- Original Articles
- Open Access
A Genetic Approach to Mapping the p53 Binding Site in the MDM2 Protein
© Picower Institute Press 1997
- Accepted: 29 January 1997
- Published: 1 April 1997
The MDM2 oncoprotein binds to the tumor suppressor p53 and inhibits its anti-oncogenic functions.
Materials and Methods
To determine the amino acids of MDM2 that are critical for binding to p53, a modified two-hybrid screen was performed in yeast. Site-directed mutagenesis was then performed to identify MDM2 residues important for p53 interaction. Mutant MDM2 proteins were subsequently tested for their ability to bind to p53 in vitro and for their ability to regulate p53-mediated transcription in vivo.
The yeast genetic screen yielded two Mdm2 mutations (G58D and C77Y) which disrupted binding to p53 in vitro without altering the conformation of MDM2 as determined with conformation-sensitive monoclonal antibodies. Site-directed mutagenesis yielded mutations of two additional amino acids of MDM2 (D68 and V75) that prevented binding to p53 in vitro. The mutant MDM2 proteins were unable to inhibit p53-dependent transcription in vivo, which is consistent with prior indications that a physical interaction between the two proteins is required for MDM2’s inhibition of p53. Finally, the crystal structure of the MDM2-p53 complex shows that two of the four critical residues identified here contact p53 directly, while the remaining two residues play important structural roles in the MDM2 domain.
MDM2 residues G58, D68, V75, and C77 are critical for MDM2’s interaction with the p53 protein. Mutation of these residues to alanine prevents MDM2’s interaction with p53 in vitro, and MDM2’s regulation of p53’s transcriptional activity in vivo.
The Mdm2 oncogene was first identified as a gene amplified in a spontaneously transformed mouse 3T3 cell line, 3T3-DM (1). Overexpression of Mdm2 was then shown to be sufficient to cause immortalized mouse cell lines to be tumorigenic in nude mice (2). Furthermore, Mdm2 can immortalize primary rat embryo fibroblasts (REFs) alone and can transform REFs in cooperation with an activated ras gene (3). The human Mdm2 gene has been shown to be amplified or overexpressed at the mRNA level in a variety of human sarcomas, gliomas, carcinomas, and leukemias (4–12). In addition, enhanced translational efficiency has been shown to be responsible for overexpression of the MDM2 protein in some human choriocarcinoma cell lines (13), and normal tissue from a member of a cancer-prone family was shown to express high levels of MDM2 protein by immunohistochemical staining (14). Thus it is clear that overexpression of the MDM2 protein contributes to oncogenesis.
At least some of MDM2’s transforming properties are a result of its ability to bind to and inhibit the functions of the p53 tumor suppressor protein (15–17). p53 causes G1 arrest or apoptosis and may enhance DNA repair in response to DNA damage (reviewed in ref. 18). An aberrant overexpression of MDM2 in cells containing wild-type p53 compromises p53-mediated responses and enhances the tumorigenic potential of the cell.
Besides the transforming phenotype of MDM2 overexpression, MDM2 has normal p53-dependent functions in vivo. One such function is essential for embryonic development; MDM2 null mouse embryos die shortly after or at the time of implantation, and this lethality is reversed by deletion of the p53 gene. This result indicates that MDM2’s first essential function is the inhibition of p53 activity during early embryonic development (19,20). MDM2 may also perform a normal role in the p53 DNA damage response. As p53 protein levels rise after cellular insult, p53 acts as a transcriptional activator for genes involved in cell cycle arrest, apoptosis, and DNA repair (reviewed in ref. 18). In addition, p53 activates expression of MDM2, which in turn negatively regulates p53’s transcriptional activity (21,22). An autoregulatory feedback loop is thus formed, in which MDM2 may contribute to the regulation of p53-dependent growth arrest.
