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Cell Growth & Differentiation Vol. 10, 87-92, February 1999
© 1999 American Association for Cancer Research

Analysis of the Degradation Function of Mdm21

Michael H. G. Kubbutat, Robert L. Ludwig, Arnold J. Levine and Karen H. Vousden2

ABL Basic Research Program, National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, Maryland 21702-1201 [M. H. G. K., R. L. L., K. H. V.], and Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544-1014 [A. J. L.]


   Abstract
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 Abstract
Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Degradation of the p53 tumor suppressor protein has been shown to be regulated by Mdm2. In this study, we identify regions of Mdm2 that are not required for p53 binding but are essential for degradation. Mdm2 mutants lacking these regions function in a dominant negative fashion, stabilizing endogenous p53 in cells by interfering with the degradative function of the endogenous Mdm2. p53 protein stabilized in this way does not strongly enhance the expression of p21Waf1/Cip1, the product of a p53-responsive gene, supporting the model in which binding of Mdm2 to the NH2-terminal domain of p53 inhibits interaction with other components of the basal transcriptional machinery. Interestingly, COOH-terminal truncations of Mdm2 that retain p53 binding but fail to mediate its degradation are also stabilized themselves. Because Mdm2, like p53, is normally an unstable protein that is degraded through the proteasome, this result suggests a direct link between the regulation of Mdm2 and p53 stability.


   Introduction
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 Abstract
 Introduction
Results
 Discussion
 Materials and Methods
 References
 
The p53 tumor suppressor protein plays a critical role in preventing tumorigenesis, and the activation of p53 by stress such as DNA damage or oncogene activation results in either cell cycle arrest or apoptotic cell death (1) . Inhibition of cell growth is an important mechanism by which p53 prevents the proliferation of cells that harbor potentially oncogenic changes, and most human cancers show evidence of a loss of p53 function (2) . Reactivation of p53 function in such tumor cells efficiently inhibits cell growth, providing an extremely attractive therapeutic approach.

In light of the inhibitory effects of p53 on cell growth, it is evident that there must be mechanisms that control p53 function during normal cell growth and development. At least some of the regulation of p53 occurs at the level of protein stability; p53 is normally a short-lived protein that is rapidly degraded through the proteasome, and activation of a p53 response involves stabilization and rapid elevation of p53 protein levels (3) . Recently, the Mdm2 protein has been shown to play a role in regulating p53 stability by targeting p53 for degradation, an activity that depends on the interaction between the two proteins (4, 5, 6) . The exact contribution of Mdm2 to p53 degradation is not known, although there is evidence that Mdm2 can function as a ubiquitin ligase for p53 in vitro (7) . Mdm2 has also been shown to shuttle from the nucleus to the cytoplasm, and this activity contributes to the degradation of p53 (8) . Mdm2 is a transcriptional target of p53 (9) and therefore functions in a negative regulatory feedback loop in which p53 activates the expression of Mdm2, which in turn inactivates p53 both by binding to and obscuring the trans-activation domain of p53 (10, 11, 12) and by targeting p53 for degradation. The importance of Mdm2 in regulating p53 is dramatically illustrated by the observation that the deletion of Mdm2 in mice results in extremely early embryonic lethality, which is efficiently rescued by the deletion of p53 (13 , 14) . Furthermore, inhibition of the p53/Mdm2 interaction in cells expressing low levels of wild-type p53 results in p53 stabilization and activation of the p53 response (6) .

In addition to the clear role for Mdm2 in the regulation of p53, several other p53-independent activities have also been described. Overexpression of Mdm2 in the mammary gland of transgenic mice led to the uncoupling of S phase from mitosis that was independent of p53 (15) , and cell cycle arrest activities of Mdm2 have also been reported (16) . Mdm2 shows transforming activities in the absence of p53 binding (17) , and this may be related to an ability of Mdm2 to affect transcription (18 , 19) . These other mechanisms of Mdm2 function are much less well understood than those depending on p53, but other important cell growth-regulatory proteins have been shown to interact with Mdm2. These include pRB, another major tumor suppressor gene product (20) , and E2F-1, a transcription factor essential for cell cycle progression whose activity is regulated directly by interaction with pRB (21) .

