Mdm2 is required for maintenance of the nephrogenic niche

The balance between nephron progenitor cell (NPC) renewal, survival and differentiation ultimately determines nephron endowment and thus susceptibile to chronic kidney disease and hypertension. Embryos lacking the p53-E3 ubiquitin ligase, Murine double minute 2 (Mdm2), die secondary to p53-mediated apoptosis and growth arrest, demonstrating the absolute requirement of Mdm2 in embryogenesis. Although Mdm2 is required in the maintenance of hematopoietic stem cells, its role in renewal and differentiation of stem/progenitor cells during kidney organogenesis is not well defined. Here we examine the role of the Mdm2-p53 pathway in NPC renewal and fate in mice. The Six2-GFP::Cre^tg/+ mediated inactivation of Mdm2 in the NPC (NPC^Mdm2^−/−) results in perinatal lethality. NPC^Mdm2^−/− neonates have hypo-dysplastic kidneys, patchy depletion of the nephrogenic zone and pockets of superficially placed, ectopic, well-differentiated proximal tubules. NPC^Mdm2−/− metanephroi exhibit thinning of the progenitor GFP⁺/Six2⁺ population and a marked reduction or loss of progenitor markers Amphiphysin, Cited1, Sall1 and Pax2. This is accompanied by aberrant accumulation of phospho-γH2AX and p53, and elevated apoptosis together with reduced cell proliferation. E13.5–E15.5 NPC^Mdm2−/− kidneys show reduced expression of Eya1, Pax2 and Bmp7 while the few surviving nephron precursors maintain expression of Wnt4, Lhx1, Pax2, and Pax8. Lineage fate analysis and section immunofluorescence revealed that NPC^Mdm2−/− kidneys have severely reduced renal parenchyma embedded in an expanded stroma. Six2-GFP::Cre^tg/+; Mdm2^f/f mice bred into a p53 null background ensures survival of the GFP-positive, self-renewing progenitor mesenchyme and therefore restores normal renal development and postnatal survival of mice. In conclusion, the Mdm2-p53 pathway is essential to the maintenance of the nephron progenitor niche.     

Reciprocally interacting domains of protein phosphatase 1 and focal adhesion kinase

The Murine double-minute clone 2 (Mdm2) onco-protein is the principal regulator of the tumour suppressor, p53. Mdm2 acts as an E3-type ubiquitin ligase that mediates the ubiquitylation and turnover of p53 under normal, unstressed circumstances. In response to cellular stress, such as DNA damage, the Mdm2–p53 interaction is disrupted. Part of the mechanism of uncoupling p53 from Mdm2-mediated degradation involves hypo-phosphorylation of a cluster of phosphorylated serine residues in the central acidic domain of Mdm2. Here, we show that two of the residues within this domain that are phosphorylated in vivo, Ser-260 and Ser-269, are phosphorylated by CK2 in vitro. Treatment of cells with the CK2 inhibitor, 4,5,6,7-tetrabromo-2-azabenzimidazole (TBB), leads to the induction of p53 and downstream targets of p53 including Mdm2 itself and p21. These data are consistent with the idea that CK2-mediated phosphorylation of Mdm2 may regulate Mdm2-mediated p53 turnover.     

Mdm2, but not Mdm4, protects terminally differentiated smooth muscle cells from p53-mediated caspase-3-independent cell death

p53 is a potent inhibitor of cell growth and an inducer of apoptosis. During embryonic development, Mdm2 and Mdm4 inhibit the growth suppressive activities of p53. However, whether tight surveillance of p53 activity is required in quiescent cells is unknown. To test this, conditional inactivation of mdm2 and mdm4 was carried out in smooth muscle cells (SMCs). Upon SMC-specific inactivation of mdm2, and not of mdm4, mice rapidly became ill and died. Necropsy showed small intestinal dilation, and histological analyses indicated a severe reduction in the number of intestinal SMCs. Increased p53 levels and activity were detected in the remaining SMCs, and the phenotype was completely rescued on a p53-null background. Interestingly, intestinal SMCs are caspase-3-negative and therefore did not undergo caspase-3-dependent apoptotic cell death. Together, Mdm2, but not Mdm4, prevents accumulation of active p53 in quiescent SMCs and thereby the induction of p53-mediated caspase-3-independent cell death.     

