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TRIAD1 inhibits MDM2-mediated p53 ubiquitination and degradation
FEBS Letters 586 (2012) 3057–3063
journal homepage: www.FEBSLetters.org
TRIAD1 inhibits MDM2-mediated p53 ubiquitination and degradation Seunghee Bae a, Jin Hyuk Jung a,b, Karam Kim a, In-Sook An c, Soo-Yeon Kim c, Jae Ho Lee d, In-Chul Park e, Young-Woo Jin f, Su-Jae Lee g, Sungkwan An a,c,⇑ a
Molecular-Targeted Drug Research Centre, Konkuk University, Seoul 143-701, Republic of Korea Section on Developmental and Stem Cell Biology, Department of Medicine, Joslin Diabetes Center, Harvard Medical School, Boston, MA 02215, USA c Korea Institute for Skin and Clinical Sciences, Konkuk University, Seoul 143-701, Republic of Korea d Laboratory of Molecular Oncology, Cheil General Hospital & Women’s Healthcare Center, Kwandong University, College of Medicine, Seoul 100-380, Republic of Korea e Laboratory of Functional Genomics, Korea Institute of Radiological and Medical Sciences, Seoul 139-706, Republic of Korea f Division of Radiation Effect Research, Radiation Health Research Institute of KHNP, Seoul 132-703, Republic of Korea g Department of Chemistry, Hanyang University, Seoul 133-791, Republic of Korea b
a r t i c l e
i n f o
Article history: Received 8 May 2012 Revised 4 July 2012 Accepted 5 July 2012 Available online 20 July 2012 Edited by Varda Rotter Keywords: p53 MDM2 Ubiquitination TRIAD1
a b s t r a c t 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 p53MDM2 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) Mdm2 physically interacts with Triad1 by anti tag coimmunoprecipitation (View interaction) p53 physically interacts with Mdm2 by anti tag coimmunoprecipitation (View interaction) Triad1 binds to p53 by pull down (View interaction) Mdm2 physically interacts with p53 by anti tag coimmunoprecipitation (View interaction) p53 physically interacts with Triad1 by anti tag coimmunoprecipitation (View interaction) Ó 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
1. Introduction The ubiquitination-mediated proteasomal degradation pathway is a predominant regulatory mechanism that promotes rapid p53 degradation. Although acetylation and phosphorylation are key processes associated with p53 control during cellular stress, p53 ubiquitination is directly related to its stability because it disrupts interactions with p53-specific ubiquitin E3 ligases [1]. Several studies have investigated the antagonistic action of MDM2 in the MDM2-p53 axis by using chemical antagonists and other molecules involved in the signaling pathways. Nutlin3 is a Abbreviations: ARF, ADP ribosylation factor; GST, glutathione S-transferase; MDM2, murine double minute; p53, tumor suppressor protein 53; RING, really interesting new gene; TRIAD1, two RING fingers and DRIL 1 ⇑ Corresponding author at: Molecular-Targeted Drug Research Centre and Korea Institute for Skin and Clinical Sciences, Konkuk University, 1 Hwayang-dong, Gwangjin-gu, Seoul 143-701, Republic of Korea. Fax: +82 2 3437 4055. E-mail address: [email protected] (S. An).
synthetic antagonist of MDM2 that stabilizes p53 by inhibiting the p53-MDM2 interaction [2]. ADP ribosylation factor (ARF), a tumor suppressor that is deleted in several cancers, activates p53 by inhibiting its interaction with MDM2 [3,4], and ribosomal proteins such as L11, L23, and L5 inhibit MDM2 activity and disrupt p53 regulation [5]. Ubiquitination can also regulate the p53-MDM2 axis. The herpes virus-associated ubiquitin-specific protease is capable of deubiquitinating both p53 and MDM2 and regulates MDM2 stability [6]. Moreover, HDM2 is ubiquitinated by P300/CBP-associated factor (PCAF) and undergoes proteolysis via a proteasome-dependent mechanism that results in p53 activation [7]. Thus, mechanisms that govern the interaction between p53 and MDM2 are important for p53 activity regulation. We previously reported two RING fingers and DRIL 1 (TRIAD1)mediated apoptosis in various cell lines. In particular, we showed that p53 is indispensible for TRIAD1-mediated apoptosis in several cancer cell lines and that TRIAD1 induces p53 transactivation [8]. However, a definitive mechanism for TRIAD1-mediated p53
0014-5793/$36.00 Ó 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.febslet.2012.07.022
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activation has not been elucidated. In the present study, we demonstrate that TRIAD1 induces p53 activation by promoting the dissociation of p53 from MDM2. These results indicate a novel TRIAD1-p53-MDM2 regulatory model. 2. Materials and methods 2.1. Plasmids Human TRIAD1 cDNA and fragments (1–246 amino acids [a.a.] and 247–493 a.a.) were subcloned into pcDNA3.1-Myc/His (Invitrogen, Carlsbad, CA), pEGFP-C1 (Clontech, Mountain View, CA), and pGEX6p (Promega, Madison, WI) vectors. Human MDM2 cDNA was subcloned into a pCMV-Tag2-FLAG vector (Stratagene, La Jolla, CA). Human p53 cDNA and fragments (1–61 a.a., 1–92 a.a., 1–292 a.a., 1–359 a.a., and 1–393 a.a.) were cloned into pcDNA3.1-HA (Invitrogen) and pGEX6p (Promega) vectors. RING domain-mutant MDM2 (MDM2-C438A) was generated using a QuikChange sitedirected mutagenesis kit (Stratagene) according to the manufacturer’s instructions. The p53 C135Y mutant was kindly provided by Dr. Carl G. Maki (University of Chicago, Chicago, IL). The HA-ubiquitin (HA-Ub) plasmid pMT123 was kindly provided by Dr. Dirk Bohmann (University of Rochester, Rochester, NY). 2.2. Antibodies and reagents Antibodies against p53 (DO-1 and FL-393), HA (Y-11 and F-7), MDM2 (SMP14 and D-12), TRIAD1 (F-23), and ubiquitin (P4D1), as well as normal mouse and rabbit IgGs and protein A/G PLUS-agarose beads were from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-actin and anti-FLAG antibodies were purchased from Sigma–Aldrich (St. Louis, MO). Anti-Myc-tag and anti-GFP antibodies were purchased from Cell Signaling Technology (Danvers, MA). 2.3. Cell culture and transfection Wild-type mouse embryonic fibroblasts (MEFs), p53/ MEF, and p53/mdm2/ MEF cells were maintained in Dulbecco’s minimum essential media (Invitrogen) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (Invitrogen). U2OS cells were maintained in RPMI 1640 media with 10% FBS and 1% penicillin/streptomycin. Transient transfections were carried out using HilyMax (Dojindo Laboratories, Kumanoto, Japan) according to the manufacturer’s instructions. 2.4. TRIAD1 stable knockdown For the shRNA-mediated knockdown of TRIAD1, pairs of synthetic DNA oligos (forward, 50 -GGACCGGTGGGAGGACTATTACGT GGGAGTACTCGAGTAC-30 ; reverse, 50 -CCGGAATTCCCAAAAAGAGGACTATTACGTGGGAGTACTCGA-GTAC-30 ) were annealed in 50-ll volumes with 20 lM of each oligo in PCR buffer, 250 lM dNTP, and 1.25 units of TaKaRa Ex-Taq DNA polymerase (TaKaRa Bio Inc., Ohtsu, Japan). The samples were incubated at 95 °C for 4 min, 30 °C for 10 min, and 72 °C for 60 min. The shRNA duplex oligos were cloned into pLKO.1 puro (Addgene, Cambridge, MA). A TRIAD1 stable knockdown cell line was established using lentiviral gene-transfer. Briefly, 293T cells were cotransfected with TRIAD1 shRNA lentiviral transfer, pCMV-delta8.9, and pVpackVSV-G plasmids (Stratagene). At 48 h after transfection, viral particle-containing medium was collected and used to infect U2OS cells, which were incubated with 1 lg/ml puromycin to establish TRIAD1-knockdown cells.
