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• W2021818279 abstract "Protooncogene Ski was identified based on its ability to transform avian fibroblasts in vitro. In support of its oncogenic activity, SKI was found to be overexpressed in a variety of human cancers, although the exact molecular mechanism(s) responsible for its oncogenic activity is not fully understood. We found that SKI can negatively regulate p53 by decreasing its level through up-regulation of MDM2 activity, which is mediated by the ability of SKI to enhance sumoylation of MDM2. This stimulation of MDM2 sumoylation is accomplished through a direct interaction of SKI with SUMO-conjugating enzyme E2, Ubc9, resulting in enhanced thioester bond formation and mono-sumoylation of Ubc9. A mutant SKI defective in transformation fails to increase p53 ubiquitination and is unable to increase MDM2 levels and to increase mono-sumoylation of Ubc9, suggesting that the ability of SKI to enhance Ubc9 activity is essential for its transforming function. These results established a detailed molecular mechanism that underlies the ability of SKI to cause cellular transformation while unraveling a novel connection between sumoylation and tumorigenesis, providing potential new therapeutic targets for cancer. Protooncogene Ski was identified based on its ability to transform avian fibroblasts in vitro. In support of its oncogenic activity, SKI was found to be overexpressed in a variety of human cancers, although the exact molecular mechanism(s) responsible for its oncogenic activity is not fully understood. We found that SKI can negatively regulate p53 by decreasing its level through up-regulation of MDM2 activity, which is mediated by the ability of SKI to enhance sumoylation of MDM2. This stimulation of MDM2 sumoylation is accomplished through a direct interaction of SKI with SUMO-conjugating enzyme E2, Ubc9, resulting in enhanced thioester bond formation and mono-sumoylation of Ubc9. A mutant SKI defective in transformation fails to increase p53 ubiquitination and is unable to increase MDM2 levels and to increase mono-sumoylation of Ubc9, suggesting that the ability of SKI to enhance Ubc9 activity is essential for its transforming function. These results established a detailed molecular mechanism that underlies the ability of SKI to cause cellular transformation while unraveling a novel connection between sumoylation and tumorigenesis, providing potential new therapeutic targets for cancer. Ski was found more than 20 years ago as the only transforming oncogene discovered through in vitro viral replication assay (1Li Y. Turck C.M. Teumer J.K. Stavnezer E. Unique sequence, ski, in Sloan-Kettering avian retroviruses with properties of a new cell-derived oncogene.J. Virol. 1986; 57: 1065-1072Crossref PubMed Google Scholar). It was shown to be able to transform chicken and quail embryo fibroblasts as evidenced by the overgrowth of virally infected cells in monolayer culture and anchorage-independent colony formation in soft agar, hallmarks of cellular transformation. Consistent with its role as an oncoprotein, SKI was found to be overexpressed in a variety of human cancers, including melanoma (2Medrano E.E. Repression of TGF-β signaling by the oncogenic protein SKI in human melanomas. Consequences for proliferation, survival, and metastasis.Oncogene. 2003; 22: 3123-3129Crossref PubMed Scopus (75) Google Scholar), leukemia (3Ritter M. Kattmann D. Teichler S. Hartmann O. Samuelsson M.K. Burchert A. Bach J.P. Kim T.D. Berwanger B. Thiede C. Jäger R. Ehninger G. Schäfer H. Ueki N. Hayman M.J. Eilers M. Neubauer A. Inhibition of retinoic acid receptor signaling by Ski in acute myeloid leukemia.Leukemia. 2006; 20: 437-443Crossref PubMed Scopus (56) Google Scholar), colorectal (4Buess M. Terracciano L. Reuter J. Ballabeni P. Boulay J.L. Laffer U. Metzger U. Herrmann R. Rochlitz C. Amplification of SKI is a prognostic marker in early colorectal cancer.Neoplasia. 