Conservation of all three p53 family members and Mdm2 and Mdm4 in the cartilaginous fish

Analysis of the genome of the elephant shark (Callorhinchus milii), a member of the cartilaginous fishes (class Chondrichthyes), reveals that it encodes all three members of the p53 gene family, p53, p63 and p73, each with clear homology to the equivalent gene in bony vertebrates (class Osteichthyes). Thus, the gene duplication events that lead to the presence of three family members in the vertebrates dates to before the Silurian era. It also encodes Mdm2 and Mdm4 genes but does not encode the p19^Arf gene. Detailed comparison of the amino acid sequences of these proteins in the vertebrates reveals that they are evolving at highly distinctive rates, and this variation occurs not only between the three family members but extends to distinct domains in each protein.Key words: p53, p63, p73, Mdm2, Mdm4, elephant shark     

The Mdm2 and p53 genes are conserved in the Arachnids

The p53 protein and its negative regulator the ubiquitin E3 ligase Mdm2 have been shown to be conserved from the Placazoan to man. In common with D.melanogaster and C.elegans, there is a single copy of the p53 gene in T.adhaerens, while in the vertebrates three p53-like genes can be found: p53 , p63 and p73. The Mdm2 gene is not present within the fully sequenced and highly annotated genomes of C.elegans and D.melanogaster. However, it is present in the Placazoan and the presence of multiple distinct p53 genes in the Sea anemone N.vectensis led us to examine the genomes of other phyla for p53 and Mdm2-like genes. We report here the discovery of an Mdm2-like gene and two distinct p53 like genes in the Arachnid Ioxodes scapularis (Northern Deer Tick). The two predicted Deer Tick p53 proteins are much more highly related to the human p53 protein in sequence than are the fruit fly and nematode proteins. One of the Deer tick genes encodes a p53 protein that is initiated within the DNA binding domain of p53 and shows remarkable homology to the newly described N-terminally truncated delta isoforms of human and zebrafish p53.     

Mdm2 and p53 are highly conserved from placozoans to man

The p53 protein is the most commonly mutated tumor suppressor gene in man. Understanding of its evolutionary origins have been enhanced by the recent discovery of p53 family genes in the sea anemone Nematostella vectensis. This amino acid sequence conservation has been reflected in biological activity since the early p53 proteins, like their human counterparts, are responsible for DNA damage-induced cellular apoptosis, albeit restricted to the germ cell compartment in model organisms such as the nematode and fruit fly. In vertebrates from zebrafish to man the function of p53 is tightly and absolutely constrained by a negative regulator Mdm2. However the Mdm2 gene has not been detected in the genome of the model nematode (C. elegans) and insect (D. melanogaster) species. We have found that the p53 gene and the Mdm2 gene are present in Placozoans, one of the simplest of all free living multi-cellular organisms, implying that both proteins arose much earlier in evolution than previously thought. Detailed sequence analysis shows the exceptional retention of key features of both proteins from man to placazoan implying that the p53-Mdm2 interaction and its regulation have been conserved from a basal eumetazoan since the pre-cambrian era over one billion years ago.     

Downregulation of Mdm2 and Mdm4 enhances viral gene expression during adenovirus infection

