiments, and binding assays were described elsewhere (Zhang and Xiong, 1999). In Vivo Ubiquitin Assay Cells were lysed in buffer (pH 7.8) containing 6 M guanidine-HCl. The lysates were centrifuged at 13,000 ⫻ g for 30 min. The supernatant was incubated with nickel-charged agarose beads (Ni-NTA SUPERFLOW, Qiagen) for 1 hr. The beads were washed twice with the buffer (ph 7.8) containing 8 M Urea, followed by washing twice with buffer (pH 6.0) containing 8 M Urea. The beads were dissolved in 2% SDS buffer for SDS-PAGE after washing with PBS. RNA Analysis For monitoring ribosomal RNA processing, pulse-chase experiments using [methyl-3H]-methionine were carried out due to the methylation of ribosomal RNA precursors and rapid turnover of the cellular pool of methionine, which donates 3H-methyl (Strezoska et al., 2000). Cells were starved of methionine for 30 min on 60 mm plates with 1 ml of methionine-free medium and were labeled with 50 ␮Ci of L-[methyl-3H]-methionine (Amersham-Pharmacia) for 30 min. Cold methionine (15 ␮g/ml) was added to chase the label for various lengths of times. After washing with PBS, the total RNA was purified by an RNA isolation system (Promega) and the incorporated radioactivity was measured by a liquid scintillation counter. Equal amounts of radioactivity were loaded onto 1% agrose denaturing gel containing 0.55 M formaldehyde. The RNA was fractionated and transferred onto a Zeta-probe blotting membrane (Bio-Rad). The

Inhibition of Nucleolar B23 by ARF 1163

membrane was dried and sprayed by EN3HANCE (Perkin Elmer) and exposed to Hyperfilm MP (Amersham Pharmacia) at -80⬚C with an intensifying screen for 3 days. The Northern analysis was described elsewhere (Itahana et al., 2003). Full-length B23 cDNA was used as a probe; 15 ␮g of total RNA was loaded in each lane. RNA Interference The siRNA oligonucleotides, targeting nucleotides 103 to 125 relative to the translation initiation codon of human B23 (5⬘UGAU GAAAAUGAGCACCAGTT3⬘) (Colombo et al., 2002), and a control scrambled siRNA oligonucleotides were synthesized by Dharmacon (Lafayette, CO). siRNA oligonucleotides (150 nM) in OPTI-MEM (Invitrogen) were transfected by Lipofectamine-plus (Invitrogen) for 5 hr following manufacturer’s protocol. After transfection, an equal amount of DMEM containing 20% FBS was added, and medium was replaced with DMEM containing 10% FBS after 20 hr. Cells were lysed 3 days after transfections. All cultures were performed without antibiotics. Acknowledgments We thank Drs. Judith Campisi, Guillermina Lozano, Sandy Chang, Mien-Chie Hung, and Michael Van Dyke for providing materials; Drs. Wei-Ya Xia, Tomoo Iwakuma, and Kevin O’Keefe for technical support. Y. Z. is a recipient of a Career Award in Biomedical Science from the Burroughs Wellcome Fund and a Howard Temin Award from National Cancer Institute. This study was supported by the M.D. Anderson Research Trust Fund and NIH grant (to Y.Z.). Received: July 14, 2003 Revised: October 1, 2003 Accepted: October 2, 2003 Published: November 20, 2003 References Bates, S., Phillips, A.C., Clark, P., Stott, F., Peters, G., Ludwig, R., and Vousden, K.H. (1998). p14ARF links the tumor suppressors RB and p53. Nature 395, 124–125. Colombo, E., Marine, J.C., Danovi, D., Falini, B., and Pelicci, P.G. (2002). Nucleophosmin regulates the stability and transcriptional activity of p53. Nat. Cell Biol. 4, 529–533. Conlon, I., and Raff, M. (1999). Size control in animal development. Cell 96, 235–244. de Stanchina, E., McCurrach, M.E., Zindy, F., Shieh, S.