[Cell Cycle 3:7, 900-904; July 2004]; ©2004 Landes Bioscience
Mdmx and Mdm2
Review: Spotlight on p53
Brothers in Arms?
2Department
of Molecular and Cell Biology; Leiden University Medical Center; Leiden, The Netherlands
*Correspondence to: Aart G Jochemsen; LUMC; Department Molecular and Cell Biology; P.O Box 9503; 2300 RA Leiden, The Netherlands; Tel.: +31.715276136; Fax: +31.715276284; Email:
[email protected] Received 05/27/04; Accepted 06/02/04
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For Molecular Cancer Biology; Flanders Interuniversity Institute for Biotechnology (VIB); Ghent, Belgium
The p53 tumor suppressor pathway is inactivated in most if not all human tumors. In about 50% of the cases this is accomplished directly by gene mutations. The tumors that retain wild type p53 frequently show defects either in effector target genes, or in the expression of p53 regulatory proteins. The Mdm2 protein is generally considered THE master regulator of the p53 tumor suppressor activity. Recently, however, the Mdm2-related protein Mdmx is taking the stage in the p53-Mdm2-Mdmx play. We summarize here observations unambiguously assigning a critical role for the Mdmx protein in the regulation of p53 function during development and tumor formation.
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ABSTRACT
Jean-Christophe Marine1 Aart G. Jochemsen2,*
MDMX IS STRUCTURALLY RELATED TO MDM2
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Previously published online as a Cell Cycle E-Publication: http://www.landesbioscience.com/journals/cc/abstract.php?id=998
p53, Mdmx, Mdm2, Mdm4, tumorigenesis
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ACKNOWLEDGEMENTS
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KEY WORDS
The p53 tumor-suppressor functions prevent cancer development by inhibiting the proliferation of stressed or abnormal cells. Mice lacking p53 show a high incidence of tumor development, and germline mutation of one p53 allele in humans gives rise to the Li-Fraumeni cancer-susceptibility syndrome.1,2 p53 functions as a transcription factor to promote several antiproliferative responses, including cell cycle checkpoints, cellular senescence, and apoptosis.3 Since p53 is such a potent inhibitor of cell growth, restraint of p53 activity during embryonic development is of paramount importance. Central to this process is the Mdm2 protein as exemplified by the observation that mdm2-null embryos die in uteri and their embryonic lethality is completely overcome by deletion of the functional p53 gene product.4,5 Mdm2-null embryos die because of apoptosis initiated as early as blastocyst stage (3.5 days post-coitum, dpc).6 In addition, Mdm2 is a critical survival factor, limiting the apoptotic function of p53, in a subset of adult homeostatic tissues.7 Finally, expression of the mdm2 gene is induced in response to UV light exposure and ionizing radiation, and the resulting high levels of Mdm2 ensure that the activity of p53 returns to its low basal levels in surviving cells (reviewed in ref. 8). Consistent with the existence of a negative feedback loop, induction of Mdm2 expression in these conditions is p53-dependent. The Mdmx protein was originally identified because of its ability to interact with p539 and later, as a Mdm2 partner.10 Human Mdmx and Mdm2 mRNA encode structurally related proteins of 490 and 491 amino acids, respectively11 (Fig. 1). The greatest similarity between the two proteins is found at the N-terminal end, a region encompassing the p53-binding domain. Importantly, the amino acids required for interaction with p53 are strictly conserved in Mdm2 and Mdmx proteins.12 Conversely, the same amino acids in p53 are required for both Mdmx/p53 and Mdm2/p53 interactions.13 Another well-conserved region is a typical zinc-binding domain, so called RING-finger domain, located at the C-terminal end of both proteins. The integrity of these domains is essential for the ability of the two proteins to heterodimerize.10,14 Another zinc-binding motif, a Zinc-finger, whose function is largely unknown, is conserved between the two proteins. A central region in both Mdm2 and Mdmx has a high content of acidic amino acids, however, no significant similarity between these two domains was observed. Although a consensus nucleolar location signal (NoLS) is present within the RING domain of both Mdmx and Mdm2,15,16,17 the nuclear localization and nuclear export signals present in Mdm2 are not found in Mdmx suggesting that the subcellular localization of Mdmx is determined by interaction with other proteins, e.g., Mdm2 (see below). Finally, acetylation of Mdm2 occurs primarily within its RING finger domain and in particular on two key lysines (K466 and K467).18 Acetylation of Mdm2 on these residues appears to modulate its activity and might also affect its cellular distribution since K466 and 467 are situated within the NoLS. In Mdmx, these residues are not conserved, arguing against a possible regulation of localization and/or function by acetylation.
