Physical and Functional Interactions of Neuronal Growth Suppressor Necdin with p53
1999; Elsevier BV; Volume: 274; Issue: 23 Linguagem: Inglês
10.1074/jbc.274.23.16242
ISSN1083-351X
AutoresHideo Taniura, Kuniharu Matsumoto, Kazuaki Yoshikawa,
Tópico(s)Epigenetics and DNA Methylation
ResumoNecdin is expressed in virtually all postmitotic neurons, and ectopic expression of this protein suppresses cell proliferation. Necdin, like the retinoblastoma protein, interacts with cell cycle promoting proteins such as simian virus 40 large T antigen, adenovirus E1A, and the transcription factor E2F1. Here we demonstrate that necdin interacts with the tumor suppressor protein p53 as well. The yeast two-hybrid and in vitro binding analyses revealed that necdin bound to a narrow region (amino acids 35–62) located between the MDM2-binding site and the proline-rich region in the amino-terminal domain of p53. The electrophoretic mobility shift assay showed that necdin supershifted a complex between p53 and its binding DNA, implying that the p53-necdin complex is competent for DNA binding. In p53-deficient osteosarcoma SAOS-2 cells, necdin markedly suppressed p53-dependent activation of the p21/WAF promoter. Necdin and p53 inhibited cell growth in an additive manner as assessed by the colony formation of SAOS-2 cells, suggesting that necdin does not affect p53-mediated growth suppression. On the other hand, necdin inhibited p53-induced apoptosis of osteosarcoma U2OS cells. Thus, necdin can be a growth suppressor that targets p53 and modulates its biological functions in postmitotic neurons. Necdin is expressed in virtually all postmitotic neurons, and ectopic expression of this protein suppresses cell proliferation. Necdin, like the retinoblastoma protein, interacts with cell cycle promoting proteins such as simian virus 40 large T antigen, adenovirus E1A, and the transcription factor E2F1. Here we demonstrate that necdin interacts with the tumor suppressor protein p53 as well. The yeast two-hybrid and in vitro binding analyses revealed that necdin bound to a narrow region (amino acids 35–62) located between the MDM2-binding site and the proline-rich region in the amino-terminal domain of p53. The electrophoretic mobility shift assay showed that necdin supershifted a complex between p53 and its binding DNA, implying that the p53-necdin complex is competent for DNA binding. In p53-deficient osteosarcoma SAOS-2 cells, necdin markedly suppressed p53-dependent activation of the p21/WAF promoter. Necdin and p53 inhibited cell growth in an additive manner as assessed by the colony formation of SAOS-2 cells, suggesting that necdin does not affect p53-mediated growth suppression. On the other hand, necdin inhibited p53-induced apoptosis of osteosarcoma U2OS cells. Thus, necdin can be a growth suppressor that targets p53 and modulates its biological functions in postmitotic neurons. In the vertebrate central nervous system, neurons withdraw from the cell cycle immediately after differentiation from their proliferative precursors, termed neuroepithelial stem cells. Differentiated neurons are absolutely incompetent to divide even in the presence of extracellular stimuli that promote cell cycle progression of proliferative cells. Therefore, the permanent arrest of the cell cycle is the most fundamental feature displayed by differentiated neurons. However, little is known about the molecular mechanism whereby neurons exit from the cell cycle and remain quiescent all of their lives. In proliferative cells, the cell cycle is strictly controlled by various regulatory proteins. Among them, E2F1 is a principal transcription factor that controls cell cycle progression of dividing cells (reviewed in Ref. 1Adams P.D. Kaelin W.G. Cancer Biol. 1995; 6: 99-108Crossref PubMed Scopus (138) Google Scholar). In the G1 phase, E2F1 is inactivated by interacting with retinoblastoma protein (Rb). 