A Novel Cellular Protein (MTBP) Binds to MDM2 and Induces a G1 Arrest That Is Suppressed by MDM2
2000; Elsevier BV; Volume: 275; Issue: 41 Linguagem: Inglês
10.1074/jbc.m004252200
ISSN1083-351X
AutoresMark T. Boyd, Nikolina Vlatković, Dale S. Haines,
Tópico(s)Epigenetics and DNA Methylation
ResumoThe MDM2 protein, through its interaction with p53, plays an important role in the regulation of the G1 checkpoint of the cell cycle. In addition to binding to and inhibiting the transcriptional activation function of the p53 protein, MDM2 binds, inter alia, to RB and the E2F-1·DP-1 complex and in so doing may promote progression of cells into S phase. Mice transgenic for Mdm2 possess cells that have cell cycle regulation defects and develop an altered tumor profile independent of their p53 status. MDM2 also blocks the growth inhibitory effects of transforming growth factor-β1 in a p53-independent manner. We show here that a novel growth regulatory molecule is also the target of MDM2-mediated inhibition. Using a yeast two-hybrid screen, we have identified a gene that encodes a novel cellular protein (MTBP) that binds to MDM2. MTBP can induce G1 arrest, which in turn can be blocked by MDM2. Our results suggest the existence of another growth control pathway that may be regulated, at least in part, by MDM2. The MDM2 protein, through its interaction with p53, plays an important role in the regulation of the G1 checkpoint of the cell cycle. In addition to binding to and inhibiting the transcriptional activation function of the p53 protein, MDM2 binds, inter alia, to RB and the E2F-1·DP-1 complex and in so doing may promote progression of cells into S phase. Mice transgenic for Mdm2 possess cells that have cell cycle regulation defects and develop an altered tumor profile independent of their p53 status. MDM2 also blocks the growth inhibitory effects of transforming growth factor-β1 in a p53-independent manner. We show here that a novel growth regulatory molecule is also the target of MDM2-mediated inhibition. Using a yeast two-hybrid screen, we have identified a gene that encodes a novel cellular protein (MTBP) that binds to MDM2. MTBP can induce G1 arrest, which in turn can be blocked by MDM2. Our results suggest the existence of another growth control pathway that may be regulated, at least in part, by MDM2. DNA-binding domain activation domain hemagglutinin rapid amplification of cDNA ends fluorescence-activated cell sorting In tumors, loss of function either of p53 itself (1Hollstein M. Shomer B. Greenblatt M. Soussi T. Hovig E. Montesano R. Harris C.C. Nucleic Acids Res. 1996; 24: 141-146Crossref PubMed Scopus (456) Google Scholar, 2Nigro J.M. Baker S.J. Preisinger A.C. Jessup J.M. Hostetter R. Cleary K. Bigner S.H. Davidson N. Baylin S. Devilee P. Glover T. Collins F.S. Weston A. Modali R. Harris C.C. Vogelstein B. Nature. 1989; 342: 705-708Crossref PubMed Scopus (2689) Google Scholar) or of the p53-dependent pathway that activates G1 arrest is one of the major and most frequent molecular events (reviewed in Ref. 3Sherr C.J. Genes Dev. 1998; 12: 2984-2991Crossref PubMed Scopus (666) Google Scholar). p53 function may be compromised directly via genetic mutation and/or deletion of the p53 gene (4Baker S.J. Fearon E.R. Nigro J.M. Hamilton S.R. Preisinger A.C. Jessup J.M. van Tuinen P. Ledbetter D.H. Barker D.F. Nakamura Y. White R. Vogelstein B. Science. 1989; 244: 217-221Crossref PubMed Scopus (1817) Google Scholar) and indirectly by changes in the regulation or level of the MDM2 protein (5Oliner J.D. Kinzler K.W. Meltzer P.S. George D.L. Vogelstein B. Nature. 