2004; 6: 207-212Crossref PubMed Scopus (58) Google Scholar), pancreatic (5Heider T.R. Lyman S. Schoonhoven R. Behrns K.E. Ski promotes tumor growth through abrogation of transforming growth factor-β signaling in pancreatic cancer.Ann. Surg. 2007; 246: 61-68Crossref PubMed Scopus (43) Google Scholar), esophageal (6Fukuchi M. Nakajima M. Fukai Y. Miyazaki T. Masuda N. Sohda M. Manda R. Tsukada K. Kato H. Kuwano H. Increased expression of c-Ski as a co-repressor in transforming growth factor-β signaling correlates with progression of esophageal squamous cell carcinoma.Int. J. Cancer. 2004; 108: 818-824Crossref PubMed Scopus (78) Google Scholar), and gastric (7Takahata M. Inoue Y. Tsuda H. Imoto I. Koinuma D. Hayashi M. Ichikura T. Yamori T. Nagasaki K. Yoshida M. Matsuoka M. Morishita K. Yuki K. Hanyu A. Miyazawa K. Inazawa J. Miyazono K. Imamura T. SKI and MEL1 cooperate to inhibit transforming growth factor-β signal in gastric cancer cells.J. Biol. Chem. 2009; 284: 3334-3344Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar) cancers. Although there is scarce evidence to suggest that SKI can transform mammalian cells except melanocytes (8Barkas, A., (1986) Ph.D. dissertation. New York UniversityGoogle Scholar), a reduction of SKI through small interfering RNA technology lessens the tumorigenic properties of cancer cells (9Chen D. Lin Q. Box N. Roop D. Ishii S. Matsuzaki K. Fan T. Hornyak T.J. Reed J.A. Stavnezer E. Timchenko N.A. Medrano E.E. SKI knockdown inhibits human melanoma tumor growth in vivo.Pigment Cell Melanoma Res. 2009; 22: 761-772Crossref PubMed Scopus (35) Google Scholar). Transgenic mice overexpressing Ski show overgrowth of type II muscle fibers but no enhanced tumor formation (10Sutrave P. Kelly A.M. Hughes S.H. Ski can cause selective growth of skeletal muscle in transgenic mice.Genes Dev. 1990; 4: 1462-1472Crossref PubMed Scopus (141) Google Scholar). Mice lacking the Ski gene result in early postnatal lethality with exencephaly caused by failed closure of the cranial neural tube during neurulation as well as a host of developmental abnormalities (11Berk M. Desai S.Y. Heyman H.C. Colmenares C. Mice lacking the ski proto-oncogene have defects in neurulation, craniofacial, patterning, and skeletal muscle development.Genes Dev. 1997; 11: 2029-2039Crossref PubMed Scopus (165) Google Scholar). Humans diagnosed with a haploid deficiency of SKI due to 1p36 deletion display similar phenotypes as shown in mice with a constitutional lack of Ski gene (12Colmenares C. Heilstedt H.A. Shaffer L.G. Schwartz S. Berk M. Murray J.C. Stavnezer E. Loss of the SKI proto-oncogene in individuals affected with 1p36 deletion syndrome is predicted by strain-dependent defects in Ski−/− mice.Nat. Genet. 2002; 30: 106-109Crossref PubMed Scopus (113) Google Scholar). The connection of the SKI oncoprotein with the TGFβ signaling pathway was established a decade ago by the finding that SKI can physically interact with Smad proteins, including Smad2, -3, and -4 (13Luo K. Stroschein S.L. Wang W. Chen D. Martens E. Zhou S. Zhou Q. The Ski oncoprotein interacts with the Smad proteins to repress TGFβ signaling.Genes Dev. 1999; 13: 2196-2206Crossref PubMed Scopus (389) Google Scholar, 14Sun Y. Liu X. Eaton E.N. Lane W.S. Lodish H.F. Weinberg R.A. Interaction of the Ski oncoprotein with Smad3 regulates TGF-β signaling.Mol. Cell. 1999; 4: 499-509Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar, 15Xu W. Angelis K. Danielpour D. Haddad M.M. Bischof O. Campisi J. Stavnezer E. Medrano E.E. Ski acts as a co-repressor with Smad2 and Smad3 to regulate the response to type β transforming growth factor.Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 5924-5929Crossref PubMed Scopus (180) Google Scholar). Smad proteins are the central mediators of TGFβ signaling pathways transmitting signals of the activated receptor at the plasma membrane to the nucleus (16Kretzschmar M. Massagué J. SMADs. Mediators and regulators of TGF-β signaling.Curr. Opin. Genet. Dev. 1998; 8: 103-111Crossref PubMed Scopus (431) Google Scholar). Upon activation of receptor through ligand binding, type I receptor kinase is activated and phosphorylates Smad protein that subsequently oligomerizes with Smad4, and the complex translocates to the nucleus to regulate target gene transcription. SKI regulates the TGFβ signaling at multiple levels; it interacts with the Smad proteins and inhibits the transcriptional activation of target genes, likely by recruiting the nuclear corepressor complex. Indeed, SKI has been found to be a component of the nuclear corepressor complex capable of inhibiting the transcriptional activation of reporter constructs (17Nomura T. Khan M.M. Kaul S.C. Dong H.D. Wadhwa R. Colmenares C. Kohno I. Ishii S. Ski is a component of the histone deacetylase complex required for transcriptional repression by Mad and thyroid hormone receptor.Genes Dev. 1999; 13: 412-423Crossref PubMed Scopus (251) Google Scholar). In addition, it has also been found that SKI can interact with the type I TGFβ receptor directly and can lead to repression of the receptor activity (18Ferrand N. Atfi A. Prunier C. The oncoprotein c-Ski functions as a direct antagonist of the transforming growth factor-β type I receptor.Cancer Res. 2010; 70: 8457-8466Crossref PubMed Scopus (29) Google Scholar). Because TGFβ signaling is a major cellular pathway that negatively regulates epithelial cell proliferation, the transforming capability of SKI is at least partially derived from its ability to neutralize the inhibition of cell proliferation by the TGFβ pathway. However, in the initial characterization of Ski oncogene in avian fibroblast cells, TGFβ signaling was thought to be promoting Ski-induced transformation by inhibiting Ski-induced myogenic differentiation (19Colmenares C. Stavnezer E. The ski oncogene induces muscle differentiation in quail embryo cells.Cell. 1989; 59: 293-303Abstract Full Text PDF PubMed Scopus (150) Google Scholar). Thus, TGFβ signaling cooperates with Ski to transform avian fibroblast cells. This apparent contradiction of the role of TGFβ signaling in Ski-induced cellular transformation has not been fully reconciled. Small ubiquitin-like modifier (SUMO) 3The abbreviation used is: SUMOsmall ubiquitin-like modifier. has been found to be involved in a variety of cellular processes, including intracellular trafficking, stress response, DNA replication, DNA damage repair, as well as transcriptional regulation (20Geiss-Friedlander R. Melchior F. Concepts in sumoylation. A decade on.Nat. Rev. Mol. Cell Biol. 2007; 8: 947-956Crossref PubMed Scopus (1353) Google Scholar). Four versions of SUMO, 1–4, have been identified. SUMO1–3 is expressed in the precursor forms and requires proteolytic activation to expose the invariant Gly-Gly motif at the C terminus of the mature SUMO for target conjugation. Much like the cellular ubiquitination process, SUMO activation enzyme E1, SUMO-conjugating enzyme E2, and SUMO ligase enzyme E3 are required to transfer the SUMO moiety to target proteins. SUMO E1 activates the SUMO through thioester bond formation between C-terminal Gly of SUMO and the catalytic cysteine residue of E1; the activated SUMO is then transferred to E2 with a similar thioester bond. Although E2 itself can transfer SUMO to target proteins, SUMO ligase E3 through enhancing efficiency and substrate specificity facilitates this process by transferring SUMO from the high energy bond to the ϵ-position of lysine of the target protein. Unlike ubiquitin modification, the main function of sumoylation is not a signal of destruction of the target through proteasomal degradation, instead it plays a role in a variety of cellular processes. SUMO modification in some cases has been shown to antagonize ubiquitination through competitive modification (21Bae S.H. Jeong J.W. Park J.A. Kim S.H. Bae M.K. Choi S.J. Kim K.W. Sumoylation increases HIF-1α stability and its transcriptional activity.Biochem. Biophys. Res. Commun. 