Several lines of evidence suggest that MDM2 may also have roles independent of p53, some of which may contribute to its oncogenic potential. While the NH2-terminus of MDM2 is sufficient for binding and inhibiting p53, the protein contains several other domains that are highly conserved at the amino acid level (23,24). These include a central acidic domain required for interaction with the ribosomal subunit L5 and its associated 5S ribosomal RNA, and a COOH-terminal RING finger that mediates binding to specific structural or sequence motifs in RNA (25–27). In addition, MDM2 contains both nuclear import and nuclear export signal sequences and can shuttle back and forth between the nucleus and the cytoplasm (JCR, DAF, AJL unpublished data). The functional significance of these domains for cellular activities of MDM2 remains unclear.
Several additional observations indicate that MDM2 may have p53-independent functions. MDM2 has physical and functional interactions with the pRB tumor suppressor and with the E2F1/DP1 S phase-promoting transcriptional activator. MDM2’s interaction with pRB disrupts the pRb—E2F complex and releases functional E2F; its interaction with E2F increases E2F-dependent transcription (28,29). Finally, a number of sarcomas, leukemias, and carcinomas have been described that harbor both p53 mutations that inactivate its function and MDM2 protein overproduction (10–12). Individuals with such sarcomas had a much lower survival rate than those with sarcomas containing either one of the two alterations alone, suggesting that overexpression of MDM2 may have tumor-promoting functions that are independent of p53 (10).
In order to analyze the p53 binding domain of MDM2 and to create tools with which to separate p53-dependent and -independent MDM2 functions, a genetic approach was used. p53 and a fragment of MDM2 were co-expressed in yeast, and a genetic screen was performed to detect Mdm2 mutations that prevented interaction between the two proteins. The inability of the resulting mutant MDM2 proteins to interact with p53 was subsequently confirmed in vitro. Site-directed mutagenesis was then performed to identify additional residues of MDM2 critical for p53 binding. Several of the mutant MDM2 proteins were tested in mammalian cell culture for their ability to bind p53 and inhibit p53-dependent transactivation of a luciferase reporter gene. Finally, analysis of the crystal structure of the MDM2–p53 complex indicates that two classes of Mdm2 mutations were generated: those that alter MDM2 residues that directly contact p53 and those that alter residues that play important structural roles for the MDM2 domain that binds to p53 (30).
The Saccharomyces cerevisiae strain used for the isolation of Mdm2 mutations was YCE1086-15B (MATa trp1 leu2 ura3 his3 ade2 isogenic with S288C), which had been modified by the integration of pCE136 (GALp::p53), and the introduction of pSH18-34 (lexAp::lacZ).
pSH18-34, a gift of S. Hanes and R. Brent, is a yeast episomal plasmid that places the lacZ gene under the control of the lexA operator, (similar to plasmids described in 31). pCE136 is a yeast-integrating plasmid that expresses wild-type human p53 under the control of the GAL1/10 promoter. It was synthesized by cloning an existing GAL1/10::p53 promoter fusion into pRS405 (32) as an XbaI/HindIII fragment. The original GAL1/10::p53 promoter fusion was a gift of S. Ramos and J. Broach. pCE136 was integrated into YCE1086-15B by linearizing inside LEU2 with AflII, followed by integrative transformation. The integrants were confirmed by Southern blotting and by demonstrating 2:2 segregation of leucine prototrophy following meiosis. To make pCE151, encoding the lexA::MDM2 fusion protein, human Mdm2 cDNA (23), a gift of J. Chen, was PCR amplified from codons −5 to 115, and then cloned as an EcoRI-XhoI fragment into pEG202 (32), a gift of R. Brent and E. Golemis. The oligos used in the PCR amplification were (5′ oligo) GACTGAGAATTCGTGAGGAGCAG GCAAATG and (3′ oligo) GACTGACTCGAGCTA TGATTCCTGCTGATTGACTACTAC. The newly introduced restriction sites are underlined. pCE151 was mutagenized with hydroxylamine as described by Rose et al. (33).