1The abbreviations used are: Rb, retinoblastoma protein; MBP, maltose-binding protein; BrdUrd, bromodeoxyuridine; PWS, Prader-Willi syndrome. During G1-S transition, this interaction disappears by phosphorylation of Rb, and released E2F1 activates transcription of its target genes that are indispensable for DNA replication. Thus, the Rb-E2F1 system is thought to govern exit from or passage through the cell cycle (reviewed in Refs. 2Nevins J.R. Science. 1992; 258: 424-429Crossref PubMed Scopus (1365) Google Scholar and 3Weinberg R.A. Cell. 1995; 81: 323-330Abstract Full Text PDF PubMed Scopus (4312) Google Scholar). Several previous studies have suggested that the Rb-E2F1 system is also involved in the growth arrest associated with neuronal differentiation (4Lee E.Y.-H.P. Chang C.-Y. Hu N. Wang Y.-C.J. Lai C.-C. Herrup K. Lee W.-H. Bradley A. Nature. 1992; 359: 288-294Crossref PubMed Scopus (1125) Google Scholar, 5Jacks T. Fazeli A. Schmitt E.M. Bronson R.T. Goodell M.A. Weinberg R.A. Nature. 1992; 359: 295-300Crossref PubMed Scopus (1518) Google Scholar, 6Slack R.S. Hamel P.A. Bladon T.S. Gill R.M. McBurney M.W. Oncogene. 1993; 8: 1585-1591PubMed Google Scholar). Necdin is a 325-amino acid residue protein encoded in a cDNA sequence isolated from the library of neurally differentiated murine embryonal carcinoma P19 cells (7Maruyama K. Usami M. Aizawa T. Yoshikawa K. Biochem. Biophys. Res. Commun. 1991; 178: 291-296Crossref PubMed Scopus (139) Google Scholar). The necdin gene is expressed in postmitotic neurons derived from embryonal carcinoma cells but not in transformed cell lines originating from neuroblastomas and pheochromocytomas even after they are induced to differentiate (8Aizawa T. Maruyama K. Kondo H. Yoshikawa K. Dev. Brain Res. 1992; 68: 265-274Crossref PubMed Scopus (81) Google Scholar). In developing mouse brain, the necdin gene is constitutively expressed in neurons from early embryonic stages (e.g. embryonic day 10 at the forebrain) until late adult periods, whereas necdin mRNA is undetectable in neuroepithelial stem cells in the neural tube (8Aizawa T. Maruyama K. Kondo H. Yoshikawa K. Dev. Brain Res. 1992; 68: 265-274Crossref PubMed Scopus (81) Google Scholar, 9Uetsuki T. Takagi K. Sugiura H. Yoshikawa K. J. Biol. Chem. 1996; 271: 918-924Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). These observations suggest that necdin is expressed in postmitotic neurons that are differentiated from their precursor cells in an irreversible manner. Ectopic expression of necdin in NIH 3T3 cells suppresses the cell growth without affecting cell viability (10Hayashi Y. Matsuyama K. Takagi K. Sugiura H. Yoshikawa K. Biochem. Biophys. Res. Commun. 1995; 213: 317-324Crossref PubMed Scopus (79) Google Scholar). Intriguingly, necdin binds to viral oncoproteins such as SV40 large T antigen and adenovirus E1A (11Taniura H. Taniguchi N. Hara M. Yoshikawa K. J. Biol. Chem. 1998; 273: 720-728Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). Moreover, necdin interacts with the transcription factor E2F1 and suppresses E2F1-dependent transcription (11Taniura H. Taniguchi N. Hara M. Yoshikawa K. J. Biol. Chem. 1998; 273: 720-728Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). These characteristics of necdin resemble those of Rb, although these proteins are structurally dissimilar. Both necdin and Rb bind to the COOH-terminal transactivation domain of E2F1 (11Taniura H. Taniguchi N. Hara M. Yoshikawa K. J. Biol. Chem. 1998; 273: 720-728Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). This transactivation domain is also a target of MDM2, a cellular oncogene product that binds to SV40 large T antigen (12Brown D.R. Deb S. Munoz R.M. Subler M.A. Deb S.P. Mol. Cell. Biol. 1993; 13: 6849-6857Crossref PubMed Scopus (56) Google Scholar). The necdin-binding site on E2F1 is located in a close proximity to the MDM2-binding site (11Taniura H. Taniguchi N. Hara M. Yoshikawa K. J. Biol. Chem. 1998; 273: 720-728Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 13Martin K. Trouche D. Hagemeier C. Sorensen T.S. La Thangue N.B. Kouzarides T. Nature. 1995; 375: 691-694Crossref PubMed Scopus (452) Google Scholar), although MDM2, unlike necdin, enhances E2F1-mediated transcriptional activation (13Martin K. Trouche D. Hagemeier C. Sorensen T.S. La Thangue N.B. Kouzarides T. Nature. 