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The authors wish to thank Erik Meulmeester and Davide Danovi critical reading of the manuscript.
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MDMX IS A NON-REDUNDANT p53 NEGATIVE REGULATOR
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Figure 1.Comparison of Mdm2 and Mdmx protein structure. The p53 binding domain, Zinc finger- (Zn) and RING finger domain are well conserved. Percentage identity between these domain is indicated. NLS, Nuclear Localization Signal; NES, Nuclear Export Signal; NoLS, Nucleolar Localization Signal. Further explanations in the text.
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Because of its similarity with Mdm2 and its ability to inhibit p53-induced transcription following overexpression, Mdmx was hypothesized to act as a novel negative regulator of p53.9,20 This view was only recently confirmed by loss of function studies in mice. Two mutant mouse lines were generated and characterized by three independent groups. These studies all concluded that, similarly to Mdm2, Mdmx acts in vivo as an essential, non-redundant, negative regulator of p53 during embryonic development. Indeed, the functional relationship between Mdmx and p53 was genetically demonstrated by the observation that a p53-null state completely rescues the early-embryonic-lethal phenotype associated with mdmx deficiency (Fig. 2).21-23 In the first reported mdmx mutant mouse, exons 3–5 encoding for most of the p53 binding domain (amino acids 27–96) were deleted by homologous recombination in embryonic stem cells. This mutation caused embryo lethality as early as E7.5 and was essentially due to severe proliferation defects. In striking contrast to mdm2 loss of expression, deletion of exon 3–5 of mdmx did not cause increased apoptosis.21 Notably, the authors reported that this allele is not a null but still produces, through alternative splicing, a truncated Mdmx protein retaining the RING finger domain and deleted from its N-terminal p53 binding domain. This observation suggests that direct p53 binding is essential for Mdmx-mediated p53-inhibitory activity in vivo. The second mdmx mutant mouse line was generated by random insertional mutagenesis, with a viral insertion mapped in intron 1 between exon 1 and 2.22 The resulting mdmx-homozygous mutants died between 9.5 and 11.5 dpc and were characterized by overall growth deficiency and massive p53-dependent apoptosis in the neuroepithelium. Thus, these two mutations lead to overlapping (cell proliferation block of the mutant cells) but also distinct phenotypes. Apoptosis was not detected in the first reported mutation probably due to the early lethality associated with this mutation. Indeed, the mutant embryos found at E8.5 did not develop neural ectoderm, tissue in which apoptosis was specifically detected in the subsequently characterized mdmx-mutants. However, it is still unclear what causes the difference in the timing of death. In the first reported mutation, interpretation of the results might be hampered by the expression of the short Mdmx product, which retains the RING finger domain. This short Mdmx protein might retain its ability to interact with Mdm2 and could affect its activity and/or stability (see below). The second mutation is expected to be a mdmx-null mutation since (i) a reduction of more than 10-fold in the transcription level of the locus was observed in the mutants by quantitative-RT PCR analysis
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Conservation between Mdmx and Mdm2 at the genomic level is also remarkable.19 Exons 4-12 of mdm2 are structurally well conserved in mdmx, with exon 12 being the last exon and encoding for the entire RING finger domain in both proteins. A significant divergence is observed, however, at the 5' end of the genes. Only one non-coding exon was found in the mdmx locus instead of two for mdm2. Also, the intron between exon 1 and 2 in mdmx is about 6 kb, while in mdm2 the first three exons are within 1 kb. Finally and most importantly, in contrast to mdm2, no p53 response elements have been found in the mdmx locus, and no p53-dependent induction of mdmx expression was detected after DNA damage.9
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MDMX AND MDM2
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Figure 2.Complete rescue of the Mdmx-dependent embryonic lethality on a p53-null background. mdmx-homozygous mutant mice die around E10.5 in utero (a representative mutant embryo is shown on the left). Concomitant loss of p53 rescues the lethality associated with mdmx loss (a viable mdmx/p53 double knock-out mouse is shown on the right).
