The alternative product from the human CDKN2A locus, p14ARF, participates in a regulatory feedback loop with p53 and MDM2
1998; Springer Nature; Volume: 17; Issue: 17 Linguagem: Inglês
10.1093/emboj/17.17.5001
ISSN1460-2075
AutoresFrancesca J. Stott, Stewart Bates, Marion C. James, Beth B. McConnell, Maria Starborg, Sharon Brookes, Ignacio Palmero, Kevin M. Ryan, Eiji Hara, Karen H. Vousden, Gordon Peters,
Tópico(s)DNA Repair Mechanisms
ResumoArticle1 September 1998free access The alternative product from the human CDKN2A locus, p14ARF, participates in a regulatory feedback loop with p53 and MDM2 Francesca J. Stott Francesca J. Stott Imperial Cancer Research Fund Laboratories, P.O. Box 123, 44 Lincoln's Inn Fields, London, WC2A 3PX UK Search for more papers by this author Stewart Bates Stewart Bates NCI-Frederick Cancer Research and Development Center, Building 560, Room 22-96, West 7th Street, Frederick, MD, 21702-1201 USA Search for more papers by this author Marion C. James Marion C. James Imperial Cancer Research Fund Laboratories, P.O. Box 123, 44 Lincoln's Inn Fields, London, WC2A 3PX UK Search for more papers by this author Beth B. McConnell Beth B. McConnell Imperial Cancer Research Fund Laboratories, P.O. Box 123, 44 Lincoln's Inn Fields, London, WC2A 3PX UK Search for more papers by this author Maria Starborg Maria Starborg Imperial Cancer Research Fund Laboratories, P.O. Box 123, 44 Lincoln's Inn Fields, London, WC2A 3PX UK Search for more papers by this author Sharon Brookes Sharon Brookes Imperial Cancer Research Fund Laboratories, P.O. Box 123, 44 Lincoln's Inn Fields, London, WC2A 3PX UK Search for more papers by this author Ignacio Palmero Ignacio Palmero Imperial Cancer Research Fund Laboratories, P.O. Box 123, 44 Lincoln's Inn Fields, London, WC2A 3PX UK Centro Nacional de Biotecnologia, Campus Universidad Autonoma, Cantoblanco, 28049 Madrid, Spain Search for more papers by this author Kevin Ryan Kevin Ryan NCI-Frederick Cancer Research and Development Center, Building 560, Room 22-96, West 7th Street, Frederick, MD, 21702-1201 USA Search for more papers by this author Eiji Hara Eiji Hara Imperial Cancer Research Fund Laboratories, P.O. Box 123, 44 Lincoln's Inn Fields, London, WC2A 3PX UK Department of Preventive Medicine, 22nd Department of Surgery, Prefectural University of Medicine, Kawaramachi-Hirokoji, Kamigyo-ku, Kyoto, 602 Japan Search for more papers by this author Karen H. Vousden Karen H. Vousden NCI-Frederick Cancer Research and Development Center, Building 560, Room 22-96, West 7th Street, Frederick, MD, 21702-1201 USA Search for more papers by this author Gordon Peters Corresponding Author Gordon Peters Imperial Cancer Research Fund Laboratories, P.O. Box 123, 44 Lincoln's Inn Fields, London, WC2A 3PX UK Search for more papers by this author Francesca J. Stott Francesca J. Stott Imperial Cancer Research Fund Laboratories, P.O. Box 123, 44 Lincoln's Inn Fields, London, WC2A 3PX UK Search for more papers by this author Stewart Bates Stewart Bates NCI-Frederick Cancer Research and Development Center, Building 560, Room 22-96, West 7th Street, Frederick, MD, 21702-1201 USA Search for more papers by this author Marion C. James Marion C. James Imperial Cancer Research Fund Laboratories, P.O. Box 123, 44 Lincoln's Inn Fields, London, WC2A 3PX UK Search for more papers by this author Beth B. McConnell Beth B. McConnell Imperial Cancer Research Fund Laboratories, P.O. Box 123, 44 Lincoln's Inn Fields, London, WC2A 3PX UK Search for more papers by this author Maria Starborg Maria Starborg Imperial Cancer Research Fund Laboratories, P.O. Box 123, 44 Lincoln's Inn Fields, London, WC2A 3PX UK Search for more papers by this author Sharon Brookes Sharon Brookes Imperial Cancer Research Fund Laboratories, P.O. Box 123, 44 Lincoln's Inn Fields, London, WC2A 3PX UK Search for more papers by this author Ignacio Palmero Ignacio Palmero Imperial Cancer Research Fund Laboratories, P.O. Box 123, 44 Lincoln's Inn Fields, London, WC2A 3PX UK Centro Nacional de Biotecnologia, Campus Universidad Autonoma, Cantoblanco, 28049 Madrid, Spain Search for more papers by this author Kevin Ryan Kevin Ryan NCI-Frederick Cancer Research and Development Center, Building 560, Room 22-96, West 7th Street, Frederick, MD, 21702-1201 