2.5. Ubiquitination assays In vivo ubiquitination assay was performed as previously described [27]. Briefly, transfected and MG132-treated cells were washed with PBS and then lysed in 200 ll of SDS-containing lysis buffer (50 mM Tris–HCl, pH 7.5, 70 mM b-mercaptoethanol, 0.5% SDS) by vortexing and boiling for 10 min at 95 °C. The lysates were diluted with 800 ll of NP-40 lysis buffer (50 mM Tris–HCl, pH 7.5; 150 mM NaCl; 1% NP-40; 1 mM DTT; 1 mM EDTA; 10 lg lysozyme; and 0.5 mM PMSF). The lysates were immunoprecipitated with indicated antibody and protein agarose A/G PLUS-Agarose beads. The beads were washed five times in NP-40 buffer and then boiled in SDS–PAGE sample buffer. The bound ubiquitinated p53 proteins were analyzed by immunoblotting using an indicated antibody. 2.6. In vitro binding assay GST and GST-TRIAD1 proteins were expressed in Escherichia coli BL21 at 25 °C with 0.1 mM IPTG for overnight. The cells were collected and resuspended in NP-40 lysis buffer. The recombinant proteins were isolated and eluted using glutathione-Sepharose 4B beads (GE Healthcare, Uppsala, Sweden) according to the manufacturer’s instructions. 35S-labeled p53 protein obtained by in vitro transcription/translation was incubated with 20 ll 50% glutathione-Sepharose 4B-bound GST or GST-TRIAD1 recombinant protein for 3 h at 37 °C in binding buffer (30 mM Tris–HCl, pH 8.0; 0.1 mM EDTA; 0.1 mM NaCl; 1 mM DTT; 1% NP-40; and 0.5 mM PMSF), washed five times, and boiled in SDS-sample buffer. The bound proteins were separated by SDS–PAGE and visualized by autoradiography. 2.7. Immunoprecipitation and immunoblotting For the immunoprecipitation assay, cell lysates in CHAPS buffer (0.5% CHAPS; 10 mM Tris–HCl, pH 7.5; 1 mM MgCl2; 1 mM EGTA; 5 mM b-mercaptoethanol; 10% glycerol) were incubated overnight with the indicated antibodies at 4 °C. After adding protein A/G PLUS-agarose beads (Santa Cruz Biotechnology), the cell lysates were further incubated for 2 h, and washed extensively. The immunoblotting assay was carried out as previously described. Western blot band intensities were compared with the Java-based image-processing program ImageJ (National Institutes of Health, Bethesda, MD), according to the software developers’ protocol. Briefly, the developed X-ray films were scanned on a flatbed scanner. The blot images were opened with ImageJ, each lane in the blot was selected, and the band densities were measured. All p53 band intensities were normalized to the respective b-actin intensity to quantify relative p53 expression. These values are presented as a ratio and graphically represented. 2.8. Cell viability assays Cell proliferation ratios were measured using an MTS assay kit (Promega) according to the manufacturer’s instructions. Briefly, cells (1 103) were transfected with GFP, GFP-TRIAD1, HA-p53, or FLAG-MDM2 expression plasmids and further cultured for 48 h. MTS/PMS solution was added to each well and incubated at 37 °C for 2 h. Absorbance was measured at 490 nm using a microplate reader. The cell proliferation ratio relative to that of control cells was graphically represented. 2.9. Colony formation assays Cells (4 104) were transfected with the GFP, GFP-TRIAD1, HAp53, or FLAG-MDM2 plasmids, incubated for 24 h, and selected
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with G418 (500 lg/ml) for 14 days. Colonies were fixed and stained with 0.1% crystal violet. 2.10. Statistical analysis Statistical analysis was performed using two-tailed Student’s t-tests. Values of P < 0.05 were considered significant. 3. Results 3.1. TRIAD1 is involved in p53 activation following DNA damage We previously demonstrated that TRIAD1 induces apoptosis by p53 activation [8]. These results led to us to evaluate the specific role of TRIAD1 by measuring p53 accumulation in camptothecin (CPT)-exposed TRIAD1-knockdown U2OS cells. Stable TRIAD1knockdown U2OS cells were generated by using lentiviral shTRIAD1 (see Section 2). Knockdown was confirmed by quantitative RT-PCR (data not shown). We observed that p53 protein levels increased following CPT treatment in U2OS control cells in a dosedependent manner (left, Fig. 1A). However, these increased levels of p53 protein were largely attenuated in TRIAD1-knockdown U2OS cells (right, Fig. 1A). Furthermore, the expression levels of targets downstream of p53, such as p21 and PUMA, were attenuated by TRIAD1 knockdown following CPT exposure (Fig. 1B). We also observed that ectopic TRIAD1 expression substantially increased p53 protein levels in U2OS cells (Fig. 1C). Collectively, these results indicate that TRIAD1 is required for p53 activation following DNA damage.