1992; 358: 80-83Crossref PubMed Scopus (1824) Google Scholar). The Mdm2 gene, itself a transcriptional target of p53 (6Barak Y. Juven T. Haffner R. Oren M. EMBO J. 1993; 12: 461-468Crossref PubMed Scopus (1193) Google Scholar, 7Juven T. Barak Y. Zauberman A. George D.L. Oren M. Oncogene. 1993; 8: 3411-3416PubMed Google Scholar, 8Wu X. Bayle J.H. Olson D. Levine A.J. Genes Dev. 1993; 7: 1126-1132Crossref PubMed Scopus (1664) Google Scholar), encodes a protein (MDM2) that is a critical negative regulator of p53 function (9Finlay C.A. Mol. Cell. Biol. 1993; 13: 301-306Crossref PubMed Scopus (311) Google Scholar, 10Momand J. Zambetti G.P. Olson D.C. George D. Levine A.J. Cell. 1992; 69: 1237-1245Abstract Full Text PDF PubMed Scopus (2828) Google Scholar). Mdm2 was originally discovered as an oncogene that was amplified on mouse double minute chromosomes (11Cahilly-Snyder L. Yang-Feng T. Francke U. George D.L. Somatic Cell Mol. Genet. 1987; 13: 235-244Crossref PubMed Scopus (310) Google Scholar,12Fakharzadeh S.S. Trusko S.P. George D.L. EMBO J. 1991; 10: 1565-1569Crossref PubMed Scopus (634) Google Scholar). Mdm2 was later found to be amplified and overexpressed in a variety of human cancers (5Oliner J.D. Kinzler K.W. Meltzer P.S. George D.L. Vogelstein B. Nature. 1992; 358: 80-83Crossref PubMed Scopus (1824) Google Scholar, 13Ladanyi M. Cha C. Lewis R. Jhanwar S.C. Huvos A.G. Healey J.H. Cancer Res. 1993; 1: 16-18Google Scholar, 14Reifenberger G. Liu L. Ichimura K. Schmidt E.E. Collins V.P. Cancer Res. 1993; 53: 2736-2739PubMed Google Scholar). MDM2 binds to the transcriptional activation domain of p53 and thus inhibits this function of p53 (15Chen J. Marechal V. Levine A.J. Mol. Cell. Biol. 1993; 13: 4107-4114Crossref PubMed Scopus (632) Google Scholar, 16Oliner J.D. Pietenpol J.A. Thiagalingam S. Gyuris J. Kinzler K.W. Vogelstein B. Nature. 1993; 362: 857-860Crossref PubMed Scopus (1326) Google Scholar). Moreover, MDM2 binding to p53 regulates the stability of the p53 protein such that p53 is ubiquitinated and is then degraded by the proteasome (17Haupt Y. Barak Y. Oren M. EMBO J. 1996; 15: 1596-1606Crossref PubMed Scopus (206) Google Scholar, 18Kubbutat M.H. Jones S.N. Vousden K.H. Nature. 1997; 387: 299-303Crossref PubMed Scopus (2880) Google Scholar). This, together with the observed effect on p53 function, has led to a model in which an autoregulatory loop connects MDM2 and p53 (6Barak Y. Juven T. Haffner R. Oren M. EMBO J. 1993; 12: 461-468Crossref PubMed Scopus (1193) Google Scholar, 8Wu X. Bayle J.H. Olson D. Levine A.J. Genes Dev. 1993; 7: 1126-1132Crossref PubMed Scopus (1664) Google Scholar). MDM2 inhibits both p53-mediated G1 arrest and apoptosis (17Haupt Y. Barak Y. Oren M. EMBO J. 1996; 15: 1596-1606Crossref PubMed Scopus (206) Google Scholar, 19Chen J. Wu X. Lin J. Levine A.J. Mol. Cell. Biol. 1996; 16: 2445-2452Crossref PubMed Scopus (335) Google Scholar). p53 induces G1 arrest by promoting transcriptional up-regulation of the cyclin-dependent kinase inhibitor p21Waf1/Cip1 (20Waldman T. Kinzler K.W. Vogelstein B. Cancer Res. 1995; 55: 5187-5190PubMed Google Scholar). Therefore, it is likely that MDM2 prevents p53 from inducing G1 arrest by inhibiting p53-dependent transcriptional activation. MDM2 can prevent p53-mediated apoptosis, and this has been shown to be dependent on the ability of MDM2 to inhibit transcriptional repression by p53 (21Hsieh J.K. Chan F.S. O'Connor D.J. Mittnacht S. Zhong S. Lu X. Mol. Cell. 1999; 3: 181-193Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar). Moreover, a previously identified interaction with RB (22Xiao Z.X. Chen J. Levine A.J. Modjtahedi N. Xing J. Sellers W.R. Livingston D.M. Nature. 