A Regulatory Loop Composed of RAP80-HDM2-p53 Provides RAP80-enhanced p53 Degradation by HDM2 in Response to DNA Damage
2009; Elsevier BV; Volume: 284; Issue: 29 Linguagem: Inglês
10.1074/jbc.m109.013102
ISSN1083-351X
AutoresJun Yan, Daniel Menéndez, Xiao-Ping Yang, Michael A. Resnick, Anton M. Jetten,
Tópico(s)Ubiquitin and proteasome pathways
ResumoThe ubiquitin interaction motif-containing protein RAP80 plays a key role in DNA damage response signaling. Using genomic and functional analysis, we established that the expression of the RAP80 gene is regulated in a DNA damage-responsive manner by the master regulator p53. This regulation occurs at the transcriptional level through a noncanonical p53 response element in the RAP80 promoter. Although it is inducible by p53, RAP80 is also able to regulate p53 through an association with both p53 and the E3 ubiquitin ligase HDM2, providing HDM2-dependent enhancement of p53 polyubiquitination. Depletion of RAP80 by small interfering RNA stabilizes p53, which, following DNA damage, results in an increased transactivation of several p53 target genes as well as greater apoptosis. Consistent with these observations, exogenous expression of RAP80 selectively inhibits p53-dependent transactivation of target genes in an mdm2-dependent manner in MEF cells. Thus, we identify a new DNA damage-associated role for RAP80. It can function in an autoregulatory loop consisting of RAP80, HDM2, and the p53 master regulatory network, implying an important role for this loop in genome stability and oncogenesis. The ubiquitin interaction motif-containing protein RAP80 plays a key role in DNA damage response signaling. Using genomic and functional analysis, we established that the expression of the RAP80 gene is regulated in a DNA damage-responsive manner by the master regulator p53. This regulation occurs at the transcriptional level through a noncanonical p53 response element in the RAP80 promoter. Although it is inducible by p53, RAP80 is also able to regulate p53 through an association with both p53 and the E3 ubiquitin ligase HDM2, providing HDM2-dependent enhancement of p53 polyubiquitination. Depletion of RAP80 by small interfering RNA stabilizes p53, which, following DNA damage, results in an increased transactivation of several p53 target genes as well as greater apoptosis. Consistent with these observations, exogenous expression of RAP80 selectively inhibits p53-dependent transactivation of target genes in an mdm2-dependent manner in MEF cells. Thus, we identify a new DNA damage-associated role for RAP80. It can function in an autoregulatory loop consisting of RAP80, HDM2, and the p53 master regulatory network, implying an important role for this loop in genome stability and oncogenesis. To assure genome integrity, all cellular organisms contain systems that can monitor and repair a variety of DNA lesions. The DNA damage response (DDR) 4The abbreviations used are: DDRDNA damage responseUIMubiquitin interaction motifE3ubiquitin-protein isopeptide ligasesiRNAsmall interfering RNAREresponse elementGSTglutathione S-transferaseChIPchromatin immunoprecipitationntnucleotide(s)WTwild typeMEFmouse embryonic fibroblastDOXOdoxorubicinERestrogen receptorGygrayIRionizing radiationCREBcAMP-response element-binding protein. in mammals is a highly dynamic and coordinated network that involves a plethora of proteins that sense damage and transduce signals to execute cellular responses, including cell cycle checkpoints, DNA repair mechanisms, cellular senescence, and apoptosis (1.Bartek J. Lukas J. Curr. Opin. Cell Biol. 2007; 19: 238-245Crossref PubMed Scopus (596) Google Scholar, 2.Harper J.W. Elledge S.J. Mol. Cell. 2007; 28: 739-745Abstract Full Text Full Text PDF PubMed Scopus (1299) Google Scholar, 3.Kastan M.B. Bartek J. 