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3.2. TRIAD1 regulates p53 stability p53 activation is closely dependent on its stability, which is tightly regulated by various mechanisms [9,10]. Based on the observation that TRIAD1 is required for p53 activation (Fig. 1), we measured p53 stability following the ectopic expression of TRIAD1 in U2OS cells to determine whether it is regulated by TRIAD1. Indeed, p53 stability was increased in TRIAD1-transfected U2OS cells (Fig. 2A). Conversely, kinetic analyses of p53 stability following cycloheximide (CHX) treatment in TRIAD1-knockdown U2OS cells revealed that p53 half-life was shorter in TRIAD1knockdown cells than in control cells (Fig. 2B). Together, these results indicate that p53 stabilization is influenced by TRIAD1 expression. 3.3. TRIAD1 antagonizes MDM2-mediated p53 degradation Given that p53 stability is mainly regulated by the RING domain-containing E3 ubiquitin ligase MDM2 [11,12], we examined whether TRIAD1 might functionally antagonize MDM2 activity. The exogenous expression levels of both wild-type and transcriptionally mutant p53 (p53 C135Y) were increased following TRIAD1 overexpression, indicating that TRIAD1-induced p53 accumulation was independent of p53 activity (Fig. 3A). Next, we examined whether TRIAD1-mediated p53 stabilization might antagonize the activities of MDM2 E3 ligase or other E3 ligases that target p53. Interestingly, cotransfection of HA-tagged p53 and Myctagged TRIAD1 plasmids into p53/ mdm2/ MEF cells did not induce p53 accumulation compared with single HA-tagged p53
Fig. 1. Ablation of TRIAD1 reduces p53 activation following DNA damage. (A) Ablation of TRIAD1 attenuates accumulation of p53 after camptothecin (CPT) treatment. Immunoblots were performed using indicated antibodies after treatment of control and TRIAD1 knockdown U2OS cells with 5 lM CPT. (B) Ablation of TRIAD1 attenuates expression levels of genes downstream of p53 following CPT treatment. Immunoblots were performed using indicated antibodies after CPT treatment of control and TRIAD1 knockdown cells containing shTRIAD1. (C) Overexpression of TRIAD1 induced p53 accumulation in a concentration-dependent manner. The graph represents the means and standard deviations of the relative p53 intensities obtained from triplicate experiments.
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Fig. 2. p53 half-life is regulated by TRIAD1. (A) Ectopic expression of TRIAD1 increases p53 stability. At 24 h after transfection with pcDNA3 control or Myc-tagged TRIAD1 plasmid, U2OS cells were treated with CHX (150 lg/ml) as indicated. Endogenous levels of p53 were measured by immunoblotting, and residual cellular levels of p53 were quantitated by densitometry. The values are an average of two independent experiments. (B) Ablation of TRIAD1 reduces the stability of p53. Stable TRIAD1-knockdown U2OS cells were incubated with cycloheximide (CHX, 150 lg/ml) for the indicated times. The values are an average of two independent experiments.
Fig. 3. TRIAD1 inhibits MDM2-mediated p53 degradation. (A) TRIAD1-induced p53 accumulation is independent of p53 activity. p53/ MEF cells were cotransfected with plasmids as indicated. At 48 h after transfection, cell extracts were subjected to immunoblotting with the indicated antibodies. (B) TRIAD1 blocks MDM2-mediated p53 degradation. p53/mdm2/ MEF cells were cotransfected with the indicated plasmids. At 48 h after transfection, cell extracts were subjected to immunoblotting with the indicated antibodies. (C) TRIAD1 inhibits MDM2-mediated p53 ubiquitination. After transfection, p53 ubiquitination was analyzed by immunoblotting (with an anti-HA antibody, right panel) using immunoprecipitates obtained with the anti-FLAG antibody.
transfection. On the contrary, MDM2-induced p53 degradation was completely inhibited by TRIAD1 expression (Fig. 3B). Consistent with these results, TRIAD1 completely eliminated MDM2-mediated p53 ubiquitination (Fig. 3C). Collectively, these data indicate that TRIAD1 promotes p53 stability and activity, most likely by antagonizing MDM2 activity.
3.4. TRIAD1 inhibits the interaction between p53 and MDM2 A basic requirement for p53 degradation is the direct interaction between p53 and MDM2. Because TRIAD1 interacts with p53 in vitro (Fig. S1), we evaluated the interaction between p53 and MDM2 following TRIAD1 transfection. As shown in Fig. 4A and B,
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Fig. 4. The N-terminal region of TRIAD1 inhibits the interaction between p53 and MDM2. (A and B) The interaction between p53 and MDM2 is inhibited by TRIAD1. p53/ mdm2/ MEF cells were transfected with the indicated plasmids. At 24 h after transfection, cells were treated with 1 lM MG132 for 12 h. Immunoprecipitates were immunoblotted with anti-HA or anti-FLAG antibody. Whole cell lysates (WCLs) were analyzed by immunoblotting using the indicated antibodies as controls (lower panel). (C) The TRIAD1 fragment (1–246 amino acids) is indispensible for binding p53 to inhibit the interaction between p53 and MDM2. TRIAD1 fragment constructs were generated as represented in the left panel. p53/mdm2/ MEF cells were transfected with the indicated plasmids. Anti-HA immunoprecipitates were immunoblotted with anti-FLAG and anti-GFP antibodies (upper panel). a.a., amino acids.
the interaction between p53 and MDM2 was substantially decreased following TRIAD1 transfection. We next identified which region of TRIAD1 could affect p53-MDM2 interaction. Because a previous in vitro study demonstrated that RING mutant MDM2 does not affect the MDM2-p53 interaction [13], we performed a series of immunoprecipitation experiments using RING mutant MDM2 constructs (Fig. 4C). In particular, the N-terminus of TRIAD1 (1–246) was found to be critical for the dissociation of p53 from MDM2 (Fig. 4C). Interestingly, the interaction between p53 and MDM2 is orchestrated by the interaction of TRIAD1 with p53; full-length TRIAD1 and the N-terminus of TRIAD1 (1–246) were capable of binding to p53 and inhibiting its interaction with MDM2, whereas the C-terminus of TRIAD1 (247–493) was unable to do either (Fig. 4C). These results indicate that TRIAD1 hinders MDM2-mediated p53 ubiquitination by inhibiting the interaction between p53 and MDM2.