Western Analysis of Yeast Colonies
Liquid cultures were grown in the appropriate selective media. 1.5 ml of each culture was pelleted and resuspended in 50 µ1 of 2 × SDS sample buffer (125 mM Tris-HCL, pH 6.8; 4% SDS; 30% glycerol; 0.002% bromophenol blue; 2% β-mercaptoethanol). Samples were then frozen at −70°C and boiled for 5 min to lyse the cells. Half of each sample was run on 8% polyacrylamide gels and transferred to Hybond-ECL nitrocellulose (Amersham Life Sciences). Membranes were blocked for 1 hr in wash buffer (PBS; 0.1% Tween 20) plus 5% milk, followed by a quick rinse in wash buffer. The membranes were then hybridized with a 2000-fold dilution of α-lexA polyclonal sera (a gift from Erica Golemis) in wash buffer with 3% BSA. After several washes, the membranes were incubated with a 500-fold dilution of protein A-peroxidase (Boehringer Mannheim) in wash buffer plus 5% milk. After extensive washing, lexA fusion proteins were visualized by ECL detection (Amersham Life Sciences). To control for loading of total protein in each lane, the membranes were then probed with a 5000-fold dilution of polyclonal sera against Kar2 protein (a gift from Mark Rose).
Approximately 5 µg of each plasmid was sequenced using Sequenase Version 2.0 kits (U.S. Biochemical). Primers used were 5′-GAGCTTCA CCATTGAAGG-3′ derived from the lexA coding sequence, 5′-TAAATCATAAGAAATTCG-3′ from pCE151 sequences, and 5′GACTACTACCAAGTT CCTG-3′ from Mdm2 sequence.
In Vitro Labeling and Analysis of Translation Products
Mdm2 fragments or full-length clones were expressed and [35S]-labeled in a coupled transcription and translation system (TnT Coupled Reticulocyte Lysate Systems from Promega), using T7 polymerase.
Immunoprecipitation of translation products was performed with 4–10 µ1 of each labeled lysate and 30 µ1 of 50% protein A-sepharose slurry with either 2 µ1 of anti-human MDM2 polyclonal serum or 200 µ1 of monoclonal antibodies (SV40 T antigen-specific 419 as a negative control; human MDM2-specific 3G5 and 4B2 ). Total volume was brought to 300 µ1 with the addition of lysis buffer (50 mM Tris-HCL, pH 7.5; 150 mM NaCl; 0.5% NP40; 1 mM EDTA; added fresh: 1 mM DTT, 100 µM PMSF, 1 µM Pepstatin, 1 µM E-64). Tubes were then rotated at 4°C for at least 2 hr. Extensive washes were done, three with SNNTE (5% sucrose; 1% NP40; 0.5 M NaCl; 50 mM Tris-HCl, pH 7.5; 5 mM EDTA) and one with RIPA buffer (50 mM Tris-HCl, pH 7.5; 150 mM NaCl; 1% Triton X-100; 0.1% SDS; 1% sodium deoxycholate). Proteins were eluted by boiling in SDS sample buffer and were then separated by SDS-PAGE and visualized by autoradiography.
Binding to p53 was assessed by incubating the same labeled lysates with 25 µg of GST::p53 (1–82) or 25 µg of negative control fusion GST::CSB (1–242) purified from E. coli on Glutathione Sepharose 4B (Pharmacia Biotech). Total volume was 400 µ1, and they were rotated at room temperature for 2 to 3 hr. Three washes were performed with PBS and one with lysis buffer. The beads were resuspended in SDS sample buffer and boiled for 5 min before separation by SDS-PAGE. Results were visualized by autoradiography.
Site-directed Mutagenesis of Mdm2
Site-directed mutagenesis was performed using the Transformer Site-Directed Mutagenesis Kit (Clontech). All clones were sequenced to verify that the correct mutation was made.
Cell Culture and Transfection Techniques
Saos-2 cells were grown in DMEM plus 15% fetal calf serum. Cells were transfected by electroporation with 1 µg of luciferase reporter plasmid, 30 ng of pRC/p53, 3 µg of pCMV plasmids (with wild-type, mutant, or no Mdm2), and 6 µg of carrier DNA as follows. Approximately 1.2 × 106 cells in 0.4 ml media were mixed with the DNA. Electroporation was performed on a Gene Pulser (BioRad) at 960 µFD and 0.23 V. Luciferase assays were performed 12 hr after transfection using the Enhanced Luciferase Assay Kit (Analytical Luminescence Laboratory) as described in the protocols supplied by the manufacturer.