USA Search for more papers by this author Eiji Hara Eiji Hara Imperial Cancer Research Fund Laboratories, P.O. Box 123, 44 Lincoln's Inn Fields, London, WC2A 3PX UK Department of Preventive Medicine, 22nd Department of Surgery, Prefectural University of Medicine, Kawaramachi-Hirokoji, Kamigyo-ku, Kyoto, 602 Japan Search for more papers by this author Karen H. Vousden Karen H. Vousden NCI-Frederick Cancer Research and Development Center, Building 560, Room 22-96, West 7th Street, Frederick, MD, 21702-1201 USA Search for more papers by this author Gordon Peters Corresponding Author Gordon Peters Imperial Cancer Research Fund Laboratories, P.O. Box 123, 44 Lincoln's Inn Fields, London, WC2A 3PX UK Search for more papers by this author Author Information Francesca J. Stott1, Stewart Bates2, Marion C. James1, Beth B. McConnell1, Maria Starborg1, Sharon Brookes1, Ignacio Palmero1,3, Kevin Ryan2, Eiji Hara1,4, Karen H. Vousden2 and Gordon Peters 1 1Imperial Cancer Research Fund Laboratories, P.O. Box 123, 44 Lincoln's Inn Fields, London, WC2A 3PX UK 2NCI-Frederick Cancer Research and Development Center, Building 560, Room 22-96, West 7th Street, Frederick, MD, 21702-1201 USA 3Centro Nacional de Biotecnologia, Campus Universidad Autonoma, Cantoblanco, 28049 Madrid, Spain 4Department of Preventive Medicine, 22nd Department of Surgery, Prefectural University of Medicine, Kawaramachi-Hirokoji, Kamigyo-ku, Kyoto, 602 Japan *Corresponding author. E-mail: [email protected] The EMBO Journal (1998)17:5001-5014https://doi.org/10.1093/emboj/17.17.5001 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The two distinct proteins encoded by the CDKN2A locus are specified by translating the common second exon in alternative reading frames. The product of the α transcript, p16INK4a, is a recognized tumour suppressor that induces a G1 cell cycle arrest by inhibiting the phosphorylation of the retinoblastoma protein by the cyclin-dependent kinases, CDK4 and CDK6. In contrast, the product of the human CDKN2A β transcript, p14ARF, activates a p53 response manifest in elevated levels of MDM2 and p21CIP1 and cell cycle arrest in both G1 and G2/M. As a consequence, p14ARF-induced cell cycle arrest is p53 dependent and can be abrogated by the co-expression of human papilloma virus E6 protein. p14ARF acts by binding directly to MDM2, resulting in the stabilization of both p53 and MDM2. Conversely, p53 negatively regulates p14ARF expression and there is an inverse correlation between p14ARF expression and p53 function in human tumour cell lines. However, p14ARF expression is not involved in the response to DNA damage. These results place p14ARF in an independent pathway upstream of p53 and imply that CDKN2A encodes two proteins that are involved in tumour suppression. Introduction The CDKN2A locus on human chromosome 9p21 (Kamb et al., 1994; Nobori et al., 1994) and the cognate loci on mouse chromosome 4 (Jiang et al., 1995; Quelle et al., 1995a) and rat chromosome 5 (Swafford et al., 1997) encode two distinct proteins translated from alternatively spliced mRNAs (see diagram in Figure 1A). The cyclin-dependent kinase inhibitor from which the locus takes its name (also known as p16INK4a) is specified by an RNA comprising exons 1α, 2 and 3 (Serrano et al., 1993; Kamb et al., 1994; Nobori et al., 1994) referred to as the α transcript. The alternative product, designated ARF for ‘alternative reading frame’, is encoded by the slightly smaller β transcript that comprises exons 1β, 2 and 3 (Duro et al., 1995; Jiang et al., 1995; Mao et al., 1995; Quelle et al., 1995b; Stone et al., 1995b). The primary amino acid sequences of ARF and p16INK4a are completely unrelated since they are produced by translating the common exon 2 sequences in different reading frames. Exon 1β bears no homology to exon 1α and, therefore, has the features of a distinct gene that has become inserted between the tandemly linked genes encoding p16INK4a and its close relative p15INK4b (reviewed in Larsen, 1996; Sidransky, 1996). Figure 1.Organization of the CDKN2A locus and sequence of p14ARF. The genomic organization of the human CDKN2A gene is depicted in (A), with exons represented by boxes and coding domains by shading. The sequences encoding p16INK4a are in grey and those encoding