Hs2st mediated kidney mesenchyme induction regulates early ureteric bud branching

Heparan sulfate proteoglycans (HSPGs) are central modulators of developmental processes likely through their interaction with growth factors, such as GDNF, members of the FGF and TGFβ superfamilies, EGF receptor ligands and HGF. Absence of the biosynthetic enzyme, heparan sulfate 2-O-sulfotransferase (Hs2st) leads to kidney agenesis. Using a novel combination of in vivo and in vitro approaches, we have reanalyzed the defect in morphogenesis of the Hs2st^−/− kidney. Utilizing assays that separately model distinct stages of kidney branching morphogenesis, we found that the Hs2st^−/− UB is able to undergo branching and induce mesenchymal-to-epithelial transformation when recombined with control MM, and the isolated Hs2st null UB is able to undergo branching morphogenesis in the presence of exogenous soluble pro-branching growth factors when embedded in an extracellular matrix, indicating that the UB is intrinsically competent. This is in contrast to the prevailing view that the defect underlying the renal agenesis phenotype is due to a primary role for 2-O sulfated HS in UB branching. Unexpectedly, the mutant MM was also fully capable of being induced in recombination experiments with wild-type tissue. Thus, both the mutant UB and mutant MM tissue appear competent in and of themselves, but the combination of mutant tissues fails in vivo and, as we show, in organ culture. We hypothesized a 2OS-dependent defect in the mutual inductive process, which could be on either the UB or MM side, since both progenitor tissues express Hs2st. In light of these observations, we specifically examined the role of the HS 2-O sulfation modification on the morphogenetic capacity of the UB and MM individually. We demonstrate that early UB branching morphogenesis is not primarily modulated by factors that depend on the HS 2-O sulfate modification; however, factors that contribute to MM induction are markedly sensitive to the 2-O sulfation modification. These data suggest that key defect in Hs2st null kidneys is the inability of MM to undergo induction either through a failure of mutual induction or a primary failure of MM morphogenesis. This results in normal UB formation but affects either T-shaped UB formation or iterative branching of the T-shaped UB (possibly two separate stages in collecting system development dependent upon HS). We discuss the possibility that a disruption in the interaction between HS and Wnts (e.g. Wnt 9b) may be an important aspect of the observed phenotype. This appears to be the first example of a defect in the MM preventing advancement of early UB branching past the first bifurcation stage, one of the limiting steps in early kidney development.     

Six1 regulates Grem1 expression in the metanephric mesenchyme to initiate branching morphogenesis

Urinary tract morphogenesis requires subdivision of the ureteric bud (UB) into the intra-renal collecting system and the extra-renal ureter, by responding to signals in its surrounding mesenchyme. BMP4 is a mesenchymal regulator promoting ureter development, while GREM1 is necessary to negatively regulate BMP4 activity to induce UB branching. However, the mechanisms that regulate the GREM1-BMP4 signaling are unknown. Previous studies have shown that Six1-deficient mice lack kidneys, but form ureters. Here, we show that the tip cells of Six1^−/− UB fail to form an ampulla for branching. Instead, the UB elongates within Tbx18- and Bmp4-expressing mesenchyme. We find that the expression of Grem1 in the metanephric mesenchyme (MM) is Six1-dependent. Treatment of Six1^−/− kidney rudiments with GREM1 protein restores ampulla formation and branching morphogenesis. Furthermore, we demonstrate that genetic reduction of BMP4 levels in Six1^−/− (Six1^−/−; Bmp4^+/−) embryos restores urinary tract morphogenesis and kidney formation. This study uncovers an essential function for Six1 in the MM as an upstream regulator of Grem1 in initiating branching morphogenesis.     