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Recognition of DNA damage and propagation of the DDR signal involves the recruitment and assembly of many DDR mediators and effectors, including BRCA1, at sites flanking damage (2.Harper J.W. Elledge S.J. Mol. Cell. 2007; 28: 739-745Abstract Full Text Full Text PDF PubMed Scopus (1299) Google Scholar, 8.Paull T.T. Rogakou E.P. Yamazaki V. Kirchgessner C.U. Gellert M. Bonner W.M. Curr. Biol. 2000; 10: 886-895Abstract Full Text Full Text PDF PubMed Scopus (1701) Google Scholar). Recruitment occurs in a hierarchical manner and is dependent on a number of post-translational modifications including phosphorylation, ubiquitination, and acetylation (2.Harper J.W. Elledge S.J. Mol. Cell. 2007; 28: 739-745Abstract Full Text Full Text PDF PubMed Scopus (1299) Google Scholar, 9.Huen M.S. Chen J. Cell. Res. 2008; 18: 8-16Crossref PubMed Scopus (155) Google Scholar, 10.Yan J. Jetten A.M. Cancer Lett. 2008; 271: 179-190Crossref PubMed Scopus (66) Google Scholar). RAP80 (receptor-associated protein 80 or UIMC1) is associated with the BRCA1-BARD1-ccdc98(Abraxas) complex and plays a key role in the translocation of this complex to DNA damage sites (10.Yan J. Jetten A.M. Cancer Lett. 2008; 271: 179-190Crossref PubMed Scopus (66) Google Scholar, 11.Kim H. Chen J. Yu X. Science. 2007; 316: 1202-1205Crossref PubMed Scopus (445) Google Scholar, 12.Sobhian B. Shao G. Lilli D.R. Culhane A.C. Moreau L.A. Xia B. Livingston D.M. Greenberg R.A. Science. 2007; 316: 1198-1202Crossref PubMed Scopus (549) Google Scholar, 13.Wang B. Matsuoka S. Ballif B.A. Zhang D. Smogorzewska A. Gygi S.P. Elledge S.J. Science. 2007; 316: 1194-1198Crossref PubMed Scopus (569) Google Scholar, 14.Yan J. Kim Y.S. Yang X.P. Li L.P. Liao G. Xia F. Jetten A.M. Cancer Res. 2007; 67: 6647-6656Crossref PubMed Scopus (139) Google Scholar). This translocation involves recognition of K63-linked polyubiquitin chains of histones H2A and H2AX by the ubiquitin interaction motifs (UIMs) within RAP80 (10.Yan J. Jetten A.M. Cancer Lett. 2008; 271: 179-190Crossref PubMed Scopus (66) Google Scholar, 15.Kolas N.K. Chapman J.R. Nakada S. Ylanko J. Chahwan R. Sweeney F.D. Panier S. Mendez M. Wildenhain J. Thomson T.M. Pelletier L. Jackson S.P. Durocher D. Science. 2007; 318: 1637-1640Crossref PubMed Scopus (707) Google Scholar, 16.Wang B. Elledge S.J. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 20759-20763Crossref PubMed Scopus (365) Google Scholar, 17.Huen M.S. Grant R. Manke I. Minn K. Yu X. Yaffe M.B. Chen J. Cell. 2007; 131: 901-914Abstract Full Text Full Text PDF PubMed Scopus (817) Google Scholar, 18.Mailand N. Bekker-Jensen S. Faustrup H. Melander F. Bartek J. Lukas C. Lukas J. Cell. 2007; 131: 887-900Abstract Full Text Full Text PDF PubMed Scopus (918) Google Scholar). The tumor suppressor p53 plays a key part in DDR signaling. It functions as a master regulator that controls a broad transcriptional network activated in response to various types of cellular and environmental stress (19.Brooks C.L. Gu W. Mol. Cell. 2006; 21: 307-315Abstract Full Text Full Text PDF PubMed Scopus (697) Google Scholar). Activation of p53, along with the subsequent induction of its target genes, plays a critical role in the regulation of cell cycle control and apoptosis to assure genome integrity (20.Rodier F. Campisi J. Bhaumik D. Nucleic Acids Res. 2007; 35: 7475-7484Crossref PubMed Scopus (296) Google Scholar). Disruption of p53 can compromise repair of DNA damage resulting in chromosome abnormalities, ultimately leading to oncogenesis. Mutations