Successful viral replication entails elimination or bypass of host antiviral mechanisms. Here, we show that shRNA-mediated knockdown of murine double minute (Mdm2) and its paralog Mdm4 enhanced the expression of early and late viral gene products during adenovirus (HAdV) infection. Remarkably, whereas the expression of HAdV genes was low in p53-deficient mouse embryonic fibroblasts (p53KO MEFs), the HAdV early gene products were efficiently expressed in Mdm2/p53 double-knockout (DKO) and Mdm4/p53 DKO MEFs, and viral capsid proteins were produced in Mdm2/p53 DKO MEFs. Thus, Mdm2 and Mdm4 seem to have potent antiviral property. In cells infected with wt HAdV or a mutant virus lacking the E1B-55K gene (dl1520), both Mdm2 and Mdm4 were rapidly depleted, whereas replication-deficient mutant viruses (Ad-GFP) or ΔpTP with deletions within the coding sequence of preterminal binding protein failed to induce their downregulation. Reduced expression of Mdm2 and Mdm4 was not due to general shutoff of host protein synthesis. Additionally, expression of a dominant-negative mutant of Cul5 did not affect Mdm2/Mdm4 downregulation. Thus, viral replication but not the presence of E1B-55K is required for Mdm2/Mdm4 degradation. Surprisingly, treatment of HAdV-infected cells with proteasome inhibitor MG132 only partially restored the protein levels of Mdm2 and Mdm4, suggesting that they may also be downregulated through an additional mechanism independent of proteasome. Interestingly, cyclin D1 and p21 appear to be downregulated similarly during HAdV infection. Collectively, our work provides the first biochemical evidence for antiviral function of Mdm2 and Mdm4 and that viruses employ efficient countermeasure to ensure viral replication.     

Downregulation of Mdm2 and Mdm4 enhances viral gene expression during adenovirus infection

Successful viral replication entails elimination or bypass of host antiviral mechanisms. Here, we show that shRNA-mediated knockdown of murine double minute (Mdm2) and its paralog Mdm4 enhanced the expression of early and late viral gene products during adenovirus (HAdV) infection. Remarkably, whereas the expression of HAdV genes was low in p53-deficient mouse embryonic fibroblasts (p53KO MEFs), the HAdV early gene products were efficiently expressed in Mdm2/p53 double-knockout (DKO) and Mdm4/p53 DKO MEFs, and viral capsid proteins were produced in Mdm2/p53 DKO MEFs. Thus, Mdm2 and Mdm4 seem to have potent antiviral property. In cells infected with wt HAdV or a mutant virus lacking the E1B-55K gene (dl1520), both Mdm2 and Mdm4 were rapidly depleted, whereas replication-deficient mutant viruses (Ad-GFP) or ΔpTP with deletions within the coding sequence of preterminal binding protein failed to induce their downregulation. Reduced expression of Mdm2 and Mdm4 was not due to general shutoff of host protein synthesis. Additionally, expression of a dominant-negative mutant of Cul5 did not affect Mdm2/Mdm4 downregulation. Thus, viral replication but not the presence of E1B-55K is required for Mdm2/Mdm4 degradation. Surprisingly, treatment of HAdV-infected cells with proteasome inhibitor MG132 only partially restored the protein levels of Mdm2 and Mdm4, suggesting that they may also be downregulated through an additional mechanism independent of proteasome. Interestingly, cyclin D1 and p21 appear to be downregulated similarly during HAdV infection. Collectively, our work provides the first biochemical evidence for antiviral function of Mdm2 and Mdm4 and that viruses employ efficient countermeasure to ensure viral replication.Key words: adenovirus (HAdV), antiviral mechanism, virus-host interaction, Mdm2, Mdm4, mouse embryonic fibroblast (MEF), DNA-damage response, cell cycle, p21, cyclin D1     

The p53 and Mdm2 families in cancer.

Cells within an organism are occasionally exposed to either intracellular or environmental stress. Such stress often has genotoxic potential that enhances the probability of cancer. Two gene families, the p53 family (p53, p63 and p73) and the Mdm2 family (Mdm2 and MdmX), serve as major integrators of the signals generated by genotoxic and oncogenic stress. Their co-ordinated modulation ensures an optimal response to stress and decreases the likelihood of cancer. Work over the past year has provided better understanding of the p53-Mdm2 module that lies in the heart of this regulatory network, and of the intricate interplay between the various members of the network.     

