[Show abstract][Hide abstract] ABSTRACT: Many cancers express mutant p53 proteins that have lost wild-type tumor suppressor activity and, in many cases, have acquired oncogenic functions that can contribute to tumor progression. These activities of mutant p53 reflect interactions with several other proteins, including the p53 family members p63 and p73. Mutations in p53 that affect protein conformation (such as R175H) show strong binding to p63 and p73, whereas p53 mutants that only mildly affect the conformation (such as R273H) bind less well. A previously described aggregation domain of mutant p53 is not required for p63 or p73 binding; indeed, mutations within this region lead to the acquisition of a mutant p53 phenotype-including a conformational shift, p63/p73 binding and the ability to promote invasion. The activity of wild-type p53 is regulated by an interaction with MDM2 and we have investigated the potential role of MDM2 in the mutant p53/p63/p73 interactions. Both mutant p53 and p73 bind MDM2 well, whereas p63 binds much more weakly. We found that MDM2 can inhibit p63 binding to p53R175H but enhances the weaker p53R273H/p73 interaction. These effects on the interactions are reflected in an ability of MDM2 to relieve the inhibition of p63 by p53R175H, but enhance the inhibition of p73 activity by p53R175H and R273H. We propose a model in which MDM2 competes with p63 for binding to p53R175H to restore p63 activity, but forms a trimeric complex with p73 and p53R273H to more strongly inhibit p73 function.Oncogene advance online publication, 24 November 2014; doi:10.1038/onc.2014.359.
[Show abstract][Hide abstract] ABSTRACT: The p53-inducible protein TIGAR (Tp53-induced Glycolysis and Apoptosis Regulator) functions as a fructose-2,6-bisphosphatase (Fru-2,6-BPase), and through promotion of the pentose phosphate pathway, increases NADPH production to help limit reactive oxygen species (ROS). Here, we show that under hypoxia, a fraction of TIGAR protein relocalized to mitochondria and formed a complex with hexokinase 2 (HK2), resulting in an increase in HK2 activity. Mitochondrial localization of TIGAR depended on mitochondrial HK2 and hypoxia-inducible factor 1 (HIF1α) activity. The ability of TIGAR to function as a Fru-2,6-BPase was independent of HK2 binding and mitochondrial localization, although both of these activities can contribute to the full activity of TIGAR in limiting mitochondrial ROS levels and protecting from cell death.
Proceedings of the National Academy of Sciences 11/2012; 109(50). DOI:10.1073/pnas.1206530109 · 9.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The p53 tumour suppressor protein is a transcription factor that prevents oncogenic progression by activating the expression of apoptosis and cell-cycle arrest genes in stressed cells. The stability of p53 is tightly regulated by ubiquitin-dependent degradation, driven mainly by the ubiquitin ligase MDM2. In this study, we have identified USP42 as a DUB that interacts with and deubiquitinates p53. USP42 forms a direct complex with p53 and controls level of ubiquitination during the early phase of the response to a range of stress signals. Although we do not find a clear role for USP42 in controlling either the basal or fully activated levels of p53, the function of USP42 is required to allow the rapid activation of p53-dependent transcription and a p53-dependent cell-cycle arrest in response to stress. These functions of USP42 are likely to contribute to the repair and recovery of cells from mild or transient damage.
The EMBO Journal 11/2011; 30(24):4921-30. DOI:10.1038/emboj.2011.419 · 10.43 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The ASPP (apoptosis-stimulating protein of p53) family of proteins can function in the nucleus to modulate the transcriptional activity of p53, with ASPP1 and ASPP2 contributing to the expression of apoptotic target genes. In this study, we describe a new function for cytoplasmic ASPP1 in controlling YAP (Yes-associated protein)/TAZ. ASPP1 can inhibit the interaction of YAP with LATS1 (large tumor suppressor 1), a kinase that phosphorylates YAP/TAZ and promotes cytoplasmic sequestration and protein degradation. This function of ASPP1 therefore enhances nuclear accumulation of YAP/TAZ and YAP/TAZ-dependent transcriptional regulation. The consequence of YAP/TAZ activation by ASPP1 is to inhibit apoptosis, in part through the down-regulation of Bim expression, leading to resistance to anoikis and enhanced cell migration. These results reveal a potential oncogenic role for cytoplasmic ASPP1, in contrast to the tumor-suppressive activity described previously for nuclear ASPP1.