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(Marine et al., unpublished observation), (ii) the remaining transcripts are expected to be translated with a very low efficiency. Accordingly, extensive protein analyses failed to detect Mdmx protein expression in the mdmx/p53 double-null mice, rendering unlikely, even if not excluding, the possibility of a hypomorphic allele.22 Clearly, this mouse model turned out to be very useful to demonstrate a direct role for Mdmx in regulating both cell cycle arrest and apoptotic functions of p53.22 The common conclusions from these studies are that (i) similarly to Mdm2, impaired Mdmx expression results in an embryonic lethal phenotype as a result of deregulated p53 activity. However, the timing and the mechanism of embryonic death are quite different if Mdm2 or Mdmx activity is impaired. (ii) Mdm2 cannot substitute for Mdmx loss underlining an important role for Mdmx to keep p53 activity in check at least during embryogenesis. A comparable role for Mdmx in adult tissues remains to be demonstrated. Earlier lethality in mdm2-mutant mice as compared to mdmxnull mice allows at least two different scenarios to be pictured. One possibility is that Mdm2 is indeed the master p53 regulator and can, at least to some extent, function in the absence of Mdmx. Mdmx would then be a cofactor of Mdm2, only required in specific tissues or, in a given tissue, at a particular phase of the differentiation program. A particularly attractive model proposes that Mdmx would be required during the massive expansion phase of the progenitor cells in a given cell type. These cells indeed undergo rapidly multiple DNA replication cycles with little time for DNA repair mechanisms to operate, conditions which would favor p53 activation. In this context, Mdmx would assist Mdm2 in keeping p53 activity down.
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MDMX AND MDM2
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Mdm2 inhibits p53 activity by various means. Mdm2 directly blocks p53 transcriptional ability, possibly by preventing the recuitment of essential cofactors. In addition, Mdm2 ubiquitin ligase activity induces mono- and, when strongly overexpressed, polyubiquitination of p53 on a series of lysines clustered at the C-terminal end of p53. This activity of Mdm2 stimulates both the nuclear export and proteolytic destruction of p53. In addition, Mdm2 favors the recruitment of histone deacetylase including HDAC1 to the p53 transcriptional complex and this recruitment may not only decrease transcriptional activity but also induce p53 degradation (reviewed in refs. 25, 26 and refs. therein). Regulation of p53 Stability. In contrast to Mdm2, Mdmx does not act as an E3 ubiquitin ligase and can not stimulate degradation of p53.27-30 Both the RING finger domains and a middle region spanning the acidic domain determine this difference in activity.31,32 Several studies even suggested that high Mdmx levels could rescue p53 from Mdm2-mediated degradation14,27-29 and that this effect was largely dependent on the ability of Mdmx to interact with Mdm2. However, conclusions from these experiments cannot be drawn unambiguously. Indeed, one potential problem is that in most of these original studies the Mdmx protein contained a tag at the C-terminal end and we now know that such tags affect the regulation of Mdmx stability by Mdm233 (de Graaf, Jochemsen, unpublished observations) and possibly other biochemical and functional properties of the protein. Mdmx interacts with Mdm2 through their respective RING domains and most transfection studies indicate that Mdmx stabilizes Mdm2, by interfering with Mdm2 auto-ubiquitination.14,29 Thus, Mdmx may simply render the Mdm2 protein sufficiently stable to function at its full potential under specific conditions. Indeed, knocking-down Mdmx expression by siRNAs in U2OS resulted in decreased Mdm2 and increased p53 levels.34 Furthermore, Mdm2 modulates Mdmx cellular localization by recruiting it into the nucleus under specific contexts, such as following DNA damage. 20,34,35Based on these observations, the mutual dependence model was proposed.34 In this model Mdmx would stimulate the p53-regulatory activities of Mdm2, both the inhibition of the transcriptional activity and proteolytic destruction. The p53-binding domain of Mdmx appears dispensable for this activity. Although quite attractive, this model is inconsistent with several other observations. First, loss of expression of full length Mdmx in vivo is not compensated by the expression of a short hypomorphic Mdmx product retaining the entire RING finger domain.21 Secondly, Linares et al. suggest that Mdmx is stimulating the Mdm2-mediated ubiquitination of p53 and also self-ubiquitination of Mdm2.30 Indeed, in their study,
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HOW DOES MDMX INHIBIT p53 ACTIVITY?