Despite the requirement for an interaction between p53 and Mdm2 for degradation, it is apparent that these activities are separable. We have recently described a series of COOH-terminal p53 deletions that retain the ability to interact with Mdm2 but are resistant to degradation (22) . In the present study, we describe the characterization of a series of Mdm2 mutants for their ability to interact with and degrade p53.


   Results
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 Abstract
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The COOH Terminus of Mdm2 Is Important for p53 Degradation in Cells.
Degradation of p53 by Mdm2 depends on the ability of the two proteins to form an interaction (4 , 5) , although our previous studies showed that regions of Mdm2 distinct from the p53 binding are also important for Mdm2-mediated degradation of p53 (4) . In light of the recent identification of Mdm2 as a ubiquitin ligase for p53 in vitro (7) , we sought to determine the contribution of the COOH terminus of Mdm2, which is necessary for ubiquitin ligase activity, to the function of Mdm2 in vivo.

We examined the activity of a series of Mdm2 mutants, which are shown in Fig. 1ACitation . Cotransfection of these mutants into U2OS cells with p53 demonstrated that several of them had lost the ability to degrade p53 (Fig. 1B)Citation compared to the wild-type Mdm2 protein. As shown previously, deletion of the p53 binding region in the NH2 terminus of Mdm2 ({Delta}58–89) and deletion of a region distinct from the p53 binding domain ({Delta}222–437) destroyed the ability to degrade p53. Two COOH-terminal truncations of Mdm2 (6–339 and 1–440) also led to the loss of ability to degrade p53. To confirm that loss of activity of the Mdm2 mutants was not a reflection of inadequate expression, transfected cell lysates were analyzed for Mdm2 protein levels by Western blot (Fig. 1B)Citation . These results showed very clearly that whereas the wild-type and p53 nonbinding ({Delta}58–89) Mdm2 proteins were expressed at low levels, the three COOH-terminal mutants that failed to degrade p53 ({Delta}222–437, 6–339, and 1–440) were expressed at significantly higher levels than the wild-type functional protein, indicating that their inability to reduce p53 stability was not simply a consequence of inadequate expression.



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  		Fig. 1. A, the structure of Mdm2 and the position of the mutants used in this study are shown. B, Western blot analysis of Mdm2 and p53 protein levels after cotransfection into U2OS cells. Wild-type p53 was cotransfected with each of the indicated Mdm2 mutants and a plasmid expressing GFP to demonstrate equal transfection efficiencies. Arrows show Mdm2 proteins. C, Western blot analysis of p53 and Mdm2 levels after cotransfection of wild-type Mdm2 or the Mdm2 point mutant 464Ala with wild-type p53 into U2OS cells.

 
The extreme COOH terminus of Mdm2 has been shown to be necessary for ubiquitin ligase function, with a role for a cysteine residue within the RING finger domain. To analyze the importance of this residue to the ability of Mdm2 to target p53 for degradation in vivo, we constructed Mdm2 mutant with a substitution of this cysteine residue at position 464 to alanine, which was previously shown to abolish the ubiquitin ligase function of Mdm2 in vitro (7) . Cotransfections with p53 into U2OS cells showed that this point mutation also rendered the protein unable to target p53 for degradation (Fig. 1C)Citation ; similar results were seen using Mdm2 proteins with a mutation of this cysteine to histidine (data not shown).

To confirm that COOH-terminal deletion mutants of Mdm2 that lost the ability to degrade p53 retained the ability to bind p53, we carried out coprecipitation experiments to detect Mdm2/p53 complexes (Fig. 2)Citation . This analysis confirmed previously published observations (23) , showing a clear interaction between wild-type Mdm2 and the three COOH-terminal mutants ({Delta}222–437, 6–339, and 1–440). In contrast, deletion of NH2-terminal Mdm2 sequences ({Delta}58–89) rendered the protein unable to interact with p53. Relatively low levels of p53 are coprecipitated with wild-type Mdm2 (Fig. 2B)Citation because the levels of p53 are much lower in these cells due to enhanced degradation (Fig. 2A)Citation .



