MDM2 is a major regulator of p53 by acting as a ubiquitin E3 ligase. The central acidic domain and C-terminal RING domain of MDM2 are both indispensable for ubiquitination of p53. Our previous study suggested that ATM phosphorylation of MDM2 near the C terminus inhibits RING domain oligomerization, resulting in p53 stabilization after DNA damage. We present here evidence that these modifications allosterically regulate the functions of both acidic domain and RING domain of MDM2. Using chemical cross-linking, we show that the MDM2 RING domain forms oligomers including dimer and higher-order complexes in vivo. RING domain dimerization efficiency is negatively regulated by upstream sequence. ATM-mediated phosphorylation of the upstream sequence further inhibits RING dimerization. Forced oligomerization of MDM2 partially overcomes the inhibitory effect of phosphorylation and stimulates p53 ubiquitination. Furthermore, the ability of MDM2 acidic domain to bind p53 core domain and induce p53 misfolding are also suppressed by the same C-terminal ATM sites after DNA damage. These results suggest that the acidic domain and RING domain of MDM2 are both allosterically coupled to the intervening ATM sites, which enables the same modification to regulate multiple MDM2 functions critical for p53 ubiquitination.The p53 pathway is critical for maintenance of genomic stability and preventing development of cancer. The most notable feature of p53 is its stabilization after exposure to a wide range of stress signals such as hyperproliferation, nucleotide depletion, and DNA damage. These responses may be essential for its function as a tumor suppressor (14). In normal cells, p53 is present at very low levels due to rapid degradation through the ubiquitin-dependent proteasome pathway. p53 turnover is regulated by MDM2, which binds p53 and functions as an ubiquitin E3 ligase to promote p53 degradation by the proteasome (15,16,21). Additional E3 ligases such as Pirh2 and Cop1 have also been implicated as regulators of p53 turn over (12,24). However, their physiological significance in p53 regulation and stress response still remain to be established. Current evidence suggests that MDM2 is a major and indispensable regulator of p53 level (18,32).MDM2 and p53 interact through their N-terminal domains in a high-affinity binding and through their central domains in a weak binding (48). Upon complex formation, the MDM2 C-terminal RING domain recruits ubiquitin-conjugating enzyme E2 that performs the transfer of activated ubiquitin to p53 lysine residues. To date, the understanding of molecular mechanisms that lead to p53 stabilization by different pathways remain incomplete. An important group of MDM2 regulators are proteins that bind to the central acidic domain, such as ARF, L5, L11, and L23 (37, 50). These basic proteins are important for mediating mitogenic stress and ribosomal stress signals to p53. They have been shown to inhibit MDM2-mediated p53 ubiquitination, but little is known about the mechanisms. They may act by neutralizing ...
Callous-unemotional (CU) traits (i.e., lack of empathy/guilt, uncaring attitudes) are believed to be a developmental antecedent to adult psychopathy and identify antisocial youth at risk for severe and persistent aggression. The psychosocial histories of antisocial and aggressive individuals with psychopathic traits are characterized by abusive or unaffectionate parenting; however, there is a gap in the literature regarding the unique impact of these factors on adolescent offenders. The purpose of the present study was to examine the contribution of maternal warmth and affection (i.e., care) to dimensions of CU traits and aggression, after accounting for the influence of various types of childhood maltreatment. We investigated this aim in a sample of 227 urban male adolescent offenders housed in residential facilities. Results indicated that low maternal care was significantly associated with greater total CU traits and uncaring and callousness dimensions, even after controlling for the effects of various types of childhood abuse and neglect. Furthermore, there was a significant interaction between CU traits and care, such that aggression was highest among youths scoring high on CU traits who were exposed to low levels of maternal care. These findings draw attention to the importance of maternal bonding to CU traits and related aggressive behaviors among antisocial youth.
MDM2 is a key regulator of the p53 tumor suppressor acting primarily as an E3 ubiquitin ligase to promote its degradation. MDM2 also inhibits p53 transcriptional activity by recruiting histone deacetylase and corepressors to p53. Here, we show that immunopurified MDM2 complexes have significant histone H3-K9 methyltransferase activity. The histone methyltransferases SUV39H1 and EHMT1 bind specifically to MDM2 but not to its homolog MDMX. MDM2 mediates formation of p53-SUV39H1/EHMT1 complex capable of methylating H3-K9 in vitro and on p53 target promoters in vivo. Furthermore, MDM2 promotes EHMT1-mediated p53 methylation at K373. Knockdown of SUV39H1 and EHMT1 increases p53 activity during stress response without affecting p53 levels, whereas their overexpression inhibits p53 in an MDM2-dependent manner. The p53 activator ARF inhibits SUV39H1 and EHMT1 binding to MDM2 and reduces MDM2-associated methyltransferase activity. These results suggest that MDM2-dependent recruitment of methyltransferases is a novel mechanism of p53 regulation through methylation of both p53 itself and histone H3 at target promoters.