1995; 375: 694-698Crossref PubMed Scopus (574) Google Scholar) was shown to be able to regulate this effect. By binding to MDM2, RB forms a stable ternary complex with p53, and this prevents the MDM2-promoted degradation of p53. The ternary complex can promote p53-dependent apoptosis, but not p53-mediated transactivation. The autoregulatory relationship between p53 and MDM2 suggests that MDM2 overexpression may be oncogenic because of the resulting inactivation of p53 (8Wu X. Bayle J.H. Olson D. Levine A.J. Genes Dev. 1993; 7: 1126-1132Crossref PubMed Scopus (1664) Google Scholar). This conclusion is supported by studies of human tumors that show that, in the majority of cases, either p53 is mutated/deleted or MDM2 is overexpressed (23Leach F.S. Tokino T. Meltzer P. Burrell M. Oliner J.D. Smith S. Hill D.E. Sidransky D. Kinzler K.W. Vogelstein B. Cancer Res. 1993; 53: 2231-2234PubMed Google Scholar). That a primary function ofMdm2 is indeed its ability to negatively regulatep53 is further supported by studies of allelic knockouts of these genes in mice. Mice that possess a homozygous deletion ofMdm2 die at around day 5 of embryogenesis, whereas mice that possess a homozygous deletion of both Mdm2 andp53 are viable and develop normally (24Jones S.N. Roe A.E. Donehower L.A. Bradley A. Nature. 1995; 378: 206-208Crossref PubMed Scopus (1078) Google Scholar, 25Montes de Oca Luna R. Wagner D.S. Lozano G. Nature. 1995; 378: 203-206Crossref PubMed Scopus (1217) Google Scholar). No differences have been detected between thesep53 −/− andp53 −/−,Mdm2 −/− mice in terms of the rate or spectrum of tumors developed (26Jones S.N. Sands A.T. Hancock A.R. Vogel H. Donehower L.A. Linke S.P. Wahl G.M. Bradley A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14106-14111Crossref PubMed Scopus (78) Google Scholar). Also, no differences could be detected between the embryonic fibroblasts derived from these animals in terms of their growth or cell cycle characteristics. Collectively, these observations might suggest that the only function of Mdm2 is to regulate p53 activity, and perhaps during normal development, this is indeed the case. However, the situation appears to be different when MDM2 is expressed at abnormally high levels. Experiments in which MDM2 was overexpressed in NIH3T3 cells have shown that naturally occurring splice variants of MDM2 that lack the ability to bind to p53 are still able to transform these cells (27Sigalas I. Calvert A.H. Anderson J.J. Neal D.E. Lunec J. Nat. Med. 1996; 2: 912-917Crossref PubMed Scopus (252) Google Scholar). Further support for the idea that MDM2 has p53-independent effects derives from studies of transgenic mice. Mice transgenic for anMdm2 gene expressed from a β-lactoglobulin promoter exhibited abnormal mammary development, with cells becoming polyploid together with a multinucleate morphology, suggestive of DNA synthesis in the absence of mitosis (28Lundgren K. Montes de Oca Luna R. McNeill Y.B. Emerick E.P. Spencer B. Barfield C.R. Lozano G. Rosenberg M.P. Finlay C.A. Genes Dev. 1997; 11: 714-725Crossref PubMed Scopus (212) Google Scholar). The same results were obtained in both wild-type p53 animals and animals with a homozygous deletion of p53. In addition, recent studies using a different transgenic system with multiple copies of the whole Mdm2gene being used to generate mice that overexpress MDM2 from theMdm2 promoter have shown that these animals develop a different spectrum of tumors, cf. p53 null mice (29Jones S.N. Hancock A.R. Vogel H. Donehower L.A. Bradley A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15608-15612Crossref PubMed Scopus (321) Google Scholar). The same effect of MDM2 overexpression was observed regardless of the p53 status of these animals. Finally, in support of the existence of p53-independent effects of MDM2 upon overexpression, it has recently been shown that Mdm2 has the ability to abrogate the growth inhibitory activities of transforming growth factor-β1. This effect was p53-independent in cells in culture (30Sun P. Dong P. Dai K. Hannon G.J. Beach D. Science. 