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J Biol Chem, Vol. 275, Issue 8, 5733-5738, February 25, 2000


p76MDM2 Inhibits the Ability of p90MDM2 to Destabilize p53*

Mary Ellen PerryDagger , Susan M. Mendrysa§, Leslie J. Saucedo§, Paul Tannous, and Marisa Holubar

From the Department of Oncology, McArdle Laboratory for Cancer Research, Madison, Wisconsin 53706

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The mdm2 oncogene encodes p90MDM2, which binds to and inactivates the p53 tumor suppressor protein. p90MDM2 inhibits p53 by blocking the transcriptional activation domain of p53 as well as by stimulating its degradation. Recently, we showed that another product of the wild-type mdm2 gene, p76MDM2, lacks the first 49 amino acids of p90MDM2 and cannot bind p53. Here, we report that, like p90MDM2, p76MDM2 is expressed in both the nuclear and cytoplasmic compartments. Overexpression of p76MDM2 antagonizes the ability of p90MDM2 to stimulate the degradation of p53 and leads to an increase in the levels and activity of p53. Seven murine tissues express an alternatively spliced mdm2 mRNA that can encode p76MDM2 but not p90MDM2, as well as the normally spliced mdm2 mRNA that encodes both MDM2 proteins. All seven tissues express both MDM2 proteins. p90MDM2 is much more abundant than p76MDM2 in the testis, brain, heart, and kidney. However, in those tissues known to undergo p53-mediated apoptosis in response to gamma -irradiation, the thymus, spleen, and intestine, the levels of the MDM2 proteins are roughly equivalent. Our results indicate that the ratio of the two MDM2 proteins may regulate the response of tissues to DNA damage.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The wild-type murine double minute 2 (mdm2)1 gene encodes p90MDM2, an important regulator of the p53 tumor suppressor protein (1, 2). p90MDM2 inhibits p53 both by directly blocking the transactivation domain of p53 and by stimulating the degradation of p53 (3-6). Both of these effects of p90MDM2 require that it bind to p53 (6, 7). Multiple lines of evidence suggest that the function of p53 is down-regulated by p90MDM2. Microinjection of antibodies that disrupt the interaction between p90MDM2 and p53 in cultured cells leads to enhanced levels of p53 and to an arrest of cell division (8). The ability of p90MDM2 to inhibit p53 appears to be essential for development because mice lacking mdm2 die in utero, whereas those lacking both mdm2 and p53 survive (1, 2). Inhibition of p53 function by p90MDM2 may also contribute to tumorigenesis because mdm2 is overexpressed in many tumors that contain wild-type p53 genes (9-11). In addition, the association between p90MDM2 and p53 may determine the level of p53 in cells responding to DNA damage (reviewed in Ref. 12). Following exposure of cells to DNA-damaging agents, binding of p90MDM2 to p53 is disrupted and p53 accumulates (13, 14). The increase in the level of p53 results in the increased expression of p53-responsive genes, including mdm2 (15, 16). The increase in the amount of p90MDM2 in cells responding to DNA damage may facilitate the recovery of normal levels of p53 protein (6).

Until recently, p90MDM2 was the only protein known to be a bona fide product of the wild-type mdm2 gene (17). It has long been known that multiple MDM2 proteins are expressed in some tumor cell lines, but it has not been clear whether these proteins are products of normal mRNAs (18, 19). We previously demonstrated that, in addition to p90MDM2, a smaller MDM2 protein, p76MDM2, is expressed in wild-type mouse embryo fibroblasts (MEFs) but not in p53/mdm2-null MEFs (17). In addition, the testis expresses an alternatively spliced mdm2 mRNA that, when expressed in COS-1 cells, produces p76MDM2 but not p90MDM2 (17, 20). Together, these observations indicate that p76MDM2 is a bona fide product of the mdm2 gene.