Mdm4 loss in the intestinal epithelium leads to compartmentalized cell death but no tissue abnormalities

Mdm4 is a critical inhibitor of the p53 tumor suppressor. Mdm4 null mice die early during embryogenesis due to increased p53 activity. In this study, we explore the role that Mdm4 plays in the intestinal epithelium by crossing mice carrying the Mdm4 floxed allele to mice with the Villin Cre transgene. Our data show that loss of Mdm4 (Mdm4^intΔ) in this tissue resulted in viable animals with no obvious morphological abnormalities. However, these mutants displayed increased p53 levels and apoptosis exclusively in the proliferative compartment of the intestinal epithelium. This phenotype was completely rescued in a p53 null background. Notably, the observed compartmentalized apoptosis in proliferative intestinal epithelial cells was not due to restricted Mdm4 expression in this region. Thus, in this specific cellular context, p53 is negatively regulated by Mdm4 exclusively in highly proliferative cells.     

Mdmx enhances p53 ubiquitination by altering the substrate preference of the Mdm2 ubiquitin ligase

mdm2 and mdmx oncogenes play essential yet non-redundant roles in synergistic inactivation of the tumor suppressor, p53. While Mdm2 inhibits p53 activity mainly by augmenting its ubiquitination, the functional role of Mdmx on p53 ubiquitination remains obscure. In transfected H1299 cells, Mdmx augmented Mdm2-mediated ubiquitination of p53. In in vitro ubiquitination assays, the Mdmx/Mdm2 heteromeric complex, in comparison to the Mdm2 homomer, showed enhanced ubiquitinase activity toward p53 and the reduced auto-ubiquitination of Mdm2. Alteration of the substrate specificity via binding to Mdmx may contribute to efficient ubiquitination and inactivation of p53 by Mdm2.

Structured summary

MINT-7219995: P53 (uniprotkb:P04637) physically interacts (MI:0914) with Ubiquitin (uniprotkb:P62988) by anti bait coimmunoprecipitation (MI:0006)MINT-7220023: Ubiquitin (uniprotkb:P62988) physically interacts (MI:0914) with P53 (uniprotkb:P04637) by pull down (MI:0096)     

The ups and downs of p53 regulation in hematopoietic stem cells

Hematopoietic stem cells provide an indispensible source for replenishing the blood with all its constituents throughout the organism's lifetime. Mice with a compromised hematopoietic stem cell compartment cannot survive. p53, a major tumor suppressor gene, has been implicated in regulation of hematopoiesis. In particular, p53 plays a role in homeostasis by regulating HSC quiescence and self renewal. We recently utilized a hypomorphic p53^515C allele in conjunction with Mdm2, a negative regulator of p53 to gain insights into the role of p53 in hematopoietic regulation. Our analyses revealed that p53^515C/515CMdm2^-/- double mutant mice die soon after birth due to hematopoietic failure. Further mechanistic studies revealed that in the absence of Mdm2, ROS induced postnatal p53 activity depletes hematopoietic stem cells, progenitors and differentiated cells.     

The ups and downs of p53 regulation in hematopoietic stem cells

Hematopoietic stem cells provide an indispensible source for replenishing the blood with all its constituents throughout the organism''s lifetime. Mice with a compromised hematopoietic stem cell compartment cannot survive. p53, a major tumor suppressor gene, has been implicated in regulation of hematopoiesis. In particular, p53 plays a role in homeostasis by regulating HSC quiescence and self renewal. We recently utilized a hypomorphic p53^515C allele in conjunction with Mdm2, a negative regulator of p53, to gain insights into the role of p53 in hematopoietic regulation. Our analyses revealed that p53^515C/515CMdm2^−/− double mutant mice die soon after birth due to hematopoietic failure. Further mechanistic studies revealed that in the absence of Mdm2, ROS-induced postnatal p53 activity depletes hematopoietic stem cells, progenitors and differentiated cells.Key words: HSC, reactive oxygen species, ROS, p53, Mdm2     