Genes & development 11/2010; 24(21):2430-9. DOI:10.1101/gad.1954310 · 10.80 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: p53 is a tumor suppressor protein whose function is frequently lost in cancers through missense mutations within the Tp53 gene. This results in the expression of point-mutated p53 proteins that have both lost wild-type tumor suppressor activity and show gain of functions that contribute to transformation and metastasis. Here, we show that mutant p53 expression can promote invasion, loss of directionality of migration, and metastatic behavior. These activities of p53 reflect enhanced integrin and epidermal growth factor receptor (EGFR) trafficking, which depends on Rab-coupling protein (RCP) and results in constitutive activation of EGFR/integrin signaling. We provide evidence that mutant p53 promotes cell invasion via the inhibition of TAp63, and simultaneous loss of p53 and TAp63 recapitulates the phenotype of mutant p53 in cells. These findings open the possibility that blocking alpha5/beta1-integrin and/or the EGF receptor will have therapeutic benefit in mutant p53-expressing cancers.
[Show abstract][Hide abstract] ABSTRACT: The conjugation of proteins with ubiquitin plays numerous regulatory roles through both proteasomal-dependent and nonproteasomal-dependent functions. Alterations in ubiquitylation are observed in a wide range of pathologic conditions, including numerous malignancies. For this reason, there is great interest in targeting the ubiquitin-proteasome system in cancer. Several classes of proteasome inhibitors, which block degradation of ubiquitylated proteins, are widely used in research, and one, Bortezomib, is now in clinical use. Despite the well-defined and central role of the ubiquitin-activating enzyme (E1), no cell permeable inhibitors of E1 have been identified. Such inhibitors should, in principle, block all functions of ubiquitylation. We now report 4[4-(5-nitro-furan-2-ylmethylene)-3,5-dioxo-pyrazolidin-1-yl]-benzoic acid ethyl ester (PYR-41) as the first such inhibitor. Unexpectedly, in addition to blocking ubiquitylation, PYR-41 increased total sumoylation in cells. The molecular basis for this is unknown; however, increased sumoylation was also observed in cells harboring temperature-sensitive E1. Functionally, PYR-41 attenuates cytokine-mediated nuclear factor-kappaB activation. This correlates with inhibition of nonproteasomal (Lys-63) ubiquitylation of TRAF6, which is essential to IkappaB kinase activation. PYR-41 also prevents the downstream ubiquitylation and proteasomal degradation of IkappaBalpha. Furthermore, PYR-41 inhibits degradation of p53 and activates the transcriptional activity of this tumor suppressor. Consistent with this, it differentially kills transformed p53-expressing cells. Thus, PYR-41 and related pyrazones provide proof of principle for the capacity to differentially kill transformed cells, suggesting the potential for E1 inhibitors as therapeutics in cancer. These inhibitors can also be valuable tools for studying ubiquitylation.
Cancer Research 11/2007; 67(19):9472-81. DOI:10.1158/0008-5472.CAN-07-0568 · 9.33 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A family of 5-deazaflavin derivatives has been synthesised using a two-step convergent strategy. The biological activity of these compounds was evaluated in cells, by assessing their ability to stabilize and activate p53. These compounds may act as low molecular weight inhibitors of the E3 activity of HMD2 in tumours that retain wild-type p53. Importantly, we have demonstrated that the nitro group present in all three of the original lead compounds [1-3 (HL198C-E)] is not essential for observation of this biological activity.
[Show abstract][Hide abstract] ABSTRACT: The p53 tumor suppressor protein is regulated by its interaction with HDM2, which serves as a ubiquitin ligase (E3) to target p53 for degradation. We have identified a family of small molecules (HLI98) that inhibits HDM2's E3 activity. These compounds show some specificity for HDM2 in vitro, although at higher concentrations effects on unrelated RING and HECT domain E3s are detectable, which could be due, at least in part, to effects on E2-ubiquitin thiol-ester levels. In cells, the compounds allow the stabilization of p53 and HDM2 and activation of p53-dependent transcription and apoptosis, although other p53-independent toxicity was also observed.
Cancer Cell 07/2005; 7(6):547-59. DOI:10.1016/j.ccr.2005.04.029 · 23.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: p53 stability is regulated by HDM2, a RING domain protein that acts as an E3 ligase to ubiquitinate p53 and target its degradation. Phosphorylation of HDM2 on serine 166 by AKT has been shown to enhance HDM2 activity and promote the degradation of p53. Here, we show that MAPKAP kinase 2 (MK2) can phosphorylate HDM2 on serine 157 and 166 in vitro. Treatment of cells with anisomycin, which activates MK2, also results in phosphorylation of HDM2 on serine 157 and 166 in vivo. Mutation of the MK2 phosphorylation sites in HDM2 to aspartic acid renders HDM2 slightly more active in the degradation of p53, and mouse cells deficient for MK2 show reduced Mdm2 phosphorylation and elevated levels of p53 protein. Together, our results suggest that MK2 may act to dampen the extent and duration of the p53 response.
