TRIAD1 inhibits MDM2-mediated p53 ubiquitination and degradation

Murine double minute (MDM2) is an E3 ligase that promotes ubiquitination and degradation of tumor suppressor protein 53 (p53). MDM2-mediated regulation of p53 has been investigated as a classical tumorigenesis pathway. Here, we describe TRIAD1 as a novel modulator of the p53-MDM2 axis that induces p53 activation by inhibiting its regulation by MDM2. Ablation of TRIAD1 attenuates p53 levels activity upon DNA damage, whereas ectopic expression of TRIAD1 promotes p53 stability by inhibiting MDM2-mediated ubiquitination/degradation. Moreover, TRIAD1 binds to the C-terminus of p53 to promote its dissociation from MDM2. These results implicate TRIAD1 as a novel regulatory factor of p53-MDM2.Structured summary of protein interactions:p53 physically interacts with Mdm2 and Triad1 by anti tag coimmunoprecipitation (View Interaction: 1, 2, 3)Mdm2physically interacts with Triad1 by anti tag coimmunoprecipitation (View interaction)p53physically interacts with Mdm2 by anti tag coimmunoprecipitation (View interaction)Triad1binds to p53 by pull down (View interaction)Mdm2physically interacts with p53 by anti tag coimmunoprecipitation (View interaction)p53physically interacts with Triad1 by anti tag coimmunoprecipitation (View interaction)     

Triptolide sensitizes TRAIL-induced apoptosis in prostate cancer cells via p53-mediated DR5 up-regulation

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising agent for cancer therapy. However, a number of prostate cancer cells exhibit high resistance to TRAIL effect. In this study, we found that Triptolide, a Chinese medicine, significantly sensitizes prostate cancer cells to TRAIL-mediated cellular apoptosis by up-regulating DR5 expression. Triptolide treatment can suppress Akt/Hdm2 signaling pathway, and lead to p53 accumulation, thereby up-regulating DR5 expression. Taken together, all evidences indicate that Triptolide may become a promising therapeutic agent that prevents the progression of prostate cancer.     

MDM2-HDAC1-mediated deacetylation of p53 is required for its degradation

The tumor suppressor p53 is stabilized and activated in response to cellular stress through post-translational modifications including acetylation. p300/CBP-mediated acetylation of p53 is negatively regulated by MDM2. Here we show that MDM2 can promote p53 deacetylation by recruiting a complex containing HDAC1. The HDAC1 complex binds MDM2 in a p53-independent manner and deacetylates p53 at all known acetylated lysines in vivo. Ectopic expression of a dominant-negative HDAC1 mutant restores p53 acetylation in the presence of MDM2, whereas wild-type HDAC1 and MDM2 deacetylate p53 synergistically. Fibroblasts overexpressing a dominant negative HDAC1 mutant display enhanced DNA damage-induced p53 acetylation, increased levels of p53 and a more pronounced induction of p21 and MDM2. These results indicate that acetylation promotes p53 stability and function. As the acetylated p53 lysine residues overlap with those that are ubiquitylated, our results suggest that one major function of p53 acetylation is to promote p53 stability by preventing MDM2-dependent ubiquitylation, while recruitment of HDAC1 by MDM2 promotes p53 degradation by removing these acetyl groups.     

XPC promotes MDM2-mediated degradation of the p53 tumor suppressor

Although ubiquitin receptor Rad23 has been implicated in bringing ubiquitylated p53 to the proteasome, how Rad23 recognizes p53 remains unclear. We demonstrate that XPC, a Rad23-binding protein, regulates p53 turnover. p53 protein in XPC-deficient cells remains ubiquitylated, but its association with the proteasome is drastically reduced, indicating that XPC regulates a postubiquitylation event. Furthermore, we found that XPC participates in the MDM2-mediated p53 degradation pathway via direct interaction with MDM2. XPC W690S pathogenic mutant is specifically defective for MDM2 binding and p53 degradation. p53 is known to become stabilized following UV irradiation but can be rendered unstable by XPC overexpression, underscoring a critical role of XPC in p53 regulation. Elucidation of the proteolytic role of XPC in cancer cells will help to unravel the detailed mechanisms underlying the coordination of DNA repair and proteolysis.     