Defective p53 post-translational modification required for wild type p53 inactivation in malignant epithelial cells with mdm2 gene amplification

Mdm2 gene amplification occurs in benign and chemotherapy-responsive malignant tumors with wtp53 genes as well as in breast and epithelial cancers. Mdm2 amplification in benign tumors suggests that it is not sufficient for p53 inactivation in cancer, implying that other defects in the p53 pathway are required for malignancy. We investigated mechanisms of wtp53 protein inactivation in malignant conversion of epithelial cells by comparing clonally related initiated cells with their derivative cancerous cells that have mdm2 amplification. Deficiencies in p53 accumulation and activities in response to DNA damage were not due simply to Mdm2 destabilization of p53 protein, but to continued association of DNA-bound p53 with Mdm2 protein and lack of binding and acetylation by p300 protein. The aberrant interactions were not because of mdm2 amplification alone, because DNA-bound p53 protein from initiated cells failed to bind ectopically expressed Mdm2 or endogenous overexpressed Mdm2 from cancerous cells. Phosphorylations of endogenous p53 at Ser18, -23, or -37 were insufficient to dissociate Mdm2, because each was induced by UV in cancerous cells. Interestingly, phospho-mimic p53-T21E did dissociate the Mdm2 protein from DNA-bound p53 and recovered p300 binding and p21 induction in the cancerous cells. Thus wtp53 in malignant cells with mdm2 amplification can be inactivated by continued association of DNA-bound p53 protein with Mdm2 and failure of p300 binding and acetylation, coupled with a defect in p53 phosphorylation at Thr21. These findings suggest therapeutic strategies that address both p53/Mdm2 interaction and associated p53 protein defects in human tumors that have amplified mdm2 genes.     

Mdm35p imports Ups proteins into the mitochondrial intermembrane space by functional complex formation

Ups1p, Ups2p, and Ups3p are three homologous proteins that control phospholipid metabolism in the mitochondrial intermembrane space (IMS). The Ups proteins are atypical IMS proteins in that they lack the two major IMS‐targeting signals, bipartite presequences and cysteine motifs. Here, we show that Ups protein import is mediated by another IMS protein, Mdm35p. In vitro import assays show that import of Ups proteins requires Mdm35p. Loss of Mdm35p led to a decrease in steady state levels of Ups proteins in mitochondria. In addition, mdm35Δ cells displayed a similar phenotype to ups1Δups2Δups3Δ cells. Interestingly, unlike typical import machineries, Mdm35p associated stably with Ups proteins at a steady state after import. Demonstrating that Mdm35p is a functional component of Ups–Mdm35p complexes, restoration of Ups protein levels in mdm35Δ mitochondria failed to restore phospholipid metabolism. These findings provide a novel mechanism in which the formation of functional protein complexes drives mitochondrial protein import.     