p14ARF in black. (B) The amino acid sequences of human p14ARF and mouse p19ARF are shown in single letter code and split into exon 1β- and exon 2-encoded domains. The percentage identity in each exon is indicated. Identical residues are indicated by asterisks, and a gap has been introduced to maximize regional alignment. Download figure Download PowerPoint Most of the current information about ARF relates to the mouse homologue. The mouse β transcript was first noted during attempts to isolate cDNAs encoding mouse p16INK4a (Quelle et al., 1995a) and subsequently shown to specify a protein of 169 amino acids, designated p19ARF, that has no obvious relatives in the current databases (Quelle et al., 1995b). Quelle et al. went on to demonstrate that although mouse p19ARF does not interfere directly with the function of cyclin-dependent kinases, it nevertheless invokes a cell cycle arrest when ectopically expressed in rodent cells (Quelle et al., 1995b). The most striking facet of the cell cycle arrest is the accumulation of cells with both a G1 and G2/M DNA content, with apparent exclusion of cells in S-phase. It was also noted that the levels of p19ARF were generally higher in cells that had sustained mutations in the p53 tumour suppressor gene or in which p53 had been functionally compromised by overexpression of MDM2 (Quelle et al., 1995b). Much less is known about the human equivalent of p19ARF. The sequence of the human CDKN2A exon 1β was originally deduced from a combination of genomic DNA analysis, cDNA cloning and PCR-based approaches (Duro et al., 1995; Mao et al., 1995; Stone et al., 1995b). In the genomic DNA, the open reading frame continues for some distance upstream of the ATG that aligns with the start of the mouse p19ARF-coding sequence. However, as this ATG is in a favourable context for translation initiation and is the first ATG in the presumptive transcript (Mao et al., 1995; Stone et al., 1995b), it is generally assumed that the human protein starts at this point. The encoded protein would therefore be predicted to comprise 132 amino acids with a molecular weight of 13 902 Da (see Figure 1B) and, by analogy to the corresponding mouse protein (p19ARF), we refer to the human homologue as p14ARF (Duro et al., 1995; Jiang et al., 1995; Mao et al., 1995; Quelle et al., 1995b; Stone et al., 1995b). The mouse and human proteins show only 50% identity over the region of overlap (Figure 1B), but transfection experiments have indicated that a cDNA representing the human β transcript can also induce a cell cycle arrest (Liggett et al., 1996; Arap et al., 1997). The functional characterization of ARF has understandably been influenced by the approaches taken to study p16INK4a and its relatives which bind directly and specifically to the cyclin-dependent kinases CDK4 and CDK6, thereby inhibiting their ability to promote cell cycle progression via the phosphorylation of the retinoblastoma gene product, pRb (Sherr and Roberts, 1995; Weinberg, 1995; Sherr, 1996; Ruas and Peters, 1998). Thus, ectopic expression of p16INK4a or p15INK4b causes cells to accumulate with a G0/G1 DNA content, but cells that lack functional pRb are resistant to these effects (Guan et al., 1994; Okamoto et al., 1994; Koh et al., 1995; Lukas et al., 1995; Medema et al., 1995; Stone et al., 1995a). Conversely, cells that lack INK4 function are likely to have a proliferative advantage, and p16INK4a is now recognized as a major tumour suppressor implicated in a wide variety of tumour types (reviewed in Sherr, 1996; Ruas and Peters, 1998). Significantly, both the α transcript and p16INK4a accumulate when primary cells are propagated in tissue culture, suggesting that p16INK4a may participate in the G1 arrest associated with replicative senescence (Alcorta et al., 1996; Hara et al., 1996; Loughran et al., 1996; Reznikoff et al., 1996; Wong and Riabowol, 1996; Palmero et al., 1997; Zindy et al., 1997). As well as explaining why p16INK4a, rather than other members of the INK4 family, acts as a tumour suppressor, a role in senescence would account for the higher frequency of p16INK4a alterations noted in tumour cell lines as opposed to primary tumours (Cairns et al., 1994; Spruck et al., 1994), since there would be a strong selection against the