Etoposide (VP-16) sensitizes p53-deficient human non-small cell lung cancer cells to caspase-7-mediated apoptosis

Human non-small-cell-lung-cancer (NSCLC) cells of _p53-null genotype were exposed to low-dosage topoisomearse II inhibitor etoposide (VP-16). The cellular proliferation rate could be effectively inhibited by VP-16 in dose-dependent manner. The effective drug concentration for growth inhibition could be as low as 0.5 M and the apoptotic phenotype became evident 48 h later. In H1299 cells, VP-16-induced cytotoxic effect was demonstrated associated with apoptosis that disappeared when restored with wild-type p53. Cell cycle analysis revealed that, upon VP-16 induction, cell death began with growth arrest by accumulating cells at the G₂-M phase. The cells at sub-G₁ phase increased at the expense of those at G₂-M transition state. To assess the regulation of cell cycle modulators, western blot analysis of H1299 cell lysates showed the release of apoptosis initiator, cytochrome c and apaf-1 hours following drug induction. The cleavage of downstream effectors, procaspase-9 and procaspase-7, but not procaspase-3, was accompanied with proteolysis of poly-(ADP-ribose) polymerase (PARP). VP-16-activated procaspase-7 cleavage was abrogated in cells with ectopically expressed p53.On the other hand, the inhibited procaspase-7 fragmentation by caspase-specific inhibitor reversed apoptotic phenotype caused by drug induction. Thus, VP-16-induced apoptotic cell death was contributed by caspase-7 activation in_p53-deficient human NSCLC cells.     

Polo-like kinase-1 phosphorylates MDM2 at Ser260 and stimulates MDM2-mediated p53 turnover

The E3 ubiqutin ligase, murne double-minute clone 2 (MDM2), promotes the degradation of p53 under normal homeostatic conditions. Several serine residues within the acidic domain of MDM2 are phosphorylated to maintain its activity but become hypo-phosphorylated following DNA damage, leading to inactivation of MDM2 and induction of p53. However, the signalling pathways that mediate these phosphorylation events are not fully understood. Here we show that the oncogenic and cell cycle-regulatory protein kinase, polo-like kinase-1 (PLK1), phosphorylates MDM2 at one of these residues, Ser260, and stimulates MDM2-mediated turnover of p53. These data are consistent with the idea that deregulation of PLK1 during tumourigenesis may help suppress p53 function.

Structured summary

MINT-7266353: MDM2 (uniprotkb:Q00987) physically interacts (MI:0915) with PLK1 (uniprotkb:P53350) by pull down (MI:0096)MINT-7266344, MINT-7266329: MDM2 (uniprotkb:Q00987) physically interacts (MI:0915) with PLK1 (uniprotkb:P53350) by anti bait coimmunoprecipitation (MI:0006)MINT-7266250: PLK1 (uniprotkb:P53350) phosphorylates (MI:0217) p53 (uniprotkb:P04637) by protein kinase assay (MI:0424)MINT-7266241, MINT-7266318: PLK1 (uniprotkb:P53350) phosphorylates (MI:0217) MDM2 (uniprotkb:P23804) by protein kinase assay (MI:0424)MINT-7266231, MINT-7266805, MINT-7266264, MINT-7266299: PLK1 (uniprotkb:P53350) phosphorylates (MI:0217) MDM2 (uniprotkb:Q00987) by protein kinase assay (MI:0424)     

Proteasome activator PA28 gamma regulates p53 by enhancing its MDM2-mediated degradation

Downregulation of p53 by MDM2-mediated proteasomal degradation makes cells resistant to apoptosis. The MDM2-p53 interaction is well characterized, but the mechanisms that regulate the interaction are not well understood. Here, we show that PA28gamma, a proteasome activator that inhibits apoptosis and promotes cell cycle progression through unknown mechanisms, exerts an effect as a cofactor in the MDM2-p53 interaction. The polymer form of PA28gamma interacts with both MDM2 and p53 proteins and facilitates their physical interaction. This promotes ubiquitination- and MDM2-dependent proteasomal degradation of p53, limiting its accumulation and resulting in inhibited apoptosis after DNA damage. Elimination of endogenous PA28gamma in human cancer cells abrogates MDM2-mediated p53 degradation, increases the activity of p53, and enhances apoptosis. These findings reveal the mechanism by which PA28gamma affects apoptosis and proliferation. Manipulation of the level of PA28gamma, an approach that would regulate the cellular content of p53, may improve the efficacy of current cancer therapies.     

AKT-dependent phosphorylation of Niban regulates nucleophosmin- and MDM2-mediated p53 stability and cell apoptosis

Although Niban is highly expressed in human cancer cells, the cellular functions of Niban remain largely unknown. We demonstrate here that ultraviolet irradiation induces phosphorylation of Niban at S602 by AKT, which increases the association of Niban with nucleophosmin and disassociation of nucleophosmin from the MDM2 complex. This leads to the promotion of MDM2-p53 interaction and subsequent p53 degradation, thereby providing an antiapoptotic effect. Conversely, depletion of or deficiency in Niban expression promotes stabilization of p53 with increased cell apoptosis. Our findings illustrate a pivotal role for AKT-mediated phosphorylation of Niban in protecting cells from genotoxic stress-induced cell apoptosis.     