Length matters: C-terminal tails regulate Mdm2–MdmX complexes

Comment on: Dolezelova P, et al. Cell Cycle 2012; 11:953–62Mechanisms controlling the p53 regulatory network remain the focus of numerous investigations in hopes of identifying more robust cancer therapies. Both Mdm2 and MdmX are found overexpressed in tumors with wild-type p53 and represent a key molecular device modulating p53 function. Thus, examining the interplay between these three proteins becomes highly relevant in the search for new pharmacological interventions in oncology.Mdm2 is a RING-type E3 ubiquitin ligase capable of forming homo-oligomers and hetero-oligomerization with MdmX via the extreme C termini of their RING domains. Since its discovery 15 years ago, MdmX has been assigned many roles in the regulation of p53, either on its own or in concert with Mdm2. While clearly an essential negative regulator or p53 in development, its lack of intrinsic ubiquitin ligase activity has made the mechanism of p53 regulation more elusive than in the case of Mdm2. The capacity of MdmX to stimulate Mdm2-mediated p53 ubiquitination was first reported in 2003.¹ Subsequent biochemical comparisons of the activity of Mdm2–MdmX complexes showed that not only does the presence of MdmX in the complex alter the substrate specificity of the holo-enzyme, it also allows for poly-ubiquitin chain formation on p53 (modification required for nuclear exclusion and degradation of p53).^2-4In vitro observations describing the importance of the MdmX RING domain in regulation of p53 turnover have now gained in vivo experimental support from the two knock-in animal models.^5,6 Consistent with the notion that MdmX is an essential component of p53 polyubiquitination/proteasomal degradation pathway, mice expressing either a point mutant in the MdmX RING domain or a RING domain deletion mutant succumbed to a p53-dependent embryonic lethality. These data implicate the RING domain of MdmX as the sole region of importance in the ability of MdmX to regulate p53 and, by extension, the Mdm2-MdmX complex (and not the Mdm2 homodimer), as the principle negative regulator of p53 activity during development.The growing body of evidence describing the presence of MdmX in the complex as crucial for target selectivity as well as the processivity of the holoezyme somewhat flies in the face of the existing structural data. Two published structures of the Mdm2 homodimer and Mdm2/MdmX heterodimer indicate virtually no difference in the complexes.^7,8 In the absence of structural differences, how then are such significant differences in function accomplished? A hypothesis unifying structural and functional data is brought forth by a very intriguing study from the Uldrijan group, which systematically looks at the differences between complex formation and activity of Mdm2 and MdmX.⁹ Phylogenetic analysis showed that the last cystein of the RING domain is followed by exactly 13 amino acids in all Mdm orthologs of vertebrate origin. Based on this, the authors hypothesized that not only the sequence of the C-terminal tails, but also their exact length are of central importance to the function of the complexes. Subsequent investigation of the ability of Mdm2 and MdmX proteins, which have been extended at the C terminus by 5, 14 or 18 amino acids, was designed to test the importance of the length of the C-terminal extensions. To the researchers surprise, when examined based on their ability to hetero-oligomerize and ubiquitinate p53, Mdm2 proteins behaved differently depending on whether the oligomeric partner was Mdm2 or MdmX. Dolezelova et al. present unexpected experimental evidence for the heterocomplex being structurally and functionally distinct from the Mdm2 homodimer, while providing a mechanism for the observed in vivo functional differences between the complexes. Although the work casts slight doubt on the complete accuracy of the existing structures, it nicely aligns with the above-mentioned results, showing the singular importance of the MdmX RING domain in the activity of the holoenzyme. In light of these results, additional structural studies that will take in to account reported differences between the complexes will undoubtedly be informative and contribute to our understanding of the biochemistry of RING-type ubiquitin ligases and the mechanisms regulating p53 in cells.     

A high-throughput screen measuring ubiquitination of p53 by human mdm2

Tumor suppressor p53 is typically maintained at low levels in normal cells. In response to cellular stresses, such as DNA damage, p53 is stabilized and can stimulate responses leading to cell cycle arrest or apoptosis. Corresponding to its central role in preventing propagation of damaged cells, mutation or deletion of p53 is found in nearly 50% of all human tumors. Mdm2 (mouse-d-minute 2) and its human ortholog (hmdm2 or hdm2) catalyze the ubiquitination of p53, targeting it for degradation via the proteosome. Thus, the activity of mdm2 is inversely correlated with p53 levels. Based on this, inhibition of human mdm2 activity by a small-molecule therapeutic will lead to net stabilization of p53 and be the basis for development of a novel cancer therapeutic. Previous high-throughput screening assays of mdm2 measured the autoubiquitination activity of mdm2, which occurs in the absence of an acceptor substrate such as p53. The major drawback to this approach is that inhibitors of mdm2 autoubiquitination may lead to a net stabilization of mdm2 and thus have the opposite effect of inhibitors that interfere with p53 ubiquitination. The authors describe the development, validation, and execution of a high-throughput screening measuring the ubiquitination of p53 by mdm2, with p53 labeled with europium and the other substrate (Ub-UbcH5b) labeled with a Cy5 on the ubiquitin. After confirming that known inhibitors are detected with this assay, it was successfully automated and used to query >600,000 compounds from the GlaxoSmithKline collection for mdm2 inhibitors.     