1995; 375: 691-694Crossref PubMed Scopus (452) Google Scholar). Furthermore, MDM2 interacts with the transactivation domain of the tumor suppressor p53 and represses the p53-driven transcriptional activity (14Oliner J.D. Pietenpol J.A. Thiagalingam S. Gyuris J. Kinzler K.W. Vogelstein B. Nature. 1993; 362: 857-860Crossref PubMed Scopus (1307) Google Scholar, 15Momand J. Zambetti G.P. Olson D.C. George D. Levine A.J. Cell. 1992; 69: 1237-1245Abstract Full Text PDF PubMed Scopus (2792) Google Scholar). There is a substantial degree of homology between the E2F1 and p53 activation domains (13Martin K. Trouche D. Hagemeier C. Sorensen T.S. La Thangue N.B. Kouzarides T. Nature. 1995; 375: 691-694Crossref PubMed Scopus (452) Google Scholar, 14Oliner J.D. Pietenpol J.A. Thiagalingam S. Gyuris J. Kinzler K.W. Vogelstein B. Nature. 1993; 362: 857-860Crossref PubMed Scopus (1307) Google Scholar), suggesting a conservation of binding sites for specific proteins. These findings prompted us to examine whether necdin also interacts with p53. In this study, we demonstrate that necdin does form a specific complex with p53 to modulate p53-mediated biological functions. We have used the yeast two-hybrid system and in vitro binding analyses to map the necdin-binding region on p53. Using cultured cells that are often adopted for functional analyses of p53, we have examined the effects of necdin on p53-mediated transcriptional activation, growth arrest, and apoptosis. The present results implicate that necdin is a novel type of growth suppressor that targets both p53 and E2F1. GAL4 DNA-binding domain vector (pGBT9) and GAL4 activation domain vector (pGAD424) were purchased fromCLONTECH. cDNAs encoding various p53 subsequences were generated from full-length p53 cDNA (a gift from Dr. T. Akiyama, University of Tokyo) by polymerase chain reaction using specific primers and inserted into pGAD424. These vectors were introduced along with pGBT9 harboring necdin cDNAs (11Taniura H. Taniguchi N. Hara M. Yoshikawa K. J. Biol. Chem. 1998; 273: 720-728Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar) intoSaccharomyces cerevisiae SFY526. Transformants were grown on leucine- and tryptophan-deficient synthetic dropout medium plates, and the colony lift filter assay for β-galactosidase activity was carried out as recommended by CLONTECH. The reaction was evaluated, and the results were separated into four ranks with the time for the appearance of blue colonies at 30 °C: +++, less than 2 h; ++, 2–6 h; +, 6–12 h; −, remaining white over 12 h. Various fragments of p53 cDNA were subcloned into pMALC2 (New England Biolabs) to make maltose-binding protein (MBP) fusion proteins. The MBP fusion proteins were affinity-purified with amylose resin as recommended by New England Biolabs. Full-length necdin cDNA was subcloned into a baculovirus transfer vector (pBlueBacHis2/B) (Invitrogen) for expression as a His-tagged fusion protein. The transfer vector and a wild-type baculovirus DNA (Bac-N-Blue AcMNPV) were introduced to Sf21 insect cells to obtain AcMNPV-Ndn. The recombinant His-tagged necdin expressed in AcMNPV-Ndn-infected Sf9 insect cells was purified using Probond metal-binding resin (Invitrogen). MBP-p53 fusion proteins (1 μg) bound to amylose resin (5 μl) was incubated with purified His-tagged necdin (100 ng) at 4 °C for 30 min in 0.5 ml of binding buffer (20 mm Tris-HCl (pH 7.5), 200 mm NaCl, and 1 mm EDTA). The resin was washed with the binding buffer, and bound proteins were eluted with 20 mm maltose. His-tagged necdin was separated by 10% SDS-polyacrylamide gel electrophoresis, transferred to Immobilon membrane (Millipore) by electroblotting, and detected with an anti-necdin antibody (C2) (1:1000) (7Maruyama K. Usami M. Aizawa T. Yoshikawa K. Biochem. Biophys. Res. Commun. 