knocking down Mdmx by siRNA, in U2OS and MCF-7, increased both p53 and Mdm2 levels. Thirdly, we observed no significant effect on neither Mdm2 nor p53 levels after knocking-down Mdmx in MCF-7 cells.36 More importantly, despite increased p53-acetylation and increased expression of p53 target genes, only a very modest increase in p53 levels was observed in mdmx knock-out embryos.36 Finch et al. proposed a very different model which invokes the ability of Mdmx to regulate p53 expression at the transcription level, based on the observation that increased steady-state p53 mRNA was detected in heterozygote mouse embryonic fibroblasts (MEFs).23 However, these experiments were carried out in cultured MEFs, cells that undergo high oxygen concentration-dependent stress complicating the interpretation of the results.37 In these conditions, both the DNA-damage machinery and p53 are activated. In sharp contrast with this model, quantitative-PCR analysis on fresh embryos revealed a two-fold decrease in the p53 mRNA steady state level in mdmx-homozygous mutants (Marine et al., unpublished observations). Regulation of p53 Transcriptional Activity. The first reported activity of Mdmx is the inhibition of p53-induced transcription, both of luciferase reporter genes and of endogenous p53 targets.9,38 This effect is dependent on the p53-binding domain of Mdmx.9,27 The ability of Mdmx to inhibit p53-dependent transcription could be a consequence of inhibition of p300/CBP-mediated acetylation of p53.36,39 Importantly, this effect is Mdm2 independent, since Mdmx abrogates p300/CBP-mediated acetylation of p53 even in mdm2-null cells and this effect is also observed with a mutant of Mdmx defective in Mdm2 binding.36 Even if the mechanism by which acetylation of p53 stimulates its activity is still a matter of debate, this modification is accepted to have a positive impact on its activity and to be critical for its function as a tumor suppressor (reviewed in ref. 26). Strikingly, the pool of acetylated p53 is constitutively elevated in the mdmx-mutant cells, convincingly confirming an active role for Mdmx in the regulation of the p53 acetylation status in vivo.36 We have recently monitored changes in gene expression in various tissues from mdmx-mutant embryos using oligonucleotide microarrays analysis. Consistent with a role of Mdmx in regulating p53-transcriptional activity in vivo, our analysis revealed a significant increase in the expression of more than 20 known p53-target genes including p21 and several proapoptic genes such as bax, Apaf-1, Noxa ...(Martoriati, Doumont, Alcalay, Pelicci, Marine, submitted). In conclusion, Mdmx primarily inhibits p53 activity by interfering with its transcriptional ability. For this effect, direct interaction between Mdmx and p53 appears essential. Mdmx also interacts with Mdm2, but whether this interaction is mainly to stabilize Mdm2 and thus indirectly inhibits p53 or whether the association is more important for regulation of Mdmx activity by Mdm2 remains to be elucidated.