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  		Fig. 2. Association between Mdm2 mutants and p53. Each of the indicated Mdm2 mutants (10 µg) was cotransfected into U2OS cells with wild-type p53 (10 µg). A, Western blot analysis of total p53 protein in lysates after cotransfection with Mdm2 and mutants. B, Western blot analysis of p53 protein coimmunoprecipitated from the same lysates as in A. Mdm2 proteins were immunoprecipitated using antibody SMP-14, and p53 was detected using rabbit serum CM-1. C, Western blot analysis of total Mdm2 protein in lysates after cotransfection with Mdm2 and mutants.

 
COOH-Terminal Mdm2 Mutants Function as Dominant Negatives.
Although expression of the p53 nonbinding Mdm2 {Delta}58–89 had no effect on p53 expression, each of the Mdm2 mutants that lost degradation activity without impinging on the p53 binding domain not only failed to reduce the levels of p53 but apparently increased p53 levels compared to the expression levels seen in the absence of exogenous Mdm2 (Fig. 1B)Citation . This suggested that these Mdm2 mutants may act in a dominant negative manner, inhibiting the normal degradation by endogenous Mdm2. To determine whether these mutants showed a similar effect on endogenous p53 protein levels, we transfected wild-type p53-expressing U2OS cells with each mutant and examined the p53 levels in the transfected cells (Fig. 3)Citation . As seen with transfected p53, these analyses showed that each of the Mdm2 mutants that had lost degradation function but retained the ability to bind p53 was able to stabilize the endogenous p53 protein.



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  		Fig. 3. Stabilization of endogenous p53 by Mdm2 mutants. The indicated Mdm2 mutants were cotransfected into U2OS cells with a GFP expression plasmid and plasmid pHook1, and transfected cells were selected using the Capture-Tec kit. p53 protein levels in the transfected cells were determined by Western blotting using antibody DO-1. Equal expression of GFP confirmed the equal transfection and selection efficiencies.

 
Despite the activity of Mdm2 in inhibiting the function of p53, an inhibitor of cell growth, it has been difficult to establish cell lines that stably overexpress wild-type Mdm2, and a recent report has shown that Mdm2 has p53-independent growth-inhibitory properties (16) . Analysis of Mdm2 protein expression in U2OS or MCF-7 cells after transfections with the various Mdm2 mutants confirmed that wild-type Mdm2 could not be stably overexpressed in these cells. Interestingly, although stable expression of the p53 nonbinding Mdm2 mutant ({Delta}58–89) was also not tolerated in these cells, each of the COOH-terminal mutant Mdm2 proteins that failed to degrade p53 ({Delta}222–437, 6–339, and 1–440) could be stably expressed at high levels in the transfected cells (Fig. 4ACitation ; data not shown). As seen in the transient assays, p53 levels were significantly increased in the cells stably expressing either {Delta}222–437 or 1–440 (Fig. 4B)Citation . Because these Mdm2 mutants remain capable of binding p53, they would be expected to inactivate p53 transcriptional function through binding to the trans-activation domain at the NH2 terminus of the p53 protein, despite their ability to stabilize the p53 protein. One transcriptional target of p53 is p21Waf1/Cip1, and the elevation of p53 protein levels in response to 5 nM actinomycin D results in an enhanced expression of the p21Waf1/Cip1 protein (Fig. 5)Citation . Actinomycin D is an intercalating agent that forms cleavable complexes with DNA and induces DNA stand breaks, thus activating the p53 response (24 , 25) . As expected, clear elevation of p21Waf1/Cip1 levels was not seen in cells in which high levels of p53 protein were induced by expression of the dominant negative Mdm2 mutants {Delta}222–437 and 1–440 (Fig. 5)Citation , although increased p21Waf1/Cip1 expression was seen in response to DNA damage. These results indicate that the dominant negative Mdm2 mutants both stabilize and inactivate endogenous p53 in the absence of DNA damage and that this inactivation is alleviated after DNA damage, although the extent of the response appears to be somewhat weaker than that seen in control cells.



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  		Fig. 4. Stabilization of p53 by Mdm2 mutants in stably expressing cells. A, Western blot analysis showing expression levels of Mdm2 mutant protein {Delta}222–437 and 1–440, respectively, in stably transfected clones of U2OS cells. B, Western blot analysis showing endogenous p53 protein levels in the same clones of U2OS cells.