1998; 282: 2270-2272Crossref PubMed Scopus (185) Google Scholar). Taken together, these results all suggest that overexpression of MDM2 acts not only upon p53, but also on additional pathways. Since the mechanism(s) by which MDM2 exerts these p53-independent effects have not yet been elucidated, we have tried to identify novel MDM2-binding proteins that could help us to understand how MDM2 overexpression alters cell growth regulation. Using MDM2 as the bait in a yeast two-hybrid screen, we identified several novel MDM2-binding proteins. Further screening of these enabled us to focus on one novel gene that encodes a protein that we have called MTBP forMDM2 (two)-bindingprotein. Our results show that MTBP is capable of negatively regulating growth by inducing G1 arrest in a p53-independent manner and, moreover, that this can be suppressed by MDM2. Cells were grown in RPMI 1640 medium supplemented with 10% fetal calf serum and 1% penicillin/streptomycin/neomycin (Life Technologies, Inc.). H1299 (ATCC CRL-5803), U2OS (ATCC HTB-96), and Saos-2 (ATCC HTB-85) cells were obtained from American Type Culture Collection. pGal4-DBD-MDM2 encodes full-length mouse MDM2 cloned in-frame with the Gal4 DNA-binding domain (DBD)1 of pGBT9 (CLONTECH). pGal4-AD-3′-MTBP contains the carboxyl-terminal 380 amino acids of MTBP cloned into theXhoI site of pACT (CLONTECH). pBBV was generated by inserting an oligonucleotide containing the black beetle virus ribosome binding sequences from pBD7 (31Dasmahapatra B. Rozhon E.J. Schwartz J. Nucleic Acids Res. 1987; 15: 3933Crossref PubMed Scopus (14) Google Scholar) into theHindIII and EcoRV sites of pcDNA1-Neo (Invitrogen). pSK-BBV was generated by subcloning aHindIII/BglII DNA fragment containing the black beetle virus ribosome binding sequence from pBBV into theHindIII and BamHI sites of plasmid pBluescript SK II+ (Stratagene). Clones identified as encoding candidate MDM2-interacting molecules in the yeast two-hybrid screen analysis were amplified from pACT with GAD5 (5′-gag aga gat atc gcc aat ttt aat caa agt ggg aat att-3′) and GAD3 (5′-gag aga gcg gcc gct ttc agt atc tac gat tca tag atc tc-3′) primers and subcloned into the EcoRV and NotI sites of pBBV. pBBV-3′-MTBP was constructed by subcloning this polymerase chain reaction-generated fragment from pGal4-AD-3′-MTBP into pBBV. The pSK-MTBP construct used for in vitro translation of full-length MTBP was made by subcloning theNotI fragment from pCEP-MTBP (see below) into theNotI site of pSK-BBV. Recombinant His6-tagged MDM2 (pQE32-MDM2) was generated by cloning anEcoRV/XhoI fragment from pBBV-MDM2 encoding the full-length murine MDM2 cDNA into the SmaI site of pQE32 (QIAGEN Inc.). Recombinant His6-tagged Δ166 (pQE31-Δ166-MDM2) contains a DNA fragment of murine MDM2 lacking the first 166 amino acid residues. The fragment was amplified from pCMVNeoBam-MDM2 by polymerase chain reaction with primers MDM2-PstI (5′-gag aga ctg cag gag aac aca gat gag cta cct gg-3′) and MDM2-HindIII (5′-gag aga aag ct gtc agc tag ttg aag taa ctt agc a-3′) using rTth-XL polymerase (Perkin-Elmer) and cloned into the PstI and HindIII sites of pQE31 (QIAGEN Inc.). pCMV (pCMVNeoBam) and pMDM2 (pCMVNeoBam-MDM2) were kindly supplied by Dr. B. Vogelstein (5Oliner J.D. Kinzler K.W. Meltzer P.S. George D.L. Vogelstein B. Nature. 1992; 358: 80-83Crossref PubMed Scopus (1824) Google Scholar), and pCMVNeoBam-CD20 was kindly provided by Dr. E. Harlow (32van den Heuvel S. Harlow E. Science. 