Mechanistic studies of MDM2-mediated ubiquitination in p53 regulation

As a central regulator for cell cycle arrest, apoptosis, and cellular senescence, p53 requires multiple layers of regulatory control to ensure correct temporal and spatial functions. It is well accepted that Mdm2-mediated ubiquitination plays a crucial role in p53 regulation. In addition to proteasome-mediated degradation, ubiquitination of p53 by Mdm2 acts a key signal for its nuclear export. Nuclear export has previously been thought to require the disassociation of the p53 tetramer and exposure of the intrinsic nuclear export signal. To elucidate the molecular mechanism of degradation-independent repression on p53 by Mdm2, we have developed a two-step approach to purify ubiquitinated forms of p53 induced by Mdm2 from human cells. Surprisingly, however, we found that ubiquitination has no effect on the tetramerization/oligomerization of p53, arguing against this seemingly well accepted model. Moreover, nuclear export of p53 alone is not sufficient to completely abolish p53 activity. Ubiquitination-mediated repression of p53 by Mdm2 acts at least, in part, through inhibiting the sequence-specific DNA binding activity. Thus, our results have important implications regarding the mechanisms by which Mdm2 acts on p53.     

E2F1 inhibits MDM2 expression in a p53-dependent manner

MDM2 expression is down-regulated upon E2F1 over-expression, but the mechanism is not well defined. In the current study, we found that E2F1 inhibits MDM2 expression by suppressing its promoter activity. Although E2F1 binds to the MDM2 promoter, the inhibitory effect of E2F1 on the MDM2 promoter does not require the direct binding. We demonstrate that E2F1 inhibits MDM2 promoter activity in a p53-dependent manner. Knockdown of p53 in U2OS cells impairs the inhibitory effect of E2F1 on the MDM2 promoter. Consistent with this observation, E2F1 does not inhibit MDM2 promoter activity in p53-deficient H1299 cells, and the inhibition is restored when p53 is expressed exogenously. Both E2F1 and p53 are up-regulated after DNA damage stimulation. We show that such stimulation induces E2F1 to inhibit MDM2 promoter activity and promote p53 accumulation. Furthermore, inhibition of MDM2 by E2F1 promotes E2F1 induced apoptosis. These data suggest that E2F1 regulates the MDM2-p53 pathway by inhibiting p53 induced up-regulation of MDM2.     

Nucleocytoplasmic shuttling of p53 is essential for MDM2-mediated cytoplasmic degradation but not ubiquitination

As a shuttling protein, p53 is constantly transported through the nuclear pore complex. p53 nucleocytoplasmic transport is carried out by a bipartite nuclear localization signal (NLS) located at its C-terminal domain and two nuclear export signals (NES) located in its N- and C-terminal regions, respectively. The role of nucleocytoplasmic shuttling in p53 ubiquitination and degradation has been a subject of debate. Here we show that the two basic amino acid groups in the p53 bipartite NLS function collaboratively to import p53. Mutations disrupting individual amino acids in the NLS, although causing accumulation of p53 in the cytoplasm to various degrees, reduce but do not eliminate the NLS activity, and these mutants remain sensitive to MDM2 degradation. However, disrupting both parts of the bipartite NLS completely blocks p53 from entering the nucleus and causes p53 to become resistant to MDM2-mediated degradation. Similarly, mutations disrupting four conserved hydrophobic amino acids in the p53 C-terminal NES block p53 export and prohibit it from MDM2 degradation. We also show that colocalization of a nonshuttling p53 with MDM2 either in the nucleus or in the cytoplasm is sufficient for MDM2-induced p53 polyubiquitination but not degradation. Our data provide new insight into the mechanism and regulation of p53 nucleocytoplasmic shuttling and degradation.     