 


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  		Fig. 5. p53 response to DNA damage in cells stably expressing Mdm2 mutants. Western blot analysis showing endogenous levels of p53 and p21Waf1/Cip1 in pooled colonies of cells stably expressing Mdm2 mutants {Delta}222–437 and 1–440 before and after a 16-h treatment with 5 nM actinomycin D.

 
Effect of Mdm2 Expression on the Stability of Other Mdm2-Interacting Proteins.
In addition to p53, several other proteins have been shown to interact with Mdm2, including the tumor suppressor protein pRB and the E2F/DP transcription factor that is regulated by pRB. We therefore analyzed the sensitivity of these proteins to Mdm2-mediated degradation in assays similar to those used to detect the degradation of p53 by Mdm2. Under these conditions, Mdm2 was not able to reduce the expression of cotransfected pRB, E2F-1, or DP1 (Fig. 6A)Citation . A functional E2F transcription factor requires an interaction between E2F-1 and DP1, and this dimeric complex is negatively regulated by direct binding to pRB. We therefore considered whether sensitivity to Mdm2-mediated degradation might depend on the formation of these complexes and examined the stability of E2F-1, DP1, and pRB protein expression after a combinatorial transfection of all three partners (Fig. 6B)Citation . Once again, no evidence for degradation of any of these proteins after coexpression of Mdm2 was seen under these conditions.



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  		Fig. 6. Mdm2 does not reduce E2F1/DP1 or pRB protein levels in cotransfected cells. A, effects of Mdm2 on the expression of wild-type p53, p53{Delta}I, E2F-1, or pRB. B, effects of Mdm2 on the expression of pRB, E2F-1, and DP-1 transfected in combination. Saos2 cells were cotransfected with plasmids encoding p53 (3 µg), E2F-1 (1 µg), RB (3 µg), and HA-DP-1(3 µg) with or without murine Mdm2 encoding plasmid (9 µg).

 
COOH-Terminal Mutations of Mdm2 Affect Its Stability.
Like p53, Mdm2 is a protein with a short half-life that is normally rapidly degraded through the proteasome after ubiquitination (26) . Analysis of the Mdm2 mutants revealed the provocative observation that Mdm2 mutants that retain the ability to bind p53 but fail to target degradation are themselves expressed at high levels in both transient and stably transfected cells (Figs. 1 and 4)Citation . A growth-inhibitory domain was recently mapped to Mdm2 residues 155–324, and loss of this region in the mutant {Delta}222–437 may allow tolerance of a high expression of this mutant protein. However, we wished to determine whether there is a correlation between the ability of Mdm2 to target p53 for degradation and the stability of the Mdm2 protein itself. Assays carried out in both transient and stably transfected cells (Fig. 7Citation ; data not shown) show clearly that deletion of the COOH terminus of Mdm2 (6–339 and 1–440) resulted in the stabilization of the Mdm2 protein, increasing the half-life from 1 h to more than 4 h. However, we were unable to detect a similarly clear increase in the half-life of the Mdm2 {Delta}222–437 mutant protein.



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  		Fig. 7. Half-life of Mdm2 mutants. U2OS cells were transiently transfected with plasmids encoding the indicated Mdm2 mutants and then incubated in medium containing [35S]methionine and [35S]cysteine. Cells were harvested at the indicated times after removal of the labeled medium, and the amount of labeled Mdm2 protein immunoprecipitated at each time point was detected by autoradiography.