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In agreement with this model, ubiquitous overexpression of Mdm2 in vivo, obtained by crossing with an mdm2-transgenic mouse,24 could rescue the mdmx-null embryonic lethality (Steinman H, Jones S, personal communication). Alternatively, even if less likely, Mdmx and Mdm2 may function independently in different cell types. For instance, there is so far no evidence for a role of Mdm2 in the survival of the erythroid lineage cells. Mice expressing low endogenous levels of the Mdm2 protein from an hypomorphic allele are viable and have fairly normal red blood counts, whereas the lymphoid compartment is strongly affected in these mice.7 In contrast, impaired Mdmx activity dramatically affects primitive as well as definitive erythropoiesis.22
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FUNCTION AND REGULATION OF MDMX UNDER STRESS
Under conditions of intracellular or environmental stress, p53 activity increases. An early step in the activation is the abrogation of Mdm2-mediated inhibition, which is accomplished by several mechanisms, including (de-)phosphorylation of both p53 and Mdm2 and enhanced degradation of Mdm2. Regulation of Mdmx expression appears equally important to ensure a proper p53 response under stress conditions, at least following genotoxic stress. Treatment of cells with UV-C, IR or adriamycin results in degradation of Mdmx.33,40 Mdmx is a substrate for the ubiquitin ligase activity of
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MDMX AND MDM2
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immortalizes mouse embryonic fibroblasts in the absence of p53 mutation or loss of ARF expression. Furthermore, Mdmx prevents oncogenic Ras-induced premature senescence, and Mdmx + RasV12transformed cells are oncogenic in nude mice.36 Together these data strongly indicate that Mdmx functions as an oncogene when constitutively overexpressed in human tumors as an alternative for p53 mutation. Consequently, interfering with the Mdmx/p53 interaction might be used as a cancer therapeutic. Given the similarities of the Mdmx and Mdm2 interfaces with p53,13 a common strategy, such as the promising one recently described by Vassilev et al.,47 might be used not only to disrupt Mdm2/p53 interaction but also Mdmx/p53 association. Many tumors have been found to contain aberrantly and/or alternatively Mdm2 splicing variants. The functions of these variants are still elusive, but their expression is more common in high-grade than in low-grade tumors.48 A systematic analysis of Mdmx splicing variants in large tumor sets is still lacking. One Mdmx splicing variant, Mdmx-S, has been identified and partly characterized.49 This variant essentially encodes only the p53-binding domain and a few alternative C-terminal amino acids. Due to a higher affinity than full length Mdmx for p53 and to increased nuclear localization, Mdmx-S appears to be a very efficient inhibitor of p53 function.48,50 In addition, Mdmx-S protein is also more stable than Mdmx, possibly because this protein can no longer interact with Mdm2 and is therefore protected from Mdm2-mediated degradation. Interestingly, Riemenschneider et al. reported a relatively higher ratio of MdmxS/Mdmx in high grade gliomas.44 However, since the number of samples analyzed is low, further analyses are needed to verify the relevance of this finding.
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MDMX IS AN ONCOGENE
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Mdm2 and, indeed, Mdmx degradation is Mdm2-dependent, but does not need p53-induced expression of Mdm2.40 Somehow the ubiquitin ligase activity towards Mdmx is stimulated after DNA damage, possibly by enhancing the Mdmx/Mdm2 interaction. Ubiquitination and degradation of Mdmx only needs an intact Mdm2 RING domain, in contrast to the regulation of p53 by Mdm2. This offers the possibility of distinct regulation of p53 and Mdmx inhibition by Mdm2. In addition, Mdmx contains a caspase-3 cleavage site, and a stepwise degradation of Mdmx, e.g., after adriamycin-treatment, has been suggested. First, caspase cleavage of Mdmx, and second a proteasome-dependent degradation of the cleavage product.41 The requirement of the caspase cleavage for Mdmx degradation by Mdm2 after DNA damage is unlikely. First, because the caspase cleavage of Mdmx was suggested to be p53-dependent41 while degradation of Mdmx after DNA damage is not.40 Furthermore, a mutant Mdmx that can not be cleaved by caspases is still degraded by Mdm2.42 We recently found that also non-genotoxic stress, e.g., PALA treatment, results in p53-independent degradation of Mdmx (de Graaf, Teunisse, Jochemsen, unpublished observations). The physiological importance of Mdmx downregulation after DNA damage is not clear. On the one hand, constitutive Mdmx expression has been shown to prolong stabilisation of p53 after adriamycin-treatment of NIH3T3 cells. As a result, increased induction of p53-responsive genes, particularly Bax, and a concomitant increase in apoptosis is observed.43 However, another study indicates that sustained Mdmx expression prevents a proper p53 response after IR, since both induction of p21 and cell cycle arrest are impaired.40 The use of non-tumor versus tumor cells in the respective studies might have affected the outcome of these experiments. Additional studies are needed to elucidate the role of Mdmx downregulation in the DNA damage response.