expression of p16INK4a during the establishment of immortal clones. Immortalization of human cells is facilitated by disruption of both pRb- and p53-dependent mechanisms (Shay and Wright, 1989; Wright et al., 1989; Hara et al., 1991; Shay et al., 1991a). Whereas the accumulation of p16INK4a with population doublings probably accounts for the pRb-linked mechanism, the p53 dependence is presumably associated with the accumulation of the p21CIP1 CDK inhibitor (Noda et al., 1994; Alcorta et al., 1996). Through its ability to inhibit G1-specific cyclin-dependent kinase complexes and to bind to proliferating cell nuclear antigen (PCNA), p21CIP1 is believed to be a major executor of the p53-dependent cell cycle arrest that occurs in response to DNA damage (Brugarolas et al., 1995; Deng et al., 1995; reviewed in Sherr and Roberts, 1995; Levine, 1997). It has also been implicated directly in senescence by experiments showing that targeted disruption of p21CIP1 can extend the lifespan of human diploid fibroblasts (Brown et al., 1997) and that activation of the RAS–RAF signalling pathway can elicit a cell cycle arrest via the up-regulation of p21CIP1 (Lloyd et al., 1997; Sewing et al., 1997; Woods et al., 1997). However, the relative contributions of p21CIP1 and p16INK4a in senescence have yet to be evaluated fully, and fibroblasts derived from mice with targeted disruptions of either gene fail to undergo senescence (Deng et al., 1995; Serrano et al., 1996). Interestingly, recent evidence suggests that this is also true for mouse cells with a specific disruption of exon 1β, further complicating the interpretation (Kamijo et al., 1997). Since there are inherent differences in the immortalization frequencies of mouse and human cells and presumably in the underlying mechanisms, we considered it important to characterize the expression patterns and biological properties of the human homologue of ARF. Like its mouse counterpart, human p14ARF is located in nuclear speckles in asynchronously growing cells, and ectopic expression of p14ARF causes cells to accumulate with both a G1 and G2/M DNA content, in stark contrast to the arrest induced by p16INK4a in the same cells. Significantly, the arrest invoked by p14ARF is accompanied by up-regulation of p53, p21CIP1 and MDM2, and p53-negative tumour cells or cells expressing the human papilloma virus (HPV) E6 protein are insensitive to the effects of p14ARF. Activation of p53 by p14ARF appears to reflect a direct interaction between p14ARF and MDM2 that inhibits the MDM2-mediated degradation of p53. Elevated expression of p14ARF therefore leads to the accumulation of both MDM2 and p53 by affecting the balance between transcriptional activation and protein turnover. In turn, the expression of endogenous p14ARF is negatively regulated by p53, and we show that in a panel of human tumour cell lines there is an inverse correlation between p14ARF expression and p53 status that has parallels with the feedback loop observed between p16INK4a expression and pRb status in human cells (Li et al., 1994b; Hara et al., 1996; Palmero et al., 1997). However, there is no evidence that the β transcript is involved in a DNA damage response. We therefore favour a model in which p14ARF is a component of a separate pathway upstream of p53 such that targeted disruption of exon 1β is likely to reproduce some but not all of the phenotypic characteristics of p53-nullizygous mice. These results are discussed in relation to the possible role of ARF as a tumour suppressor. Results Detection of human p14ARF Using the known sequence to predict suitable primers, a cDNA corresponding to the human CDKN2A β transcript was generated by reverse transcription and PCR using RNA obtained from TIG3 human diploid fibroblasts. The DNA sequence predicts a protein of 132 amino acids that terminates prematurely compared with the mouse homologue (Figure 1B). Polyclonal antisera were generated against a synthetic peptide corresponding to the C-terminal 15 amino acids of human p14ARF and validated in a number ways. For example, the antisera were capable of immunoprecipitating [35S]methionine-labelled p14ARF generated by coupled in vitro transcription and translation (not shown). In Western blots, the antisera detected a 14 kDa protein in cells