3.5. TRIAD1 interacts with the C-terminus of p53 Because TRIAD1 interacts with p53 and induces its dissociation from MDM2 (Fig. 4), we performed experiments to identify the p53 domain that is critical for TRIAD1 interaction. Several fragmented p53 constructs were prepared as indicated (Fig. 5A). Interestingly, in vitro binding assays using these fragmented constructs with a GST-TRIAD1 fusion protein revealed that the C-terminus of p53 is critical for the interaction between p53 and TRIAD1 (Fig. 5B). 3.6. TRIAD1 negatively regulates MDM2 function by inhibiting p53mediated cell growth suppression Because TRIAD1 induces the dissociation of p53 from MDM2, we evaluated whether TRIAD1 restores p53 activation in presence of MDM2. We cotransfected p53/ mdm2/ MEF cells with p53 or
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Fig. 5. The C-terminus of p53 interacts with TRIAD1. (A) Schematic representation of the generated p53 fragment constructs. The p53 fragment constructs were cloned into pcDNA3.1 vectors. (B) In vitro pull-down assay showing the interactions between in vitro-translated, 35S-labeled p53 fragments, and GST or GST-TRIAD1 after a 1-h incubation at room temperature. Bound proteins were detected by autoradiography. a.a., amino acids.
its fragmented mutant forms and MDM2 in the presence or absence of TRIAD1. We found that TRIAD1 reduces cell viability through p53 activation in the presence of ectopic MDM2 expression (Fig. 6A). The cytotoxic function of TRIAD1 was found to be defective in p53/ mdm2/ MEF cells (Fig. S2). Moreover, the Nterminal fragment of TRIAD1, which is capable of interacting with p53, induced p53-mediated cellular growth inhibition, whereas the
C-terminal fragment did not. These results indicate that TRIAD1 induces p53-mediated cellular growth inhibition by binding to p53, thereby inhibiting its interaction with MDM2. We also evaluated whether TRIAD1 restores p53-mediated clonogenic growth inhibition in the presence of MDM2. As shown in Fig. 6B, p53 transfection inhibited clonogenic growth, which was restored by TRIAD1 transfection in the presence of MDM2.
Fig. 6. TRIAD1 restores p53-mediated cell growth inhibition in the presence of MDM2. (A) p53/mdm2/ MEF cells were transfected with the indicated plasmids. At 72 h after transfection, cell viability was determined by three independent MTS assays. (B) p53/mdm2/ MEF cells were cotransfected with plasmids for HA-p53, FLAG-MDM2, GFP-TRIAD1, GFP-TRIAD1 (a.a. 1–246), GFP-TRIAD1 (a.a. 247–493), or control GFP. Colonies were counted 2 week after seeding using a standard clonogenic assay. a.a., amino acids.
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4. Discussion
References
We previously demonstrated that TRIAD1 expression induces p53 activation and subsequent apoptosis [8]. The present work examines the role of TRIAD1 in p53 stabilization and activation. We found that p53 protein levels were lower and p53 activity was decreased in TRIAD1-knockdown cells compared to control U2OS cells. Cotransfection of TRIAD1 and p53 failed to induce p53 activation in p53/ mdm2/ MEF cells, however, transfection of TRIAD1 and MDM2 inhibited MDM2-mediated p53 degradation, indicating that TRIAD1-mediated p53 activation is related to the MDM2-antagonizing function of TRIAD1. Indeed, MDM2 suppression of p53-mediated cell growth inhibition was blocked by TRIAD1 expression. Furthermore, although TRIAD1 expression did not inhibit p53/ mdm2/ MEF cell viability, it did affect cell viability in MDM2-transfected cells. Several p53-interacting proteins that function as MDM2 antagonists have been reported. For example, ARF, L5, L11, and L23 can interact with MDM2 [5] and inhibit MDM2-mediating p53 ubiquitination/degradation. However, TRIAD1 interacts with and stabilizes p53 but not MDM2. Although the transactivation domain located in the N-terminus of p53 is important for MDM2 interaction [14], a recent report suggests that the C-terminus of p53, which contains multiple lysine residues, is also important for this interaction [15]. In the present study, we demonstrated that TRIAD1 binds to the C-terminus of p53 and inhibits MDM2 interaction, as well as MDM2-mediated ubiquitination. Thus, the C-terminus of p53 is important for regulation of its activity, not only via post-translational modifications but also through its interaction with TRIAD1. Recently, TRIAD1 transcription was found to be directly promoted by the transcription factor HoxA10 and induces anti-proliferative effects in acute myeloid leukemia (AML) [16]. In addition, HoxA10 reduces breast cancer cell invasiveness by upregulating p53 protein; however, the mechanism is unknown [17]. Therefore, our results extend the possibility that HoxA10-mediated p53 upregulation is related to HoxA10-mediated TRIAD1 expression. Although ectopic TRIAD1 expression induces apoptosis in several cell lines [18], it is possible that TRIAD1 also mediates differentiation for the following reasons: (1) TRIAD1 expression levels are greatly increased following treatment with all-trans retinoic acid (ATRA), which affects cellular differentiation [19,20]; (2) growth factor independent-1 (Gfi-1) transcription repressor, which is known to be stabilized by TRIAD1, is critical for B cell and myeloid cell development and neutrophil differentiation [21,22]; and (3) p53 activation regulates differentiation in several cell lines [23–25]. Moreover, modulation of the p53-MDM2 loop by nutlin decreases preosteoclast differentiation [26]. Thus, the physiological effects of TRIAD1 on p53 activation warrant further investigation.
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(2008) P53 and Pten control neural and glioma stem/progenitor cell renewal and differentiation. Nature 455, 1129–1133. [26] Zauli, G., Rimondi, E., Corallini, F., Fadda, R., Capitani, S. and Secchiero, P. (2007) MDM2 antagonist Nutlin-3 suppresses the proliferation and differentiation of human pre-osteoclasts through a p53-dependent pathway. J. Bone Miner. Res. 22, 1621–1630. [27] Bae, S., Kim, S.Y., Jung, J.H., Yoon, Y., Cha, H.J., Lee, H., Kim, K., Kim, J., An, I.S., Kim, J., Um, H.D., Park, I.C., Lee, S.J., Nam, S.Y., Jin, Y.W., Lee, J.H. and An, S. (2012) Akt is negatively regulated by the MULAN E3 ligase. Cell Res. 22, 873– 885.