p76MDM2 lacks the N-terminal 49 amino acids of p90MDM2 (17). p76MDM2 can be expressed via internal initiation at codon 50 in the mdm2 mRNA that also directs the synthesis of p90MDM2 (17). In addition, p76MDM2, but not p90MDM2, can be expressed from an alternatively spliced mdm2 mRNA lacking exon 3 (17). In the alternatively spliced mRNA, the AUG that gives rise to p90MDM2 is missing, and codon 50 in the normally spliced mRNA is the first AUG. Epitope mapping has confirmed that p76MDM2 lacks a portion of the domain of p90MDM2 involved in interaction with p53 (17, 21). Therefore, p76MDM2 cannot bind to p53 or inhibit the transcriptional activation function of p53 (7, 17). However, p90MDM2 and p76MDM2 do share several sequence motifs and therefore may share interactions with other proteins. Both p90MDM2 and p76MDM2 contain nuclear import and export signals (19, 22), an RNA-binding domain (23), and a ubiquitin ligase domain (24). It is known that three proteins, L5, p19ARF, and p300, interact with regions of p90MDM2 contained within p76MDM2 (23, 25-27). Because p76MDM2 is identical to 90% of the coding sequence for p90MDM2, it is predicted that p76MDM2 binds these proteins as well as unidentified proteins that interact with p90MDM2. Interaction of p76MDM2 with partners of p90MDM2 may allow p76MDM2 and p90MDM2 to perform similar functions or to compete with each other for regulatory factors.

Expression of both MDM2 proteins is enhanced following exposure of cultured cells to ultraviolet light (17). Although the induction of p90MDM2 is thought to facilitate the return of p53 to normal levels, it is unclear what role p76MDM2 plays in the DNA damage response. Because p90MDM2 must bind p53 in order to stimulate its degradation, and because p76MDM2 cannot bind p53 (7, 17), it is unlikely that p76MDM2 shares the ability of p90MDM2 to target p53 for degradation. However, if p76MDM2 were to bind a key regulatory partner of p90MDM2, it could affect the function of p90MDM2 by influencing the availability of such a factor. For example, p76MDM2 could stabilize p53 by sequestering a factor that enhances the ability of p90MDM2 to stimulate the degradation of p53, such as p300 (26). Alternatively, p76MDM2 could increase the ability of p90MDM2 to stimulate the degradation of p53 by binding a regulatory factor that inhibits p90MDM2 function, such as p19ARF (25, 27). If either scenario is correct, the induction of p76MDM2 expression by p53 in response to DNA damage may indicate that p76MDM2 contributes to the mechanism through which p53 regulates its own levels and activity. Therefore, we tested whether overexpression of p76MDM2 antagonizes or enhances the ability of p90MDM2 to stimulate the degradation of p53.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cell Culture-- Cells were grown in Dulbecco's modified Eagle's medium with 10% fetal calf serum (Summit) supplemented with penicillin and streptomycin. (10)1 and (10)3 cells are immortal murine fibroblasts that lack p53 (28). 3T3DM cells contain amplified copies of the mdm2 gene (19). The U2OS human osteosarcoma cell line expresses wild-type p53 (29) and was obtained from the ATCC. Early passage p53-null and p53/mdm2-null MEFs were kind gifts from Steve Jones (University of Massachusetts).

Calcium Phosphate Transfection-- All transfections were done using the calcium phosphate protocol (30). The plasmids used were as follows: pC53C1N, encoding wild-type human p53 (31); pCMV5B, encoding wild-type murine p76MDM2; pCMV5F, encoding p90MDM2 and a small amount of p76MDM2; pCMV5X2, encoding wild-type murine p90MDM2 and a minor amount of p76MDM2 (17); and pGFP1929, in which expression of the green fluorescent protein (GFP) was under the control of the cytomegalovirus (CMV) promoter (a gift from Ashok Aiyar and Bill Sugden, McArdle Laboratory). Stable cell lines overexpressing p76MDM2 or p90MDM2 were created by co-transfecting U2OS cells with pCMV5B or pCMV5F and pSV2NEO, which encodes aminoglycoside phosphotransferase and confers resistance to the drug Geneticin. The transfected cells were placed under selection in 1 mg/ml Geneticin (Calbiochem) for 2 weeks. Colonies were isolated and screened for overexpression of p76MDM2. To measure transient transfection efficiencies in MEFs, we added 1 µg of pGFP1929 to each transfection and quantitated the GFP-positive cells by flow cytometry.

Immunofluorescence-- (10)1 cells were fixed for 10 min at room temperature in 100% methanol and stored at 4 °C in phosphate buffered saline. For detection of MDM2, the polyclonal 628 antiserum was diluted 1:200 (32). The primary antibody was detected using fluorescein isothiocyanate-conjugated goat anti-rabbit antibody (Vector) at a dilution of 1:1000. The cells were photographed with a Zeiss Axiophot photomicroscope.