that had been engineered to express the β cDNA under the control of an isopropyl-β-D-thiogalactopyranoside (IPTG)-inducible promoter (see below). Although the mouse protein migrates anomalously in SDS–PAGE, possibly due to the high arginine content (Quelle et al., 1995b), the shorter human protein migrates as expected at 14 kDa and is clearly distinguishable from p16INK4a. Following affinity purification on immobilized peptide, the antisera were able to detect endogenous p14ARF by immunofluorescence. As illustrated in Figure 2, staining of the 5637 bladder carcinoma cell line revealed a distinctive punctate pattern as well as staining of larger more diffuse bodies (Figure 2A). By direct comparison with phase contrast images, it is clear that these correspond to nucleoli (Figure 2C and D). The staining could be competed by excess peptide (Figure 2B) and was present in almost all cells (Figure 2E and F). Less distinct staining was noted in mitotic figures. A similar staining pattern was detected in U2OS cells that expressed inducible p14ARF, and in these cells positive staining was dependent on the addition of IPTG (Figure 2G and H). This pattern of staining is virtually identical to that seen with mouse p19ARF (Quelle et al., 1995b) and has recently been described for the human protein in HeLa and HS27 cells (Della Valle et al., 1997). Figure 2.Localization of p14ARF by immunofluorescence. Logarithmically growing monolayer cultures were fixed and stained with an affinity-purified polyclonal antiserum against the C-terminal 15 residues of human p14ARF. (A) 5637 bladder carcinoma cells; (B) the same cells with the primary antibody pre-adsorbed against excess peptide antigen; (C and D) matching immunofluorescence and phase contrast images of 5637 cells; (E and F) the same cells at lower magnification; (G) U2OS cells expressing IPTG-inducible p14ARF; (H) the same cells without IPTG induction. (A–D) and (G–H) were photographed at 100× magnification, whereas (E) and (F) were at 20× magnification. Download figure Download PowerPoint Cell cycle arrest by inducible expression of p14ARF Mouse p19ARF has previously been shown to arrest cells in both G1 and G2/M phases when ectopically expressed using retroviral vectors (Quelle et al., 1995b), but the nature of the arrest elicited by human ARF has not been defined (Liggett et al., 1996; Arap et al., 1997). To address this issue and to compare directly the properties of p14ARF and p16INK4a, the U2OS osteosarcoma cell line, which is wild-type for both p53 and pRb (Diller et al., 1990), was used to establish clones expressing regulatable forms of the corresponding cDNAs, based on the LAC SWITCH™ expression system (Stratagene). With the β cDNA, all six of the clonal lines tested (NARF1–NARF6) were found to express p14ARF upon addition of IPTG, whereas only two of the 30 lines transfected with the α cDNA (EH1 and EH2) showed inducible expression of p16INK4a. In EH1 cells, the expression of p16INK4a was activated by as little as 0.01 mM IPTG and was maximal at between 0.3 and 1.0 mM IPTG, whereas the expression of p14ARF in NARF2 cells showed a steeper dose responsiveness, with maximal levels achieved with 0.1 mM IPTG (Figure 3A). Similar results were obtained with different clones, and two clones of p14ARF-expressing cells, NARF1 and NARF2, were used interchangably in subsequent experiments. When the same cell lysates were immunoblotted for pRb protein (Figure 3A), it was clear that the phosphorylation of pRb, as judged by the presence of the more slowly migrating forms, was inhibited by the induction of either p16INK4a or p14ARF. The degree of inhibition paralleled the dose responsiveness of p16INK4a and p14ARF expression (Figure 3A). Figure 3.Cell cycle arrest by inducible expression of p14ARF and p16INK4a. (A) EH1 and NARF1 cells were treated with increasing amounts of IPTG as indicated, and the expression of p16INK4a and p14ARF monitored by immunoblotting. The same samples were also immunoblotted with an antibody to pRb to determine the extent of phosphorylation. (B) FACS profiles of EH1 and NARF1 cells either untreated or treated for 48 h with 1 mM IPTG. Download figure Download PowerPoint Since the inhibition of pRb phosphorylation is generally equated with a G1-phase arrest, we also