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. 1999 May;19(5):3257-66.
doi: 10.1128/MCB.19.5.3257.

MDM2 suppresses p73 function without promoting p73 degradation

X Zeng  1 , L Chen, C A Jost, R Maya, D Keller, X Wang, W G Kaelin Jr, M Oren, J Chen, H Lu
Affiliations

MDM2 suppresses p73 function without promoting p73 degradation

X Zeng et al. Mol Cell Biol. 1999 May.

Abstract

The newly identified p53 homolog p73 can mimic the transcriptional activation function of p53. We investigated whether p73, like p53, participates in an autoregulatory feedback loop with MDM2. p73 bound to MDM2 both in vivo and in vitro. Wild-type but not mutant MDM2, expressed in human p53 null osteosarcoma Saos-2 cells, inhibited p73- and p53-dependent transcription driven by the MDM2 promoter-derived p53RE motif as measured in transient-transfection and chloramphenicol acetyltransferase assays and also inhibited p73-induced apoptosis in p53-null human lung adenocarcinoma H1299 cells. MDM2 did not promote the degradation of p73 but instead disrupted the interaction of p73, but not of p53, with p300/CBP by competing with p73 for binding to the p300/CBP N terminus. Both p73alpha and p73beta stimulated the expression of the endogenous MDM2 protein. Hence, MDM2 is transcriptionally activated by p73 and, in turn, negatively regulates the function of this activator through a mechanism distinct from that used for p53 inactivation.