1991; 178: 291-296Crossref PubMed Scopus (139) Google Scholar) and peroxidase-conjugated anti-rabbit IgG (Cappel) using chemiluminescence method (Renaissance, NEN Life Science Products). Protein concentrations were determined by the Bradford method (Bio-Rad). The oligonucleotide probe containing the p53-binding site (5′-TACAGAACATGTCTAAGCATGCTGGGG-3′) was labeled with [γ-32P]ATP (Amersham Pharmacia Biotech) using T4 polynucleotide kinase. cDNAs encoding Myc tag p53 proteins were cloned into pRc/CMV (Invitrogen) for expression of Myc-tagged p53 (amino acids 1–393) (pRc-Myc-p53), its NH2-terminal deletion mutants p53 (amino acids 55–393) (pRc-Myc-p53 (55–393)), and p53 (amino acids 75–393) (pRc-Myc-p53 (75–393)). The Myc tag was added to the p53 subsequences using a 6 × Myc tag plasmid (a gift from Dr. M. W. McBurney, University of Ottawa). The expression vectors were transfected into SAOS-2 cells by the calcium phosphate method (16Graham F.L. van der Eb A.J. Virology. 1973; 52: 456-467Crossref PubMed Scopus (6495) Google Scholar). Nuclear extracts (2–4 μg of protein) were prepared by the small scale extraction method (17Schreiber E. Matthias P. Muller M.M. Schaffner W. Nucleic Acids Res. 1989; 17: 6419Crossref PubMed Scopus (3917) Google Scholar) and incubated in a reaction mixture (20 μl) containing 15 mm Hepes (pH 7.9), 1 mm EDTA, 4% Ficoll 400, 4 mg/ml bovine serum albumin, 50 mm KCl, 1 mm dithiothreitol, 1 μg of sonicated salmon sperm DNA, and the 32P-labeled oligonucleotide probe. For supershift analysis, the reaction mixture was incubated at 4 °C for 60 min with hybridoma culture media (1:10) containing anti-Myc (9E10) and anti-E1A (M73) antibodies or with purified His-tagged necdin (100 ng). DNA binding activities were analyzed by electrophoresis in a 4% nondenaturing polyacrylamide gel run in a buffer (25 mm Tris borate (pH 8.2), 50 mm EDTA) at 4 °C. Combinations of expression vectors of pRc-necdin, pRc-Myc-p53, and pRc-Myc-p53 (75–393) were transfected into SAOS-2 cells, and nuclear extracts were prepared 48 h after transfection. Aliquots (40 μg of protein) of extracts were incubated for 2 h at 4 °C with the anti-Myc antibody (1:5) or with an anti-necdin antibody C2 (1:125) in 250 μl of a buffer containing 20 mm Tris-HCl (pH 7.5), 200 mm NaCl, 0.1% Triton X-100, 1 mm EDTA, 0.1 mm phenylmethylsulfonyl fluoride. The antibody-protein complexes were pelleted with protein A-Sepharose (Amersham Pharmacia Biotech), separated by SDS-polyacrylamide gel electrophoresis, and analyzed by immunoblotting as described above. A 2.4-kilobase pair fragment of human p21/WAF1 promoter (18Datto M.B. Yu Y. Wang X.F. J. Biol. Chem. 1995; 270: 28623-28628Abstract Full Text Full Text PDF PubMed Scopus (400) Google Scholar) (a gift from Dr. T. Akiyama, University of Tokyo) was subcloned into theHindIII site of the luciferase reporter vector pGL2-basic (Promega). To construct the expression vectors for Myc-p53 (amino acids 1–37) (pRc-Myc-p53 (1Adams P.D. Kaelin W.G. 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Chem. 1998; 273: 720-728Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar) were transfected into ∼70% confluent SAOS-2 cells in 35-mm dishes by the calcium phosphate method (16Graham F.L. van der Eb A.J. Virology. 1973; 52: 456-467Crossref PubMed Scopus (6495) Google Scholar). Transfectants were harvested 36 h after transfection, and luciferase activities were measured with a luminometer (Lumat LB9501, Berthold) using a reagent kit (Toyo Ink, Tokyo). Transfection efficiency was normalized with co-transfected LacZ reporter plasmid (pRc-LacZ) (11Taniura H. Taniguchi N. Hara M. Yoshikawa K. J. Biol. Chem. 1998; 273: 720-728Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). The colony formation assay using SAOS-2 cells was carried out as described previously (11Taniura H. Taniguchi N. Hara M. Yoshikawa K. J. Biol. Chem. 1998; 273: 720-728Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). SAOS-2 cells grown in 60-mm dishes were transfected with pRc-Myc-p53 (5 μg), pRc-necdin (5 μg), or both (5 μg each) by the calcium phosphate method (16Graham F.L. van der Eb A.J. Virology. 