-Y., Ferbeyre, G., Samuelson, A.V., Prives, C., Roussel, M.F., Sherr, C.J., and Lowe, S.W. (1998). E1A signaling to p53 involves the p19ARF tumor suppressor. Genes Dev. 12, 2434–2442. Feuerstein, N., and Mond, J.J. (1987). Numatrin, a nuclear matrix protein associated with induction of proliferation in B lymphocytes. J. Biol. Chem. 262, 11389–11397. Feuerstein, N., Spiegel, S., and Mond, J.J. (1988). The nuclear matrix protein, numatrin (B23), is associated with growth factor-induced mitogenesis in Swiss 3T3 fibroblasts and with T lymphocyte proliferation stimulated by lectins and anti-T cell antigen receptor antibody. J. Cell Biol. 107, 1629–1642. Freedman, D.A., and Levine, A.J. (1998). Nuclear export is required for degradation of endogenous p53 by MDM2 and human papillomavirus E6. Mol. Cell. Biol. 18, 7288–7293. Hadjiolova, K.V., Normann, A., Cavaille, J., Soupene, E., Mazan, S., Hadjiolov, A.A., and Bachellerie, J.P. (1994). Processing of truncated mouse or human rRNA transcribed from ribosomal minigenes transfected into mouse cells. Mol. Cell. Biol. 14, 4044–4056. Hemmati, P.G., Gillissen, B., von Haefen, C., Wendt, J., Starck, L., Guner, D., Dorken, B., and Daniel, P.T. (2002). Adenovirus-mediated overexpression of p14(ARF) induces p53 and Bax-independent apoptosis. Oncogene 21, 3149–3161. Herrera, J.E., Savkur, R., and Olson, M.O. (1995). The ribonuclease activity of nucleolar protein B23. Nucleic Acids Res. 23, 3974–3979. Hingorani, K., Szebeni, A., and Olson, M.O. (2000). Mapping the

functional domains of nucleolar protein B23. J. Biol. Chem. 275, 24451–24457. Honda, R., and Yasuda, H. (1999). Association of p19ARF with MDM2 inhibits ubiquitin ligase activity of MDM2 for tumor suppressor p53. EMBO J. 18, 22–27. Itahana, K., Zou, Y., Itahana, Y., Martinez, J.L., Beausejour, C., Jacobs, J.J., Van Lohuizen, M., Band, V., Campisi, J., and Dimri, G.P. (2003). Control of the replicative life span of human fibroblasts by p16 and the polycomb protein Bmi-1. Mol. Cell. Biol. 23, 389–401. Johnston, G.C., Pringle, J.R., and Hartwell, L.H. (1977). Coordination of growth with cell division in the yeast Saccharomyces cerevisiae. Exp. Cell Res. 105, 79–98. Kamb, A., Gruis, N.A., Weaver-Feldhaus, J., Liu, Q., Harshman, K., Tavitgian, S.V., Stockert, E., Day, R.S., Johnson, B.E., and Skolnick, M.H. (1994). A cell cycle regulator potentially involved in genesis of many tumor types. Science 264, 436–440. Kamijo, T., Zindy, F., Roussel, M.F., Quelle, D.E., Downing, J.R., Ashmun, R.A., Grosveld, G., and Sherr, C.J. (1997). Tumor suppression at the mouse INK4a locus mediated by the alternative reading frame product p19ARF. Cell 91, 649–659. Kamijo, T., Weber, J.D., Zambetti, G., Zindy, F., Roussel, M., and Sherr, C.J. (1998). Functional and physical interaction of the ARF tumor suppressor with p53 and MDM2. Proc. Natl. Acad. Sci. USA 95, 8292–8297. Korgaonkar, C., Zhao, L., Modestou, M., and Quelle, D.E. (2002). ARF function does not require p53 stabilization or Mdm2 relocalization. Mol. Cell. Biol. 22, 196–206. Kressler, D., Linder, P., and de La Cruz, J. (1999). Protein transacting factors involved in ribosome biogenesis in Saccharomyces cerevisiae. Mol. Cell. Biol. 19, 7897–7912. Lischwe, M.A., Richards, R.L., Busch, R.K., and Busch, H. (1981). Localization of phosphoprotein C23 to nucleolar structures and to the nucleolus organizer regions. Exp. Cell Res. 136, 101–109. Llanos, S., Clark, P.A., Rowe, J., and Peters, G. (2001). Stabilization of p53 by p14ARF without relocation of MDM2 to the nucleolus. Nat. Cell Biol. 3, 445–452. Matsuoka, M., Kurita, M., Sudo, H., Mizumoto, K., Nishimoto, I., and Ogata, E. (2003). Multiple domains of the mouse p19(ARF) tumor suppressor are involved in p53-independent apoptosis. Biochem. Biophys. Res. Commun. 