Phospholabeling-- Following transfection with pCMV5, pCMV5B, or pCMV5F (17), (10)3 cells were labeled with 32PO4 (250 µCi/ml; NEN Life Science Products) for 4 h in phosphate-free medium with 2% dialyzed fetal calf serum. Cells were rinsed in Tris-buffered saline and immediately lysed. Samples were normalized for incorporation of radiolabel and 107 cpm were incubated with a polyclonal antibody (266) raised in this laboratory against a histidine-tagged fusion protein of murine MDM2 purified from Escherichia coli as first described by Olson et al. (33).

Mice-- Wild-type 129/Sv mice were obtained from the Jackson Laboratory and housed in the American Association for the Accreditation of Laboratory Animal Care-approved McArdle Laboratory Animal Care Facility. Following sacrifice, animals were dissected, and tissues frozen immediately in liquid nitrogen. Tissue samples were subsequently stored at -80 °C.

RT-PCR-- Total RNA from tissues was prepared using Tri reagent (Molecular Research Center, Inc.) according to the manufacturer's instructions. First-strand synthesis was performed using oligo-dT as a primer. The PCR was performed as described previously, using primers complementary to exons 2 and 4 (17). The positive controls were mdm2 cDNAs containing or lacking exon 3 (20).

Immunoprecipitation/Western Analysis-- Cultured cells were lysed as described (34). Protein concentrations were determined by either of the Bio-Rad Protein Assays (Bradford or Lowry). For immunoprecipitation of p53, the CM5 polyclonal antibody (Vector Laboratories) was used because it immunoprecipitates human p53 quantitatively from lysates of cells (35). To detect p53, the DO-1 antibody (Oncogene Science) was used at a dilution of 1:1000. For analysis of MDM2 protein levels, the polyclonal antibody 628 (32) was used for immunoprecipitation, and the monoclonal antibody 2A10 (21) was used for detection as described (17). Both primary antibodies were revealed using sheep anti-mouse Ig conjugated to horseradish peroxidase (Amersham Pharmacia Biotech) and enhanced chemiluminescence (ECL) (Amersham Pharmacia Biotech). As a control for p53, human p53 was translated from pSKp53 (a gift from Jiandong Chen, University of South Florida) with the TNT coupled reticulocyte lysate system of Promega. Murine tissues were lysed in radioimmune precipitation buffer (RIPA) (34). As a negative control, we used testis tissue from a p53/mdm2-null mouse of mixed C57BL/6-129/Sv background (a gift from Steve Jones, University of Massachusetts) because mice lacking mdm2 die in utero, whereas those lacking both mdm2 and p53 live (1, 2). The positive control for MDM2 proteins was a lysate from 3T3DM cells, which have amplified copies of the mdm2 gene (19).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

p76MDM2 Is a Phosphoprotein Distributed in the Nucleus and the Cytoplasm-- p76MDM2 and p90MDM2 may bind some of the same proteins and participate in some of the same regulatory pathways because all of the sequence of p76MDM2 is contained within p90MDM2 (Fig. 1A) (17). The majority of p90MDM2 protein localizes to the nucleus (22, 33), but it can shuttle from the nucleus to the cytoplasm (22). Both its ability to localize to the nucleus and its ability to shuttle are important for the functional interactions of p90MDM2 (22, 31, 36, 37). Because p76MDM2, like p90MDM2, contains both nuclear import and export signals, we determined the subcellular distribution of p76MDM2. We transiently expressed the plasmid pCMV5B, which encodes p76MDM2, in immortal p53-null fibroblasts that express low levels of endogenous MDM2 proteins (17) and detected p76MDM2 by indirect immunofluorescence using a polyclonal antibody against MDM2 (Fig. 1B). There was no detectable staining in cells transfected with the vector alone. In contrast, strong staining was apparent in cells transfected with the expression vector encoding p76MDM2. The staining revealed that in some cells, p76MDM2 was present in the nucleus. In other cells, p76MDM2 was in the nucleus and in the cytoplasm. The ability of p76MDM2 to accumulate in both compartments indicates that it could interact with both nuclear and cytoplasmic proteins.


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  		Fig. 1.   p76MDM2 shares some properties with p90MDM2. A, schematic of the domains of p90MDM2 and p76MDM2. Proteins known to bind regions of p90MDM2 contained within p76MDM2 are shown at the top. The white boxes indicate nuclear localization (NLS) and export (NES) signals, an acidic domain, a zinc finger, and a RING finger. The ubiquitin ligase function of p90MDM2 requires a cysteine residue within the RING finger (24). B, immunofluorescent detection of p76MDM2. p53-null (10)1 cells were transfected with either an empty vector (pCMV5) or with pCMV5B, which expresses p76MDM2 but not p90MDM2. The cells were fixed, and p76MDM2 was detected using a polyclonal antibody against MDM2 (antibody 628) and a fluorescein isothiocyanate-conjugated secondary antibody. C, phospholabeling of p76MDM2. p53-null (10)3 cells were transfected with pCMV5B, which encodes p76MDM2, pCMV5F, which encodes mostly p90MDM2, or the pCMV5 vector alone and incubated in 32PO4 for 4 h. MDM2 proteins were immunoprecipitated with polyclonal antibody 266 and visualized by autoradiography.