2004; 324: 394-400Crossref PubMed Scopus (164) Google Scholar, 22Desterro J.M. Rodriguez M.S. Hay R.T. SUMO-1 modification of IκBα inhibits NF-κB activation.Mol. Cell. 1998; 2: 233-239Abstract Full Text Full Text PDF PubMed Scopus (911) Google Scholar, 23Sun Y. Perera J. Rubin B.P. Huang J. SYT-SSX1 (Synovial Sarcoma Translocated) regulates PIASy to cause overexpression of NCOA3.J. Biol. Chem. 2011; 286: 18623-18632Abstract Full Text Full Text PDF PubMed Scopus (6) Google Scholar). Another difference between ubiquitination and sumoylation is their respective core enzymes that carry out the modifications. It appears that there is only one E2 enzyme for sumoylation, although there are many E2s that can fulfill the ubiquitin-conjugating activity. Similar to regulation by ubiquitination, there are also proteases, sentrin-specific proteases, that can hydrolytically remove the SUMO moiety, and thus reverse the modification of sumoylation. These sentrin-specific proteases are also responsible for the activation of SUMO precursor by removing their C-terminal peptides to expose the Gly-Gly motif. Like many biological processes, the core machinery of SUMO modification has also been found to be regulated by other proteins. For example, RSUME and SF2/ASF have been found to enhance SUMO E2 enzyme Ubc9 to regulate target sumoylation (24Carbia-Nagashima A. Gerez J. Perez-Castro C. Paez-Pereda M. Silberstein S. Stalla G.K. Holsboer F. Arzt E. RSUME, a small RWD-containing protein, enhances SUMO conjugation and stabilizes HIF-1α during hypoxia.Cell. 2007; 131: 309-323Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar, 25Pelisch F. Gerez J. Druker J. Schor I.E. Muñoz M.J. Risso G. Petrillo E. Westman B.J. Lamond A.I. Arzt E. Srebrow A. The serine/arginine-rich protein SF2/ASF regulates protein sumoylation.Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 16119-16124Crossref PubMed Scopus (46) Google Scholar), although Rhes can enhance cross-sumoylation between E1 and Ubc9 (26Subramaniam S. Mealer R.G. Sixt K.M. Barrow R.K. Usiello A. Snyder S.H. Rhes, a physiologic regulator of sumoylation, enhances cross-sumoylation between the basic sumoylation enzymes E1 and Ubc9.J. Biol. Chem. 2010; 285: 20428-20432Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). small ubiquitin-like modifier. Several prominent proteins involved in tumorigenesis have been found to be modified by sumoylation. For example, p53 can be modified by sumoylation with either enhancement or reduction of its activity (27Hock A. Vousden K.H. Regulation of the p53 pathway by ubiquitin and related proteins.Int. J. Biochem. Cell Biol. 2010; 42: 1618-1621Crossref PubMed Scopus (57) Google Scholar). Similarly, MDM2, the main ubiquitin E3 ligase for p53, has been found to be sumoylated (28Miyauchi Y. Yogosawa S. Honda R. Nishida T. Yasuda H. Sumoylation of Mdm2 by protein inhibitor of activated STAT (PIAS) and RanBP2 enzymes.J. Biol. Chem. 2002; 277: 50131-50136Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar), and this sumoylation was shown to be associated with increased activity of MDM2 (29Lee M.H. Lee S.W. Lee E.J. Choi S.J. Chung S.S. Lee J.I. Cho J.M. Seol J.H. Baek S.H. Kim K.I. Chiba T. Tanaka K. Bang O.S. Chung C.H. SUMO-specific protease SUSP4 positively regulates p53 by promoting Mdm2 self-ubiquitination.Nat. Cell Biol. 2006; 8: 1424-1431Crossref PubMed Scopus (64) Google Scholar). MDM2 can be self-ubiquitinated and degraded. Sumoylation of MDM2 decreases its self-ubiquitination and degradation, whereas de-sumoylation leads to enhanced MDM2 self-ubiquitination. UV damage can decrease MDM2 sumoylation through a SUMO protease SUSP4. MDM2 sumoylation requires its N-terminal domain of amino acids 40–59 (30Buschmann T. Lerner D. Lee C.G. Ronai Z. The Mdm-2 amino terminus is required for Mdm2 binding and SUMO-1 conjugation by the E2 SUMO-1 conjugating enzyme Ubc9.J. Biol. Chem. 2001; 276: 40389-40395Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). We report here that SKI is capable of regulating sumoylation of MDM2 through its ability to interact with and regulate SUMO E2 enzyme Ubc9, resulting in enhanced MDM2 activity and decreased p53 protein. This novel function of SKI is likely a major molecular mechanism for its oncogenic activity as p53 is