Targeting RING domains of Mdm2–MdmX E3 complex activates apoptotic arm of the p53 pathway in leukemia/lymphoma cells

Reactivation of tumor-suppressor p53 for targeted cancer therapy is an attractive strategy for cancers bearing wild-type (WT) p53. Targeting the Mdm2–p53 interface or MdmX ((MDM4), mouse double minute 4)–p53 interface or both has been a focus in the field. However, targeting the E3 ligase activity of Mdm2–MdmX really interesting new gene (RING)–RING interaction as a novel anticancer strategy has never been explored. In this report, we describe the identification and characterization of small molecule inhibitors targeting Mdm2–MdmX RING–RING interaction as a new class of E3 ligase inhibitors. With a fluorescence resonance energy transfer-based E3 activity assay in high-throughput screening of a chemical library, we identified inhibitors (designated as MMRis (Mdm2–MdmX RING domain inhibitors)) that specifically inhibit Mdm2–MdmX E3 ligase activity toward Mdm2 and p53 substrates. MMRi6 and its analog MMRi64 are capable of disrupting Mdm2–MdmX interactions in vitro and activating p53 in cells. In leukemia cells, MMRi64 potently induces downregulation of Mdm2 and MdmX. In contrast to Nutlin3a, MMRi64 only induces the expression of pro-apoptotic gene PUMA (p53 upregulated modulator of apoptosis) with minimal induction of growth-arresting gene p21. Consequently, MMRi64 selectively induces the apoptotic arm of the p53 pathway in leukemia/lymphoma cells. Owing to the distinct mechanisms of action of MMRi64 and Nutlin3a, their combination synergistically induces p53 and apoptosis. Taken together, this study reveals that Mdm2–MdmX has a critical role in apoptotic response of the p53 pathway and MMRi64 may serve as a new pharmacological tool for p53 studies and a platform for cancer drug development.Activation of tumor-suppressor p53 as a targeted non-genotoxic cancer therapy has been pursued for many years,^{1, 2} because p53 possesses potent tumor-suppressing activity in vivo.^{3, 4, 5} p53 can inhibit cancer cell growth by cell cycle arrest or terminate their proliferation by inducing apoptosis and senescence.⁶ The p53-based therapy is particularly attractive for cancer types including retinoblastoma, neuroblastoma and leukemia/lymphoma in which p53 is rarely mutated⁷ and p53-dependent apoptotic pathway is a predominant endpoint.^{8, 9, 10} Except for cancer-selected p53 mutations, the p53 activity is mainly inhibited by p53-binding proteins Mdm2 and MdmX ((MDM4), mouse double minute 4) in normal and cancer cells.^{11, 12} Prior focus of p53 reactivation strategy has been on targeting the Mdm2–p53 and/or MdmX–p53 interface. This has led to the discovery of a list of potent Mdm2–p53 inhibitors¹³ with several compounds of this class being advanced to phase I clinical trials in hematological neoplasia and solid tumors.² However, the therapeutic effects of these Mdm2–p53 inhibitors can be attenuated by MdmX overexpression.^{14, 15, 16} Although peptide inhibitors with dual functions of inhibiting both Mdm2–p53 and MdmX–p53 interactions will overcome this problem and enhance p53-dependent cancer killing;^{17, 18} these inhibitors will not inhibit Mdm2 E3 ligase activity toward non-p53 targets such as retinoblastoma protein (RB), p21 and DAXX (death domain-associated protein),^{19, 20, 21} which to a different extent contributes to the p53-dependent biological effects.Recent genetic studies indicated that really interesting new gene (RING) domains of Mdm2 and MdmX are required for in vivo inhibition of p53 activity during development.^{22, 23, 24} MdmX was reported to stimulate Mdm2-mediated p53 multiple monoubiquitination using glutathione S-transferase (GST) fusion Hdm2 proteins.^{25, 26} Using non-GST Hdm2 proteins in in vitro biochemical assays, we found that MdmX–Mdm2 RING–RING interaction is essential for p53 polyubiquitination and proteasome-dependent degradation.²⁶ These findings established that Mdm2–MdmX complex is the key regulator of p53 activity and Mdm2–MdmX RING–RING interaction is a critical but an unexplored interface for drug targeting.²⁷ Identification of E3 ligase inhibitors for cancer therapy presents a huge opportunity but with great challenges.²⁸ In this report, we describe successful identification and characterization of small molecule inhibitors for the E3 ligase activity of Mdm2–MdmX E3 complex. Among seven specific MMRis (Mdm2–MdmX RING domain inhibitors), MMRi64 was followed up in detail in this report. MMRi64 has several unique features that distinguish it from Mdm2–p53 inhibitor Nutlin3a. MMRi64 disrupts Mdm2–MdmX interaction in vitro and inhibits the E3 ligase activity of Mdm2–MdmX without affecting the E3 ligase activity of Mdm2 RING domain homodimers. MMRi64 induces p53 accumulation without induction of Mdm2 and p21 in lymphoma cells, which is distinct from the effects of Nutlin3a. Finally, MMRi64 induces PUMA (p53 upregulated modulator of apoptosis) but strongly downregulates MdmX and Mdm2, consequently activating the apoptotic arm of the p53 pathway in leukemia/lymphoma cells without the induction of growth arrest.     