1993; 262: 2050-2054Crossref PubMed Scopus (989) Google Scholar). MTBP (pCEP-MTBP-HA) contains full-length murine cDNA for MTBP excised from the pCR-XL-TOPO vector (see below) and cloned into the NotI site of pCEP (Invitrogen). p53 contains full-length human p53 cloned into the pCEP vector. The anti-p53 antibody Ab-1 (PAb421), the anti-MDM2 antibody Ab-1 used for Western blotting (IF2), and the anti-β-galactosidase antibody Ab-1 (200-193) were purchased from Oncogene Research Products. The anti-MDM2 antibody used for immunoprecipitation (SMP14) and the antibody used to detect p21Waf1/Cip1 (F-5) were purchased from Santa Cruz Biotechnology, Inc., and the anti-hemagglutinin A (HA) antibodies used to detect HA-tagged MTBP (12CA5 and 16B12) were purchased from Roche Molecular Biochemicals and BAbCO, respectively. The anti-CD20 antibody leu16 was purchased from Becton Dickinson, and fluorescein isothiocyanate-conjugated anti-mouse IgG was obtained from Pierce. We utilized the MatchmakerTM system (CLONTECH) to screen a mouse T-cell lymphoma library (ML4001AE) and to assess interactions between the Gal4-DBD-MTBP and Gal4-AD-MDM2 deletion mutants. MDM2 deletion mutants were prepared as described (52Vlatkovic N. Guerrera S. Li Y. Linn S. Haines D.S. Boyd M.T. Nucleic Acids Res. 2000; 18: 3581-3586Crossref Google Scholar). We used the Marathon RACETM system (CLONTECH) to amplify the 5′- and 3′-ends of MTBP from a murine B-cell cDNA. Briefly, total cellular RNA was prepared from murine SP2 cells (ATCC CRL-1646) using RNAzolTM (MBI), and poly(A)+ RNA was isolated from this using OligotexTM beads (QIAGEN Inc.). 5′-RACE was performed using the gene-specific oligonucleotides GSP-1 (5′-tga aga ata agg ttc aac tgt acc-3′) and GSP-2 (5′-cag ctt tca cgg tgt ctg ttt g-3′). Polymerase chain reaction was performed with rTth-XL polymerase, and the products were cloned into pCR2.1 (Invitrogen). 3′-RACE was also performed and confirmed the termination codon identified in the yeast two-hybrid screen. Sequencing was performed using dye terminators and an ABI Model 373 sequencer. Homology to Boi1p and Boi2p was identified using the FASTA program to examine theSaccharomyces cerevisiae data base at Stanford University. The full-length cDNA for MTBP was prepared by polymerase chain reaction amplification with the oligonucleotides MTBP-5′-NotI (5′-gag aga gcg gcc gcg gcg cga aga gga tgg atc ggt act tgc tg-3′) and MTBP-3′-HA-NotI (5′-gag aga gcg gcc gcc tac agg gag gcg taa tcg ggc aca tcg tag ggg tat ttc ttg ctc atc ttt tct acc acc-3′) using rTth-XL polymerase, and the product was cloned into pCR-XL-TOPO (Invitrogen). For in vitrobinding assays, MDM2 and Δ166-MDM2 were expressed in XL-1 bacteria (Stratagene) from the pQE32-MDM2 and pQE31-Δ166-MDM2 constructs, respectively; captured on Ni2+-agarose (QIAGEN Inc.); and washed with buffers B–D as described by the manufacturer. Prior to all binding reactions, protein captured on beads was run on an SDS-polyacrylamide gel and analyzed by both Western blotting and staining with Coomassie Blue. 100 μl of washed beads were then mixed with 10 μl of in vitro translated protein (TNT, Promega) for 3 h at 30 °C, followed by washing three times with Dignam buffer D (33Dignam J.D. Lebovitz R.M. Roeder R.G. Nucleic Acids Res. 1983; 11: 1475-1489Crossref PubMed Scopus (9586) Google Scholar) supplemented with 75 mm imidazole. Beads were then resuspended in loading buffer and analyzed by SDS-polyacrylamide gel electrophoresis and fluorography using AmplifyTM(Amersham Pharmacia Biotech). Cells were transfected either by the calcium phosphate-DNA coprecipitation method (34Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar) or using FUGENE-6TM (Roche Molecular Biochemicals) according to the manufacturer's instructions. For immunoprecipitation experiments, cells were typically transfected with 10 μg