an important tumor suppressor, and a mechanism that leads to its destruction will greatly contribute to cellular transformation. LNCaP was cultured in RPMI 1640 medium; H1299, U2OS, HEK293, 293T, HepG2, and mink lung epithelial cells were cultured in Dulbecco's modified Eagle's medium; HCT116 and HCT116 p53−/− cells were in McCoy's 5A medium, and all were supplemented with 10% fetal calf serum, 2 mm glutamine, penicillin, and streptomycin at 37 °C with 5% CO2. Chemical siRNA for Ski, PIAS1, and PIAS3 were from IDT Inc. with the following sequences: Ski, sense 5′-rCrCrArGrUrArArGrGrArGrArCrUrUrGrArArArUrUrCrAGA-3′ and antisense 5′-rUrCrUrGrArArUrUrUrCrArArGrUrCrUrCrCrUrUrArCrUrGrGrUrU-3′; PIAS1, sense 5′-rUrGrGrUrUrArUrGrArGrCrCrUrUrArGrArGrUrUrUrCrUGA-3′ and antisense 5′-rUrCrArGrArArArCrUrCrUrArArGrGrCrUrCrArUrArArCrCrArUrU-3′; and PIAS3, sense 5′-rCrArCrUrGrArUrCrArArGrGrArGrArArArUrUrGrArCrUGC-3′ and antisense 5′-rGrCrArGrUrCrArArUrUrUrCrUrCrCrUrUrGrArUrCrArGrUrGrCrC-3′. Dharmafect was used to transfect siRNA into cells. Expression plasmids were introduced into cells with BioT transfection reagent (Bioland Inc.) according to the instruction of the vendor, and empty vector DNA was used to provide equal amount of DNA in each transfection. PCR primers for p53 target gene quantification are as follows: p21, forward 5′-CTGGAGACTCTCAGGGTCGAA-3′ and reverse 5′-GGATTAGGGCTTCCTCTTGGA-3′; Noxa, forward 5′-CTCTTTCCTCCTCGCCACTT-3′ and reverse 5′-CGTGCACCTCCTGAGAAAAC-3′; Tsp1, 5′-GGGAAGAAAATCATGGCTGA-3′ and reverse 5′-GGTCGCACGTTCTAGGAGTC-3′; Mdm2 mRNA (p2 promoter), forward 5′-CGATTGGAGGGTAGACCTGT-3′ and reverse 5′-GGTCTCTTGTTCCGAAGCTG-3′; Mdm2 (last exon), forward 5′-CAGACGGGGACTAGCTTTTG-3′ and reverse 5′-AGGTTGCAGTGAGCCAAGAT-3′; and Gapdh, forward 5′-CATGGGTGTGAACCATGAGA-3′ and reverse 5′-CAGTGATGGCATGGACTGTG-3′. RNA was isolated using Qiagen RNeasy kit, and cDNA was generated using SuperscriptII reverse transcriptase (Invitrogen). Anti-SKI, p53, and Ubc9 antibodies were from Santa Cruz Biotechnology. Anti-FLAG M2 antibody was from Sigma (A1205). Anti-HA antibody was from Roche Applied Science. Anti-SUMO1 antibody was from Epitomics. For immunoprecipitation and immunoblotting, cells were collected by scraping or trypsinization and lysed in lysis buffer (20 mm KCl, 150 mm NaCl, 1% IGEPAL, 50 mm Tris-HCl (pH 7.5), 50 mm NaF, 1 mm EGTA, 1 mm DTT, and 1× protease inhibitor mixture (Roche Applied Science) and 10% glycerol). Immunoprecipitations were performed with appropriate antibody and protein A- or G-Sepharose (Upstate Biotechnology). Beads were washed three times in lysis buffer, and immunoprecipitated proteins were separated on SDS-PAGE followed by Western blotting with primary and horseradish peroxidase-conjugated secondary antibodies (Bio-Rad). Immunoreactive proteins were visualized by enhanced chemiluminescence (SuperSignal West Femto, Pierce). CellTiter Glo (Promega Inc.) was used to measure relative cell number according to the manufacturer. For FACS analysis to gauge the sub-G1 DNA content, the cells were collected and fixed in 70% ethanol followed by RNase treatment and 1 μg/ml propidium iodide staining, and the cells were then analyzed by FACS. Expression plasmid for His6-MDM2 was transfected into 293T cells together with other expression plasmids. 40 h after transfection, cells were collected and lysed in 150 μl of RIPA buffer (regular buffer + 0.1% SDS + 0.5% sodium deoxycholate) followed by centrifugation. The supernatant was mixed with 8 m guanidine hydrochloride to obtain 6 m guanidine hydrochloride with 20 mm sodium phosphate buffer (pH 7.5), 20 mm imidazole, 1 mm DTT. This lysate was centrifuged at room temperature for 10 min, and the supernatant was mixed with 20 μl of nickel-nitrilotriacetic acid beads in 50% bead slurry. After binding for 3 h, the beads were washed three times with urea buffer (8 m urea with 20 mm sodium phosphate buffer (pH 7.5)) followed by incubation with sample buffer at 95 °C for 10 min. The supernatant was then separated by SDS-PAGE followed by immunoblot