We next asked whether p76MDM2, like p90MDM2, is phosphorylated. p53-null fibroblasts were transfected with pCMV5F and pCMV5B, expressing p90MDM2 and p76MDM2, respectively. The cells were then radiolabeled with inorganic phosphate, and MDM2 proteins were immunoprecipitated using a polyclonal antibody against MDM2 (Fig. 1C). No phosphoproteins were detected in immunoprecipitates from cells transfected with the vector alone. Cells transfected with pCMV5B expressed a 76-kDa phosphoprotein, whereas those transfected with the pCMV5F expressed a 90-kDa phosphoprotein. These results show that p76MDM2, like p90MDM2, can be phosphorylated in cells. The finding that p76MDM2, like p90MDM2, is a phosphoprotein that can reside in both the nucleus and the cytoplasm supports the notion that these two MDM2 proteins may interact with some of the same proteins.

p76MDM2 Interferes with the Ability of p90MDM2 to Stimulate the Degradation of p53-- Based on the similar characteristics of p76MDM2 and p90MDM2, we reasoned that increased levels of p76MDM2 might disrupt protein interactions that regulate the ability of p90MDM2 to destabilize p53. Two cellular proteins, p300 and p19ARF, have been found to regulate the ability of p90MDM2 to stimulate the degradation of p53 (25-27). Because p76MDM2 lacks the ability to bind p53 (7, 17), it is unlikely for p76MDM2 to target p53 for degradation. However, if p76MDM2 competes with p90MDM2 for binding to a regulatory protein, the level of p76MDM2 may influence the ability of p90MDM2 to decrease the level of p53. Therefore, we tested whether overexpression of p76MDM2 could either augment or interfere with the ability of p90MDM2 to decrease the level of p53. To test whether p76MDM2 could affect the level of p53 protein, we compared the amount of p53 protein in p53-null (10)1 cells transfected with a plasmid encoding p53 (pC53C1N) to the amount of p53 in cells transfected with both pC53C1N and a plasmid encoding p76MDM2 (pCMVB). The levels of p53 were determined using immunoprecipitation followed by Western analysis. p53 levels were increased when pCMVB was co-transfected with pC53C1N (Fig. 2A, lanes 2-4). Co-transfection of a plasmid encoding p90MDM2 resulted in lower levels of p53 in the cells (Fig. 2A, compare lane 2 with lane 5), indicating that p76MDM2 and p90MDM2 have opposing effects on the level of p53. Analysis of the level of MDM2 proteins indicated that the level of p90MDM2 was higher in cells transfected with the plasmid expressing p76MDM2 (Fig. 2B, lanes 2-4) (17, 20). Because mdm2 is a p53-responsive gene, the increase in p90MDM2 expression could be the result of increased p53 function in the presence of p76MDM2.


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  		Fig. 2.   p76MDM2 increases the level of p53. p53-null (10)1 cells were transfected with the vector alone (lane 1) or with 1 µg (1 ×) of a vector encoding wild-type human p53 (pC53C1N). Some plates of cells also received 6 µg (1 ×) or 18 µg (3 ×) of a plasmid encoding murine p76MDM2 (pCMV5B) or 2 µg (1 ×) of a plasmid encoding p90MDM2 (pCMV5X2). The total amount of plasmid transfected was normalized using the pCMV5 vector. The cells were harvested and lysed 48 h after transfection. A, equivalent amounts of cellular protein were incubated with the CM5 polyclonal antibody against p53. Following transfer to nitrocellulose, p53 was revealed using DO-1 and ECL. The slower migrating product of the p53 protein is likely to be a degradation product (47). B, MDM2 proteins were immunoprecipitated with the 628 polyclonal antibody and detected using 2A10 and ECL.