Saos-2 cells were electroporated with 10 µg of various pCMV plasmids as described above, and were plated into 6-well dishes with cover slips. Twelve hours following transfection, the cells were rinsed with PBS and fixed for 5 min in cold methanol. Cells were then rinsed several times with PBS, before a 20-min block in 10% goat serum in BT-PBS (PBS with 0.1% Tween 20 and 1% BSA). Coverslips were then incubated for 50 min with 2A9 MDM2 monoclonal supernatants and washed extensively with BT-PBS. The secondary antibody, biotin-SP-conjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories), was diluted 500-fold in 10% goat serum BT-PBS and incubated on the cells for 40 min. After extensive washing, the cells were incubated for 15 min with a 1000-fold dilution of streptavidin-fluorescein isothiocyanate conjugate (Gibco BRL) and washed again. Cells were then mounted and viewed using a confocal microscope.
Yeast Screen for MDM2 Mutants Deficient in p53 Interaction
Summary of yeast screen results
Colonies that retest white
7.9 × 10−3
6.9 × 10−4
2.5 × 10−4
Mutation in MDM2
1.1 × 10−4
Testing of Mutant MDM2 1–115 Fragments In Vitro
One possible explanation for the inability of the mutant MDM2 proteins to interact with p53 is misfolding of the mutant peptides. To address this concern, the mutant MDM2 fragments were tested for their recognition by two conformation-dependent MDM2 monoclonal antibodies. Antibodies 4B2 and 3G5 have epitopes that map to the NH2-terminus of MDM2 (amino acids 6–58 and 58–89, respectively), and neither antibody recognizes the denatured form of the MDM2 protein (23). Interestingly, 3G5 can only bind to free MDM2 and cannot precipitate MDM2-p53 complexes, which suggests that its epitope is within the p53-binding site (23). These two monoclonal antibodies, MDM2-specific polyclonal sera and a monoclonal antibody that recognizes SV40 large T antigen (as a negative control) were used to immunoprecipitate the in vitro-labeled wild-type and mutant MDM2 fragments from the same lysates used in the p53-binding assay described above. The MDM2 polyclonal sera but not the negative control antibody recognized the mutant and wild-type MDM2 proteins as expected. In addition, both MDM2-specific conformation-dependent antibodies recognized the G58D and the C77Y mutants, as well as the wild-type protein, indicating that the two MDM2 mutant forms are not grossly misfolded (Fig. 2B).
Site-directed Mutagenesis of Full-length Mdm2
Summary of mutagenesis and binding assays with MDM2
In Vivo Regulation
Full-length MDM2 Mutants Fail to Regulate the Transcriptional Activity of p53 In Vivo
The expression and proper localization of these inactive MDM2 mutant proteins were verified by indirect immunofluorescence. Wild-type and mutant MDM2 proteins were detected in approximately the same percentage of cells in each transfected population. In addition, mutant MDM2 proteins were localized to the nucleus as was the wild-type protein (data not shown).
Analysis of a Crystal Structure of the MDM2-p53 Complex
The other two residues identified in this mutational analysis as important for the MDM2-p53 interaction are found in the hydrophobic lining of the MDM2 cleft and directly contact p53 residues that were determined by prior genetic analysis of p53 to be critical for binding to MDM2 (30,34; Fig. 6B). G58, a component of the α helix that forms one side of the cleft, makes van der Waals contacts with both residues F19 and W23 of p53 and is in a highly compact region of the complex (Fig. 6B). Mutating this residue to aspartic acid (G58D of the yeast genetic screen) or to alanine (G58A from site-directed mutagenesis) could prevent proper docking of the p53 peptide. An additional MDM2 residue that was identified as critical for the MDM2-p53 interaction is V75, one of the hydrophobic residues of the middle β sheet that caps one end of the cleft. This valine residue makes van der Waals contacts with F19 of p53. As the highly conservative change of valine to alanine at this site (V75A from site-directed mutagenesis) prevented the binding of p53, it is probable that even subtle changes in this hydrophobic pocket of the NH2-terminus of MDM2 are sufficient to destabilize the interaction between MDM2 and p53 (Fig. 6B).