The p53 isoforms are differentially modified by Mdm2

The discovery that the single p53 gene encodes several different p53 protein isoforms has initiated a flurry of research into the function and regulation of these novel p53 proteins. Full-length p53 protein level is primarily regulated by the E3-ligase Mdm2, which promotes p53 ubiquitination and degradation. Here, we report that all of the novel p53 isoforms are ubiquitinated and degraded to varying degrees in an Mdm2-dependent and -independent manner, and that high-risk human papillomavirus can degrade some but not all of the novel isoforms, demonstrating that full-length p53 and the p53 isoforms are differentially regulated. In addition, we provide the first evidence that Mdm2 promotes the NEDDylation of p53β. Altogether, our data indicates that Mdm2 can distinguish between the p53 isoforms and modify them differently.     

The p53 isoforms are differentially modified by Mdm2

The discovery that the single p53 gene encodes several different p53 protein isoforms has initiated a flurry of research into the function and regulation of these novel p53 proteins. Full-length p53 protein level is primarily regulated by the E3-ligase Mdm2, which promotes p53 ubiquitination and degradation. Here, we report that all of the novel p53 isoforms are ubiquitinated and degraded to varying degrees in an Mdm2-dependent and -independent manner, and that high-risk human papillomavirus can degrade some but not all of the novel isoforms, demonstrating that full-length p53 and the p53 isoforms are differentially regulated. In addition, we provide the first evidence that Mdm2 promotes the NEDDylation of p53β. Altogether, our data indicates that Mdm2 can distinguish between the p53 isoforms and modify them differently.     

Nucleolar Arf sequesters Mdm2 and activates p53

The Ink4/Arf locus encodes two tumour-suppressor proteins, p16Ink4a and p19Arf, that govern the antiproliferative functions of the retinoblastoma and p53 proteins, respectively. Here we show that Arf binds to the product of the Mdm2 gene and sequesters it into the nucleolus, thereby preventing negative-feedback regulation of p53 by Mdm2 and leading to the activation of p53 in the nucleoplasm. Arf and Mdm2 co-localize in the nucleolus in response to activation of the oncoprotein Myc and as mouse fibroblasts undergo replicative senescence. These topological interactions of Arf and Mdm2 point towards a new mechanism for p53 activation.     

Tight regulation of p53 activity by Mdm2 is required for ureteric bud growth and branching

Mdm2 (Murine Double Minute-2) is required to control cellular p53 activity and protein levels. Mdm2 null embryos die of p53-mediated growth arrest and apoptosis at the peri-implantation stage. Thus, the absolute requirement for Mdm2 in organogenesis is unknown. This study examined the role of Mdm2 in kidney development, an organ which develops via epithelial–mesenchymal interactions and branching morphogenesis. Mdm2 mRNA and protein are expressed in the ureteric bud (UB) epithelium and metanephric mesenchyme (MM) lineages. We report here the results of conditional deletion of Mdm2 from the UB epithelium. UB^mdm2−/− mice die soon after birth and uniformly display severe renal hypodysplasia due to defective UB branching and underdeveloped nephrogenic zone. Ex vivo cultured UB^mdm2−/− explants exhibit arrested development of the UB and its branches and consequently develop few nephron progenitors. UB^mdm2−/− cells have reduced proliferation rate and enhanced apoptosis. Although markedly reduced in number, the UB tips of UB^mdm2−/−metanephroi continue to express c-ret and Wnt11; however, there was a notable reduction in Wnt9b, Lhx-1 and Pax-2 expression levels. We further show that the UB^mdm2−/− mutant phenotype is mediated by aberrant p53 activity because it is rescued by UB-specific deletion of the p53 gene. These results demonstrate a critical and cell autonomous role for Mdm2 in the UB lineage. Mdm2-mediated inhibition of p53 activity is a prerequisite for renal organogenesis.     