301, 1000–1010. Neufeld, T.P., and Edgar, B.A. (1998). Connections between growth and the cell cycle. Curr. Opin. Cell Biol. 10, 784–790. Nobori, T., Mlura, K., Wu, D.J., Lois, A., Takabayashi, K., and Carson, D.A. (1994). Deletion of the cyclin-dependent kinase-4 inhibitor gene in multiple human cancers. Nature 368, 753–756. Nozawa, Y., Van Belzen, N., Van der Made, A.C., Dinjens, W.N., and Bosman, F.T. (1996). Expression of nucleophosmin/B23 in normal and neoplastic colorectal mucosa. J. Pathol. 178, 48–52. Okuda, M., Horn, H.F., Tarapore, P., Tokuyama, Y., Smulian, A.G., Chan, P.K., Knudsen, E.S., Hofmann, I.A., Snyder, J.D., Bove, K.E., et al. (2000). Nucleophosmin/B23 is a target of CDK2/cyclin E in centrosome duplication. Cell 103, 127–140. Olson, M.O., Wallace, M.O., Herrera, A.H., Marshall-Carlson, L., and Hunt, R.C. (1986). Preribosomal ribonucleoprotein particles are a major component of a nucleolar matrix fraction. Biochemistry 25, 484–491. Olson, M.O., Hingorani, K., and Szebeni, A. (2002). Conventional and nonconventional roles of the nucleolus. Int. Rev. Cytol. 219, 199–266. Palmero, I., Pantoja, C., and Serrano, M. (1998). p19ARF links the tumor suppressor p53 and Ras. Nature 395, 125–126. Pomerantz, J., Schreiber-Agus, N., Liegeois, N.J., Silverman, A., Alland, L., Chin, L., Potes, J., Chen, K., Orlow, I., and DePinho, R.A. (1998). The INK4a tumor suppressor gene product, p19Arf, interacts with MDM2 and neutralizes MDM2⬘s inhibition of p53. Cell 92, 713–723. Quelle, D.E., Zindy, F., Ashmun, R., and Sherr, C.J. (1995). Alterative reading frames of the INK4a tumor suppressor gene encode two unrelated proteins capable of inducing cell cycle arrest. Cell 83, 993– 1000.

Molecular Cell 1164

Radfar, A., Unnikrishnan, I., Lee, H.-W., DePinho, R., and Rosenberg, N. (1998). p19Arf induces p53-dependent apoptosis during Abelson virus-mediated pre-B cell transformation. Proc. Natl. Acad. Sci. USA 95, 13194–13199. Roth, J., Dobbelstein, M., Freedman, D.A., Shenk, T., and Levine, A.J. (1998). Nucleo-cytoplasmic shuttling of the hdm2 oncoprotein regulates the levels of the p53 protein via a pathway used by the human immunodeficiency virus rev protein. EMBO J. 17, 554–564. Ruas, M., and Peters, G. (1998). The p16INK4a/CDK2A tumor suppresor and its relatives. Biochim. Biophys. Acta 1378, F115–F177. Ruggero, D., and Pandolfi, P.P. (2003). Does the ribosome translate cancer? Nat. Rev. Cancer 3, 179–192. Savkur, R.S., and Olson, M.O. (1998). Preferential cleavage in preribosomal RNA byprotein B23 endoribonuclease. Nucleic Acids Res. 26, 4508–4515. Serrano, M., Lin, A.W., McCurrach, M.E., Beach, D., and Lowe, S.W. (1997). Oncogenic ras provokes premature cell senescence associated with accumulation of p53 and p16INK4a. Cell 88, 593–602. Sherr, C.J. (1998). Tumor surveillance via the ARF-p53 pathway. Genes Dev. 12, 2984–2991. Spector, D.L., Ochs, R.L., and Busch, H. (1984). Silver staining, immunofluorescence, and immunoelectron microscopic localization of nucleolar phosphoproteins B23 and C23. Chromosoma 90, 139–148. Stommel, J.M., Marchenko, N.D., Jimenez, G.S., Moll, U.M., Hope, T.J., and Wahl, G.M. (1999). A