The increased level of p53 protein suggests that p76MDM2 may antagonize the ability of p90MDM2 to stimulate the degradation of p53. One established mechanism of stabilizing p53 is to inhibit the interaction between p53 and p90MDM2 (8). To determine whether p76MDM2 could abrogate the interaction between p53 and p90MDM2, the immortal p53-null (10)1 cell line was transfected as described above and the amount of p90MDM2 co-immunoprecipitating with p53 was determined. No p90MDM2 precipitated with the p53-specific antibody when lysates from cells transfected with the vector alone were analyzed (Fig. 3, lane 1). The amount of p90MDM2 co-immunoprecipitating with p53 from cells expressing p76MDM2 (Fig. 3, lanes 3 and 4) was greater than the amount co-immunoprecipitating with p53 from cells lacking p76MDM2 (Fig. 3, lane 2) because the levels of both p53 and p90MDM2 are elevated. The co-immunoprecipitation data show that p76MDM2 does not abrogate the interaction between p90MDM2 and p53.


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  		Fig. 3.   p76MDM2 does not inhibit the interaction between p90MDM2 and p53. (10)1 cells were transfected exactly as described in the legend to Fig. 2. Equivalent amounts of cellular protein were incubated with the CM5 polyclonal antibody against p53. MDM2 proteins bound to p53 were detected using 2A10 and ECL.

Next, we assessed whether the ability of p76MDM2 to increase the level of p53 was dependent on p90MDM2 expression. We transfected pC53C1N into p53/mdm2-null MEFs and determined whether expression of p76MDM2 increased the level of p53 in the presence and absence of exogenous p90MDM2. Expression of p76MDM2 did not increase the level of exogenous p53 in cells lacking p90MDM2 (Fig. 4, lanes 2 and 3). However, expression of exogenous p90MDM2 in p53/mdm2-null MEFs restored the ability of p76MDM2 to increase p53 levels (Fig. 4, lanes 4-9). Increasing amounts of p90MDM2 diminish the effect of p76MDM2. These results indicate that p76MDM2 inhibits the ability of p90MDM2 to stimulate the degradation of p53.


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  		Fig. 4.   p76MDM2 increases the level of p53 in the presence of p90MDM2. MEFs lacking both p53 and MDM2 (p53/mdm2-null MEFs) were transfected with vector alone (lane 1) or with 100 ng of a plasmid encoding p53 (1 ×). Some plates of cells also received 100 ng, 300 ng or 1 µg of a plasmid encoding p90MDM2 (1 ×, 3 ×, or 10 ×) and 10 µg of a plasmid encoding p76MDM2 (1 ×), as indicated. All samples received 1 µg of a plasmid expressing GFP, to determine the transfection efficiencies. Forty-eight h after transfection, cells were harvested, and both transfection efficiencies and protein concentrations were determined. The amount of protein added to each immunoprecipitation reaction was normalized for the transfection efficiency. The CM5 antibody was used to immunoprecipitate p53, and DO-1 was used to detect it.

The stability of p53 is increased in the presence of DNA breaks, and there is evidence that it is enhanced by transfection of DNA into cells (38, 39). To eliminate the possibility that the increase of p53 in the presence of p76MDM2 was due to DNA breaks introduced during the transfection procedure, we compared the effects of transient and stable overexpression of p76MDM2 on the levels of endogenous p53. Murine p76MDM2 was introduced into the human U2OS cell line, and subclones overexpressing p76MDM2 were identified following selection. Our results indicate that stable expression of murine p76MDM2 was as effective as transient expression in increasing the level of endogenous p53 protein in U2OS cells (Fig. 5A, lanes 5 and 8). In contrast, both transient and stable expression of murine p90MDM2 resulted in a decrease in p53 protein levels (Fig. 5A, lanes 4 and 7). Thus, the transient transfection protocol does not contribute to the changes in the level of p53 caused by p76MDM2 or p90MDM2.


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  		Fig. 5.   Stable expression of p76MDM2 increases the levels of endogenous p53 and p90MDM2. U2OS cells were transfected with the pCMV5 vector alone or with pCMV5B encoding p76MDM2 or with pCMV5X2 encoding p90MDM2. After 48 h, the cells were either harvested (transient) or placed under selection in G418 (stable) as described under "Experimental Procedures." Equivalent amounts of cellular protein (50 µg) were loaded, and the gel was transferred to nitrocellulose. A, p53 was detected as described in the legend to Fig. 2. p53 protein translated in vitro was used as a control for the detection of p53. B, MDM2 proteins were detected using antibody 2A10 and ECL. Arrows indicate endogenous human p90MDM2, exogenous murine p90MDM2, and exogenous murine p76MDM2. The protein that migrates between human and murine p90MDM2 is likely to be a cross-reactive protein that is not a product of the mdm2 gene (17).