Haploinsufficiency of Mdm2 and Mdm4 in tumorigenesis and development

The tumor suppressor p53 is inactivated by multiple mechanisms that include mutations of the p53 gene itself and increased levels of the p53 inhibitors MDM2 and MDM4. Mice lacking Mdm2 or Mdm4 exhibit embryo-lethal phenotypes that are completely rescued by concomitant deletion of p53. Here we show that Mdm2 and Mdm4 haploinsufficiency leads to increased p53 activity, exhibited as increased sensitivity to DNA damage and decreased transformation potential. Moreover, in in vivo tumor development, Emu-myc Mdm4+/- mice show a delayed onset of B-cell lymphomas compared to Emu-myc mice. Additionally, Mdm2+/- Mdm4+/- double-heterozygous mice are not viable and exhibit defects in hematopoiesis and cerebellar development. The defects in Mdm2+/- Mdm4+/- mice are corrected by deletion of a single p53 allele. These findings highlight the exquisite sensitivity of p53 to Mdm2 and Mdm4 levels and suggest that some cell types may be more sensitive to therapeutic drugs that inhibit the Mdm-p53 interaction.     

Mdm2 and Mdm4 loss regulates distinct p53 activities

Mutational inactivation of p53 is a hallmark of most human tumors. Loss of p53 function also occurs by overexpression of negative regulators such as MDM2 and MDM4. Deletion of Mdm2 or Mdm4 in mice results in p53-dependent embryo lethality due to constitutive p53 activity. However, Mdm2(-/-) and Mdm4(-/-) embryos display divergent phenotypes, suggesting that Mdm2 and Mdm4 exert distinct control over p53. To explore the interaction between Mdm2 and Mdm4 in p53 regulation, we first generated mice and cells that are triple null for p53, Mdm2, and Mdm4. These mice had identical survival curves and tumor spectrum as p53(-/-) mice, substantiating the principal role of Mdm2 and Mdm4 as negative p53 regulators. We next generated mouse embryo fibroblasts null for p53 with deletions of Mdm2, Mdm4, or both; introduced a retrovirus expressing a temperature-sensitive p53 mutant, p53A135V; and examined p53 stability and activity. In this system, p53 activated distinct target genes, leading to apoptosis in cells lacking Mdm2 and a cell cycle arrest in cells lacking Mdm4. Cells lacking both Mdm2 and Mdm4 had a stable p53 that initiated apoptosis similar to Mdm2-null cells. Additionally, stabilization of p53 in cells lacking Mdm4 with the Mdm2 antagonist nutlin-3 was sufficient to induce a cell death response. These data further differentiate the roles of Mdm2 and Mdm4 in the regulation of p53 activities.     

miR-661 downregulates both Mdm2 and Mdm4 to activate p53

The p53 pathway is pivotal in tumor suppression. Cellular p53 activity is subject to tight regulation, in which the two related proteins Mdm2 and Mdm4 have major roles. The delicate interplay between the levels of Mdm2, Mdm4 and p53 is crucial for maintaining proper cellular homeostasis. microRNAs (miRNAs) are short non-coding RNAs that downregulate the level and translatability of specific target mRNAs. We report that miR-661, a primate-specific miRNA, can target both Mdm2 and Mdm4 mRNA in a cell type-dependent manner. miR-661 interacts with Mdm2 and Mdm4 RNA within living cells. The inhibitory effect of miR-661 is more prevalent on Mdm2 than on Mdm4. Interestingly, the predicted miR-661 targets in both mRNAs reside mainly within Alu elements, suggesting a primate-specific mechanism for regulatory diversification during evolution. Downregulation of Mdm2 and Mdm4 by miR-661 augments p53 activity and inhibits cell cycle progression in p53-proficient cells. Correspondingly, low miR-661 expression correlates with bad outcome in breast cancers that typically express wild-type p53. In contrast, the miR-661 locus tends to be amplified in tumors harboring p53 mutations, and miR-661 promotes migration of cells derived from such tumors. Thus, miR-661 may either suppress or promote cancer aggressiveness, depending on p53 status.     

p53-Independent Down-Regulation of Mdm2 in Human Cancer Cells Treated with Adriamycin

