Nucleolar Arf sequesters Mdm2 and activates p53

Arf relocalizes Mdm2 to the nucleolus. p19^Arf was originally found to localize to nuclear speckles ¹ that are now recognized as nucleoli ¹⁷. Exogenous Arf blocks cell-cycle progression in wild-type and Arf-null cells but is without effect in cells lacking functional p53 (ref. 3, 17). The amino-terminal domain of p19 ^Arf (residues 1−62; referred to here as Arf N62) is necessary and sufficient for binding to Mdm2 and for cell-cycle arrest, whereas the p19^Arf carboxy terminus (residues 63−169; Arf 1−62) is dispensable ^{18, 27}. To understand the significance of Arf localization, we first microinjected plasmids encoding green fluorescent protein (GFP)-tagged forms of mouse p19^Arf and various Arf mutants into MEFs of different genotypic backgrounds (wild-type, p53-null, Mdm2/p53 double-null and Arf-null backgrounds). GFP alone accumulated in the cytoplasm and nucleoplasm of injected wild-type cells (data not shown), but GFP-tagged Arf and GFP-tagged Arf N62 localized to the nucleolus (Fig. 1a, b). In contrast, GFP-tagged Arf 1−62 was excluded from the nucleolus and localized to both the nucleoplasm and the cytoplasm of wild-type cells (Fig. 1c). Nucleolar localization of GFP−Arf occurred in MEFs of all genotypes tested and therefore did not depend upon the presence of endogenous Arf, p53 or Mdm2 proteins.

Figure 1. Subcellular localization of p19Arf and relocalization of HDM2 in cells expressing exogenous Arf proteins. a−j, Serum-starved wild-type MEFs at passage 7 were microinjected with cDNAs encoding GFP-tagged Arf (a, d), GFP-tagged Arf N62 (b, e, g−j) or GFP-tagged Arf 1−62 (c, f). a−c, Cells were fixed 6 h after injection and visualized for GFP expression (green) using an FITC filter. DAPI staining of corresponding nuclei is shown in d−f. g, Cells were fixed 6 h after injection and visualized for GFP expression using an FITC filter. This image is the result of confocal analysis of a 0.25-µm optical section, showing GFP-tagged Arf N62. h, Confocal analysis of a 0.25-µm optical section, showing labelling with an anti-fibrillarin antibody. i, j, Labelling for both GFP-tagged Arf N62 and fibrillarin shows non-overlapping (red and green, i) and overlapping (yellow, i, j) regions. k−p, Serum-starved Arf-null (k, l) and wild-type (n−p) MEFs were microinjected with T7-epitope-tagged HDM2 plasmid DNA in the absence (k) or presence (l, n−p ) of cDNA encoding GFP-tagged Arf N62. Cells were fixed 12 h after injection and analysed by indirect immunofluorescence using a monoclonal antibody to T7 followed by rhodamine-conjugated anti-mouse immunoglobulin (k, l, o) and for GFP expression using an FITC filter (n). Confocal analysis of co-expressed GFP-tagged Arf N62 (green, n) and HDM2 (red, o) in a 0.25-µm optical section shows overlapping regions (yellow, p). m, Passage-12 (p12) wild-type MEFs were microinjected with T7-epitope-tagged HDM2. These cells were fixed 12 h after injection and analysed by indirect immunofluorescence using monoclonal anti-T7 antibody followed by rhodamine anti-mouse immunoglobulin. q−s, Co-transfection of NIH3T3 cells with GFP−Arf (q) and HDM2 (r) similarly results in overlapping regions (yellow, s). Scale bars represent 10 µm (a−f), 5 µm (g−j, n−p) or 7 µm (k−m). Full Figure and legend (64K)

Figure 2. Nucleolar accumulation of Arf and Mdm2 as MEFs approach replicative senescence. Wild-type MEFs propagated in culture on a 3T9 protocol (see Methods) were seeded onto coverslips at the indicated passages (p5, p10 or p15). a−f , m−o, Cells were fixed and analysed for localization of endogenous Arf (a−c), Mdm2 (d−f) and p53 (m−o ) using antibody to the Arf C terminus, a monoclonal anti-Mdm2 antibody (2A10) or monoclonal antibody 421 to p53, followed by either biotinylated anti-rabbit immunoglobulin and streptavidin-conjugated Texas Red (a− c) or FITC-conjugated anti-mouse immunoglobulin (d−f, m−o). g−i, Arf and Mdm2 co-localized in overlapping regions in nucleoli (yellow), whereas p53 remained in the nucleoplasm ( m−o). j−l, p−r, Nuclei were visualized by Hoechst staining of DNA. Exposure times were 10 s for a−c and m−o and 50 s for d−f. Full Figure and legend (30K)