Acknowledgements We thank Dr. van der Reijden (Radboud University Nijmegen Medical Centre, Netherlands) for providing us with the GFP-TRIAD1 plasmids. This work was supported by a grant from the Ministry of Education, Science, and Technology (Grant 20110028646) of the Republic of Korea. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.febslet.2012.07. 022.
journal homepage: www.FEBSLetters.org
TRIAD1 inhibits MDM2-mediated p53 ubiquitination and degradation Seunghee Bae a, Jin Hyuk Jung a,b, Karam Kim a, In-Sook An c, Soo-Yeon Kim c, Jae Ho Lee d, In-Chul Park e, Young-Woo Jin f, Su-Jae Lee g, Sungkwan An a,c,⇑ a
Molecular-Targeted Drug Research Centre, Konkuk University, Seoul 143-701, Republic of Korea Section on Developmental and Stem Cell Biology, Department of Medicine, Joslin Diabetes Center, Harvard Medical School, Boston, MA 02215, USA c Korea Institute for Skin and Clinical Sciences, Konkuk University, Seoul 143-701, Republic of Korea d Laboratory of Molecular Oncology, Cheil General Hospital & Women’s Healthcare Center, Kwandong University, College of Medicine, Seoul 100-380, Republic of Korea e Laboratory of Functional Genomics, Korea Institute of Radiological and Medical Sciences, Seoul 139-706, Republic of Korea f Division of Radiation Effect Research, Radiation Health Research Institute of KHNP, Seoul 132-703, Republic of Korea g Department of Chemistry, Hanyang University, Seoul 133-791, Republic of Korea b
a r t i c l e
i n f o
Article history: Received 8 May 2012 Revised 4 July 2012 Accepted 5 July 2012 Available online 20 July 2012 Edited by Varda Rotter Keywords: p53 MDM2 Ubiquitination TRIAD1
a b s t r a c t 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 p53MDM2 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) Mdm2 physically interacts with Triad1 by anti tag coimmunoprecipitation (View interaction) p53 physically interacts with Mdm2 by anti tag coimmunoprecipitation (View interaction) Triad1 binds to p53 by pull down (View interaction) Mdm2 physically interacts with p53 by anti tag coimmunoprecipitation (View interaction) p53 physically interacts with Triad1 by anti tag coimmunoprecipitation (View interaction) Ó 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
1. Introduction The ubiquitination-mediated proteasomal degradation pathway is a predominant regulatory mechanism that promotes rapid p53 degradation. Although acetylation and phosphorylation are key processes associated with p53 control during cellular stress, p53 ubiquitination is directly related to its stability because it disrupts interactions with p53-specific ubiquitin E3 ligases [1]. Several studies have investigated the antagonistic action of MDM2 in the MDM2-p53 axis by using chemical antagonists and other molecules involved in the signaling pathways. Nutlin3 is a Abbreviations: ARF, ADP ribosylation factor; GST, glutathione S-transferase; MDM2, murine double minute; p53, tumor suppressor protein 53; RING, really interesting new gene; TRIAD1, two RING fingers and DRIL 1 ⇑ Corresponding author at: Molecular-Targeted Drug Research Centre and Korea Institute for Skin and Clinical Sciences, Konkuk University, 1 Hwayang-dong, Gwangjin-gu, Seoul 143-701, Republic of Korea. Fax: +82 2 3437 4055. E-mail address: [email protected] (S. An).
synthetic antagonist of MDM2 that stabilizes p53 by inhibiting the p53-MDM2 interaction [2]. ADP ribosylation factor (ARF), a tumor suppressor that is deleted in several cancers, activates p53 by inhibiting its interaction with MDM2 [3,4], and ribosomal proteins such as L11, L23, and L5 inhibit MDM2 activity and disrupt p53 regulation [5]. Ubiquitination can also regulate the p53-MDM2 axis. The herpes virus-associated ubiquitin-specific protease is capable of deubiquitinating both p53 and MDM2 and regulates MDM2 stability [6]. Moreover, HDM2 is ubiquitinated by P300/CBP-associated factor (PCAF) and undergoes proteolysis via a proteasome-dependent mechanism that results in p53 activation [7]. Thus, mechanisms that govern the interaction between p53 and MDM2 are important for p53 activity regulation. We previously reported two RING fingers and DRIL 1 (TRIAD1)mediated apoptosis in various cell lines. In particular, we showed that p53 is indispensible for TRIAD1-mediated apoptosis in several cancer cell lines and that TRIAD1 induces p53 transactivation [8]. However, a definitive mechanism for TRIAD1-mediated p53
0014-5793/$36.00 Ó 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.febslet.2012.07.022
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activation has not been elucidated. In the present study, we demonstrate that TRIAD1 induces p53 activation by promoting the dissociation of p53 from MDM2. These results indicate a novel TRIAD1-p53-MDM2 regulatory model. 2. Materials and methods 2.1. Plasmids Human TRIAD1 cDNA and fragments (1–246 amino acids [a.a.] and 247–493 a.a.) were subcloned into pcDNA3.1-Myc/His (Invitrogen, Carlsbad, CA), pEGFP-C1 (Clontech, Mountain View, CA), and pGEX6p (Promega, Madison, WI) vectors. Human MDM2 cDNA was subcloned into a pCMV-Tag2-FLAG vector (Stratagene, La Jolla, CA). Human p53 cDNA and fragments (1–61 a.a., 1–92 a.a., 1–292 a.a., 1–359 a.a., and 1–393 a.a.) were cloned into pcDNA3.1-HA (Invitrogen) and pGEX6p (Promega) vectors. RING domain-mutant MDM2 (MDM2-C438A) was generated using a QuikChange sitedirected mutagenesis kit (Stratagene) according to the manufacturer’s instructions. The p53 C135Y mutant was kindly provided by Dr. Carl G. Maki (University of Chicago, Chicago, IL). The HA-ubiquitin (HA-Ub) plasmid pMT123 was kindly provided by Dr. Dirk Bohmann (University of Rochester, Rochester, NY). 2.2. Antibodies and reagents Antibodies against p53 (DO-1 and FL-393), HA (Y-11 and F-7), MDM2 (SMP14 and D-12), TRIAD1 (F-23), and ubiquitin (P4D1), as well as normal mouse and rabbit IgGs and protein A/G PLUS-agarose beads were from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-actin and anti-FLAG antibodies were purchased from Sigma–Aldrich (St. Louis, MO). Anti-Myc-tag and anti-GFP antibodies were purchased from Cell Signaling Technology (Danvers, MA). 