[Show abstract][Hide abstract] ABSTRACT: The HDM2 protein plays an important role in regulating the stability and function of the p53 tumor suppressor protein. In this report, we show that the ribosomal protein L11 can interact with HDM2 and inhibit HDM2 function, thus leading to the stabilization and activation of p53. The inhibition of HDM2 activity by L11 shows some similarity to the previously described activity of ARF, and expression of either ARF or L11 can induce a p53 response. Enhancement of the interaction between endogenous L11 and HDM2 following treatment of cells with low levels of actinomycin-D suggests that the HDM2/L11 interaction represents a novel pathway for p53 stabilization in response to perturbations in ribosome biogenesis.
Cancer Cell 07/2003; 3(6):577-87. DOI:10.1016/S1535-6108(03)00134-X · 23.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The HDM2 protein is a key regulator of the tumour suppressor, p53. Control of HDM2 function is critical for normal cell proliferation and stress responses, and it is becoming evident that multiple modifications of HDM2 can regulate its function within cells. In this study we show that HDM2 associated with the serine-threonine kinase, Akt, in response to growth factor stimulation of human primary cells. This association was concurrent with phosphorylation of Akt (at Ser 473), and resulted in elevated expression of HDM2 and enhanced nuclear localization. However, analysis of HDM2 proteins mutated at the consensus Akt recognition sites at serines 166 and 186 indicated that modification at these residues was not sufficient for the increased expression of the protein, which was blocked by the PI3 kinase inhibitor LY294002. Tryptic peptide and mutational analyses revealed evidence for an Akt phosphorylation site in HDM2 additional to the two consensus sites.
[Show abstract][Hide abstract] ABSTRACT: The growth inhibitory functions of p53 are controlled in unstressed cells by rapid degradation of the p53 protein. One of the principal regulators of p53 stability is MDM2, a RING finger protein that functions as an E3 ligase to ubiquitinate p53. MDM2 promotes p53 nuclear export, and in this study, we show that ubiquitination of the C terminus of p53 by MDM2 contributes to the efficient export of p53 from the nucleus to the cytoplasm. In contrast, MDM2 did not promote nuclear export of the p53-related protein, p73. p53 nuclear export was enhanced by overexpression of the export receptor CRM1, although no significant relocalization of MDM2 was seen in response to CRM1. However, nuclear export driven by CRM1 overexpression did not result in the degradation of p53, and nuclear export was not essential for p53 degradation. These results indicate that MDM2 mediated ubiquitination of p53 contributes to both nuclear export and degradation of p53 but that these activities are not absolutely dependent on each other.
[Show abstract][Hide abstract] ABSTRACT: Mdm2 has been shown to regulate p53 stability by targeting the p53 protein for proteasomal degradation. We now report that Mdm2 is a ubiquitin protein ligase (E3) for p53 and that its activity is dependent on its RING finger. Furthermore, we show that Mdm2 mediates its own ubiquitination in a RING finger-dependent manner, which requires no eukaryotic proteins other than ubiquitin-activating enzyme (E1) and an ubiquitin-conjugating enzyme (E2). It is apparent, therefore, that Mdm2 manifests an intrinsic capacity to mediate ubiquitination. Mutation of putative zinc coordination residues abrogated this activity, as did chelation of divalent cations. After cation chelation, the full activity could be restored by addition of zinc. We further demonstrate that the degradation of p53 and Mdm2 in cells requires additional potential zinc-coordinating residues beyond those required for the intrinsic activity of Mdm2 in vitro. Replacement of the Mdm2 RING with that of another protein (Praja1) reconstituted ubiquitination and proteasomal degradation of Mdm2. However, this RING was ineffective in ubiquitination and proteasomal targeting of p53, suggesting that there may be specificity at the level of the RING in the recognition of heterologous substrates.
[Show abstract][Hide abstract] ABSTRACT: 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 p21(Waf1/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.
Cell growth & differentiation: the molecular biology journal of the American Association for Cancer Research 03/1999; 10(2):87-92.