of each plasmid, and proteins were extracted 48–72 h post-transfection. Transfected cells were harvested, and the cell pellet was lysed in immunoprecipitation buffer (50 mm HEPES, pH 7.5, 150 mm NaCl, 10% glycerol, 0.1% Triton X-100, and 0.5 mg/ml bovine serum albumin) in the presence of protease inhibitors (1–2 μg/ml aprotinin, 1–2 μg/ml leupeptin, 1 μg/ml pepstatin A, 100 μg/ml soybean trypsin inhibitor (Roche Molecular Biochemicals), and 1 mm phenylmethylsulfonyl fluoride) for 10 min on ice. The lysate was clarified by centrifugation for 10 min at 4 °C, and the concentration of total proteins was determined by the Bio-Rad protein assay. Between 1 and 5 mg of protein were then precleared by incubation with 50 μl of protein G-Sepharose (Amersham Pharmacia Biotech) for 1 h at 4 °C. The precleared lysate was incubated with 1 μg of primary antibody for 1 h at 4 °C, followed by incubation with 50 μl of protein G-Sepharose for 2 h at 4 °C. Immunoprecipitated complexes were washed three times with immunoprecipitation buffer; resuspended in 30 μl of protein sample buffer (0.1 m Tris-HCl, pH 6.8, 4% SDS, 0.2% bromphenol blue, 20% glycerol, and 0.5 mdithiothreitol); and subjected to SDS-polyacrylamide gel electrophoresis, followed by transfer to Hybond ECL membrane (Amersham Pharmacia Biotech). Following incubation with primary antibodies and subsequently with horseradish peroxidase-conjugated anti-mouse IgG (Amersham Pharmacia Biotech), the signal was detected by enhanced chemiluminescence with RenaissanceTM (NEN Life Science Products). Saos-2 and U2OS cells were transfected using FUGENE-6 with the indicated plasmids. Cells were harvested and analyzed by FACS essentially as described (19Chen J. Wu X. Lin J. Levine A.J. Mol. Cell. Biol. 1996; 16: 2445-2452Crossref PubMed Scopus (335) Google Scholar). Briefly, nocodazole was added to the indicated cells at 50 ng/ml for 12 h prior to harvesting. Cells were harvested 48–72 h after the addition of FUGENE-6·DNA complexes and washed with Dulbecco's phosphate-buffered saline containing 1% bovine serum albumin. CD20-positive cells were detected using anti-CD20 antibody and fluorescein isothiocyanate-conjugated anti-mouse IgG. Cells were fixed in ethanol and then stained with propidium iodide. Cells were analyzed using a FACScanTM (Becton Dickinson) and LYSIS-II software. H1299 cells were transfected using either the calcium phosphate precipitation method or FUGENE-6. Typically for the calcium phosphate precipitation procedure, 24 h after removal of precipitates, hygromycin B (Roche Molecular Biochemicals) was added to a final concentration of 200 μg/ml. Cells were maintained under selective conditions for 72 h, washed, and refed with hygromycin-free complete medium. Nocodazole (Sigma) was added as indicated at a concentration of 20 ng/ml, 16 h before cells were harvested for analysis. For colony formation assays, cells were transfected with the indicated plasmids; and 24–48 h after the addition of DNA, hygromycin B was added at to final concentration of 200 μg/ml. Cells were refed every 3 days with medium containing hygromycin B until colonies were visible. For some experiments, cells were stained with Giemsa. We have used a yeast two-hybrid screen (35Fields S. Song O. Nature. 