Topors Functions as an E3 Ubiquitin Ligase with Specific E2 Enzymes and Ubiquitinates p53

Abstract

The human topoisomerase I- and p53-binding protein topors contains a highly conserved, N-terminal C3HC4-type RING domain that is homologous to the RING domains of known E3 ubiquitin ligases. We demonstrate that topors functions in vitro as a RING-dependent E3 ubiquitin ligase with the E2 enzymes UbcH5a, UbcH5c, and UbcH6 but not with UbcH7, CDC34, or UbcH2b. Additional studies indicate that a conserved tryptophan within the topors RING domain is required for ubiquitination activity. Furthermore, both in vitro and cellular studies implicate p53 as a ubiquitination substrate for topors. Similar to MDM2, overexpression of topors results in a proteasome-dependent decrease in p53 protein expression in a human osteosarcoma cell line. These results are similar to the recent finding that a Drosophila topors orthologue ubiquitinates the Hairy transcriptional repressor and suggest that topors functions as a ubiquitin ligase for multiple transcription factors. The human topoisomerase I- and p53-binding protein topors contains a highly conserved, N-terminal C3HC4-type RING domain that is homologous to the RING domains of known E3 ubiquitin ligases. We demonstrate that topors functions in vitro as a RING-dependent E3 ubiquitin ligase with the E2 enzymes UbcH5a, UbcH5c, and UbcH6 but not with UbcH7, CDC34, or UbcH2b. Additional studies indicate that a conserved tryptophan within the topors RING domain is required for ubiquitination activity. Furthermore, both in vitro and cellular studies implicate p53 as a ubiquitination substrate for topors. Similar to MDM2, overexpression of topors results in a proteasome-dependent decrease in p53 protein expression in a human osteosarcoma cell line. These results are similar to the recent finding that a Drosophila topors orthologue ubiquitinates the Hairy transcriptional repressor and suggest that topors functions as a ubiquitin ligase for multiple transcription factors. Topors was originally discovered in a screen for proteins that bind to the N terminus of topoisomerase I (1Haluska P.H. Saleem A. Rasheed Z. Ahmed F. Su E.W. Liu L.F. Rubin E.H. Nucleic Acids Res. 1999; 27: 2538-2544Crossref PubMed Scopus (74) Google Scholar) and was also identified in a screen for proteins that interact with p53 (denoted p53BP3) (2Zhou R. Wen H. Ao S.Z. Gene (Amst.). 1999; 235: 93-101Crossref PubMed Scopus (41) Google Scholar). Furthermore, topors was identified in an assay for RING domain proteins that are expressed in normal lung tissue (denoted LUN) (3Chu D. Kakazu N. Gorrin-Rivas M.J. Lu H.P. Kawata M. Abe T. Ueda K. Adachi Y. J. Biol. Chem. 2001; 276: 14004-14013Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). While topors is widely expressed in normal human tissues, topors mRNA and protein levels are commonly decreased or undetectable in colon adenocarcinomas and cell lines (4Saleem A. Dutta J. Malegaonkar D. Rasheed F. Rasheed Z. Rajendra R. Marshall H. Luo M. Li H. Rubin E.H. Oncogene. 2004; 23: 5293-5300Crossref PubMed Scopus (37) Google Scholar). The topors protein contains an N-terminal C3HC4-type RING domain that is conserved in orthologues from various species (5Rasheed Z.A. Saleem A. Ravee Y. Pandolfi P.P. Rubin E.H. Exp. Cell Res. 2002; 277: 152-160Crossref PubMed Scopus (45) Google Scholar) and is closely related in sequence to the RING domains of known E3 ubiquitin ligases such as the herpesvirus protein ICP0 and Cbl (6Joazeiro C.A. Wing S.S. Huang H. Leverson J.D. Hunter T. Liu Y.C. Science. 