 

   Discussion
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 Abstract
 Introduction
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In this study, we describe mutations within Mdm2 that affect the ability to target p53 for degradation. These appear to fall into three classes: (a) mutations within the NH2 terminus of p53 that prevent interaction with p53; (b) mutations within the central growth-inhibitory domain of Mdm2 that retain p53 binding; and (c) deletion or mutation in the COOH-terminal RING finger-containing region, which prevents degradation of p53 and stabilizes the Mdm2 protein itself. The Mdm2 mutants that fail to degrade p53 while retaining the ability to bind are of particular interest, because they seem to behave in a dominant negative manner, stabilizing endogenous p53 by protection from endogenous Mdm2. The stabilization of endogenous p53 by these Mdm2 mutants strongly supports previous studies showing that Mdm2 is responsible for the turnover of p53 in undamaged cells (6) . We have also shown previously that p53 mutants that fail to bind Mdm2 show increased stability that is not further enhanced after treatment with proteasome inhibitors (4) . Taken together, these studies indicate that Mdm2-directed degradation is the principal mechanism by which p53 stability is regulated in cells. Despite stabilizing p53, these Mdm2 mutants retain the ability to block p53 activity, and enhanced transcription of the p53 target gene p21Waf1/Cip1 and cell cycle arrest is not seen. However, cells expressing dominant negative Mdm2 mutants retain some ability to respond to DNA damage by activating a p53 response, indicating that the normal mechanisms that allow p53 to become resistant to regulation by Mdm2 remain functional in these cells.

Deletion of, or a point mutation (464Ala) within the COOH-terminal RING finger domain of Mdm2 both abolishes the ability of Mdm2 to target p53 for degradation and stabilizes Mdm2 itself. These results support a role for Mdm2 as a ubiquitin ligase for p53, as reported recently (7) , and suggest the intriguing possibility that the ubiquitin ligase activity of Mdm2 also plays a role in regulating the stability of the Mdm2 protein itself. Interestingly, regulation of the protein stability of a ubiquitin ligase though auto-ubiqutination has recently been shown for the ubiqutin ligase E6-AP (27) . It should be noted, however, that the ubiquitin ligase function of Mdm2 has not yet been detected in vivo, and the mutations of the cysteine residue within the RING finger may affect additional activities of Mdm2. Alterations of the RING finger domain have been shown to prevent specific RNA binding by Mdm2 (28) , and this region may also play a role in contacting the nuclear export machine or docking to the 26S proteasome. Deletion of the central acidic region of Mdm2 in {Delta}222–437 also inhibited the degradation of p53, although this Mdm2 mutant was not so clearly more stable than wild-type p53. Deletion of this region has been shown to abolish the growth-inhibitory activity of Mdm2 (16) , and the enhanced expression of this protein may reflect, in part, a tolerance of the cell to this Mdm2 mutant. It is of interest that this region of Mdm2 has been shown to participate in the interaction with the p14ARF protein (29, 30, 31) . Interaction with p14ARF also prevents degradation of p53, suggesting that the contribution of this region of Mdm2 to p53 degradation can be lost either by deletion or by interaction with p14ARF. Although this region of Mdm2 is close to the nuclear export sequence shown to participate in the ability of Mdm2 to degrade p53, this mutant has been shown to export normally (8) ; hence, defects in nucleocytoplasmic shuttling are unlikely to contribute to the inability to degrade p53. Indeed, all the Mdm2 mutants used here retain nuclear localization and export signals and have been shown to localize predominantly to the nucleus (23) . It is possible that another protein that is important for the degradation interacts with the region of Mdm2 deleted in {Delta}222–437 and that this interaction is perturbed by both deletion of the domain or binding of p14ARF. This may represent further similarities to the degradation of p53 by E6, which requires interaction with the cell protein E6-AP to function as a ubiquitin ligase (32) .

Although reduced p53 expression is seen very clearly after coexpression of Mdm2, we were unable to detect any effect of Mdm2 on the expression levels of other Mdm2-associated proteins such as E2F1/DP1 or pRB. E2F1/DP1 is predicted to bind Mdm2 at the same site as p53 (21) , although the binding site of pRB on Mdm2 has not been reported. Mdm2 has been shown to degrade fusion proteins containing p53 sequences constituting the Mdm2 binding site (5) , and we expected to see some degradation of the E2F1/DP1 complex. However, the lack of degradation is consistent with the reported observation that Mdm2 potentiates rather than inhibits the transcriptional activity of E2F1/DP1 (21) . We have recently shown that the extreme COOH terminus of p53 contributes to the sensitivity to degradation by Mdm2 without affecting the interactions between the two proteins (22) , indicating that the binding between Mdm2 and another protein is not sufficient by itself to target degradation.