1989; 340: 245-246Crossref PubMed Scopus (4937) Google Scholar) to identify potential MDM2-binding proteins that might directly interact with MDM2 to mediate its p53-independent effects. Full-length cDNA for murine MDM2 was subcloned into a Gal4-DBD yeast expression construct and used to screen a murine T-cell lymphoma cDNA library. Fig.1 A shows that a carboxyl-terminal cDNA from a novel gene fused to the activation domain of Gal4 (Gal4-AD-3′-MTBP) interacted with Gal4-DBD-MDM2, but not with Gal4-DBD. To confirm this interaction in a different system, thein vitro translated cDNA from the yeast two-hybrid screen (pBBV-3′-MTBP) was mixed with recombinant His6-tagged MDM2. Fig. 1 B shows that pBBV-3′-MTBP encoded a peptide that can bind in vitro to MDM2. Sequence analysis of this cDNA demonstrated that it is a novel sequence that encodes a predicted peptide of 380 amino acids. Northern analysis demonstrated that the carboxyl-terminal cDNA hybridized to an mRNA of ∼3 kilobases (Fig.2 A and data not shown). We therefore cloned the rest of the cDNA for this gene using a RACE-based strategy. Analysis of 5′-RACE products from mRNA obtained from a murine B-cell line demonstrated that several clones possessed an authentic 5′-end; they were identical and terminated upstream of a single long open reading frame that was in frame with the clone identified in the yeast two-hybrid screen. The sequence of this clone has been deposited in the GenBankTM/EBI Data Bank (AJ278508). This cDNA encodes a protein with a predicted molecular mass of 104 kDa, and we have given this gene the nameMtbp (MDM2 (two)-binding protein). Data base analysis detected two yeast genes whose protein products possessed significant homology to MTBP: BOI1 andBOI2 (36Bender L. Lo H.S. Lee H. Kokojan V. Peterson V. Bender A. J. Cell Biol. 1996; 133: 879-894Crossref PubMed Scopus (78) Google Scholar, 37Matsui Y. Matsui R. Akada R. Toh-e A. J. Cell Biol. 1996; 133: 865-878Crossref PubMed Scopus (64) Google Scholar). The two proteins encoded by these genes (Boi1p and Boi2p, respectively) exhibit an overall amino acid identity of 38%, but this is concentrated into four regions (I–IV) that possess identities of 71, 65, 78, and 69%, respectively. Both Boi1p and Boi2p inhibit growth in yeast when expressed at high levels. The homology between Boi1p and MTBP and between Boi2p and MTBP is 21.2 and 21% amino acid identities in alignments of 401 and 400 amino acids, respectively, and is entirely contained within the carboxyl-terminal regions of all three proteins. Fig. 1 Cshows the FASTA-generated alignment of Boi2p and MTBP. Domain 3 of Boi2p is a proline-rich area that is essential for binding to the second SH3 region of Bem1p. The corresponding region of MTBP is also proline-rich. It is noteworthy that the growth inhibitory function of Boi2p is entirely contained within the carboxyl-terminal moiety of the protein. Apart from expressed sequence tags, no other substantial homologies to MTBP could be identified.Figure 2MTBP is expressed in a variety of tissues, and full-length MTBP binds to MDM2 in vitro and in transfected mammalian cells. A, shown are the results from Northern analysis of MTBP expression. The blot was exposed for 4 days at −70 °C. Longer exposures demonstrated low level expression in peripheral blood lymphocytes (PBL) and slightly higher levels in colon and prostate tissues. B, an in vitro binding assay was performed using full-length MTBP. The full-length protein bound to murine MDM2 and not to the XL-1 negative control. IVT indicates 10% of the in vitro translated input for binding. C, shown are the results from immunoprecipitation and Western analysis of H1299 cells transfected with the indicated plasmids. Cells were harvested 48–72 h after removal of precipitates and feeding, followed by immunoprecipitation with an MDM2-specific antibody (SMP14), an HA-specific antibody to detect MTBP (16B12), or an isotype-matched control antibody. Western analysis of the immunoprecipitates was performed with either an MDM2-specific antibody (Ab-1), an HA-specific antibody to detect MTBP (12CA5), or an anti-mouse IgG-specific serum to determine the relative efficiency of the immunoprecipitation.