checked the cell cycle distribution of the inducible cell clones by propidium iodide staining and flow cytometry. As shown in Figure 3B, asynchronously growing EH1 and NARF2 cells had a normal cell cycle profile, with cells distributed in the G1, S and G2/M fractions. Upon addition of 1 mM IPTG, the EH1 cells, expressing inducible p16INK4a, arrested with a G1 DNA content as expected. In contrast, the NARF2 cells expressing inducible p14ARF accumulated in both the G1 and G2/M phases (Figure 3B). More detailed analyses were performed to determine the time scale, dose responsiveness and reversibility of the arrests imposed by either p14ARF or p16INK4a. Changes in cell cycle profiles were apparent as early as 6 h after IPTG addition and were essentially maximal after 24 h in the case of p14ARF, whereas the proportion of p16INK4a-expressing cells in G1 continued to increase up to 48 h (not shown). The effects were more pronounced at higher concentrations of IPTG, reflecting the dose responsiveness of p14ARF or p16INK4a induction, and in both cases cells treated with 1 mM IPTG for 48 h were able to resume normal cycling upon removal of the inducer (not shown). Activation of p53 and p21 by ectopic expression of p14ARF The ability of p14ARF to arrest cells in the G1 and G2/M phases of the cycle suggested that the effects might be mediated via p53 (Agarwal et al., 1995). Lysates prepared from NARF2 and EH1 cells treated with increasing concentrations of IPTG (as in Figure 3A) were immunoblotted with antibodies against p53, p21CIP1 and MDM2. As shown in Figure 4A, the levels of p53 itself were clearly increased upon induction of p14ARF in NARF2 cells, but there was no obvious effect on p53 upon induction of p16INK4a in EH1 cells. More dramatic induction by p14ARF was observed for p21CIP1 and MDM2, both of which are targets for transcriptional activation by p53 (Barak et al., 1993; El-Deiry et al., 1993; Wu et al., 1993), and the effects were broadly in line with the dose dependence of p14ARF expression. No changes in p21CIP1 and MDM2 levels were observed in EH1 cells. These data clearly imply that elevated levels of p14ARF cause the functional activation of p53. Figure 4.Up-regulation of p53, p21CIP1 and MDM2 by p14ARF. Lysates prepared from EH1 and NARF1 cells treated with increasing doses of IPTG (as in Figure 3A) were immunoblotted with antibodies against p53, p21CIP1 and MDM2, as indicated. The results for p16INK4a and p14ARF are reproduced from Figure 3A. Download figure Download PowerPoint Direct binding of p14ARF and MDM2 In other settings, such as the response to DNA damage, a rapid increase in p53 levels is brought about through stabilization of the protein. In undamaged cells, the p53 protein is turned over rapidly at least in part through interaction with MDM2 which targets p53 for ubiquitination and proteasome-mediated degradation (Haupt et al., 1997; Kubbutat et al., 1997). We therefore asked whether the ability of p14ARF to activate a p53 response in NARF cells occurred via a physical interaction with either MDM2 or p53. When p14ARF immunoprecipitates from NARF cells were immunoblotted with a monoclonal antibody against MDM2, clear evidence for co-precipitation was obtained, specifically in cells that had been treated with IPTG (Figure 5A). This interaction was investigated further in vitro using labelled proteins expressed by coupled transcription and translation in reticulocyte lysates. In this direct binding assay, wild-type MDM2 and a mutant form lacking the C-terminal 51 amino acids (1–440) were co-precipitated efficiently with p14ARF (Figure 5B). In contrast, the cyclin D1 control and a mutant form of MDM2 lacking residues 222–437 did not bind efficiently to p14ARF under these conditions. Note that the Δ222–437 mutant did show some residual degree of binding when the corresponding plasmid constructs were co-expressed in U2OS cells (data not shown). However, attempts to map the p14ARF-binding domain on MDM2 in this way have so far produced equivocal results. Figure 5.Physical interaction of p14ARF with MDM2 and p53. (A) Lysates prepared from NARF1 cells with and without addition of 1 mM IPTG were either analysed directly (Lysate) or immuno- precipitated with p14ARF antiserum or the pre-immune control (IgG) and