The Transcriptional Activation Function of p53 Is Increased by p76MDM2-- To ascertain whether the transcriptional activation function of p53 is enhanced by p76MDM2, we compared the levels of endogenous MDM2 in U2OS cells stably transfected with the vector to those expressing p76MDM2. Human MDM2 proteins migrate more slowly on polyacrylamide gels than do their murine counterparts (Fig. 5B), so we could distinguish between the endogenous and exogenous MDM2 proteins. Expression of human p90MDM2 correlated with the level of p53, being high when murine p76MDM2 was expressed (Fig. 5B, lanes 3 and 6) and low when murine p90MDM2 was expressed (Fig. 5B, lanes 2 and 5). These results demonstrate that p76MDM2 can increase the level of p90MDM2 by activating p53.

p76MDM2 Is Expressed in Vivo-- If p76MDM2 is a physiologically important product of the mdm2 gene, it must be expressed in vivo. We first asked whether p76MDM2 was expressed in the testis because an mdm2 cDNA derived from testis mRNA lacks exon 3 and directs the synthesis of p76MDM2, but not p90MDM2, when expressed in COS-1 cells (17, 20). Although the testis also expresses normally spliced mRNAs that direct the synthesis of both p90MDM2 and p76MDM2 in COS-1 cells (17, 20), we reasoned that expression of the alternatively spliced mdm2 mRNA would result in a significant amount of p76MDM2 in the testis if this protein is expressed in vivo. To determine whether p76MDM2 is expressed in the testis, we performed Western analysis following immunoprecipitation of MDM2 proteins from wild-type 129/Sv mice. We compared the mobilities of the resulting proteins to bona fide p90MDM2 and p76MDM2 from 3T3DM cells that contain amplified copies of the mdm2 gene (19, 33) (Fig. 6). Two immunoreactive proteins, with mobilities expected for p90MDM2 and p76MDM2, were expressed in the testis of wild-type mice (Fig. 6). No immunoreactive proteins were detected in the immunoprecipitates from the testis of p53/mdm2-null mice, indicating that both proteins are products of the mdm2 gene.


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  		Fig. 6.   p76MDM2 is expressed in murine testis. The 628 polyclonal antibody was used to immunoprecipitate MDM2 proteins from the 3T3DM cell line (10 µg) or from the testes of p53/mdm2-null and wild-type mice (1.5 mg). Following Western transfer, MDM2 proteins were revealed using the 2A10 monoclonal antibody and ECL.

An Alternatively Spliced mdm2 mRNA Is Expressed in Vivo-- Expression of an mdm2 mRNA lacking exon 3, which does not encode p90MDM2, indicates the testis has a mechanism to express p76MDM2 independently of p90MDM2. To determine whether other murine tissues use this mechanism of expressing p76MDM2, we performed RT-PCR on total RNA from the intestine, brain, thymus, spleen, heart, and kidney. A schematic of the assay is shown in Fig. 7A. For controls, we used two cDNAs from the testis that reflect both normally spliced and alternatively spliced mdm2 mRNAs (20). Our analysis indicates that all seven tissues expressed normally spliced mdm2 mRNA as well as the alternatively spliced mdm2 RNA lacking exon 3 (Fig. 7B). Therefore, all these tissues possess an alternative splicing mechanism for producing p76MDM2 independently of p90MDM2. This RT-PCR assay does not differentiate between RNAs initiated at the P1 and P2 promoters (Fig. 7A), so these results do not allow us to make a prediction about the relative amounts of the two MDM2 proteins in these tissues because the two normally spliced mRNAs from the two promoters give rise to different relative amounts of p90MDM2 and p76MDM2 (17, 20).


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  		Fig. 7.   An alternatively spliced mdm2 mRNA lacking exon 3 is expressed in multiple murine tissues. A, schematic of murine mdm2 mRNAs and RT-PCR assay. Shown are the first four exons of the mdm2 mRNAs arising from transcriptional initiation at the p53-independent (P1) and p53-responsive (P2) promoters. The primers (arrows) in the RT-PCR assay are complementary to exons 2 and 4. B, results of RT-PCR analysis of mdm2 mRNAs from murine tissues. Total RNA from each of the indicated tissues was reverse transcribed and subjected to PCR. Controls are mdm2 cDNAs lacking (-exon 3) and containing exon 3 (+exon 3) (20). The PCR was performed under nonquantitative conditions.