The intestinal epithelium compensates for p53-mediated cell death and guarantees organismal survival

Mdm2 is the major inhibitor of the p53 tumor suppressor. Loss of Mdm2 in mice or in specific tissues of the mouse always yields p53-dependent lethal phenotypes. However, the role of Mdm2 in tissues with high turnover capacity is unknown. We have engineered mice lacking Mdm2 in the intestinal epithelium using the Cre/LoxP system. Loss of Mdm2 (Mdm2(intDelta)) results in viable animals, but neonates display multiple intestinal abnormalities such as hyperplasia, enterocyte vacuolization, and inflammation. These defects correlate with a drastic increase in p53-dependent apoptosis in highly proliferative and differentiated cells. Unexpectedly, the observed phenotypes disappear with age. The tissue selects against Mdm2-null cells and increases its proliferative capacity. Additionally, the intestinal stem and progenitor cell populations are enriched leading to an increase in crypt fission events. Enhanced proliferation is achieved by activation of the canonical Wnt and EGFR-mediated Ras/MAPK pathways. While Mdm2 is a critical inhibitor of p53 in the intestinal epithelium, the tissue employs a series of processes that compensate for cell death.     

Identification of a sequence element from p53 that signals for Mdm2-targeted degradation

The binding of Mdm2 to p53 is required for targeting p53 for degradation. p73, however, binds to Mdm2 but is refractory to Mdm2-mediated degradation, indicating that binding to Mdm2 is not sufficient for degradation. By utilizing the structural homology between p53 and p73, we generated p53-p73 chimeras to determine the sequence element unique to p53 essential for regulation of its stability. We found that replacing an element consisting of amino acids 92 to 112 of p53 with the corresponding region of p73 results in a protein that is not degradable by Mdm2. Removal of amino acids 92 to 112 of p53 by deletion also results in a non-Mdm2-degradable protein. Significantly, the finding that swapping this fragment converts p73 from refractory to sensitive to Mdm2-mediated degradation supports the conclusion that the amino acids 92 to 112 of p53 function as a degradation signal. We propose that the presence of an additional protein recognizes the degradation signal and coordinates with Mdm2 to target p53 for degradation. Our finding opens the possibility of searching for the additional protein, which most likely plays a critical role in the regulation of p53 stability and therefore function.