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Figures

FIG. 1
FIG. 1
Wild-type but not mutant MDM2 inhibits the transcription mediated by p73. (A) Overexpression of MDM2 leads to decrease of p73-dependent transcription as measured by CAT activity (top panel). Saos-2 cells were transfected with expression vectors for p53 (200 ng), p73α (200 ng), and p73β (200 ng) and increasing amounts (0, 1.5 and 3.0 μg) of MDM2-expressing vectors as indicated at the top. The CAT assay was conducted with a CAT reporter plasmid driven by two copies of the p53RE motif derived from the MDM2 promoter as described in Materials and Methods. Expression of MDM2 (middle panel) and p53 and p73α/β (bottom panel) under the same conditions was detected by co-IP followed by WB with cell lysates of triplicate plates for each lane. The antibodies used for co-IP-WB are indicated above the panels. (B and C) MDM2 mutants fail to inhibit p73-dependent transcription in vivo. An experiment identical to that in panel A was carried out, except that two MDM2 mutants, G58A (replacement of Gly 58 by Ala) and V75A (replacement of Val 75 by Ala), were also included. Saos-2 cells were transfected with plasmids encoding HA-p53 or HA-p73α (500 ng each) in the presence or absence of plasmids encoding either wild-type (wt) or mutant hMDM2Δ (10 μg each), as indicated, along with a p53-responsive CAT reporter plasmid (4 μg) containing two p53-binding sites derived from the ribosomal gene cluster and a cytomegalovirus-driven β-galactosidase reporter plasmid (4 μg). Sufficient pBSK was added to bring the total to 24 μg. MDM2Δ lacks residues 58 to 89 (7). The raw CAT activity was corrected for β-galactosidase activity to normalize for differences in transfection efficiency. To facilitate comparison, the activities of p53 and p73 in the absence of MDM2 were both set to 100%. Error bars indicate the standard error of the mean. (D) IP-WB analysis of MDM2 and MDM2Δ expression in transfected Saos-2 cells. The same transient transfection as that in the experiment in panel C was conducted to detect MDM2 expression levels. 2A10 was used for IP, and polyclonal MDM2 antibodies were used for WB. IgG, immunoglobulin G. (E) MDM2 inhibits p73- and p53-induced apoptosis. H1299 cells (3 × 105/60-mm dish) were transfected with plasmids encoding proteins indicated at the bottom. The amounts used per transfection are indicated (in micrograms) in the lower part of the figure. Mouse MDM2 and N-terminally deleted MDM2 (MDM2Δ) were used in this experiment. A GFP expression plasmid (50 ng) was included in all the combinations. Transfected dishes were scored by fluorescence microscopy for the appearance of cells with distinct apoptotic morphology (rounding and shrinkage), 40 or 32 h after transfection for p53 or p73α, respectively. Only GFP-positive cells were counted. The percentage of apoptotic cells shown in each column represents the mean of three independent transfections; standard deviations are indicated. The total amount of DNA was kept constant by including an appropriate amount of empty vector DNA. Columns 1 and 5 are the vector-only controls for p53 (2 μg of vector DNA) and p73α (6 μg of vector DNA), respectively.
FIG. 2
FIG. 2
p73 binds to MDM2 in vivo. (A) Coimmunoprecipitation of p73 with MDM2. (10)1 cells (5 × 105/60-mm dish) were transfected with plasmids encoding HA-p73α or no insert, in the presence or absence of plasmids encoding either wild-type or mutant hMDM2Δ, as indicated at the top. hMDM2Δ lacks residues 58 to 89 (7). In this experiment, 1.5 μg of the relevant plasmids was used, and the parental pCMV vector was added to bring the total amount of plasmid DNA to 3 μg for each transfection. The antibodies used for IP and WB are indicated. Each lane presents a result from cell extracts of three dishes. αMDM2 denotes the polyclonal anti-MDM2 antibody. The strong enhanced chemiluminescence signals of p73α were due to a longer exposure (20 min) of the membrane when blotting with ER15. Because the same membrane was probed first with ER15 and then with anti-MDM2 antibodies, the slight bands comigrating with MDM2Δ in lanes 1, 4, and 5 were the remaining signals of p73α, since this band also appeared in lane 6, where only the p73α plasmid was added, but not in lane 7, where p73α was not included. Owing to the comigration of p73α with MDM2Δ, the results for MDM2 and p73α are presented separately. (B) Co-IP of p53 with MDM2. The same assay was performed, except that MDM2 (1.5 μg) and/or p53 (1.5 μg) expression plasmids were used. The parental vector was used to bring the total amount of plasmids to 3 μg where necessary. Anti-MDM2 antibodies used for IP and WB were 2A10 and polyclonal anti-MDM2 antibodies, respectively. Each lane is representative of the results from three dishes of cells. IgG, immunoglobulin G.
FIG. 3
FIG. 3