MDM2 regulates p53 predominantly by promoting p53 ubiquitination. However, ubiquitination-independent mechanisms of MDM2 have also been implicated. Here we show that MDM2 inhibits p53 DNA binding activity in vitro and in vivo. MDM2 binding promotes p53 to adopt a mutant-like conformation, losing reactivity to antibody Pab1620, while exposing the Pab240 epitope. The acidic domain of MDM2 is required to induce p53 conformational change and inhibit p53 DNA binding. Alternate reading frame binding to the MDM2 acidic domain restores p53 wild type conformation and rescues DNA binding activity. Furthermore, histone methyl transferase SUV39H1 binding to the MDM2 acidic domain also restores p53 wild type conformation and allows p53-MDM2-SUV39H1 complex to bind DNA. These results provide further evidence for an ubiquitination-independent mechanism of p53 regulation by MDM2 and reveal how MDM2-interacting repressors gain access to p53 target promoters and repress transcription. Furthermore, we show that the MDM2 inhibitor Nutlin cooperates with the proteasome inhibitor Bortezomib by stimulating p53 DNA binding and transcriptional activity, providing a rationale for combination therapy using proteasome and MDM2 inhibitors.The tumor suppressor protein p53 encodes a transcription factor that plays a critical role in preventing malignant transformation by inducing cell cycle arrest, DNA repair, or apoptosis in response to various types of damage. Mutations or deletions in the p53 gene occur in over 50% of all human cancers, often in the centrally located sequence-specific DNA-binding domain, resulting in the unfolding and accumulation of p53 in the nucleus (1-3). The p53 core domain has poor thermostability at physiological temperatures and can undergo spontaneous denaturation (4 -6). Changes in p53 conformation can inhibit the ability of p53 to induce expression of its downstream transcriptional targets (7,8).p53 is maintained at low levels in unstressed cells with a short half-life. This is mainly achieved through the ability of MDM2 to bind p53 and act as an ubiquitin E3 ligase to promote its proteasomal degradation (9, 10). MDM2 is also a transcriptional target of p53, forming an auto-regulatory feedback loop (11,12). The importance of MDM2 in the regulation of p53 is highlighted by the fact that mice deficient in MDM2 are embryonic lethal, whereas mice deficient for both MDM2 and p53 are viable (13,14). MDMX, a p53 binding partner with sequence homology to MDM2, is also an essential negative regulator of p53 (15). Unlike MDM2, MDMX lacks ubiquitin ligase activity and is unable to target p53 for proteasomal degradation (16,17). Recent studies suggest that MDMX is a bona fide p53 transcriptional target in certain cell types (17, 18). MDMX can bind to p53 N-terminal transactivation domain and inhibit p53 transcription of target genes (19). MDMX-null mice are embryonically lethal despite expression of MDM2, suggesting a unique role for MDMX in the regulation of p53 (20). The prevailing view is that MDMX mainly functions by re...
Implications for improved behavioral health screening and coordination of university behavioral health services with veterans' health systems are discussed.
Marked aneuploidy and loss of multiple chromosomes are hallmarks of cancer, but whether these events are only present in malignant cells is not known. In prior work, we showed that approximately half of spontaneous autosomal mutants isolated directly from normal kidney epithelium arose from loss of a marker chromosome 8 containing the wild type Aprt gene. Chromosome loss was detected by loss of heterozygosity (LOH) for all chromosome 8 polymorphic loci examined (Turker et al, Aging Cell, 6:73-86, 2007). To determine whether loss of chromosome 8 reflected a larger mitotic event, LOH was examined for polymorphic loci on 11 non-selected chromosomes in Aprt mutants that lost the selected chromosome 8 homologue. LOH events were detected for one or more non-selected chromosomes in 38% of these mutants. The additional LOH events also reflected apparent chromosome loss based on the molecular analysis. Metaphase spreads from mutants that lost chromosome 8 were markedly aneuploid and chromosome painting revealed reduced levels for any chromosome shown to be lost with the LOH analysis. In contrast, LOH on non-selected chromosomes was infrequent in Aprt mutants exhibiting intragenic events or mitotic recombination for chromosome 8, and marked aneuploidy was absent. These observations suggest that the mechanism leading to chromosome loss in somatic mammalian cells is often not a simple non-disjunction event and instead could result from a single catastrophic event. They also suggest that cells with characteristics of malignancy are present in normal appearing tissue.
Lastly, thanks to Cathy Gaffney who coordinates everything for the Cancer Biology Program and was a huge help all the way through to graduation. i TABLE OF CONTENTS LIST OF FIGURES .
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