The Ratio of p76MDM2 to p90MDM2 Differs among Murine Tissues-- The high ratio of p90MDM2 to p76MDM2 in the testis is similar to that seen in many cell lines and in early passage MEFs (3, 17, 33). To determine whether the ratio of p76MDM2 to p90MDM2 varied among murine tissues, we compared the levels of the two MDM2 proteins in each of six tissues using immunoprecipitation followed by Western analysis. Both the ratio and the level of the two proteins differed among tissues (Fig. 8). Whereas p90MDM2 was more abundant than p76MDM2 in the testis, brain, heart, and kidney, the amounts of p76MDM2 and p90MDM2 were roughly equivalent in the thymus. On longer exposures, it can be seen that the levels of p76MDM2 and p90MDM2 are roughly equivalent in the spleen and intestine (data not shown). Thus, the level of p76MDM2 does not strictly correlate with the level of p90MDM2.


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  		Fig. 8.   p76MDM2 is expressed in multiple murine tissues. MDM2 proteins were immunoprecipitated and revealed as described in the legend to Fig. 6 except that 2 mg of each lysate was used. The negative control (neg.) was protein from the testis of a p53/mdm2-null mouse. In the autoradiogram presented here, the signal from the wild-type testis is saturated to reveal that there are equivalent levels of the two MDM2 proteins in the thymus but not the brain, heart, and kidney. On a longer exposure, both MDM2 proteins can be detected in the samples from the intestine and spleen.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

MDM2 and p53 have been postulated to form a negative feedback loop in which p53 regulates the expression of p90MDM2, thereby inhibiting its own activity (40, 41). p53 binds to the first intron in the mdm2 gene and stimulates the activity of an internal promoter, P2, causing an increase in the expression of at least two MDM2 proteins, p90MDM2 and p76MDM2 (17). p90MDM2 is known to inhibit the function of p53, thereby establishing the negative feedback loop (3, 40, 41). Here we show that the smaller MDM2 protein, p76MDM2, increases the level of p53 by blocking the function of p90MDM2. Thus, the induction of mdm2 expression by p53 results in increased levels of both an inhibitor, p90MDM2, and an activator, p76MDM2, of the function of p53. p53 does not regulate basal levels of expression of mdm2 in vivo, but it specifically stimulates the P2 promoter following whole body gamma -irradiation (49). Activation of the mdm2 P2 promoter results in synthesis of normally spliced mRNA that expresses a higher ratio of p90MDM2 to p76MDM2 than does normally spliced RNA from the P1 promoter (17, 42). Hence, the resulting higher ratio of p90MDM2 to p76MDM2 may facilitate reduction of p53 levels in the later stages of the damage response.

Our results demonstrate that p76MDM2 stabilizes p53 through a dominant-negative mechanism in which it interferes with the function of p90MDM2. Whereas the molecular interactions required for p76MDM2 to interfere with the function of p90MDM2 have not been determined, the experiments described here provide evidence to exclude two mechanisms. First, p76MDM2 does not stabilize p53 by decreasing the level of p90MDM2. Instead, overexpression of p76MDM2 results in an increase in the level of p90MDM2 due to stabilization of p53 protein that is transcriptionally active as evidenced by increases in both p21 and mdm2 mRNAs.2 Second, p76MDM2 does not inhibit the interaction of p90MDM2 with p53, indicating that p76MDM2 differs from viral oncoproteins such as E7 and E1A, which stabilize p53, at least in part, by inhibiting the ability of p90MDM2 to bind p53 (35, 43). Furthermore, p76MDM2 is not likely to sequester p90MDM2, as the two MDM2 proteins do not appear to dimerize based on results obtained with the 4B2 antibody, which immunoprecipitates p90MDM2 but not p76MDM2 from mixtures of the two proteins (17, 21).

Because p76MDM2 shares 90% of the sequence of p90MDM2 as well as its distribution in both the nucleus and the cytoplasm, we hypothesize that p76MDM2 inhibits the function of p90MDM2 by binding to and titrating out a key regulatory factor. The ability of increasing amounts of p90MDM2 to reduce the effect of p76MDM2 is consistent with a model in which the two MDM2 proteins compete for a regulatory factor. In addition, the inhibitory effect of p76MDM2 does not depend on a large excess of p76MDM2 relative to p90MDM2.