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Impairment of the p53 tumor suppressor pathway in tumors can be accomplished by overexpression of Mdm2. The first evidence that Mdmx could perform the same function came from analysis of a large series of gliomas. It was found that mdmx was amplified/overexpressed in 5/208 tumor samples.44 Refined mapping showed later that indeed the mdmx gene is the common amplified gene in the large amplicons.45 Overexpression of Mdmx and the appearance of alternative Mdmx proteins were found in approximately 30% of tumor cell lines, with a few exceptions correlating with wild-type p53 status.46 A recent analysis of a large series of tumors by in situ RNA hybridisation on Tissue Micro Arrays indicated overexpression of the Mdmx mRNA in a significant percentage of several tumor types, i.e., 19% (41/216) of breast carcinomas, a proportion (8/41) of which can be explained by gene amplification.36 The overexpression of Mdmx in the mdmx-amplified cases could be confirmed at the protein level by both immunohistochemistry and Western blotting analysis. Where analysed, the amplification of the mdmx gene correlated with a wild-type p53 status and lack of mdm2 amplification. The importance of Mdmx overexpression was tested in the MCF-7 breast tumor cell line, which overexpresses Mdmx and contains wild type p53. Knocking down endogenous Mdmx by siRNA technology increased p21WAF1 expression without significant increase in p53 levels. Colony assays showed that knock-down of Mdmx was incompatible with proliferation of MCF-7 cells, unless p53 levels were simultaneously decreased. Finally, constitutive expression of mouse Mdmx
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CONCLUDING REMARKS
Although it has been unequivocally established that Mdmx is an essential negative regulator of p53 activity during embryogenesis, many questions still remain. Is Mdmx function required in all cell types and/or at all differentiation stages in a given tissue? Is Mdmx function essential to protect adult homeostatic tissues from p53 activity? These questions will undoubtedly soon be answered by tissue specific functional inactivation of Mdmx and/or Mdm2 in vivo. The molecular mechanism by which Mdmx regulates p53 activity has not been fully elucidated. The direct binding of Mdmx to p53 appears to be necessary and might even be sufficient (Mdmx-S) to inhibit p53 function. If so, what is the function of the Mdmx/Mdm2 interaction? Is Mdmx indeed stabilising Mdm2 under physiological conditions and, therefore, also indirectly decreasing p53 activity? Or is the Mdmx/ Mdm2 interaction only essential for a proper regulation of Mdmx stability and subcellular localisation? The generation of better research tools, e.g., high affinity anti-Mdmx antibodies, should give the opportunity to answer these questions in the near future. Last but not least, the role of Mdmx in human tumorigenesis has to be further investigated. An oncogene function of Mdmx can no longer be denied, but what does overexpression of Mdmx mean for prognosis, and can it be used to develop specific cancer treatments? Based on our current knowledge, we would propose that perturbation of Mdmx function will turn out to be a new powerful therapeutic tool for human tumors expressing wild-type p53.