1973; 52: 456-467Crossref PubMed Scopus (6495) Google Scholar). G418 (500 μg/ml) was added to the culture medium 48 h after transfection. The cells were incubated for 14 days, fixed with 10% acetate/10% methanol for 15 min, and stained with 0.4% crystal violet in 20% ethanol for 15 min to visualize the colonies. For bromodeoxyuridine (BrdUrd) labeling, the 293 cells grown on coverslips in 35-mm dishes were transfected with pRc-LacZ (0.5 μg) and combinations of pRc-Myc-p53 (0.8 μg), pRc-necdin (1.6 μg), and pRc-necdinΔN (1.6 μg). The empty vector pRc/CMV was added to equalize the amounts of transfected DNA (4 μg/assay). BrdUrd was added to the medium (final concentration, 10 μm) 36 h after transfection, and the cells were fixed 2 h later with 70% ethanol containing 20 mm glycine-HCl (pH 2.0) for 25 min at −20 °C. BrdUrd and β-galactosidase were detected by fluorescence immunocytochemistry with an anti-BrdUrd monoclonal antibody (1:200) (Roche Molecular Biochemicals) and an anti-β-galactosidase polyclonal antibody (1:2000) (Chemicon). BrdUrd and β-galactosidase were visualized with fluorescein isothiocyanate-conjugated goat anti-mouse immunoglobulin (1:500) (Cappel) and rhodamine B-conjugated goat anti-rabbit immunoglobulin (1:500) (Cappel), respectively, using a fluorescence microscope (BX 50–34-FLAD 1, Olympus). BrdUrd-positive cells among 100 β-galactosidase-positive cells were counted. U2OS cells (∼70% confluence) grown on coverslips in 35-mm dishes and transfected with pRc-Myc-p53 and/or pRc-necdin. The cells were fixed at 48, 60, and 72 h with 4% formaldehyde (pH 7.4) for 25 min at 4 °C and treated with methanol for 20 min at 20 °C and incubated with the anti-Myc antibody (1:10) and the anti-necdin antibody C2 (1:1000) for 1 h at 20 °C. Myc-tagged p53 and necdin were visualized with fluorescein isothiocyanate-conjugated anti-mouse immunoglobulin and rhodamine B-conjugated anti-rabbit immunoglobulin, respectively. For Hoechst dye staining, the fixed cells were treated with 3.3 μm Hoechst 33342 for 15 min at 20 °C and observed with the fluorescence microscope. p53-immunopositive cells in 10 nonadjacent fields (total area, 10 mm2) were counted. Statistical significance was tested using Student's ttest. We first examined whether necdin interacts with p53 by the yeast two-hybrid assay. Because the NH2-terminal sequences of necdin fused to GAL4 DNA-binding protein stimulate the transcription even in the absence of GAL4 activation fusion proteins (11Taniura H. Taniguchi N. Hara M. Yoshikawa K. J. Biol. Chem. 1998; 273: 720-728Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar), we used an NH2-terminally truncated form of necdin (amino acids 83–325) as a DNA-binding fusion protein in this assay. As shown in Fig. 1, full-length p53 (amino acids 1–393) strongly interacted with necdin (amino acids 83–325). Because the transactivation domain of p53 is located at the NH2terminus, NH2-terminally truncated p53 mutants were tested for necdin binding activities. An MDM2-binding site-deleted mutant (amino acids 35–393) still retained the ability to bind to necdin, but further deletions (amino acids 55–393 and 75–393) failed to bind to necdin. These results suggest that necdin binds to the transactivation domain, in which a region (amino acids 35–55) is indispensable. We then tested the in vitro binding between necdin and p53 (Fig. 2). We prepared a series of p53 deletion mutants as MBP fusion proteins and confirmed that purified MBP-p53 fusion proteins had predicted sizes of polypeptides (Fig.2 A). These MBP-p53 fusion proteins were incubated with His-tagged necdin, and formation of p53-necdin complexes in vitro was examined. As shown in Fig. 2 B, both p53 (amino acids 1–393) and p53 (amino acids 35–393) bound to necdin, whereas neither p53 (amino acids 55–393) nor p53 (amino acids 75–393) had necdin binding activities. An NH2-terminal region of p53 (amino acids 1–83), which encompasses the transactivation domain, was competent to interact with necdin. An NH2-terminal subsequence of p53 (amino acids 1–37), which contains the entire MDM2-binding region, failed to interact with necdin, whereas a region (amino acids 35–62) located between the MDM2-binding site and the proline-rich region retained