Mapping the protein interaction domains for MDM2 and p73. (A) Both p73α and p73β bind to the N terminus of MDM2. The indicated GST-MDM2 fusion proteins, immobilized on Sepharose, were incubated with 10 μl of in vitro-translated 35S-labeled p73α and p73β at room temperature for 1 h. Bound proteins were analyzed as described in Materials and Methods. As a control, 5% of the input was directly loaded on the last lane. Binding between GST-HDM-425–491 and p73β was not done due to the redundancy (see lanes for GST-290-491 and GST-384-491). (B) MDM2 binds to the N-terminal domain of p73 (aa 1 to 70). A set of GST pull-down experiments similar to those in panel A were performed, except that GST-p73 deletion fusion proteins (indicated at the top) were used in this case. In lane 1, 50% of MDM2 input (35S labeled) was loaded directly. The GST-p73 truncation fusion proteins are schematically drawn at the bottom of the panel, and their expression is shown in panel D. (C) Expression of GST-MDM2 fusion proteins used in panel A. GST-MDM2 fusion proteins (10 μl; 50% slurry) were loaded directly onto an SDS–10% polyacrylamide gel and stained with Coomassie brilliant blue. (D) Expression of GST-p73 fusion proteins used in panel B. GST- or GST-p73 fusion protein-containing beads (10 μl; 50% slurry) were directly analyzed on an SDS–10% polyacrylamide gel and stained with Coomassie brilliant blue. The amounts of these proteins used in panels A and B were normalized by comparing the intensity of the bands with BSA standards; 400 ng of each of these proteins was used for GST-pull down assays.
FIG. 4
FIG. 4
MDM2 does not induce rapid proteolytic degradation of p73. H1299 cells (4 × 105/60-mm dish) were transfected with the indicated combinations of the following expression plasmids (total DNA, 3 μg): pCDNA3 vector (3 μg/dish; lanes 1), HA-tagged p73α (0.5 μg/dish; lanes 2 to 7), wild-type human p53 (0.5 μg/dish; lanes 2 to 4), human p53 mutant p53L14N,F19S [p53(14,19), 0.5 μg/dish; lanes 5 to 7], full-length mouse MDM2 (2 μg/dish; lanes 3 and 6), or N-terminally deleted mouse MDM2 (MDM2Δ, 2 μg/dish; lanes 4 and 7). At 28 h posttransfection, the cells were harvested and extracted in protein sample buffer. WB was performed with either a monoclonal anti-HA antibody (HA.11 [Babco, Berkeley, Calif.]; WB=α-HA), a mixture of the p53-specific monoclonal antibodies Pab 1801 and Pab 421 (WB=α-p53), or 2A10 (WB=α-MDM2). The endogenous human MDM2 protein of the H1299 cells, migrating slower than the mouse MDM2, is also indicated (HDM2). The fast-migrating bands below the MDM2Δ are nonspecific signals detected by anti-MDM2 antibodies in the bottom panel.
FIG. 5
FIG. 5
MDM2 blocks p73- but not p53-p300/CBP interactions. (A) MDM2 disrupts the p73-p300 interaction in an in vitro co-IP/protein competition assay. A 4-μl volume of in vitro-translated [35S]Met-labeled p73α and p73β, and 275 ng of the purified Flag-p300 protein were used in the assay. In lanes 3 and 4, 200 and 400 ng of MDM2 was added, respectively. The anti-Flag antibody used for IP is indicated at the top, and inputs (50%) of p73α and p73β as well as MDM2 (200 ng) are shown in lane 5. αp300 and αMDM2 indicate polyclonal anti-p300 and anti-MDM2 antibodies, respectively. The top panel is an autoradiogram of the same membrane for the WB in the bottom panel (the same is true for panels B to D). (B) MDM2 does not interfere with the p53-p300/CBP interaction. Experimental details were as for panel A and the corresponding test, except that 4 μl of in vitro-translated [35S]Met-labeled p53 was used. In lane 5, 50% of the p53 or MDM2 input was directly loaded. (C) The MDM2 mutant MDM2Δ does not interfere with the p73-p300 interaction. The same experiment was conducted, except that the N-terminally deleted mutant MDM2Δ was used as a competitor. For all these WB results, the strong signals were due to a longer exposure (15 min) to X-ray film. (D) Detecting p73- or p53-MDM2 complexes in the same experimental settings as those in panels A and B. The same IP-autoradiography-WB analysis was performed as described above, except the monoclonal anti-MDM2 antibody 2A10 was used for IP. The bound p73 or p53 was detected by autoradiography, and MDM2 was detected by WB with polyclonal anti-MDM2 antibodies, as indicated. The amount of p73, p53, MDM2, or p300 used in this analysis was the same as that in panels A to C.
FIG. 6
FIG. 6
p73α and p73β, like p53, induce the expression of the endogenous MDM2 protein in transfected H1299 cells. (A) Immunofluorescent staining of the endogenous MDM2. Cells were transiently transfected with p73 or p53 expression plasmids and then subjected to immunofluorescent staining with the anti-MDM2 monoclonal antibody 2A9 as described in Materials and Methods. The vectors used in the experiment are indicated at the top of each panel. Similar results were obtained in two independent experiments. (B). IP-WB analysis of the endogenous MDM2. H1299 cells (106/100-mm dish) were transfected with plasmids (15 μg/transfection) encoding p53, p73α, and p73β, respectively. MDM2 was detected by IP with 2A10 followed by WB with polyclonal anti-MDM2 antibodies. Each lane is representative of the result from three dishes of cells. Lane 1 contains the vector only.
FIG. 7
FIG. 7
Schematic presentation showing distinct mechanisms by which MDM2 inactivates p53 (A) and p73 (B). p53 with dashed lines in panel A indicates that this protein may or may not be in the ternary complex. See Discussion for details. This model does not address the suggested alternative mechanism for p53 inactivation by MDM2, namely, that MDM2 interferes with the binding of other transcription factors, e.g., hTAFII31 and hTAFII70, to the N-terminal transcription domain of p53 (37, 38, 55). Also, binding of p53 to the N terminus of p300/CBP (19, 63) in the absence of MDM2 is not shown.
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