in the p53 gene have been associated with more than half of human cancers (21.Levine A.J. Cell. 1997; 88: 323-331Abstract Full Text Full Text PDF PubMed Scopus (6759) Google Scholar). Under normal physiological conditions, p53 levels are kept low because of its ubiquitination by the E3 ubiquitin ligase HDM2 (corresponding to mouse double-minute 2 protein mdm2), resulting in its rapid turnover by proteasomes. In response to DNA damage, p53 becomes stabilized through processes that include post-translational modification of p53. HDM2 is itself a p53 target gene that can become activated after stress and lead to p53 destabilization (22.Wu X. Bayle J.H. Olson D. Levine A.J. Genes Dev. 1993; 7: 1126-1132Crossref PubMed Scopus (1645) Google Scholar, 23.Riley T. Sontag E. Chen P. Levine A. Nat. Rev. Mol. Cell Biol. 2008; 9: 402-412Crossref PubMed Scopus (1501) Google Scholar). The resulting p53-HDM2 auto-regulatory loop is of vital importance in controlling the level of p53 and its activity. In this study, we identify a new role for RAP80 as both a modulator of p53 activity and as a direct transcription target of p53 following DNA damage, mainly through a noncanonical response element (RE) sequence in its promoter. RAP80 is able to form a complex with p53 and increase HDM2-dependent polyubiquitination of p53. RAP80, therefore, expands the p53-HDM2 relationship to a DNA damage-responsive, autoregulatory RAP80-p53-HDM2 loop. pLXIN and pEGFP were purchased from BD Biosciences. pCMV-HA-Ub, pCMV-Myc-p53, pCMV-Myc-HDM2, and pCMV-HDM2 were gifts from Dr. Yue Xiong (University of North Carolina at Chapel Hill). pGEX-p53 was kindly provided by Dr. Yang Shi (Harvard University). Plasmids pC53-SN3 coding for human p53 cDNA under the control of cytomegalovirus promoter and pCMV-Neo-Bam were provided by Dr. Bert Vogelstein (Johns Hopkins University). Luciferase reporter constructs containing the p53-REs were created in pGL4.26 (luc2/miniP/Hygro) reporter vector (Promega). pRL-SV40 is a reporter plasmid coding for Renilla reniformis luciferase (Promega). More detailed information of plasmids and constructs used in this study are described in the supplemental material. Detailed information of the cell lines used is provided in the supplemental material. Where indicated, the cells were treated with doxorubicin (Sigma) (0.3 μg/ml), γ-irradiation (0.5 or 4 Gy), or UV radiation (10 or 15 J/m2) at ∼70% confluence; 24 h later cells were harvested for protein and RNA extraction. Evaluation of cellular death was assessed by annexin V-fluorescein isothiocyanate/propidium iodide apoptosis detection kit (BD Pharmigen) following the manufacturer's protocol. Protein analysis and Western blot protocols are described in the supplemental material. For RNA interference, U2OS cells were transfected with control or RAP80 siRNAs (Dharmacon and Invitrogen) following the manufacturer's suggestions. For transcriptional assays, the cells were transfected with the reporter gene in the absence or presence of expression vectors for the indicated proteins or empty expression vector (pCMV-Neo-Bam) as described previously (24.Menendez D. Inga A. Resnick M.A. Mol. Cell. Biol. 2006; 26: 2297-2308Crossref PubMed Scopus (69) Google Scholar). A more detail protocol is provided in the supplemental material. The methods for purifying GST or GST-p53 fusion proteins and their binding to [35S]methionine-labeled RAP80 were described previously (14.Yan J. Kim Y.S. Yang X.P. Li L.P. Liao G. Xia F. Jetten A.M. Cancer Res. 2007; 67: 6647-6656Crossref PubMed Scopus (139) Google Scholar). U2OS cells were transfected with wild type pLXIN-3×FLAG-RAP80 or their mutants and pcDNA3-Myc-p53 as