2.3. Cell culture and transfection Wild-type mouse embryonic fibroblasts (MEFs), p53/ MEF, and p53/mdm2/ MEF cells were maintained in Dulbecco’s minimum essential media (Invitrogen) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (Invitrogen). U2OS cells were maintained in RPMI 1640 media with 10% FBS and 1% penicillin/streptomycin. Transient transfections were carried out using HilyMax (Dojindo Laboratories, Kumanoto, Japan) according to the manufacturer’s instructions. 2.4. TRIAD1 stable knockdown For the shRNA-mediated knockdown of TRIAD1, pairs of synthetic DNA oligos (forward, 50 -GGACCGGTGGGAGGACTATTACGT GGGAGTACTCGAGTAC-30 ; reverse, 50 -CCGGAATTCCCAAAAAGAGGACTATTACGTGGGAGTACTCGA-GTAC-30 ) were annealed in 50-ll volumes with 20 lM of each oligo in PCR buffer, 250 lM dNTP, and 1.25 units of TaKaRa Ex-Taq DNA polymerase (TaKaRa Bio Inc., Ohtsu, Japan). The samples were incubated at 95 °C for 4 min, 30 °C for 10 min, and 72 °C for 60 min. The shRNA duplex oligos were cloned into pLKO.1 puro (Addgene, Cambridge, MA). A TRIAD1 stable knockdown cell line was established using lentiviral gene-transfer. Briefly, 293T cells were cotransfected with TRIAD1 shRNA lentiviral transfer, pCMV-delta8.9, and pVpackVSV-G plasmids (Stratagene). At 48 h after transfection, viral particle-containing medium was collected and used to infect U2OS cells, which were incubated with 1 lg/ml puromycin to establish TRIAD1-knockdown cells.
2.5. Ubiquitination assays In vivo ubiquitination assay was performed as previously described [27]. Briefly, transfected and MG132-treated cells were washed with PBS and then lysed in 200 ll of SDS-containing lysis buffer (50 mM Tris–HCl, pH 7.5, 70 mM b-mercaptoethanol, 0.5% SDS) by vortexing and boiling for 10 min at 95 °C. The lysates were diluted with 800 ll of NP-40 lysis buffer (50 mM Tris–HCl, pH 7.5; 150 mM NaCl; 1% NP-40; 1 mM DTT; 1 mM EDTA; 10 lg lysozyme; and 0.5 mM PMSF). The lysates were immunoprecipitated with indicated antibody and protein agarose A/G PLUS-Agarose beads. The beads were washed five times in NP-40 buffer and then boiled in SDS–PAGE sample buffer. The bound ubiquitinated p53 proteins were analyzed by immunoblotting using an indicated antibody. 2.6. In vitro binding assay GST and GST-TRIAD1 proteins were expressed in Escherichia coli BL21 at 25 °C with 0.1 mM IPTG for overnight. The cells were collected and resuspended in NP-40 lysis buffer. The recombinant proteins were isolated and eluted using glutathione-Sepharose 4B beads (GE Healthcare, Uppsala, Sweden) according to the manufacturer’s instructions. 35S-labeled p53 protein obtained by in vitro transcription/translation was incubated with 20 ll 50% glutathione-Sepharose 4B-bound GST or GST-TRIAD1 recombinant protein for 3 h at 37 °C in binding buffer (30 mM Tris–HCl, pH 8.0; 0.1 mM EDTA; 0.1 mM NaCl; 1 mM DTT; 1% NP-40; and 0.5 mM PMSF), washed five times, and boiled in SDS-sample buffer. The bound proteins were separated by SDS–PAGE and visualized by autoradiography. 2.7. Immunoprecipitation and immunoblotting For the immunoprecipitation assay, cell lysates in CHAPS buffer (0.5% CHAPS; 10 mM Tris–HCl, pH 7.5; 1 mM MgCl2; 1 mM EGTA; 5 mM b-mercaptoethanol; 10% glycerol) were incubated overnight with the indicated antibodies at 4 °C. After adding protein A/G PLUS-agarose beads (Santa Cruz Biotechnology), the cell lysates were further incubated for 2 h, and washed extensively. The immunoblotting assay was carried out as previously described. Western blot band intensities were compared with the Java-based image-processing program ImageJ (National Institutes of Health, Bethesda, MD), according to the software developers’ protocol. Briefly, the developed X-ray films were scanned on a flatbed scanner. The blot images were opened with ImageJ, each lane in the blot was selected, and the band densities were measured. All p53 band intensities were normalized to the respective b-actin intensity to quantify relative p53 expression. These values are presented as a ratio and graphically represented. 2.8. Cell viability assays Cell proliferation ratios were measured using an MTS assay kit (Promega) according to the manufacturer’s instructions. Briefly, cells (1 103) were transfected with GFP, GFP-TRIAD1, HA-p53, or FLAG-MDM2 expression plasmids and further cultured for 48 h. MTS/PMS solution was added to each well and incubated at 37 °C for 2 h. Absorbance was measured at 490 nm using a microplate reader. The cell proliferation ratio relative to that of control cells was graphically represented. 2.9. Colony formation assays Cells (4 104) were transfected with the GFP, GFP-TRIAD1, HAp53, or FLAG-MDM2 plasmids, incubated for 24 h, and selected
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with G418 (500 lg/ml) for 14 days. Colonies were fixed and stained with 0.1% crystal violet. 2.10. Statistical analysis Statistical analysis was performed using two-tailed Student’s t-tests. Values of P < 0.05 were considered significant. 3. Results 3.1. TRIAD1 is involved in p53 activation following DNA damage We previously demonstrated that TRIAD1 induces apoptosis by p53 activation [8]. These results led to us to evaluate the specific role of TRIAD1 by measuring p53 accumulation in camptothecin (CPT)-exposed TRIAD1-knockdown U2OS cells. Stable TRIAD1knockdown U2OS cells were generated by using lentiviral shTRIAD1 (see Section 2). Knockdown was confirmed by quantitative RT-PCR (data not shown). We observed that p53 protein levels increased following CPT treatment in U2OS control cells in a dosedependent manner (left, Fig. 1A). However, these increased levels of p53 protein were largely attenuated in TRIAD1-knockdown U2OS cells (right, Fig. 1A). Furthermore, the expression levels of targets downstream of p53, such as p21 and PUMA, were attenuated by TRIAD1 knockdown following CPT exposure (Fig. 1B). We also observed that ectopic TRIAD1 expression substantially increased p53 protein levels in U2OS cells (Fig. 1C). Collectively, these results indicate that TRIAD1 is required for p53 activation following DNA damage.