Mitochondrial inhibitor sensitizes non-small-cell lung carcinoma cells to TRAIL-induced apoptosis by reactive oxygen species and Bcl-XL/p53-mediated amplification mechanisms

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising agent for anticancer therapy; however, non-small-cell lung carcinoma (NSCLC) cells are relatively TRAIL resistant. Identification of small molecules that can restore NSCLC susceptibility to TRAIL-induced apoptosis is meaningful. We found here that rotenone, as a mitochondrial respiration inhibitor, preferentially increased NSCLC cells sensitivity to TRAIL-mediated apoptosis at subtoxic concentrations, the mechanisms by which were accounted by the upregulation of death receptors and the downregulation of c-FLIP (cellular FLICE-like inhibitory protein). Further analysis revealed that death receptors expression by rotenone was regulated by p53, whereas c-FLIP downregulation was blocked by Bcl-X_L overexpression. Rotenone triggered the mitochondria-derived reactive oxygen species (ROS) generation, which subsequently led to Bcl-X_L downregulation and PUMA upregulation. As PUMA expression was regulated by p53, the PUMA, Bcl-X_L and p53 in rotenone-treated cells form a positive feedback amplification loop to increase the apoptosis sensitivity. Mitochondria-derived ROS, however, promote the formation of this amplification loop. Collectively, we concluded that ROS generation, Bcl-X_L and p53-mediated amplification mechanisms had an important role in the sensitization of NSCLC cells to TRAIL-mediated apoptosis by rotenone. The combined TRAIL and rotenone treatment may be appreciated as a useful approach for the therapy of NSCLC that warrants further investigation.Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has emerged as a promising cancer therapeutic because it can selectively induce apoptosis in tumor cells in vitro, and most importantly, in vivo with little adverse effect on normal cells.¹ However, a number of cancer cells are resistant to TRAIL, especially highly malignant tumors such as lung cancer.^{2, 3} Lung cancer, especially the non-small-cell lung carcinoma (NSCLC) constitutes a heavy threat to human life. Presently, the morbidity and mortality of NSCLC has markedly increased in the past decade,⁴ which highlights the need for more effective treatment strategies.TRAIL has been shown to interact with five receptors, including the death receptors 4 and 5 (DR4 and DR5), the decoy receptors DcR1 and DcR2, and osteoprotegerin.⁵ Ligation of TRAIL to DR4 or DR5 allows for the recruitment of Fas-associated protein with death domain (FADD), which leads to the formation of death-inducing signaling complex (DISC) and the subsequent activation of caspase-8/10.⁶ The effector caspase-3 is activated by caspase-8, which cleaves numerous regulatory and structural proteins resulting in cell apoptosis. Caspase-8 can also cleave the Bcl-2 inhibitory BH3-domain protein (Bid), which engages the intrinsic apoptotic pathway by binding to Bcl-2-associated X protein (Bax) and Bcl-2 homologous antagonist killer (BAK). The oligomerization between Bcl-2 and Bax promotes the release of cytochrome c from mitochondria to cytosol, and facilitates the formation of apoptosome and caspase-9 activation.⁷ Like caspase-8, caspase-9 can also activate caspase-3 and initiate cell apoptosis. Besides apoptosis-inducing molecules, several apoptosis-inhibitory proteins also exist and have function even when apoptosis program is initiated. For example, cellular FLICE-like inhibitory protein (c-FLIP) is able to suppress DISC formation and apoptosis induction by sequestering FADD.^{8, 9, 10, 11}Until now, the recognized causes of TRAIL resistance include differential expression of death receptors, constitutively active AKT and NF-κB,^{12, 13} overexpression of c-FLIP and IAPs, mutations in Bax and BAK gene.² Hence, resistance can be overcome by the use of sensitizing agents that modify the deregulated death receptor expression and/or apoptosis signaling pathways in cancer cells.⁵ Many sensitizing agents have been developed in a variety of tumor cell models.² Although the clinical effectiveness of these agents needs further investigation, treatment of TRAIL-resistant tumor cells with sensitizing agents, especially the compounds with low molecular weight, as well as prolonged plasma half-life represents a promising trend for cancer therapy.Mitochondria emerge as intriguing targets for cancer therapy. Metabolic changes affecting mitochondria function inside cancer cells endow these cells with distinctive properties and survival advantage worthy of drug targeting, mitochondria-targeting drugs offer substantial promise as clinical treatment with minimal side effects.^{14, 15, 16} Rotenone is a potent inhibitor of NADH oxidoreductase in complex I, which demonstrates anti-neoplastic activity on a variety of cancer cells.^{17, 18, 19, 20, 21} However, the neurotoxicity of rotenone limits its potential application in cancer therapy. To avoid it, rotenone was effectively used in combination with other chemotherapeutic drugs to kill cancerous cells.²²In our previous investigation, we found that rotenone was able to suppress membrane Na⁺,K⁺-ATPase activity and enhance ouabain-induced cancer cell death.²³ Given these facts, we wonder whether rotenone may also be used as a sensitizing agent that can restore the susceptibility of NSCLC cells toward TRAIL-induced apoptosis, and increase the antitumor efficacy of TRAIL on NSCLC. To test this hypothesis, we initiated this study.     