indicated; 48 h later, the cells were collected and processed as described previously (14.Yan J. Kim Y.S. Yang X.P. Li L.P. Liao G. Xia F. Jetten A.M. Cancer Res. 2007; 67: 6647-6656Crossref PubMed Scopus (139) Google Scholar). Evaluation of RAP80 and p53 target genes mRNA levels was determined using TaqMan probe-based chemistry (Applied Biosystems), and the relative quantitative values were calculated based on the 2-ΔΔCt method following the manufacturer's instructions. ChIP assays were done as described previously (24.Menendez D. Inga A. Resnick M.A. Mol. Cell. Biol. 2006; 26: 2297-2308Crossref PubMed Scopus (69) Google Scholar) using the ChIP kit (Millipore) following the manufacturer's instructions. A more detailed protocol is provided in the supplemental material. Because the p53 protein plays an important role in DDR signaling by activating the transcription of many DDR effectors, we investigated the possibility that RAP80 might also be a target of transcriptional regulation by p53. A 4-kb region surrounding the transcription start site of the human RAP80 promoter was scanned for the presence of p53 response element sequences (p53-REs). Although the commonly accepted consensus p53-RE consists of (RRRCWWGYYY followed by a 0–13-nt spacer followed by RRRCWWGYYY) with up to approximately five mismatches, we recently established that a fully functional RE has a spacer of ≤3 nt and that a single decamer, a "noncanonical half-site" (23.Riley T. Sontag E. Chen P. Levine A. Nat. Rev. Mol. Cell Biol. 2008; 9: 402-412Crossref PubMed Scopus (1501) Google Scholar, 25.Menendez D. Inga A. Snipe J. Krysiak O. Schönfelder G. Resnick M.A. Mol. Cell. Biol. 2007; 27: 2590-2600Crossref PubMed Scopus (51) Google Scholar, 26.Jordan J.J. Menendez D. Inga A. Nourredine M. Bell D. Ma R. PLoS Genet. 2008; 4: e1000104Crossref PubMed Scopus (87) Google Scholar), could mediate transcriptional activation by p53. The CWWG core is important for p53 responsiveness, where C and G are essential and CATG is the strongest responder. Using these criteria, nine potential p53-REs were identified. Based on sequence similarities between the nine potential p53-REs with the p53-RE consensus and on observations that more than 80% of functional p53-REs are located in the proximal promoter region of target genes (23.Riley T. Sontag E. Chen P. Levine A. Nat. Rev. Mol. Cell Biol. 2008; 9: 402-412Crossref PubMed Scopus (1501) Google Scholar), the p53-RE3, -4, and -5 located in the ∼1.5-kb promoter region just upstream of the transcription start site of the RAP80 gene appeared the best candidates. As shown in Fig. 1A, the potential p53-RE3 (−1266 to −1237) has a 9-nt spacer and contains three mismatches in the first decamer, one of which was located at a critical position (27.Inga A. Storici F. Darden T.A. Resnick M.A. Mol. Cell. Biol. 2002; 22: 8612-8625Crossref PubMed Scopus (156) Google Scholar, 28.Menendez D. Inga A. Jordan J.J. Resnick M.A. Oncogene. 2007; 26: 2191-2201Crossref PubMed Scopus (30) Google Scholar, 29.Jegga A.G. Inga A. Menendez D. Aronow B.J. Resnick M.A. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 944-949Crossref PubMed Scopus (59) Google Scholar) in the CWWG core. The p53-RE4 (−1211 to −1188) contains only two mismatches in the first decamer followed by a 3-nt spacer and a perfect second decamer with a moderately responsive CWWG core (CTAG) (29.Jegga A.G. Inga A. Menendez D. Aronow B.J. Resnick M.A. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 944-949Crossref PubMed Scopus (59) Google Scholar). The p53-RE5 (−717 to −693) had a single mismatch at a critical position in the first decamer, a 4-nt spacer, and a perfect second half-site with a strong core signature (CATG). The remaining potential p53-REs have either a long spacer, mismatches in the CWWG core or a predicted weak CWWG. We therefore focused our further studies on the region containing REs 3 to 5. The complete list of potential p53-REs found in the analyzed region is presented in supplemental Table S1. Interestingly, neither of the potential p53REs found in the region analyzed of the human RAP80 are conserved in rodents (supplemental information). Consistent with this observation, the expression of RAP80 was not induced in WT p53 MEFs treated with UV radiation, and the levels remained comparable with those in treated p53−/− MEFs (Fig. 1E). The ability of these p53-REs to function as p53 targets sequences was first investigated using ChIP assays. Binding was assessed with primer pairs that amplify the regions encompassing p53-RE3/RE4 (nt −1308 to −1058) or p53-RE5 (nt −609 to −885). Colon carcinoma HCT116 p53+/+ cells were treated with doxorubicin (DOXO, 0.3 μg/ml) for 24 h or with UV radiation (10 J/m2). After cross-linking with formaldehyde, DNA-protein complexes were immunoprecipitated with anti-p53 antibody; mouse IgG provided a negative control. p53 activated by UV radiation was found to be associated with both regions, whereas p53 activated by DOXO treatment did not bind (Fig. 1B). p53 bound the region containing p53-RE5 with a nearly 2-fold greater efficiency than that of p53-RE3/4. As expected, activation of p53 by both DNA-damaging agents resulted in high p53 occupancy of the p21 promoter, a positive control. Similar results were found with cell lines U2OS and A549 (supplemental Fig. S1). Using SaOS2 cell lines containing either TET-inducible WT or G279E mutant p53 (24.Menendez D. Inga A. Resnick M.A. Mol. Cell. Biol. 2006; 26: 2297-2308Crossref PubMed Scopus (69) Google Scholar), only the expressed WT p53 was able to bind the putative sites in the 1.5-kb promoter region of RAP80 (supplemental Fig. S1). To establish that the p53-RE5 sequence is also functional in p53-mediated transactivation, the 24-bp sequence (AAGCTgGCCTccttGAACATGTCT) was cloned upstream of a minimal promoter containing a TATA box promoter element upstream of the pGL4.26 luciferase reporter. In addition, to assess the potential function of the perfect p53 half-site, 4-nt changes were introduced into the first decamer. Luciferase reporter constructs containing the p53-RE of p21, PUMA, or AIP were used as positive controls. Each of these constructs were co-transfected into p53 null SaOS2 cells with either a pCSN3 control vector or a vector that expresses WT or the G279E mutant p53. As shown in Fig. 1C, the WT p53 greatly increased transcriptional activation and most of the activation was supported by the perfect half-decamer of p53-RE5 because mutations in the first decamer had little impact on the transactivation. The level of RE5-dependent p53 transactivation was comparable with that of the moderately active PUMA and AIP p53-REs. These results led us to investigate whether DNA damage alters the expression of RAP80 in cells that express endogenous WT p53. Following UV radiation and IR treatment, RAP80 mRNA was up-regulated in p53+/+ HCT116 cells but not in the isogenic p53−/− counterpart (Fig. 1D) or in p53-deficient H1299 cells (supplemental Fig. S1). Consistent with p53 promoter occupancy results, DOXO did not induce RAP80 expression, whereas all treatments induced the expression of p21 in p53+/+ cells. UV radiation and IR, but not DOXO, also induced RAP80 expression in human lung carcinoma A549 and osteosarcoma U2OS cells (supplemental Fig. S1) containing WT p53. Finally, overexpression of p53 in the p53 null HCT116 (Fig. 1D) and