Mdm2 is a nuclear phosphoprotein which functions as a negative feedback regulator of the p53 tumor suppressor gene. In this study, we investigated the alteration of Mdm2 and p53 in three human cancer cell lines containing either a wild-type or mutant p53 gene after treatment with Adriamycin (doxorubicin, ADR), a DNA damaging agent. We found that human breast cancer MCF-7 cells containing wild-type p53 were much more susceptible to ADR compared to human breast cancer MDA-MB-231 and human prostate cancer Du-145 cells which contain mutant p53. ADR resulted in a significant dose-dependent accumulation of p53 protein in MCF-7 cells, whereas little or no influence was observed on p53 protein of the two mutant p53 cell lines. However, a significant down-regulation of Mdm2 at protein and mRNA levels was observed in these three cell lines following ADR treatment. Moreover, the decrease of Mdm2 was in both a dose- and time-dependent manner. It is interestingly noted that 5 μM is a critical dose for significant down-regulation of the Mdm2 protein. Selected proteasome inhibitors did not rescue the ADR-caused decline in the expression of Mdm2 protein. Therefore, our present results reveal that ADR can induce a down-regulation of Mdm2 via a p53-independent pathway in human cancer cells and the ubiquitin-proteasome degradation mechanism may not be involved in the decreased expression of Mdm2 protein.     

Cocompartmentalization of p53 and Mdm2 is a major determinant for Mdm2-mediated degradation of p53

The product of the Mdm2 oncogene directly interacts with p53 and promotes its ubiquitination and proteasomal degradation. Initial biological studies identified nuclear export sequences (NES), similar to that of the Rev protein from the human immunodeficiency virus, both in Mdm2 and p53. The reported phenotypes resulting from mutation of these NESs, together with results obtained using the nuclear export inhibitor leptomycin B (LMB), have led to a model according to which nuclear export of p53 (via either the NES of Mdm2 or its own NES) is required for efficient p53 degradation. In this study we demonstrate that Mdm2 can promote degradation of p53 in the nucleus or in the cytoplasm, provided both proteins are colocalized. We also investigated if nuclear export is an obligate step on the p53 degradation pathway. We find that (1) when proteasome activity is inhibited, ubiquitinated p53 accumulates in the nucleus and not in the cytoplasm; (2) Mdm2 with a mutated NES can efficiently mediate degradation of wild type p53 or p53 with a mutated NES; (3) the nuclear export inhibitor LMB can increase the steady-state level of p53 by inhibiting Mdm2-mediated ubiquitination of p53; and (4) LMB fails to inhibit Mdm2-mediated degradation of the p53NES mutant, demonstrating that Mdm2-dependent proteolysis of p53 is feasible in the nucleus in the absence of any nuclear export. Therefore, given cocompartmentalization, Mdm2 can promote ubiquitination and proteasomal degradation of p53 with no absolute requirement for nuclear to cytoplasmic transport.     

Mutational analysis of Mdm2 C-terminal tail suggests an evolutionarily conserved role of its length in Mdm2 activity toward p53 and indicates structural differences between Mdm2 homodimers and Mdm2/MdmX heterodimers

Mdm2 can mediate p53 ubiquitylation and degradation either in the form of the Mdm2 homodimer or Mdm2/MdmX heterodimer. The ubiquitin ligase activity of these complexes resides mainly in their respective RING finger domains and also requires adjacent C-terminal tails. So far, structural studies have failed to show significant differences between Mdm2 RING homodimers and Mdm2/MdmX RING heterodimers. Here, we report that not only the primary amino acid sequence, but also the length of the C-terminal tail of Mdm2 is highly conserved through evolution and plays an important role in Mdm2 activity toward p53. Mdm2 mutants with extended C termini do not ubiquitylate p53 despite being capable of forming Mdm2 homodimers through both RING-acidic domain and RING-RING interactions. All extended mutants also retained the ability to interact with MdmX, and this interaction led to reactivation of their E3 ubiquitin ligase activity. In contrast, only a subset of extended Mdm2 mutants was activated by the interaction with Mdm2 RING domain, suggesting that Mdm2 homodimers and Mdm2/MdmX heterodimers may not be structurally and functionally fully equivalent.Key words: p53, Mdm2, RING domain, ubiquitylation, ubiquitin ligase, E3