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3.2. TRIAD1 regulates p53 stability p53 activation is closely dependent on its stability, which is tightly regulated by various mechanisms [9,10]. Based on the observation that TRIAD1 is required for p53 activation (Fig. 1), we measured p53 stability following the ectopic expression of TRIAD1 in U2OS cells to determine whether it is regulated by TRIAD1. Indeed, p53 stability was increased in TRIAD1-transfected U2OS cells (Fig. 2A). Conversely, kinetic analyses of p53 stability following cycloheximide (CHX) treatment in TRIAD1-knockdown U2OS cells revealed that p53 half-life was shorter in TRIAD1knockdown cells than in control cells (Fig. 2B). Together, these results indicate that p53 stabilization is influenced by TRIAD1 expression. 3.3. TRIAD1 antagonizes MDM2-mediated p53 degradation Given that p53 stability is mainly regulated by the RING domain-containing E3 ubiquitin ligase MDM2 [11,12], we examined whether TRIAD1 might functionally antagonize MDM2 activity. The exogenous expression levels of both wild-type and transcriptionally mutant p53 (p53 C135Y) were increased following TRIAD1 overexpression, indicating that TRIAD1-induced p53 accumulation was independent of p53 activity (Fig. 3A). Next, we examined whether TRIAD1-mediated p53 stabilization might antagonize the activities of MDM2 E3 ligase or other E3 ligases that target p53. Interestingly, cotransfection of HA-tagged p53 and Myctagged TRIAD1 plasmids into p53/ mdm2/ MEF cells did not induce p53 accumulation compared with single HA-tagged p53
Fig. 1. Ablation of TRIAD1 reduces p53 activation following DNA damage. (A) Ablation of TRIAD1 attenuates accumulation of p53 after camptothecin (CPT) treatment. Immunoblots were performed using indicated antibodies after treatment of control and TRIAD1 knockdown U2OS cells with 5 lM CPT. (B) Ablation of TRIAD1 attenuates expression levels of genes downstream of p53 following CPT treatment. Immunoblots were performed using indicated antibodies after CPT treatment of control and TRIAD1 knockdown cells containing shTRIAD1. (C) Overexpression of TRIAD1 induced p53 accumulation in a concentration-dependent manner. The graph represents the means and standard deviations of the relative p53 intensities obtained from triplicate experiments.
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Fig. 2. p53 half-life is regulated by TRIAD1. (A) Ectopic expression of TRIAD1 increases p53 stability. At 24 h after transfection with pcDNA3 control or Myc-tagged TRIAD1 plasmid, U2OS cells were treated with CHX (150 lg/ml) as indicated. Endogenous levels of p53 were measured by immunoblotting, and residual cellular levels of p53 were quantitated by densitometry. The values are an average of two independent experiments. (B) Ablation of TRIAD1 reduces the stability of p53. Stable TRIAD1-knockdown U2OS cells were incubated with cycloheximide (CHX, 150 lg/ml) for the indicated times. The values are an average of two independent experiments.
Fig. 3. TRIAD1 inhibits MDM2-mediated p53 degradation. (A) TRIAD1-induced p53 accumulation is independent of p53 activity. p53/ MEF cells were cotransfected with plasmids as indicated. At 48 h after transfection, cell extracts were subjected to immunoblotting with the indicated antibodies. (B) TRIAD1 blocks MDM2-mediated p53 degradation. p53/mdm2/ MEF cells were cotransfected with the indicated plasmids. At 48 h after transfection, cell extracts were subjected to immunoblotting with the indicated antibodies. (C) TRIAD1 inhibits MDM2-mediated p53 ubiquitination. After transfection, p53 ubiquitination was analyzed by immunoblotting (with an anti-HA antibody, right panel) using immunoprecipitates obtained with the anti-FLAG antibody.
transfection. On the contrary, MDM2-induced p53 degradation was completely inhibited by TRIAD1 expression (Fig. 3B). Consistent with these results, TRIAD1 completely eliminated MDM2-mediated p53 ubiquitination (Fig. 3C). Collectively, these data indicate that TRIAD1 promotes p53 stability and activity, most likely by antagonizing MDM2 activity.
3.4. TRIAD1 inhibits the interaction between p53 and MDM2 A basic requirement for p53 degradation is the direct interaction between p53 and MDM2. Because TRIAD1 interacts with p53 in vitro (Fig. S1), we evaluated the interaction between p53 and MDM2 following TRIAD1 transfection. As shown in Fig. 4A and B,
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Fig. 4. The N-terminal region of TRIAD1 inhibits the interaction between p53 and MDM2. (A and B) The interaction between p53 and MDM2 is inhibited by TRIAD1. p53/ mdm2/ MEF cells were transfected with the indicated plasmids. At 24 h after transfection, cells were treated with 1 lM MG132 for 12 h. Immunoprecipitates were immunoblotted with anti-HA or anti-FLAG antibody. Whole cell lysates (WCLs) were analyzed by immunoblotting using the indicated antibodies as controls (lower panel). (C) The TRIAD1 fragment (1–246 amino acids) is indispensible for binding p53 to inhibit the interaction between p53 and MDM2. TRIAD1 fragment constructs were generated as represented in the left panel. p53/mdm2/ MEF cells were transfected with the indicated plasmids. Anti-HA immunoprecipitates were immunoblotted with anti-FLAG and anti-GFP antibodies (upper panel). a.a., amino acids.
the interaction between p53 and MDM2 was substantially decreased following TRIAD1 transfection. We next identified which region of TRIAD1 could affect p53-MDM2 interaction. Because a previous in vitro study demonstrated that RING mutant MDM2 does not affect the MDM2-p53 interaction [13], we performed a series of immunoprecipitation experiments using RING mutant MDM2 constructs (Fig. 4C). In particular, the N-terminus of TRIAD1 (1–246) was found to be critical for the dissociation of p53 from MDM2 (Fig. 4C). Interestingly, the interaction between p53 and MDM2 is orchestrated by the interaction of TRIAD1 with p53; full-length TRIAD1 and the N-terminus of TRIAD1 (1–246) were capable of binding to p53 and inhibiting its interaction with MDM2, whereas the C-terminus of TRIAD1 (247–493) was unable to do either (Fig. 4C). These results indicate that TRIAD1 hinders MDM2-mediated p53 ubiquitination by inhibiting the interaction between p53 and MDM2.