H1299 cells (supplemental Fig. S1) resulted in increased RAP80 mRNA expression. In agreement with these observations, RAP80 protein levels were increased after UV radiation and IR treatment reaching a maximum between 12 and 18 h post-treatment (Fig. 1F). Taken together, these results strongly suggest that RAP80 expression is controlled by p53 in a DNA damage-dependent manner through a noncanonical p53 half-site in the RAP80 promoter. We investigated whether the regulation of RAP80 by p53 might be linked directly to p53 function. This was stimulated by the observation that RAP80 interacts with estrogen receptor α (ERα) affecting its stability/activity (30.Yan J. Kim Y.S. Yang X.P. Albers M. Koegl M. Jetten A.M. Nucleic Acids Res. 2007; 35: 1673-1686Crossref PubMed Scopus (29) Google Scholar) and that HDM2, which is regulated by p53, also determines p53 stability. We first examined whether RAP80 and p53 interact. HeLa cells were co-transfected with pCMV-Myc-p53 and pLXIN-3×FLAG-RAP80 expression plasmids. An antibody against FLAG-RAP80 was able to co-immunoprecipitate p53 (Fig. 2A), suggesting that RAP80 and p53 are associated with the same protein complex. In vitro pulldown analysis was performed to further confirm this interaction. Moreover, GST-p53 fusion protein effectively pulled down in vitro translated 35S-labeled RAP80, whereas GST alone did not bind RAP80. ERα, previously shown to bind p53 (30.Yan J. Kim Y.S. Yang X.P. Albers M. Koegl M. Jetten A.M. Nucleic Acids Res. 2007; 35: 1673-1686Crossref PubMed Scopus (29) Google Scholar), was used as a positive control (Fig. 2B). The interaction of RAP80 and p53 was further confirmed with an MCF-7 cell line stably expressing FLAG-RAP80 (14.Yan J. Kim Y.S. Yang X.P. Li L.P. Liao G. Xia F. Jetten A.M. Cancer Res. 2007; 67: 6647-6656Crossref PubMed Scopus (139) Google Scholar). As shown in supplemental Fig. S2, antibodies against FLAG or RAP80 could co-immunoprecipitate endogenous p53. Finally, we demonstrated that endogenous RAP80 was able to pull down endogenous p53 in U2OS (Fig. 2C) and 293T cells (supplemental Fig. S2). To determine the region(s) important for RAP80 interaction with p53, RAP80 carboxyl-terminal deletion mutants were examined by co-immunoprecipitation. As shown in Fig. 2D and supplemental Fig. S3, the region between amino acids 122 and 204 that lacks the UIMs and two potential zinc fingers is essential for the interaction. Based on in vitro pulldown analysis with p53 deletion mutants (Fig. 2E), the DNA-binding domain (amino acids 100–200) of p53 is necessary and sufficient to bind RAP80. The p53 fragments containing the transactivation domain, oligomerization domain, and regulatory domain failed to interact with RAP80 (supplemental Fig. S4). Given that RAP80 affects the stability of another protein, ERα (30.Yan J. Kim Y.S. Yang X.P. Albers M. Koegl M. Jetten A.M. Nucleic Acids Res. 2007; 35: 1673-1686Crossref PubMed Scopus (29) Google Scholar) and that p53 ubiquitination and stability is controlled by HDM2, the major E3 ubiquitin ligase of p53, we examined whether RAP80 was associated with HDM2. Co-immunoprecipitation analysis using U2OS cells transfected with FLAG-RAP80 and Myc-HDM2 expression plasmids demonstrated that RAP80 was able to pull down HDM2 (Fig. 3A). Subsequently, we assessed the effect of RAP80 on HDM2-mediated p53 ubiquitination and proteasome-mediated degradation. U2OS cells were transfected with the plasmids indicated in Fig. 3B and treated with the proteasome inhibitor MG132 before collection. Ubiquitination was determined in immunoprecipitated p53. As shown in Fig. 3B, overexpression of RAP80 enhanced HDM2-mediated ubiquitination of