Cytokines inhibit p53-mediated apoptosis but not p53-mediated G1 arrest.

Murine erythroleukemia cells that lack endogenous p53 expression were transfected with a temperature-sensitive p53 allele. The temperature-sensitive p53 protein behaves as a mutant polypeptide at 37 degrees C and as a wild-type polypeptide at 32 degrees C. Three independent clones expressing the temperature-sensitive p53 protein were characterized with respect to p53-mediated G1 cell cycle arrest, apoptosis, and differentiation. Clone ts5.203 responded to p53 activation at 32 degrees C by undergoing G1 arrest, apoptosis, and differentiation. Apoptosis was seen in cells representative of all phases of the cell cycle and was not restricted to cells arrested in G1. The addition of a cytokine (erythropoietin, c-kit ligand, or interleukin-3) to the culture medium of ts5.203 cells blocked p53-mediated apoptosis and differentiation but not p53-mediated G1 arrest. These observations indicate that apoptosis and G1 arrest can be effectively uncoupled through the action of cytokines acting as survival factors and are consistent with the idea that apoptosis and G1 arrest represent separate functions of p53. Clones ts15.15 and tsCB3.4 responded to p53 activation at 32 degrees C by undergoing G1 arrest but not apoptosis. We demonstrate that tsCB3.4 secretes a factor with erythropoietin-like activity and that ts15.15 secretes a factor with interleukin-3 activity and suggest that autocrine secretion of these cytokines blocks p53-mediated apoptosis. These data provide a framework in which to understand the variable responses of cells to p53 overexpression.     

The Regulation of the p53-mediated Stress Response by MDM2 and MDM4

Exquisite control of the activity of p53 is necessary for mammalian survival. Too much p53 is lethal, whereas too little permits tumorigenesis. MDM2 and MDM4 are structurally related proteins critical for the control of p53 activity during development, homeostasis, and the response to stress. These two essential proteins regulate both the activation of p53 in response to stress and the recovery of cells following resolution of the damage, yet both are oncogenic when overexpressed. Thus, multiple regulatory circuits ensure that their activities are fine-tuned to promote tumor-free survival. Numerous diverse stressors activate p53, and much research has gone into trying to find commonalities between them that would explain the mechanism by which p53 becomes active. It is now clear that although these diverse stressors activate p53 by different biochemical pathways, one common feature is the effort they direct, through a variety of means, toward disrupting the functions of both MDM2 and MDM4. This article provides an overview of the relationship between MDM2 and MDM4, features the various biochemical mechanisms by which p53 is activated through inhibition of their functions, and proposes some emerging areas for investigation of the p53-mediated stress response.Regulation of the p53-mediated stress response by the essential inhibitory proteins MDM2 and MDM4 is critical for survival. In response to stressors such as ionizing radiation, p53 induces a number of potentially lethal but tumor-suppressive processes, including cell cycle arrest, senescence, and apoptosis (reviewed by Horn and Vousden 2007). Both MDM2 and MDM4 are critical to surviving the p53-mediated stress response to whole body ionizing irradiation as mice with reduced levels of either protein undergo p53-dependent death after exposure to doses of radiation that are sublethal to wild-type mice (Mendrysa et al. 2003; Terzian et al. 2007). MDM2 and MDM4 are also required to control p53 function during development, as shown by the early embryonic death of mice lacking either MDM2 or MDM4, unless they also lack p53 (Jones et al. 1995; Montes de Oca Luna et al. 1995; Parant et al. 2001; Migliorini et al. 2002).Although both MDM2 and MDM4 are essential for development, they are detrimental to long-term survival when in excess, because both are oncogenic. Both MDM2 and MDM4 confer the tumorigenic phenotype on cultured cells when experimentally overexpressed (Fakharzadeh et al. 1991; Danovi et al. 2004). In addition, targeted expression of MDM2 in the mammary gland results in tumorigenesis (Lundgren et al. 1997). In people, single nucleotide polymorphisms that reduce expression of either of the orthologs of MDM2 or MDM4 (also referred to as Hdm2 and Hdm4) correlate with increased risk for breast cancer (Bond et al. 2004; Atwal et al. 2009). Approximately 10% of human tumors have been found to overexpress either MDM2 or MDM4 and many of these express wild-type p53 (reviewed in Toledo and Wahl 2006). Because the majority of human cancers express mutant forms of p53, overexpression of MDM2 and MDM4 in the subset of tumors expressing wild-type p53 supports the notion that excessive MDM2 and MDM4 promote tumorigenesis, at least in part, by blocking p53 function. Thus, limiting the activities of MDM2 and MDM4 is important to prevent cancer.