1999; 286: 309-312Crossref PubMed Scopus (912) Google Scholar, 7Boutell C. Everett R.D. J. Biol. Chem. 2003; 278: 36596-36602Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). Recently, a Drosophila topors orthologue was shown to interact physically and genetically with the Hairy transcriptional repressor (8Secombe J. Parkhurst S.M. J. Biol. Chem. 2004; 279: 17126-17133Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar). Furthermore, the Drosophila topors protein was shown to ubiquitinate Hairy in vitro and to decrease expression of an epitope-tagged Hairy protein in co-transfection studies (8Secombe J. Parkhurst S.M. J. Biol. Chem. 2004; 279: 17126-17133Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar). Topors is also known to associate with promyelocytic leukemia (PML) 1The abbreviations used are: PML, promyelocytic leukemia; GST, glutathione S-transferase; DTT, dithiothreitol; MALDI-MS, matrix-assisted laser desorption/ionization mass spectrometry; LC-MS/MS, liquid chromatography tandem mass spectrometry; CMV, cytomegalovirus. nuclear bodies in the nuclei of exponentially growing cells (5Rasheed Z.A. Saleem A. Ravee Y. Pandolfi P.P. Rubin E.H. Exp. Cell Res. 2002; 277: 152-160Crossref PubMed Scopus (45) Google Scholar, 9Weger S. Hammer E. Engstler M. Exp. Cell Res. 2003; 290: 13-27Crossref PubMed Scopus (41) Google Scholar). Treatment with transcriptional inhibitors or with the topoisomerase I-targeting drug camptothecin results in rapid dispersion of topors to the nucleoplasm, suggesting that topors is involved in the cellular response to transcriptional perturbation (5Rasheed Z.A. Saleem A. Ravee Y. Pandolfi P.P. Rubin E.H. Exp. Cell Res. 2002; 277: 152-160Crossref PubMed Scopus (45) Google Scholar). To gain insight into the role of the topors and the conserved RING domain, we determined whether topors functions as a ubiquitin ligase. Our results indicate that topors acts as a RING domain-dependent, E3 ubiquitin ligase with specific E2 enzymes. Similar to the E3 ubiquitin ligase Cbl, a conserved tryptophan within the topors RING domain is required for ubiquitin ligase activity. Furthermore, additional in vitro and cellular studies implicate p53 as a ubiquitination substrate for topors. Expression Plasmids—A plasmid expressing a GST-N-terminal topors fusion protein (pGEX-topors) was constructed using PCR with pEGFP-topors (1Haluska P.H. Saleem A. Rasheed Z. Ahmed F. Su E.W. Liu L.F. Rubin E.H. Nucleic Acids Res. 1999; 27: 2538-2544Crossref PubMed Scopus (74) Google Scholar), the pGEX-4T3 vector (Amersham Biosciences), and primers designed to amplify the entire topors coding region. PGEXtopors-1–195 was created by digestion of pGEX-topors with DraIII and NotI. PGEX-topors-196–1045 was created similarly, with the exception that the EcoRI and DraIII sites were utilized. A plasmid expressing an N-terminal polyhistidine-topors fusion protein (pET-topors) was constructed by digestion of pKG-topors (5Rasheed Z.A. Saleem A. Ravee Y. Pandolfi P.P. Rubin E.H. Exp. Cell Res. 2002; 277: 152-160Crossref PubMed Scopus (45) Google Scholar) with SmaI and HindIII, followed by ligation into the pET-28a(+) vector (Novagen). Mutagenesis of tryptophan 131 in the topors RING domain to alanine was achieved using PCR (oligonucleotide sequences available upon request). EcoRI and DraIII sites were used to place the mutant fragment in the pGEX-topors vector. All plasmids were sequenced to confirm that the correct recombinant had been obtained. A plasmid expressing GST-tagged human MDM2 (pGEX-MDM2) was a gift from Jiandong Chen (10Chen J. Marechal V. Levine A.J. Mol. Cell. Biol. 1993; 13: 4107-4114Crossref PubMed Scopus (624) Google Scholar). Mammalian expression plasmids for human p53 (pRC/CMV-p53) and MDM2 (pCHDM1B) were kindly provided by Arnold Levine (11Lin J. Chen J. Elenbaas B. Levine A.J. Genes Dev. 1994; 8: 1235-1246Crossref PubMed Scopus (581) Google Scholar). A mammalian expression plasmid for polyhistidine-tagged ubiquitin (pMT.107) was obtained from Dirk Bohmann (12Treier M. Staszewski L.M. Bohmann D. Cell. 1994; 78: 787-798Abstract Full Text PDF PubMed Scopus (846) Google Scholar). In Vitro Ubiquitination Assays—Purification of GST fusion proteins from bacterial (BLR(DE3); Invitrogen) lysates was performed as described (13Edwards T.K. Saleem A. Shaman