p53. However, RAP80 had no effect on HDM2-p53 interaction based on reprobing the same filter with anti-HDM2 antibody. Because HDM2 self-ubiquitination was greatly enhanced (Fig. 3B), the RAP80 enhancement of p53 ubiquitination appears to result from increased HDM2 ligase activity. Additionally, several slower migrating, ubiquitinated forms of RAP80 were identified, indicating that HDM2 is also a potential E3 ligase of RAP80 (Fig. 3B). Because the UIMs of RAP80 are required for its effect on ERα ubiquitination (30.Yan J. Kim Y.S. Yang X.P. Albers M. Koegl M. Jetten A.M. Nucleic Acids Res. 2007; 35: 1673-1686Crossref PubMed Scopus (29) Google Scholar), we determined whether they are required for the regulation of p53 ubiquitination by RAP80. As shown in Fig. 3C, the UIMs are not needed because WT and RAP80ΔUIM enhanced p53 ubiquitination to similar extents (lanes 4 and 5). This effect of RAP80 on p53 ubiquitination was dependent on HDM2 expression (lane 2 versus lane 4). Moreover, RAP80 was not ubiquitinated in the absence of expressed HDM2 (lane 2 versus lane 4). However, ubiquitination of RAP80ΔUIM was greatly diminished (lane 4 and lane 5) compared with that of RAP80, indicating that the UIMs are important for optimal ubiquitination of RAP80, consistent with our previous findings (30.Yan J. Kim Y.S. Yang X.P. Albers M. Koegl M. Jetten A.M. Nucleic Acids Res. 2007; 35: 1673-1686Crossref PubMed Scopus (29) Google Scholar). Because RAP80 can mediate HDM2 ubiquitination of p53, the effect of RAP80 on p53-mediated transcriptional activation of p53 target genes and the role of HDM2 was examined. p53-deficient SaOS2 cells were co-transfected with WT p53 along with a plasmid expressing either 3×FLAG-RAP80 or the deletion mutant 3×FLAG-RAP80(N1–122) and various p53-RE transcription reporter plasmids. Western blot analysis demonstrated that all proteins were expressed at similar levels (supplemental Fig. S5). Expression of RAP80 but not mutant RAP80(N1–122) interfered with p53-dependent transcriptional activation. As shown in Fig. 4A, RAP80 inhibited p53-dependent transactivation at p53-RE(PUMA) and p53-RE(AIP) by ∼95%, and P21-RE and the artificial pG13 reporters by ∼50%; RAP80(N1–122) had no effect. In the absence of p53, there was no effect of RAP80 or RAP80 (N1–122) on the residual transcription. As expected, co-transfection of HDM2 with p53 led to reduced transactivation at target REs. Inhibition was directly related to the amount of RAP80 plasmid added (supplemental Fig. S5). Similar results were obtained when p53+/+ U2OS cells were transfected with RAP80 expression plasmid and endogenous p53 was activated by DOXO (supplemental Fig. S5). RAP80 alone was unable to mediate p53 degradation, suggesting that RAP80 interacts with both p53 and HDM2 to enhance p53 ubiquitination by HDM2. To further assess the role of HDM2, we determined the effect of RAP80 on transactivation driven by exogenous p53 in mdm2−/− p53−/− and mdm2+/+ p53−/− MEF cells. As shown in Fig. 4 (B and C), p53 transactivation at the P21 and PUMA REs was not affected by RAP80 expression in the mdm2−/− cells, whereas RAP80 expression in the mdm2+/+ MEFs resulted in 40 and 70% reductions in P21-RE- and PUMA-RE-dependent transactivation, respectively. Similar to results obtained with SAOS2 cells, the inhibition of p53 transactivation activity by RAP80 in MEFs was directly dependent on the amount of RAP80 added (supplemental Fig. S5). As expected, ectopic expression of mouse MDM2 resulted in a large reduction in p53 transactivation (Fig. 4, B and C). Thus, the RAP80 reduction of p53-mediated transactivation is dependent on MDM2. To examine the effect of RAP80 depletion on p53 protein