Evidence suggests that some of the regulatory factors of p90MDM2 can in fact bind p76MDM2. The tumor suppressor protein p19ARF, which can inhibit the ability of p90MDM2 to stimulate the degradation of p53, binds to the C-terminal half of p90MDM2, and there is evidence from co-immunoprecipitation experiments that it binds p76MDM2 (25, 27). The mechanism(s) by which p19ARF inhibits p90MDM2 function is unclear (25, 44, 45). However, it seems most likely that, were p76MDM2 to titrate p19ARF away from p90MDM2, the effect would be to enhance, not inhibit, the effect of p90MDM2 on p53. Alternatively, p76MDM2 may interfere with the interaction between p90MDM2 and the p300 transcriptional coactivator, which appears to be essential for the ability of p90MDM2 to stimulate the degradation of p53 (26). The predicted effect of p76MDM2 titrating p300 away from p90MDM2 would be to stabilize and activate p53. However, because p300 is thought to facilitate the interaction between p90MDM2 and p53 (26), we would expect to see a decrease in the amount of p90MDM2 co-immunoprecipitating with p53 if p76MDM2 blocked the interaction of p90MDM2 with p300. Instead, we see greater amounts of p90MDM2 co-immunoprecipitating with p53. Although p300 remains a candidate for the protein through which p76MDM2 regulates p90MDM2 function, it is also possible that a later step in the degradation process is inhibited. For example, p76MDM2 may inhibit the ability of p90MDM2 to transfer ubiquitin to p53 or to shuttle from the nucleus to the cytoplasm, two steps that appear to be essential for p90MDM2 to stimulate the degradation of p53 (24, 36, 37). The increased transcriptional activation function of p53 in the presence of p76MDM2 indicates that the stable p53 is not localized to the cytoplasm, but this observation does not allow us to distinguish between the possibilities raised here. It will be important to determine the step at which p76MDM2 interferes with the ability of p90MDM2 to stimulate the degradation of p53.

Examination of the levels of MDM2 proteins in several murine tissues revealed a correlation between the ratio of p76MDM2 to p90MDM2 and the sensitivity of the tissues to gamma -irradiation. The thymus, spleen, and intestine are known to rapidly accumulate p53 protein and undergo p53-mediated apoptosis in response to gamma -irradiation, whereas the kidney, brain, and heart do not (46-48). The levels of p76MDM2 are nearly equivalent to those of p90MDM2 in the thymus, spleen, and intestine, whereas they are much lower than those of p90MDM2 in the kidney, heart, and brain. The higher ratio of p76MDM2 to p90MDM2 may allow p53 protein to accumulate more rapidly in the thymus, spleen, and intestine than in the kidney, heart, and brain. A rapid increase in p53 function may trigger the apoptotic response before protective mechanisms, such as the induction of p90MDM2 expression, can become activated. We propose that the ratio of the two MDM2 proteins contributes to the kinetics at which p53 protein and activity accumulates in response to stress. Targeted overexpression of p76MDM2 in radioresistant tissues, such as the kidney and liver, would allow us to determine whether the ratio of these two MDM2 proteins is a critical influence on the response of tissues to genotoxic stress.

    ACKNOWLEDGEMENTS

We thank Steve Jones for tissue from p53/mdm2-null mice and for early passage p53-null and p53/mdm2 double-null MEFs. We are grateful to Jiandong Chen for supplying plasmids. We appreciate the intellectual contributions of Paul Lambert and Bill Sugden, who suggested adding p90MDM2 to p53/mdm2-null MEFs, and of John Petrini, Peggy Farnham, and Michael Hoffmann, who helpfully critiqued the manuscript.

    FOOTNOTES

* This work was supported by developmental funds from NCI Cancer Center Support Grant CA-07175 (to the McArdle Laboratory for Cancer Research), by NCI Grant CA-14520-26 (to the University of Wisconsin Comprehensive Cancer Center Flow Cytometry Facility), and by NCI Grant CA-70718 (to M. E. P.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: 207A McArdle Laboratory for Cancer Research, 1400 University Ave., Madison, WI 53706. Tel.: 608-265-5537; Fax: 608-262-2824; E-mail: perry@oncology.wisc.edu.

§ Supported by NCI, National Institutes of Health, Predoctoral Training Grant CA-09135.

2 L. J. Saucedo and S. E. Seavey, unpublished results.

    ABBREVIATIONS

The abbreviations used are: MDM2, murine double minute 2; CMV, cytomegalovirus; ECL, enhanced chemiluminescence; GFP, green fluorescent protein; MEF, mouse embryo fibroblast; RT, reverse transcription; PCR, polymerase chain reaction.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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