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The full-length cDNA for MTBP was then used to examine the pattern of expression of this gene by Northern blotting. Fig. 2 Ashows that MTBP was expressed in a variety of normal tissues, with the highest levels of expression in the thymus, testis, and ovary and with low or almost undetectable expression in peripheral blood lymphocytes. We also found MTBP expression in the pancreas, heart, liver, skeletal muscle, and liver and relatively low expression in the brain (data not shown). To test whether the interaction we had detected between the carboxyl-terminal region of MTBP and MDM2 also occurred with the full-length form of the protein, we performed an in vitrobinding assay using recombinant His6-MDM2 and in vitro translated MTBP as shown in Fig. 2 B. Further confirmation of the interaction between these two proteins came from our analysis of mammalian cells transfected with MDM2 and a carboxyl-terminal HA-tagged form of MTBP. Immunoprecipitation with either an anti-HA or anti-MDM2 monoclonal antibody, followed by Western blot analysis, demonstrated that the two proteins could be coprecipitated as shown in Fig. 2 C. These results suggest that a novel protein (MTBP) can bind specifically to MDM2 under these conditions. The MDM2 protein has a number of highly conserved regions, and the function of these is not fully understood (reviewed in Ref.38Freedman D.A. Wu L. Levine A.J. Cell Mol. Life Sci. 1999; 55: 96-107Crossref PubMed Scopus (497) Google Scholar). To determine the region of MDM2 that binds to MTBP, we used a series of carboxyl-terminal deletion mutants of Gal4-DBD-MDM2 and tested them for the ability to interact in yeast with Gal4-AD-MTBP. Fig. 3 A shows that the interaction could be detected with all mutants that contain the amino-terminal 304 amino acids of MDM2, but not with shorter mutants. It is important to mention here that the p53-containing construct Gal4-AD-p53 interacts with the above mutants and in addition with mutants 1–199 and 1–166 (data not shown), which suggests that the failure of MTBP to bind to these mutants of MDM2 does not merely reflect lower expression or other conformational problems. We also tested the ability of in vitro translated full-length MTBP and carboxyl-terminal MTBP (pBBV-3′-MTBP) to bind to both full-length MDM2 and a mutant that lacks the first 166 amino acids (Δ166). Δ166 did not bind to p53 (data not shown), but as shown in Fig. 3 B, bound to both full-length MTBP and pBBV-3′-MTBP. Taken together, these results suggest that a region of MDM2 bounded by amino acids 167–304 is sufficient to bind to MTBP and that an essential minimal region is bounded by amino acids 200–304 inclusive. As illustrated in Fig. 3 C, this portion of MDM2 contains a nuclear localization signal, a motif identified as a nuclear export signal, and an acidic region. Our results in both yeast (Fig.3 A) and in vitro (Fig. 3 B, left panel) suggest that the carboxyl-terminal portion of MTBP (amino acids 515–894) is sufficient for binding to MDM2. We conclude that MDM2 binds to the carboxyl-terminal 380 amino acids of MTBP and that an area of MDM2 bounded by amino acids 167–304 is sufficient for the binding interaction to occur. Several MDM2-binding proteins are regulators of cell growth; and indeed, both of the MTBP partial homologs (Boi1p and Boi2p) have been shown to have growth inhibitory activity (36Bender L. Lo H.S. Lee H. Kokojan V. Peterson V. Bender A. J. Cell Biol. 1996; 133: 879-894Crossref PubMed Scopus (78) Google Scholar, 37Matsui Y. Matsui R. Akada R. Toh-e A. J. Cell Biol. 1996; 133: 865-878Crossref PubMed Scopus (64) Google Scholar). Therefore, we investigated the effect of MTBP
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