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33. Pan Y, Chen J. MDM2 promotes ubiquitination and degradation of MDMX. Mol Cell Biol 2003; 23:5113-21. 34. Gu J, Kawai H, Nie L, Kitao H, Wiederschain D, Jochemsen AG, et al. Mutual dependence of MDM2 and MDMX in their functional inactivation of p53. J Biol Chem 2002; 277:19251-4. 35. Li C, Chen L, Chen J. DNA damage induces MDMX nuclear translocation by p53-dependent and -independent mechanisms. Mol Cell Biol 2002; 22:7562-71. 36. Danovi D, Meulmeester E, Pasini D, Migliorini D, Capra M, Frenk R, et al. Amplification of Mdmx (or Mdm4) directly contributes to tumor formation by inhibiting p53 tumor suppressor activity. Mol Cell Biol 2004; 24:5835-43. 37. Parrinello S, Samper E, Krtolica A, Goldstein J, Melov S, Campisi J. Oxygen sensitivity severely limits the replicative lifespan of murine fibroblasts. Nat Cell Biol 2003; 5:741-7. 38. Little NA, Jochemsen AG. Hdmx and Mdm2 can repress transcription activation by p53 but not by p63. Oncogene 2001; 20:4576-80. 39. Sabbatini P, McCormick F. MDMX inhibits the p300/CBP-mediated acetylation of p53. DNA Cell Biol 2002; 21:519-25. 40. Kawai H, Wiederschain D, Kitao H, Stuart J, Tsai KK, Yuan ZM. DNA damage-induced MDMX degradation is mediated by MDM2. J Biol Chem 2003; 278:45946-53. 41. Gentiletti F, Mancini F, D'Angelo M, Sacchi A, Pontecorvi A, Jochemsen AG, et al. DMX stability is regulated by p53-induced caspase cleavage in NIH3T3 mouse fibroblasts. Oncogene 2002; 21:867-77. 42. de Graaf P, Little NA, Ramos YFM, Meulmeester E, Letteboer SJ, Jochemsen AG. Hdmx protein stability is regulated by the ubiquitin ligase activity of Mdm2. J Biol Chem 2003; 278:38315-24. 43. Mancini F, Gentiletti F, D’Angelo M, Giglio S, Nanni S, D’Angelo C, et al. MDM4 (MDMX) overexpression enhances stabilization of stress-induced p53 and promotes apoptosis. J Biol Chem 2004; 279:8169-80. 44. Riemenschneider MJ, Buschges R, Wolter M, Reifenberger J, Bostrom J, Kraus JA, et al. Amplification and overexpression of the MDM4 (MDMX) gene from 1q32 in a subset of malignant gliomas without TP53 mutation or MDM2 amplification. Cancer Res 1999; 59:6091-6. 45. Riemenschneider MJ, Knobbe CB, Reifenberger G. Refined mapping of 1q32 amplicons in malignant gliomas confirms MDM4 as the main amplification target. Int J Cancer 2003; 104:752-7. 46. Ramos YFM, Stad R, Attema J, Peltenburg LT, van der Eb AJ, Jochemsen AG. Aberrant expression of HDMX proteins in tumor cells correlates with wild-type p53. Cancer Res 2001; 61:1839-42. 47. Vassilev LT, Vu BT, Graves B, Carvajal D, Podlaski F, Filipovic Z, et al. In vivo activation of the p53 pathway by small-molecule antagonists of MDM2. Science 2004; 303:844-8. 48. Bartel F, Harris LC, Wurl P, Taubert H. MDM2 and its splice variant messenger RNAs: Expression in tumors and down-regulation using antisense oligonucleotides. Mol Cancer Res 2004; 2:29-35. 49. Rallapalli R, Strachan G, Cho B, Mercer WE, Hall DJ. A novel MDMX transcript expressed in a variety of transformed cell lines encodes a truncated protein with potent p53 repressive activity. J Biol Chem 1999; 274:8299-308. 50. Rallapalli R, Strachan G, Tuan RS, Hall DJ. Identification of a domain within MDMX-S that is responsible for its high affinity interaction with p53 and high-level expression in mammalian cells. J Cell Biochem 2003; 89:563-75.
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