   Materials and Methods
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Antibodies and Plasmids.
Plasmids encoding human Mdm2 (pCHDM 1A, pCHDM{Delta}58–89, pCHDM{Delta}222–437, pCHDM6–339, and pCHDM1–440; Ref. 23 ), murine Mdm2 (pCOC Mdm2 X2; Ref. 33 ), human p53 (pCB6+p53Pro) and human mutant p53 (pCB6+ p53{Delta}I; Ref. 34 ), human E2F-1 (35) , and human HA-tagged DP-1(pCMV HADP-1; Ref. 36 ) and pCMVpRb (37) were previously described. Plasmids encoding human Mdm2 464Ala and 464His, respectively, were constructed by site-directed mutagenesis (QuikChange site-directed mutagenesis kit; Stratagene, La Jolla, CA). Mutations were confirmed by sequencing. pEGFP N1 encoding GFP and pHook1 were obtained from Clontech (Palo Alto, CA) and Invitrogen (Carlsbad, CA), respectively.

Human Mdm2-specific monoclonal antibody IF2 (which recognizes Mdm2 sequences in the NH2 terminus of the protein, somewhere between amino acids 26 and 169), p53-specific monoclonal antibody DO-1, and p21Waf1/Cip1 EA10 were obtained from Oncogene Science (Cambridge, MA); antibody against the HA tag was obtained from Boehringer Mannheim (Indianapolis, IN); rabbit anti-p53 serum CM-1 was obtained from Novocastra (Vector Laboratories, Burlingame, CA), and GFP-specific monoclonal antibody was obtained from Clontech. E2F-1 (KH95/E2F)-, pRB (G3-349)-, and Mdm2 (SMP-14)-specific antibodies were obtained from PharMingen (San Diego, CA).

Cell Culture and Transfections.
U2OS, MCF-7, and Saos-2 cells were maintained in DMEM supplemented with 10% FCS. Cells were transfected using the calcium phosphate precipitation method. Unless otherwise indicated, cells were transfected with 3 µg of Mdm2-encoding plasmid, 9 µg of p53-encoding plasmid, and 1 µg of plasmid expressing GFP. For transient transfections, cells were harvested 24 h after transfection. For the detection of endogenous p53 protein after transient transfection with Mdm2-expressing plasmids, cells were cotransfected with pHook1 plasmid (5 µg), and positively transfected cells were separated using the Capture-Tec kit (Invitrogen). To establish cell lines stably expressing Mdm2 mutant proteins, cells were selected after transfection with 0.6 mg/ml G418, and outgrowing colonies were pooled.

Protein Analysis and Measurement of Half-Life.
Western blotting and immunoprecipitation were carried out as described previously (38) . Equal loading of lysates was confirmed by Ponceau S staining. For measurement of the protein half-life incubated cells were first incubated in medium lacking methionine and cysteine and subsequently incubated in medium supplemented with 100 µCi of [35S]methionine and [35S]cysteine (ProMix; Amersham, Arlington Heights, IN) for 30 min. Cells were harvested at 0, 1, 3, and 6 h after chase with medium containing an excess amount of unlabeled methionine and cysteine. Mdm2 protein was immunoprecipitated from the lysates using monoclonal antibody SMP-14 and detected by autoradiography.


   Acknowledgments
 
We thank Ed Harlow and Kristian Helin for pRB, E2F1, and DP1 expression plasmids and Moshe Oren for mouse Mdm2 expression plasmid.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by the National Cancer Institute, Department of Health and Human Services, under contract with ABL. Back

2 To whom requests for reprints should be addressed, at ABL Basic Research Program, National Cancer Institute-Frederick Cancer Research and Development Center, Building 560, Room 22-96, West 7th Street, Frederick, MD 21702-1201. Phone: (301) 846-1726; Fax: (301) 846-1666; E-mail: vousden{at}ncifcrf.gov Back

3 The abbreviations used are: HA, hemagglutinin; GFP, green fluorescent protein. Back

Received for publication 10/22/98. Revision received 12/29/98. Accepted for publication 12/30/98.


   References
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 Abstract
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 Discussion
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 References
 

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