Figure 3. Myc induces nucleolar accumulation of Mdm2 in an Arf-dependent manner. a−x, Wild-type MEFs (passage 7; a−r) or Arf -null MEFs (s−x) infected with a retrovirus encoding a Myc−ER TM fusion protein were seeded onto coverslips and treated with 4-hydroxytamoxifen (1 µM) for 0, 24 or 48 h in complete serum-containing medium. a−r , Treated cells were fixed and analysed for endogenous Arf, Mdm2 and p53 using antibodies as described in Fig. 2 followed by either biotinylated anti-rabbit immunoglobulin and streptavidin-conjugated Texas Red (a−c) or FITC-conjugated anti-mouse immunoglobulin (d−f, m−o). Co-localization of nucleolar Arf and Mdm2 is shown in overlapping regions (yellow, h, i). Nuclei were visualized by Hoechst staining of DNA (j−l, p−r ). s−x, Arf-null MEFs were similarly stained for Mdm2 ( t) and p53 (w). Nucleoli were detected using an antibody to fibrillarin followed by biotinylated anti-human immunoglobulin and streptavidin-conjugated Texas Red (s). The exclusion of Mdm2 from nucleoli of Arf-null MEFs is seen by the absence of overlapping regions (u). Nuclei were visualized by Hoechst staining of DNA (v, x). Exposure times were 1 s for a−c, 2 s for d−f, t, and 3 s for m−o, w. Full Figure and legend (42K)

Mobilization of Mdm2 by Arf activates p53. Proteins destined for the nucleolus often contain highly basic domains required for import, and Arf contains 24% arginine residues that are spatially distributed throughout the protein. Because the first 62 amino acids of Arf are critical for its nucleolar import, we deleted a cluster of basic amino acids (residues 26−37; KFVRSRRPRTAS) from the full-length Arf protein (producing Arf 26−37). We engineered a second Arf mutant (Arf 38−52) to lack the adjacent residues (CALAFVNMLLRLER). We infected NIH3T3 cells with retroviral vectors encoding these mutants, and blotted lysate proteins with an antibody to the Arf C terminus that does not detect Arf N62 (Fig. 4a). Full-length Arf and Arf 38−52 induced growth arrest in both G1 and G2 phases (the percentages of cells in S phase in the presence of full-length Arf and Arf 38−52 were 8% and 9%, respectively, 48 h post-infection). Arf 26−37 had no effect, yielding an S-phase fraction like that obtained with the control vector (percentages of S-phase cells were 25% and 28% in the presence of Arf 26−37 and control vector, respectively). In agreement with these results, Arf 38−52 increased expression of both p53 and Mdm2 in infected cells, but Arf 26−37 did not (4). Induction of Mdm2 was p53 dependent, as cells lacking functional p53 did not undergo arrest or show increased expression of Mdm2 (ref. 19 and data not shown).

Figure 4. Induction of p53 by Arf requires nucleolar recruitment of Mdm2. a−c, NIH3T3 fibroblasts (ARF-null) were lysed 48 h after infection with retroviruses encoding p19Arf, Arf N62, Arf 26−37 or ARF 38−52. Proteins were detected by direct immunoblotting using antibodies to the p19Arf C terminus (a) or to p53 (b). BALB3T3 10(1) fibroblasts (p53-null) were used as a negative control for p53 expression. Mdm2 protein was detected by immunoprecipitation with a monoclonal antibody (2A10) (as a control, NRS was used) followed by direct immunoblotting using antibody 2A10 (c). IP, immunoprecipitate. d, Sf9 cells co-infected for 48 h with baculoviruses encoding Mdm2 together with the indicated Arf mutants were lysed and precipitated with NRS, antibody to Mdm2 (2A10) or antibody to the Arf C terminus. Proteins in immune complexes separated on denaturing gels were transferred to membranes and detected by immunoblotting using antibodies to Mdm2 (top) or the Arf C terminus (bottom). e−m, For immunofluorescence, NIH3T3 cells were transfected with plasmids encoding Arf, Arf 26−37 or Arf 38−52 together with T7-epitope-tagged HDM2. Cells were fixed and analysed for Arf and HDM2 localization using antibody to the Arf C terminus or monoclonal antibody to T7 followed by either biotinylated anti-rabbit immunoglobulin and streptavidin-conjugated Texas Red (e, h, k) or FITC-conjugated anti-mouse immunoglobulin (f, i, l). Nuclei were visualized by Hoechst staining of DNA (g, j, m). Full Figure and legend (32K)