3.5. TRIAD1 interacts with the C-terminus of p53 Because TRIAD1 interacts with p53 and induces its dissociation from MDM2 (Fig. 4), we performed experiments to identify the p53 domain that is critical for TRIAD1 interaction. Several fragmented p53 constructs were prepared as indicated (Fig. 5A). Interestingly, in vitro binding assays using these fragmented constructs with a GST-TRIAD1 fusion protein revealed that the C-terminus of p53 is critical for the interaction between p53 and TRIAD1 (Fig. 5B). 3.6. TRIAD1 negatively regulates MDM2 function by inhibiting p53mediated cell growth suppression Because TRIAD1 induces the dissociation of p53 from MDM2, we evaluated whether TRIAD1 restores p53 activation in presence of MDM2. We cotransfected p53/ mdm2/ MEF cells with p53 or
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Fig. 5. The C-terminus of p53 interacts with TRIAD1. (A) Schematic representation of the generated p53 fragment constructs. The p53 fragment constructs were cloned into pcDNA3.1 vectors. (B) In vitro pull-down assay showing the interactions between in vitro-translated, 35S-labeled p53 fragments, and GST or GST-TRIAD1 after a 1-h incubation at room temperature. Bound proteins were detected by autoradiography. a.a., amino acids.
its fragmented mutant forms and MDM2 in the presence or absence of TRIAD1. We found that TRIAD1 reduces cell viability through p53 activation in the presence of ectopic MDM2 expression (Fig. 6A). The cytotoxic function of TRIAD1 was found to be defective in p53/ mdm2/ MEF cells (Fig. S2). Moreover, the Nterminal fragment of TRIAD1, which is capable of interacting with p53, induced p53-mediated cellular growth inhibition, whereas the
C-terminal fragment did not. These results indicate that TRIAD1 induces p53-mediated cellular growth inhibition by binding to p53, thereby inhibiting its interaction with MDM2. We also evaluated whether TRIAD1 restores p53-mediated clonogenic growth inhibition in the presence of MDM2. As shown in Fig. 6B, p53 transfection inhibited clonogenic growth, which was restored by TRIAD1 transfection in the presence of MDM2.
Fig. 6. TRIAD1 restores p53-mediated cell growth inhibition in the presence of MDM2. (A) p53/mdm2/ MEF cells were transfected with the indicated plasmids. At 72 h after transfection, cell viability was determined by three independent MTS assays. (B) p53/mdm2/ MEF cells were cotransfected with plasmids for HA-p53, FLAG-MDM2, GFP-TRIAD1, GFP-TRIAD1 (a.a. 1–246), GFP-TRIAD1 (a.a. 247–493), or control GFP. Colonies were counted 2 week after seeding using a standard clonogenic assay. a.a., amino acids.
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4. Discussion
References
We previously demonstrated that TRIAD1 expression induces p53 activation and subsequent apoptosis [8]. The present work examines the role of TRIAD1 in p53 stabilization and activation. We found that p53 protein levels were lower and p53 activity was decreased in TRIAD1-knockdown cells compared to control U2OS cells. Cotransfection of TRIAD1 and p53 failed to induce p53 activation in p53/ mdm2/ MEF cells, however, transfection of TRIAD1 and MDM2 inhibited MDM2-mediated p53 degradation, indicating that TRIAD1-mediated p53 activation is related to the MDM2-antagonizing function of TRIAD1. Indeed, MDM2 suppression of p53-mediated cell growth inhibition was blocked by TRIAD1 expression. Furthermore, although TRIAD1 expression did not inhibit p53/ mdm2/ MEF cell viability, it did affect cell viability in MDM2-transfected cells. Several p53-interacting proteins that function as MDM2 antagonists have been reported. For example, ARF, L5, L11, and L23 can interact with MDM2 [5] and inhibit MDM2-mediating p53 ubiquitination/degradation. However, TRIAD1 interacts with and stabilizes p53 but not MDM2. Although the transactivation domain located in the N-terminus of p53 is important for MDM2 interaction [14], a recent report suggests that the C-terminus of p53, which contains multiple lysine residues, is also important for this interaction [15]. In the present study, we demonstrated that TRIAD1 binds to the C-terminus of p53 and inhibits MDM2 interaction, as well as MDM2-mediated ubiquitination. Thus, the C-terminus of p53 is important for regulation of its activity, not only via post-translational modifications but also through its interaction with TRIAD1. Recently, TRIAD1 transcription was found to be directly promoted by the transcription factor HoxA10 and induces anti-proliferative effects in acute myeloid leukemia (AML) [16]. In addition, HoxA10 reduces breast cancer cell invasiveness by upregulating p53 protein; however, the mechanism is unknown [17]. Therefore, our results extend the possibility that HoxA10-mediated p53 upregulation is related to HoxA10-mediated TRIAD1 expression. Although ectopic TRIAD1 expression induces apoptosis in several cell lines [18], it is possible that TRIAD1 also mediates differentiation for the following reasons: (1) TRIAD1 expression levels are greatly increased following treatment with all-trans retinoic acid (ATRA), which affects cellular differentiation [19,20]; (2) growth factor independent-1 (Gfi-1) transcription repressor, which is known to be stabilized by TRIAD1, is critical for B cell and myeloid cell development and neutrophil differentiation [21,22]; and (3) p53 activation regulates differentiation in several cell lines [23–25]. Moreover, modulation of the p53-MDM2 loop by nutlin decreases preosteoclast differentiation [26]. Thus, the physiological effects of TRIAD1 on p53 activation warrant further investigation.
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Acknowledgements We thank Dr. van der Reijden (Radboud University Nijmegen Medical Centre, Netherlands) for providing us with the GFP-TRIAD1 plasmids. This work was supported by a grant from the Ministry of Education, Science, and Technology (Grant 20110028646) of the Republic of Korea. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.febslet.2012.07. 022.