immunoblotted for MDM2. (B) In vitro binding of p14ARF and MDM2. The left hand panel shows the individual [35S]methionine-labelled translation products and the right hand panel shows the co-immunoprecipitation of labelled products with p14ARF antiserum. Wild-type MDM2 and a form lacking the C-terminal 51 residues (1–440) associated with p14ARF in this assay, whereas the Δ222–437 mutant did not. (C) Co-precipitation of p14ARF, p53 and MDM2 from transiently transfected SAOS2 cells with antiserum against p14ARF. The efficient immunoprecipitation of p53 depended on the co-expression of MDM2 and was not apparent with a deleted form of p53 (ΔI) that lacks the MDM2-binding site. Download figure Download PowerPoint The transient co-transfection assay was also used to determine whether p14ARF could form ternary complexes with p53 and MDM2. SAOS2 cells, which lack endogenous p53, were transfected with various combinations of plasmids encoding p14ARF, MDM2, p53 or a form of p53 (ΔI) lacking conserved box I that is incapable of binding MDM2 (Marston et al., 1994; Kubbutat et al., 1997). To avoid potential apoptotic effects, both forms of p53 used in this experiment were also deleted for conserved box II (kindly provided by M.Ashcroft). The p14ARF immunoprecipitates were then immunoblotted for p53 and MDM2. From these data, it was clear that p14ARF was capable of forming a three-way complex with p53 and MDM2, and that the co-precipitation of p53 was absolutely dependent on the presence of MDM2 (Figure 5C). The ΔI mutant of p53 did not enter a ternary complex with p14ARF presumably because of its inability to bind MDM2. Stabilization of p53 by expression of p14ARF We next asked whether the association of p14ARF with MDM2 had any effect on its ability to promote the degradation of p53. As shown previously (Kubbutat et al., 1997), co-transfection of MDM2 leads to a dose-dependent reduction in the amount of p53 protein detectable after 24 h (Figure 6A). This MDM2-induced turnover of p53 was completely inhibited by co-transfection of p14ARF. Similar effects were observed with mouse and human MDM2 and in U2OS and SAOS2 cells (data not shown). Significantly, there was also a marked accumulation of MDM2 in the p14ARF-transfected cells, analogous to the effects seen with inducible p14ARF in NARF cells. In the transiently transfected cells, the increased expression of MDM2 cannot be attributed to transcriptional activation by p53 since it is being expressed from a heterologous promoter. Moreover, the LLnL proteasome inhibitor, which can protect p53 from MDM2-mediated degradation (Kubbutat et al., 1997), had no additional effect on p53 levels over those attributable to co-expression of p14ARF (Figure 6B). Thus, p14ARF appears to stabilize both p53 and MDM2. Figure 6.Functional interaction of p14ARF with MDM2 and p53. (A) U2OS cells were transfected with plasmids encoding p53, MDM2 and p14ARF as indicated, and p53 and MDM2 levels were analysed by immunoblotting (Kubbutat et al., 1997). Co-transfection of 5 μg of p14ARF vector efficiently overcame the ability of MDM2 to induce degradation of p53 and resulted in the stabilization of MDM2. The lower panel shows the immunodetection of green fluorescent protein (GFP) which was included as a control for transfection efficiency. (B) An analogous experiment was performed in the presence or absence of the proteasome inhibitor LLnL. In the presence of p14ARF, LLnL failed to cause additional stabilization of p53. (C) In the absence of exogenous p53, increasing amounts of p14ARF (0, 5 and 15 μg) caused the accumulation of wild-type MDM2 as well as two mutant forms (1–440 and 6–339) lacking the C-terminal ring finger domain. GFP was used as a control for transfection efficiency. (D) A similar experiment was performed in the presence or absence of HPV16 E6. Co-expression of p14ARF caused an increase in the amount of MDM2 irrespective of the presence of E6. Download figure Download PowerPoint To explore further the effects of p14ARF on MDM2, co-transfections were carried out with two mutant forms of MDM2 (1–440 and 6–339) that lack the C-terminal domain and are therefore impaired in their ability to target p53 for ubiquitin-mediated degradation (M.Kubbuta
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