leucine-rich nuclear export signal in the p53 tetramerization domain: regulation of subcellular localization and p53 activity by NES masking. EMBO J. 18, 1660–1672. Stott, F.J., Bates, S., James, M.C., McConnell, B.B., Starborg, M., Brookes, S., Palmero, I., Ryan, K., Hara, E., Vousden, K.H., et al. (1998). The alternative product from the human CDK2A locus, p14ARF, participates in a regulatory feedback loop with p53 and MDM2. EMBO J. 17, 5001–5014. Strezoska, Z., Pestov, D.G., and Lau, L.F. (2000). Bop1 is a mouse WD40 repeat nucleolar protein involved in 28S and 5.8S RRNA processing and 60S ribosome biogenesis. Mol. Cell. Biol. 20, 5516– 5528. Sugimoto, M., Kuo, M.L., Roussel, M.F., and Sherr, C.J. (2003). Nucleolar Arf tumor suppressor inhibits ribosomal RNA processing. Mol. Cell 11, 415–424. Szebeni, A., and Olson, M.O. (1999). Nucleolar protein B23 has molecular chaperone activities. Protein Sci. 8, 905–912. Tao, W., and Levine, A.J. (1999). p19ARF stabilizes p53 by blocking nucleo-cytoplasmic shuttling of Mdm2. Proc. Natl. Acad. Sci. USA 96, 6937–6941. Tsuji, K., Mizumoto, K., Sudo, H., Kouyama, K., Ogata, E., and Matsuoka, M. (2002). p53-independent apoptosis is induced by the p19ARF tumor suppressor. Biochem. Biophys. Res. Commun. 295, 621–629. Venema, J., and Tollervey, D. (1999). Ribosome synthesis in Saccharomyces cerevisiae. Annu. Rev. Genet. 33, 261–311. Wang, D., Baumann, A., Szebeni, A., and Olson, M.O. (1994). The nucleic acid binding activity of nucleolar protein B23.1 resides in its carboxyl-terminal end. J. Biol. Chem. 269, 30994–30998. Warner, J.R. (1999). The economics of ribosome biosynthesis in yeast. Trends Biochem. Sci. 24, 437–440. Weber, J.D., Taylor, L.J., Roussel, M.F., Sherr, C.J., and Bar-Sagi, D. (1999). Nucleolar Arf sequesters Mdm2 and activates p53. Nat. Cell Biol. 1, 20–26. Weber, J.D., Jeffers, J.R., Rehg, J.E., Randle, D.H., Lozano, G., Roussel, M.F., Sherr, C.J., and Zambetti, G.P. (2000a). p53-independent functions of the p19(ARF) tumor suppressor. Genes Dev. 14, 2358–2365. Weber, J.D., Kuo, M.-L., Bothner, B., Digiammarino, E.L., Kriwacki, R.W., Roussel, M.F., and Sherr, C.J. (2000b). Cooperative signals governing ARF-MDM2 interaction and nucleolar localization of the complex. Mol. Cell. Biol. 20, 2517–2528. Yarbrough, W.G., Bessho, M., Zanation, A., Bisi, J.E., and Xiong, Y. (2002). Human tumor suppressor ARF impedes S-phase progression independent of p53. Cancer Res. 62, 1171–1177.

Yung, B.Y., and Chan, P.K. (1987). Identification and characterization of a hexameric form of nucleolar phosphoprotein B23. Biochim. Biophys. Acta 925, 74–82. Zhang, Y., and Xiong, Y. (1999). Mutation in human ARF exon 2 disrupt its nucleolar localization and impair its ability to block nuclear export of MDM2 and p53. Mol. Cell 3, 579–591. Zhang, Y., and Xiong, Y. (2001a). Control of p53 ubiquitination and nuclear export by MDM2 and ARF. Cell Growth Diff 12, 175. Zhang, Y., and Xiong, Y. (2001b). A p53 amino terminal nuclear export signal inhibited by DNA damage-induced phosphorylation. Science 292, 1910. Zhang, Y., Xiong, Y., and Yarbrough, W.G. (1998). ARF promotes MDM2 degradation and stabilizes p53: ARF-INK4a locus deletion impairs both the Rb and p53 tumor suppression pathways. Cell 92, 725–734. Zindy, F., Eischen, C.M., Randle, D.H., Kamijo, T., Cleveland, J.L., Sherr, C.J., and Roussel, M.F. (1998). Myc signaling via the ARF tumor suppressor regulates p53-dependent apoptosis and immortalization. Genes Dev. 12, 2424–2433.