[Show abstract][Hide abstract] ABSTRACT: The stability of the p53 tumor suppressor protein is regulated by interaction with Mdm2, the product of a p53-inducible gene. Mdm2-targeted degradation of p53 depends on the interaction between the two proteins and is mediated by the proteasome. We show here that in addition to the N-terminal Mdm2 binding domain, the C terminus of p53 participates in the ability of p53 to be degraded by Mdm2. In contrast, alterations in the central DNA binding domain of p53, which change the conformation of the p53 protein, do not abrogate the sensitivity of the protein to Mdm2-mediated degradation. The importance of the C-terminal oligomerization domain to Mdm2-targeted degradation of p53 is likely to reflect the importance of oligomerization of the full-length p53 protein for interaction with Mdm2, as previously shown in vitro. Interestingly, the extreme C-terminal region of p53, outside the oligomerization domain, was also shown to be necessary for efficient degradation, and deletion of this region stabilized the protein without abrogating its ability to bind to Mdm2. Mdm2-resistant p53 mutants were not further stabilized following DNA damage, supporting a role for Mdm2 as the principal regulator of p53 stability in cells. The extreme C terminus of the p53 protein has previously been shown to contain several regulatory elements, raising the possibility that either allosteric regulation of p53 by this domain or interaction between this region and a third protein plays a role in determining the sensitivity of p53 to Mdm2-directed degradation.
[Show abstract][Hide abstract] ABSTRACT: Most human cancers show perturbation of growth regulation mediated by the tumour-suppressor proteins retinoblastoma (RB) and p53 (ref. 1), indicating that loss of both pathways is necessary for tumour development. Loss of RB function leads to abnormal proliferation related to the deregulation of the E2F transcription factors, but also results in the activation of p53, which suppresses cell growth. Here we show that E2F-1 directly activates expression of the human tumour-suppressor protein p14ARF (the mouse homologue is called p19ARF), which binds to the MDM2-p53 complex and prevents p53 degradation2,5. These results complete a pathway linking abnormal proliferative signals, such as loss of RB, with the activation of a p53 response, through E2F-1 and p14ARF. They suggest that E2F-1, a protein inherently activated by cell-cycle progression, is part of a fail-safe mechanism to protect against aberrant cell growth.
[Show abstract][Hide abstract] ABSTRACT: The p53 tumor suppressor protein can adopt both latent, non-DNA binding and active, DNA binding forms, and p53 activity is thought to be regulated in cells, at least in part, through a conformational shift which leads to sequence specific DNA binding. In vitro, this allosteric regulation of DNA binding by p53 has been shown to be mediated through the C-terminus of the protein. We show here that although deletion of the C-terminal 16 amino acids of p53 did not activate DNA binding, deletion of a further eight amino acids resulted in constitutive activation of DNA binding activity. Simultaneous mutation of the three lysine residues within these eight amino acids also resulted in constitutive DNA binding activity, although this was reduced when only two of these lysines were altered. The deletion or point mutants of p53 showing constitutive DNA binding activity did not display clear evidence of DNA binding site specificity, although some binding site preference was seen with the point mutants. Each of the constitutively active p53 mutants retained transcriptional activity and induced both cell cycle arrest and apoptosis in transiently transfected cells at rates comparable with the wild type protein.
[Show abstract][Hide abstract] ABSTRACT: Introduction of simian virus 40 T antigen into rodent fibroblasts gives rise to cells that can proliferate indefinitely but are dependent upon it for maintenance of their growth once the normal mitotic life span has elapsed. Inactivation of T antigen in these immortalized cells causes rapid and irreversible cessation of growth. To determine whether this growth arrest is associated with entry into senescence, we have undertaken a genetic and biological analysis of conditionally immortal (tsa) cell lines derived by immortalizing rat embryo fibroblasts with the thermolabile tsA58 T antigen. This analysis has identified the following parallels between the tsa cells after inactivation of T antigen and senescent rat embryo fibroblasts: (i) growth arrest is irreversible; (ii) it occurs in G1 as well as G2; (iii) the G1 block can be partially overcome by stimulation with 20% fetal calf serum, but the G2 block cannot be overcome; (iv) 20% fetal calf serum induces c-fos, but c-myc is unaltered; and (v) fibronectin and p21(Waf1/Cip1/Sdi1) are upregulated upon growth arrest. These results suggest that T-antigen-immortalized fibroblasts are committed to undergo senescence but are prevented from undergoing this process by T antigen. Inactivation of T antigen removes this block and results in senescence of the cells. Thus, these cell lines may represent a powerful system for study of the molecular basis of entry into senescence.