Full-length Arf and both Arf 26−37 and Arf 38−52 bound physically to Mdm2 when the proteins were co-expressed in Sf9 cells (Fig. 4d). In contrast, Arf 1−62, which is biologically inert ²⁷, was unable to associate with Mdm2, as reported previously ¹⁸. We also used retroviral vectors to express haemagglutinin (HA)-tagged Arf mutants in DM3T3 cells, in which the Mdm2 gene is amplified on double-minute chromosomes. Immunoprecipitation of cell lysates with antibodies to Mdm2 or to the HA tag confirmed that Mdm2 interacted with both Arf 26−37 and Arf 38−52, but not with Arf 1−62 (data not shown). The fact that Arf 26−37 bound to Mdm2 but was unable to induce p53 or arrest the cell cycle prompted us to investigate its nucleolar localization. In Arf-null NIH3T3 cells transfected with the same vectors, both full-length p19^Arf and HDM2 co-localized to the nucleolus (Fig. 4e−g) as did Arf 38−52 and HDM2 (Fig. 4h−j). However, Arf 26−37 did not enter the nucleolus ( Fig. 4k) and did not mobilize HDM2 from the nucleoplasm ( Fig. 4l). Therefore, both binding to Mdm2 and the localization of Arf in the nucleolus are necessary for Arf-induced p53 stabilization, p53 activation and cell-cycle arrest.

Figure 5. Ectopically overexpressed p53 and Mdm2 prevent the nucleolar localization of Arf. Serum-starved Mdm2/p53 double-null MEFs were microinjected with plasmids encoding p53 and GFP-tagged Arf N62 in the absence or presence of T7-epitope-tagged HDM2. Cells were fixed 12 h after injection and analysed for localization of p53 (a, b), GFP-tagged ARF N62 (c, d) and HDM2 (e). The localization of HDM2 and p53 was analysed by indirect immunofluorescence using monoclonal anti-T7 and polyclonal anti-p53 antibodies followed by rhodamine-conjugated anti-mouse immunoglobulin (red) and Alexa 350 anti-rabbit immunoglobulin (blue), respectively. Alexa 350 was detected using a DAPI filter and GFP expression (green) was visualized using an FITC filter. Scale bar represents 5 µm. Full Figure and legend (42K)

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The Mdm2−p53 interaction is crucial in the sense that disruption of Mdm2 in the mouse germ line leads to early embryonic lethality unless p53 function is eliminated ^{30, 31}. Hence, maintenance of steady-state levels of p53, even in the absence of genotoxic stress or hyperproliferative signals, probably depends on Mdm2. Mdm2 has evolved to regulate the transcription ^{21, 22}, nuclear export ^{23, 24} and turnover ^{25, 32, 33} of p53 and, in principle, antagonism of any of these functions by Arf might be sufficient to explain its ability to activate p53. In response to -irradiation, p53 is stabilized through post-translational modifications, such as N-terminal phosphorylations, that weaken its binding to Mdm2 (ref. 9, 11, 34, 35). However, induction of Arf by hyperproliferative signals seems not to stabilize p53 through phosphorylation ⁵, and Arf-mediated sequestration of Mdm2 in the nucleolus provides an alternative mechanism for up-regulation of p53 levels. The accumulation of Arf in the nucleolus may help to explain why, as MEFs age, basal levels of p53 (and of p53-responsive genes, such as p21Cip1) gradually rise, and, conversely, why Arf -null MEFs do not senesce. Induction of Arf by Myc is also accompanied by the relocalization of Mdm2 to the nucleolus, underscoring the function of Arf (sequestration of Mdm2 away from p53) in gating hyperproliferative signals.

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NIH3T3 (Arf-null, p53-wild-type), BALB3T3 10(1) (Arf-wild-type, p53-null), DM3T3 (amplified Mdm2) and explanted MEF strains of the indicated genotypes ³ were infected or transfected as described ⁴. MEFs were routinely passaged on a 3T9 protocol in which 9 10⁵ cells were transferred every 3 days ³. The growth of wild-type MEFs progressively diminishes until replicative senescence at passage 15−18. For cell-cycle analyses, cells were analysed by flow cytometry 48 h after retroviral infection to determine DNA content. Spodoptera frugiperda Sf9 cells were maintained in Grace's medium supplemented with 5% fetal bovine serum and infected for 48 h with the indicated baculoviruses before lysis.