levels, U2OS cells were transfected with RAP80 siRNA or scrambled siRNA (control) and 72 h later treated with 4 Gy (Fig. 5A) of γ-irradiation. The levels of p53 were determined during the subsequent 2 h. RAP80 siRNA greatly diminished the level of RAP80 protein without affecting p53 levels. However, after irradiation the p53 levels were considerably higher in RAP80-depleted cells as compared with post-irradiation controls, consistent with RAP80 enhancing HDM2-mediated ubiquitination and degradation of p53. Interestingly, knockdown of RAP80 inhibited the level of Ser(P)15-p53, which is commonly used as a marker for p53 activation after IR damage. As expected based on the results described above, there was a significant increase in RAP80 protein starting 1 h after treatment with IR for cells transfected with scrambled with siRNA. Qualitatively similar results were obtained at a much lower dose, 0.5 Gy, which is expected to result in only a small induction of p53 (supplemental Fig. S6). Because RAP80 overexpression inhibited p53-dependent transactivation through REs from the apoptosis-related genes PUMA and AIP to a greater extent than transactivation through the RE from the cell cycle arrest gene P21 (described above), depletion of RAP80 might particularly affect regulation of apoptosis-related genes. The effect of RAP80 depletion on p53-dependent apoptosis was therefore examined in U2OS cells treated with 4 Gy or 15 J/m2 UV radiation. As shown in Fig. 5B, apoptosis induced by IR was significantly enhanced in RAP80-depleted cells compared with cells transfected with scrambled siRNA. The difference in IR-induced (versus no IR) apoptotic cells was nearly twice that for RAP80-depleted (∼16%) as compared with scrambled siRNA cells (∼9%). Similarly, there was a significant increase in apoptosis following UV radiation for the RAP80-depleted cells. The effect of RAP80 depletion on transactivation of p53 target genes after genotoxic stress was also examined in U2OS cells. Expression of the following well known p53 targets genes was evaluated by real time PCR: cell cycle arrest genes P21 and PLAGL1; apoptosis-related genes P53AIP1, APAF1, BAX, BBC3-PUMA, FAS, TP53I3-PIG3, and TP53INP1; DNA repair genes DDB2 and XPC; and the p53 gatekeeper gene HDM2. In general, the expression of most p53 target genes was elevated in RAP80-depleted cells treated with IR (Fig. 5C) or UV radiation (supplemental Fig. S6) relative to cells transfected with scrambled siRNA. Neither RAP80 knockdown or radiation affected p53 mRNA expression. At the doses used, IR had a larger effect than UV radiation. The effect was greatest for apoptosis genes, whereas the induction of P21 and HDM2 were not affected. The net impact of ionizing radiation on expression (exposure versus no exposure) was typically 1.5–3-fold greater in RAP80-depleted cells as compared with cells transfected with scrambled siRNA. At the protein level RAP80 siRNA led to no differences in p21, whereas there was a small increase in BAX in untreated and IR-treated U2OS cells (Fig. 5D). In previous studies we and other groups demonstrated that RAP80 is part of a BRCA1-BARD1-ccdc98(Abraxas) complex and promotes the translocation of these complexes to DNA damage sites and as such is involved in DSB repair and cell cycle checkpoint control following IR exposure (10.Yan J. Jetten A.M. Cancer Lett. 2008; 271: 179-190Crossref PubMed Scopus (66) Google Scholar, 11.Kim H. Chen J. Yu X. Science. 2007; 316: 1202-1205Crossref PubMed Scopus (445) Google Scholar, 12.Sobhian B. Shao G. Lilli D.R. Culhane A.C. Moreau L.A. Xia B. Livingston D.M. Greenberg R.A. Science. 20
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