The best-studied activities of the MDM2 oncoprotein are its interaction with p53 and its inhibition of p53-dependent transcription, both of which are mediated by the NH2-terminus of MDM2 (23). To better define the amino acids of MDM2 required for this interaction with p53, a genetic screen in yeast was employed to select mutations in MDM2 which interfered with its ability to bind p53. This method proved fruitful, identifying two residues that were important for this protein-protein interaction (G58D and C77Y of MDM2). Although the mutant screen certainly did not saturate the gene for all possible mutations affecting p53 binding, the mutations that were found helped to define a smaller region that was then further altered by site-directed mutagenesis. Two of the three additional residues altered in this region proved to be critical for the MDM2-p53 interaction.
The mutant MDM2 proteins were tested for their ability to bind to p53 in vitro. One mutation residing outside of the region defined by the screen did not prevent binding to p53 (i.e., the C2A mutation). This result eliminated the possibility that a disulfide bond between residues C2 and C77 of MDM2 is critical for interaction with p53. All mutations that were made between residues 58 and 77 affected the interaction of MDM2 with p53. The E69A mutant retained approximately 50% of the wild-type binding ability, while the other mutants did not bind p53 to a detectable level. These mutations include the likely less disruptive changes to alanine at the critical residues originally found in the yeast-based screen (G58A and C77A), an aspartic acid to alanine change at residue 68 (D68A), as well as the highly conservative change of valine to alanine at residue 75 (V75A).
Several of the Mdm2 mutations (G58A, D68A, and V75A) were made in the Mdm2 cDNA in mammalian expression vectors by site-directed mutagenesis to test them in vivo for their abilities to functionally interact with p53. All three of these mutant MDM2 proteins failed to inhibit p53-dependent transcriptional activation in a transient transfection system. These results indicate that the MDM2 mutations described here indeed prevent MDM2 from regulating p53.
The MDM2 protein’s p53-binding domain is highly conserved between human, mouse, and Xenopus laevis (24; Fig. 3). All of the residues shown to be important by the genetic approach described here are conserved between these three species. Since this work was initiated, the crystal structure of this MDM2-p53 complex has been determined (30). The genetic and physical approaches produced complimentary results. Two of the residues identified in the genetic screen play structural roles in MDM2’s p53-binding domain, while two residues line the hydrophobic pocket of MDM2 and directly contact p53.
MDM2 may well have p53-independent functions, some of which may be relevant to its role in oncogenesis. It will be interesting to determine if MDM2 mutants that are unable to bind and regulate p53 retain any transforming ability in wild-type p53-containing cells (3). The other possible functions of MDM2 in pRb and E2F binding (28,29), binding to the ribosomal protein L5–5S RNA complex (25), shuttling between the nucleus and the cytoplasm (JCR, DAF, AJL unpublished data), and binding to specific RNA structures (27) indicate that there are novel functions of MDM2 that remain to be explored. The mutant MDM2 proteins described here should prove useful in separating the p53-dependent and -independent roles of MDM2.
We thank R. Brent, J. Broach, J. Chen, E. Golemis, S. Hanes, H. Lu, S. Ramos, and B. Vogelstein for plasmids. We also thank E. Golemis and M. Rose for the polyclonal antibodies used in the yeast screen. Lastly, we thank P. Kussie and N. Pavletich for helpful discussions, for sharing data prior to publication, and for assistance with figures.
This work was supported by an NIH Program Project Grant to A.J.L., PO1CA41086. D.A.F. was supported by an NIH Cellular and Molecular Training Grant, 5T32GM07312. C.B.E. was supported by a Cancer Research Fund of the Damon Runyon-Walter Winchell Foundation Fellowship, DRG-1235. J.C.R. was supported by a fellowship from the German Research Foundation (DFG).
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