J.A. Dennis T. Gerigk C. Oliveros E. Gartenberg M.R. Rubin E.H. J. Biol. Chem. 2000; 275: 36181-36188Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). His fusion proteins were purified using a cobalt-based affinity resin (Talon resin, Clontech). Briefly, bacterial pellets were lysed in 500 mm NaCl, 50 mm Tris, pH 8.0 (buffer A), with 0.2 mm phenylmethylsulfonyl fluoride, 0.5 μg/ml leupeptin, and 1 μg/ml pepstatin, followed by incubation with Talon resin for 2 h at 4 °C.The beads were washed three times with buffer A containing 5 mm imidazole, then eluted with an equal volume of buffer A containing 500 mm imidazole. Eluted GST and His fusion proteins were dialyzed against a buffer containing 50 mm Tris-Cl, pH 8.0, 20% glycerol, 0.2 mm EDTA, 300 mm KCl, 0.2 mm phenylmethylsulfonyl fluoride, and 0.5 mm DTT. In vitro ubiquitination reactions were performed in a buffer containing 50 mm HEPES, pH 7.0, 5 mm MgCl2, 15 μm ZnCl2, and an ATP regenerating system (10 mm creatine phosphate, 3.5 units/ml creatine kinase (Sigma), and 0.6 unit/ml pyrophosphatase (Sigma)), with 400 ng of rabbit E1 (Boston Biochem), 400 ng of an E2, 1 μg of bovine ubiquitin or methylated bovine ubiquitin (Sigma), and 100 ng of either purified GST, GST-MDM2, GST-topors, GST-topors fragments, or His-topors. E2 enzymes were expressed in bacteria and purified as described (14Boutell C. Sadis S. Everett R.D. J. Virol. 2002; 76: 841-850Crossref PubMed Scopus (320) Google Scholar). All E2 preparations were tested to ensure that they were active in forming thiol ester conjugates with ubiquitin (data not shown). Certain reactions also included 50 ng of purified p53 (Pharmingen). Reactions were carried out at 30 °C for 60 min and were terminated by addition of SDS sample buffer (60 mm Tris-Cl, pH 6.8, 2% SDS, 10% glycerol, and 0.1% phenol red) containing 1 mm DTT. Reaction products were analyzed by SDS-PAGE and immunoblotting using polyclonal ubiquitin (Sigma) or monoclonal anti-p53 (Santa Cruz Biotechnology) antibodies. Mass Spectrometry of Ubiquitin Conjugates—In vitro ubiquitin assays were performed using the GST-1–195 topors fragment as described above. The resulting SDS-PAGE gel was stained with Coomassie dye, and a slice containing proteins migrating above Mr 175 was excised. The slice was washed sequentially with 50:50 water:acetonitrile (5 min), 50% CH3CN, 50 mm NH4HCO3 (30 min), and 50% CH3CN, 10 mm NH4HCO3 (30 min), dried using a lyophilizer, then treated with 0.01 mg/ml trypsin for 18 h at 37 °C. Extracted peptides were subjected to matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) using a Micromass TofSpec SE instrument. The extracted peptides were also subjected to LC-MS/MS analysis using a Micromass Q-Tof API mass spectrometer (W. M. Keck Foundation Laboratory, Yale University). Results were analyzed using ProFound and Mascot searches on the NCBI data base. In Vivo Ubiquitination Assays—H1299 (p53 null) and 2KO (p53–/–, mdm2–/–murine embryonic fibroblast) cells were obtained from Arnold Levine (University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, New Brunswick, NJ) and were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. Equal numbers of exponentially growing cells were seeded into 60-mm dishes and were transfected using LipofectAMINE 2000 (Invitrogen) in the presence of serum- and antibiotic-free media. Cells were co-transfected with 200 ng of pRC/CMV-p53, 500 ng of pMT.107 (expressing His-tagged ubiquitin), or empty CMV vector, and 2.5 μg of either pEGFP, pCHDM1B (expressing human MDM2), pEGFP-topors, or pEGFP-topors-231–1045 expression vectors (5Rasheed Z.A. Saleem A. Ravee Y. Pandolfi P.P. Rubin E.H. Exp. Cell Res. 2002; 277: 152-160Crossref PubMed Scopus (45) Google Scholar). In each case, the total amount of DNA transfected was 3.2 μg/60-mm dish. Twenty-four hours after transfection, MG132 was added to 4 μm, and the cells were incubated for an additional 12 h. Cells were harvested in 