the necdin binding activity. These data are schematically shown in Fig. 2 C. We have previously reported that the large T antigen, E1A, and E2F1 bind to the central domain of necdin (amino acids 83–292) (11Taniura H. Taniguchi N. Hara M. Yoshikawa K. J. Biol. Chem. 1998; 273: 720-728Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). The p53-binding region on necdin was determined using various necdin deletion mutants in the two-hybrid system (Fig.3). Both necdin (amino acids 83–325) and necdin (amino acids 102–325) strongly bound to p53, but further NH2-terminal truncations of necdin (amino acids 110–325, 167–325) failed to interact with p53. Although a COOH-terminally truncated form of necdin (amino acids 83–292) retained the ability to bind to p53, a further truncated form of necdin (amino acids 83–279) had no p53 binding activity. These results suggest that the central region of necdin is indispensable for the interaction with p53. This region coincided with the region required for the interactions with the large T antigen (11Taniura H. Taniguchi N. Hara M. Yoshikawa K. J. Biol. Chem. 1998; 273: 720-728Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). Although p53 and E2F1 share the binding domain on necdin, p53 showed a stronger necdin binding activity than E2F1 (Fig.3). We carried out the electrophoretic mobility shift assay to examine whether necdin affects the ability of p53 to bind to its specific DNA sequence (Fig.4). SAOS-2 cells transfected with cDNAs for Myc-tagged fusion proteins of p53 (amino acids 1–393), p53 (amino acids 55–393), and p53 (amino acids 75–393) expressed the products of predicted sizes (Fig. 4 A). These three p53 species, all of which contain the sequence-specific DNA-binding region, formed complexes with p53 site-carrying DNA (Fig. 4 B,lanes 3, 6, and 9). The signals of these p53-containing complexes were competed with excess amounts of the oligonucleotide (data not shown). These major bands were supershifted or diminished in density by the anti-Myc antibody (Fig. 4 B,lanes 5, 8, and 1 1) but not by the anti-E1A antibody used as a negative control (Fig. 4 B,lanes 4, 7, and 10), indicating that shifted complexes contain Myc-tagged p53 proteins. Addition of purified His-tagged necdin protein to the reaction mixture supershifted the complexes containing p53 (amino acids 1–393) and p53 (amino acids 55–393) (Fig. 4 C, lanes 2 and 4) but not the complex containing p53 (amino acids 75–393) (Fig.4 C, lane 6). These results imply that the necdin-p53 complexes are competent for DNA binding. In this analysis, the NH2-terminally truncated mutant p53 (amino acids 55–393), which failed to interact with necdin in the two-hybrid andin vitro binding assays (Figs. 1 and 2), bound to necdin. It seems likely that a potential binding site present in p53 (amino acids 55–74) is manifested by a conformational change due to p53-DNA complex formation in the mobility shift assay. We were unable to reconstitute the necdin-p53 complex competent for DNA binding by using purified MBP-p53 fusion protein instead of nuclear extracts of p53 cDNA-transfected cells (data not shown), suggesting that additional nuclear factors are required for the complex formation. We examined the effect of necdin on p53-driven transactivation using SAOS-2 cells. As shown in Fig. 5 A, necdin formed a nuclear complex with Myc-tagged p53 in SAOS-2 cells. Necdin bound to full-length p53 (amino acids 1–393) but not to the NH2-terminally truncated p53 (amino acids 75–393). We then transfected full-length necdin and p53 into SAOS-2 cells with a luciferase reporter vector driven by the p21/WAF1 promoter (18Datto M.B. Yu Y. Wang X.F. J. Biol. Chem. 1995; 270: 28623-28628Abstract Full Text Full Text PDF PubMed Scopus (400) Google Scholar), which contains the p53-binding site. As shown in Fig. 5 B, necdin had no appreciable effect on the reporter activity in the absence of co-transfected p53, whereas the p53-stimulated activity (6-fold of the basal activity) was suppressed by full-length necdin
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