HDM2 was subcloned by the polymerase chain reaction (PCR) into the XbaI−BamHI sites of the pCGT-CMV vector in-frame with two tandem T7 epitopes. Complementary DNAs for p19^Arf, Arf N62 and Arf 1−62 (27) were subcloned into the EcoRI site of the pEGFP-C1 vector (Clontech) in-frame with the C terminus of GFP. Arf mutants were constructed using mutated sense and antisense oligonucleotides complementary to wild type p19^Arf sequences as primers. Two PCR reactions were performed with template Arf cDNA (200 ng) as follows: sense 26−37 (5'-GTTTTCTTGGTGTGCGCTCTGGCT) or 38−52 (5'-AGGACAGCGAGCTTGAGAAGAGGG) mixed with T3 primer; and antisense 26−37 (5'-AGCCAGAGCGCACACCAAGAAAAC) or 38−52 (5'-CCCTCTTCTCAAGCTCGCTGTCCT) mixed with T7 primer. Reaction buffer included 10 mM Tris-HCl, 50 mM KCl, 1mM MgCl₂, 0.1% gelatin, 80 µM of each dNTP, 1 µg of each primer, and 0.5 units of Taq DNA polymerase (Stratagene). Each cycle (25 cycles total) consisted of denaturation at 95 °C for 1 min, annealing at 55 °C for 1 min, and extension at 72 °C for 2 min. PCR products were isolated on 1% agarose gels and purified (Qiagen Gel Extraction). Purified products from 26−37 and 38−52 reactions were mixed separately in reaction buffer along with T3 and T7 primers in the following two-step PCR reaction: first, denaturation at 95 °C for 1 min, annealing at 37 °C for 1 min, and extension at 72 °C for 2 min for 10 cycles; followed by second, denaturation at 95 °C for 1 min, annealing at 55 °C for 1 min, and extension at 72 °C for 2 min. Final PCR products were ligated into pCR2.1 cloning vectors, excised with Eco RI, and subcloned into the EcoRI site of pSRMSV-tkneo retroviral vector (for expression in mammalian cells) and into the EcoRI site of the pVL1393 baculovirus vector (for expression in insect Sf9 cells). The Myc−ER^TM cDNA cassette (a gift from D. Felsher, J. M. Bishop and M. McMahon) was subcloned into the pSR retroviral vector for expression in MEFs⁴. The oestrogen-receptor hormone-response domain (ER^TM) has been mutated to respond to tamoxifen but not estrogen ^{36, 37}. Treatment with 1 µM 4-hydroxytamoxifen mobilizes the fusion protein to the nucleus and results in Myc activation.

For microinjection experiments, MEFs were plated onto gridded glass cover slips, grown to subconfluency, and serum-starved in DMEM medium with 0.5% fetal calf serum for 24 h. DNA was resuspended in buffer containing 50 mM HEPES, pH 7.2, 100 mM KCl and 5 mM NaH₂PO₄ and was microinjected into nuclei. MEFs were injected with GFP−Arf, GFP−Arf-N62, GFP−Arf-1−62, HDM2 and p53 either independently or in combination as indicated in figure legends. Cells were fixed after 6 or 12 h and prepared for immunofluorescence as described¹. p53 was detected using polyclonal anti-p53 antibody (1:300 dilution; Santa Cruz) followed by Alexa 350 anti-rabbit immunoglobulin (Molecular Probes). Nucleoli were detected using anti-fibrillarin antibody (1:5; Sigma) ³⁸ followed by rhodamine-conjugated anti-human immunoglobulin (ICN). NIH3T3 cells (3 10⁵) were seeded onto coverslips and transfected³⁹ with pSRMSV-tkneo plasmids containing Arf, Arf 26−37 or Arf 38−52 in combination with pCGT-CMV-T7HDM2. Cells were fixed 48 h after transfection with methanol/acetone (1:1 v/v) and stained for 1 h with affinity-purified rabbit anti-p19^Arf antibody (0.04 mg ml⁻¹)¹ followed by 30-min exposures to biotinylated anti-rabbit immunoglobulin (Amersham) and streptavidin-conjugated Texas Red (Amersham). HDM2 was detected with monoclonal T7 antibody (Novagen) (1:400) followed by fluorescein isothiocyanate (FITC)-conjugated anti-mouse immunoglobulin (Amersham) or rhodamine-conjugated anti-mouse immunoglobulin (Sigma). Endogenous p53 was detected with monoclonal antibody 421 (1:20; Oncogene Science) followed by FITC-conjugated anti-mouse immunoglobulin. DNA was visualized with Hoechst or 4,6-diamidino-2-phenylindole (DAPI) dye. Immunofluorescence was detected using a BX50 Fluorescent microscope (Olympus) or Axiovert 135 (Zeiss). For confocal laser microscopy, a Noran confocal system was used for simultaneous collection in green and red channels of single 0.25-µm optical sections.

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