1 ml of ice-cold phosphate-buffered saline, and the cell suspension was divided into two parts; 100 μl were lysed using 1× SDS-PAGE sample loading buffer containing 10% DTT, and 900 μl were lysed in guanidine HCl buffer B (6 m guanidine HCl, 0.1% Nonidet P-40, 10 mm β-mercaptoethanol, and 5% glycerol, in phosphate-buffered saline, pH 8.0) as described (7Boutell C. Everett R.D. J. Biol. Chem. 2003; 278: 36596-36602Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). The guanidine lysates were incubated with 65 μl of equilibrated Talon resin at 4 °C for 4 h to bind His-tagged ubiquitinated proteins. The beads were washed twice each with 500 μl of buffer B, 500 μl of a 1:2 mixture of buffer B and buffer C (phosphate-buffered saline, 0.1% Nonidet P-40, 5% glycerol, and 20 mm imidazole), 500 μl of a 1:3 mixture of buffers B and C, and finally with 500 μl of buffer C. Bound proteins were analyzed by boiling the beads in SDS sample buffer with 1 mm DTT, followed by SDS-PAGE and immunoblotting using anti-p53 antibodies. Topors Induces Formation of Polyubiquitin Chains with Specific E2 Enzymes in Vitro—To test whether human topors functions as a ubiquitin ligase, we employed an in vitro assay using purified recombinant proteins expressed in bacteria (Fig. 1A). Previous work using similar assays demonstrated that many RING proteins stimulate formation of polymeric ubiquitin chains in the absence of a specific substrate (14Boutell C. Sadis S. Everett R.D. J. Virol. 2002; 76: 841-850Crossref PubMed Scopus (320) Google Scholar, 15Lorick K.L. Jensen J.P. Fang S. Ong A.M. Hatakeyama S. Weissman A.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11364-11369Crossref PubMed Scopus (944) Google Scholar). In the presence of E1 and the E2 UbcH5a, GST-topors, but not GST, stimulated formation of ubiquitin conjugates with low mobility on SDS-polyacrylamide gels (Fig. 1B). Furthermore, formation of these conjugates required the topors RING domain, since a 196–1045 fragment was inactive (Fig. 1B). Moreover, similar to other RING proteins (6Joazeiro C.A. Wing S.S. Huang H. Leverson J.D. Hunter T. Liu Y.C. Science. 1999; 286: 309-312Crossref PubMed Scopus (912) Google Scholar), a small topors fragment containing the RING domain (1–195) was sufficient for this activity (Fig. 1B). Since previous results suggest that dimerization conferred by GST could contribute to E2- or E3-type ubiquitin ligase activity (16Haldeman M.T. Xia G. PubMed Scopus Google Scholar, A. K.L. Proc. Natl. Acad. Sci. U. S. A. 2002; PubMed Scopus Google Scholar), we also a expressed fusion protein in this also stimulated the formation of low mobility ubiquitin conjugates in reactions with (Fig. To whether the low mobility ubiquitin conjugates polymeric ubiquitin we the assay using methylated to mobility ubiquitin conjugates were not methylated ubiquitin was used in the that topors formation of polymeric ubiquitin chains (Fig. with against either GST or topors demonstrated that the of the ubiquitin conjugates not topors (data not suggesting that they conjugates to either the E2, or as been for ubiquitin chains (14Boutell C. Sadis S. Everett R.D. J. Virol. 2002; 76: 841-850Crossref PubMed Scopus (320) Google Scholar). We analyzed the low mobility conjugates using and LC-MS/MS of the resulting mass highly of the ubiquitin of the the mass of a of ubiquitin in is commonly in polymeric ubiquitin chains J. D. D. G. J. D. 2003; PubMed Scopus Google Scholar) were suggesting that the low mobility or ubiquitinated or E2 with the immunoblotting these results indicate that the low mobility are polymeric ubiquitin similar to other RING the topors RING domain an E3-type ubiquitin ligase activity in the absence of a specific of ubiquitin peptides by and analysis of low mobility proteins obtained in ubiquitination reactions with a topors mass of the with a to by an as a of ubiquitination mass of the with a to by an as a of ubiquitination J. D. D. G. J. D. 2003; PubMed Scopus Google Scholar). in a we analyzed the of topors to interact with specific E2 enzymes. In the absence of ubiquitin conjugates migrating from Mr to 100 that were were commonly by E2 UbcH6 (Fig. Similar were and E2 A. Y. V. J. Biol. Chem. 1993; Full Text PDF PubMed Google Scholar, T. D. Cell. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). of topors in the formation of low mobility ubiquitin conjugates in reactions with human UbcH5a, UbcH5c, and UbcH6 but not UbcH7, CDC34, or (Fig. topors polymeric ubiquitin conjugates with but not E2 enzymes. A RING for the Ubiquitin of studies identified a tryptophan within the Cbl RING domain as required for ubiquitin ligase activity (6Joazeiro C.A. Wing S.S. Huang H. Leverson J.D. Hunter T. Liu Y.C. Science. 1999; 286: 309-312Crossref PubMed Scopus (912) Google Scholar) and as in the Cbl and the E2 N. Cell. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). of the tryptophan in the ICP0 RING domain not ubiquitination activity in vitro (14Boutell C. Sadis S. Everett R.D. J. Virol. 2002; 76: 841-850Crossref PubMed Scopus (320) Google Scholar). These results of the role of a and conserved tryptophan in the topors RING Reactions with a topors mutant of of the RING tryptophan 131 with alanine not polymeric ubiquitin chains with of the E2 enzymes that were active with the protein (Fig. These results indicate that the topors RING tryptophan is required for ubiquitin ligase activity. Topors as a Ubiquitin for p53 in that topors was to interact with p53 (2Zhou R. Wen H. Ao S.Z. Gene (Amst.). 1999; 235: 93-101Crossref PubMed Scopus (41) Google Scholar), we whether topors ubiquitinates p53 in In the presence of UbcH5a, topors of with this activity similar to results obtained with MDM2 using assay (Fig. Topors also ubiquitinated p53 in the presence of and UbcH6 (data not shown). proteins either the entire RING domain or containing an alanine for the conserved RING tryptophan were that the RING domain is required for p53 ubiquitination by topors. Furthermore, of polymeric ubiquitin chains by the topors fragment in containing this fragment not p53 ubiquitination (Fig. finding that the 196–1045 of topors is required for p53 ubiquitination and is with previous results that a of topors p53 (2Zhou R. Wen H. Ao S.Z. Gene (Amst.). 1999; 235: 93-101Crossref PubMed Scopus (41) Google Scholar). Topors Induces Ubiquitination and of p53 in whether topors ubiquitinates p53 in we performed co-transfection studies using and To of ubiquitin were treated with the MG132 to In co-transfected mobility p53 with and of were in cell lysates of cells transfected with the MDM2 vector, and to a the topors (Fig. and affinity chromatography were used to of p53 was in cells transfected with MDM2, and to a the topors plasmid (Fig. Furthermore, p53 ubiquitination was not in cells transfected with a topors plasmid (Fig. To the that ubiquitination of p53 by topors in cells was an with MDM2, similar were performed in 2KO both p53 and The results indicate that MDM2 is not required for ubiquitination of p53 by topors in (Fig. we the of topors overexpression on p53 protein cells were transfected with vectors expressing either MDM2, or the topors performed h after that to p53 protein levels decreased in cells transfected with MDM2 or topors plasmids but not the topors plasmid (Fig. C and The of MDM2 to topors on p53 protein expression is with results obtained in the cellular ubiquitination assays (Fig. Moreover, in MDM2 and topors treated with MG132 for h to p53 protein levels were similar to in that the decrease in p53 protein expression by topors and MDM2 is on (Fig. Our results indicate that topors functions as a RING domain-dependent, E3-type ubiquitin ligase with specific E2 enzymes. the E2 enzymes that were the by topors is to that of contains a RING domain that is similar to that of topors (Fig. The RING domains of topors and ICP0 are also highly similar to that of Cbl (Fig. of a Cbl and the E2 implicate within the Cbl RING domain as of E2 N. Cell. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). The in the topors and ICP0 RING domains are to of Cbl, with the exception of (Fig. These the that Cbl functions with but not (6Joazeiro C.A. Wing S.S. Huang H. Leverson J.D. Hunter T. Liu Y.C. Science. 1999; 286: 309-312Crossref PubMed Scopus (912) Google Scholar, M. T. A. M. H. A. R. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar), topors and ICP0 the Since in E2 are conserved in the ICP0 and topors RING domains (Fig. is not that these proteins similar E2 The finding that tryptophan 131 is required for topors to as a ubiquitin ligase is similar to results obtained with Cbl (6Joazeiro C.A. Wing S.S. Huang H. Leverson J.D. Hunter T. Liu Y.C. Science. 1999; 286: 309-312Crossref PubMed Scopus (912) Google Scholar). this finding with results obtained with of the RING tryptophan not ubiquitin ligase activity in vitro (14Boutell C. Sadis S. Everett R.D. J. Virol. 2002; 76: 841-850Crossref PubMed Scopus (320) Google Scholar). is that sequence the ICP0 and topors RING domains bind E2 enzymes using recent studies of a the for of RING domains and E2 enzymes by that the RING domain of in a that is that for the Cbl C. A.M. R. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). to human topors was to interact physically with topoisomerase and proteins (1Haluska P.H. Saleem A. Rasheed Z. Ahmed F. Su E.W. Liu L.F. Rubin E.H. Nucleic Acids Res. 1999; 27: 2538-2544Crossref PubMed Scopus (74) Google Scholar, R. Wen H. Ao S.Z. Gene (Amst.). 1999; 235: 93-101Crossref PubMed Scopus (41) Google Scholar, S. Hammer E. R. J. Virol. 2002; PubMed Scopus Google Scholar). Our results indicate that topors functions as a ubiquitin ligase for a Drosophila topors orthologue was shown to ubiquitinate the Hairy transcription in vitro and to of the Hairy protein (8Secombe J. Parkhurst S.M. J. Biol. Chem. 2004; 279: 17126-17133Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar). these were to ubiquitination of the Drosophila p53 orthologue by the Drosophila topors protein in These results to in the in vitro ubiquitination assays as the of an in assay but not that of and Parkhurst (8Secombe J. Parkhurst S.M. J. Biol. Chem. 2004; 279: 17126-17133Abstract Full Text Full Text PDF PubMed Scopus (13) Google or to substrate the Drosophila and human topors ubiquitin ligases been identified for the domain protein M. R.D. Cell. 1993; Full Text PDF PubMed Scopus Google Scholar), as as the RING proteins MDM2 R. H. H. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, Y. R. A. M. PubMed Scopus Google Scholar), (7Boutell C. Everett R.D. J. Biol. Chem. 2003; 278: 36596-36602Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar), Y. H. B. S. G. R. S. Cell. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar), a Y. Chen E. R. Mol. Cell. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar), and D. H. D. K. H. 2004; PubMed Scopus Google Scholar). In the protein was shown to as an ubiquitin ligase for p53 by formation C. H. Y. Science. 2003; PubMed Scopus Google Scholar). Topors is with nuclear bodies in exponentially growing cells (5Rasheed Z.A. Saleem A. Ravee Y. Pandolfi P.P. Rubin E.H. Exp. Cell Res. 2002; 277: 152-160Crossref PubMed Scopus (45) Google Scholar). Since nuclear bodies are in of M. R. C. M. M. S. Y. E. S. Pandolfi P.P. 2000; PubMed Scopus Google Scholar), S. C. Cell. Biol. 2002; PubMed Scopus Google Scholar), S. H. A. F. R. Genes Dev. 2001; PubMed Scopus Google Scholar), and ubiquitination S. C. A. Exp. Cell Res. 1999; PubMed Scopus (123) Google Scholar), is that topors is involved in the ubiquitination of p53 that are in these The amount of ubiquitinated p53 in co-transfection studies with topors MDM2 (Fig. is with ubiquitination of a of the p53 that are in to additional of the human topors protein and to the role of topors in p53 in both and normal We for and for the to