analysis. SKI is a transforming oncoprotein for chicken embryo fibroblast cells upon overexpression. Previously, we and others have established that SKI can interact with Smad proteins and negatively regulate TGFβ signal transduction (13Luo K. Stroschein S.L. Wang W. Chen D. Martens E. Zhou S. Zhou Q. The Ski oncoprotein interacts with the Smad proteins to repress TGFβ signaling.Genes Dev. 1999; 13: 2196-2206Crossref PubMed Scopus (389) Google Scholar, 14Sun Y. Liu X. Eaton E.N. Lane W.S. Lodish H.F. Weinberg R.A. Interaction of the Ski oncoprotein with Smad3 regulates TGF-β signaling.Mol. Cell. 1999; 4: 499-509Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar, 15Xu W. Angelis K. Danielpour D. Haddad M.M. Bischof O. Campisi J. Stavnezer E. Medrano E.E. Ski acts as a co-repressor with Smad2 and Smad3 to regulate the response to type β transforming growth factor.Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 5924-5929Crossref PubMed Scopus (180) Google Scholar), and it was widely thought that this inhibition of TGFβ signaling is the underlying mechanism of its transforming activity. Although this mechanistic explanation is plausible, we suspected that other mechanism(s) might also be involved in the oncogenic activity of SKI. Therefore, we examined in cells with overexpression of SKI the abundance of p53 protein, one of the essential tumor suppressor proteins in regulating cell proliferation and differentiation. As shown in Fig. 1A, overexpression of SKI in mink lung epithelial cells leads to a decrease of p53, and this reduction is independent of the status of TGFβ signaling (data not shown). Consistent with this reduction, the protein level of p21waf1, one of the p53 target genes, also decreases. Moreover, this reduced p53 level is likely a result of decreased metabolic stability as its half-life is apparently reduced in cells with overexpression of SKI (Fig. 1B). These findings therefore suggested the possibility that a higher level of SKI results in a reduction of p53, thus resulting in cellular transformation. We further tested whether in a transient overexpression system SKI can also cause a reduction of p53. As shown in Fig. 1C, in human colorectal carcinoma HCT-116 cells with or without endogenous Tp53 (see supplemental Fig. 1), overexpression of SKI by itself cannot decrease p53 levels; however, SKI can enhance the reduction of p53 caused by the wild type MDM2, the main ubiquitin E3 ligase for p53, but not the RING finger mutant of MDM2 (C464A) (31Honda R. Tanaka H. Yasuda H. Oncoprotein MDM2 is a ubiquitin ligase E3 for tumor suppressor p53.FEBS Lett. 1997; 420: 25-27Crossref PubMed Scopus (1597) Google Scholar). These results suggest that a higher level of SKI can result in an MDM2-dependent p53 reduction likely through enhanced p53 degradation. To rule out the possibility that SKI can only regulate p53 in an overexpression system, we also examined the impact of SKI depletion on endogenous p53 activity. As shown in Fig. 1D, siRNA-mediated loss-of-function of SKI in the prostate cancer cell line LNCaP results in elevated p53 levels and enhanced apoptosis as determined by cell number enumeration through measurement of ATP levels as well as of sub-G1 DNA content in a fluorescent-activated cell sorter (FACS) analysis (Fig. 1, E and F). Consistent with these findings, we also found that a subset of p53 target genes are up-regulated in response to a reduction of SKI and up-regulation of p53 (Fig. 1G). Taken together, these results strongly suggest that SKI with both physiological and quasi-physiological expressions can negatively regulate p53 through MDM2, and this aspect of SKI is likely a major contributor for its transforming activity. p53 is a major tumor suppressor protein that is inactivated in many human cancers. Nearly half of the human cancers harbor mutations of Tp53, whereas p53 is inactivated in a large proportion of tumors without Tp53 mutation through a multitude of mechanisms. For example, overexpression of MDM2, loss of the p14Arf protein, which is a negative regular of MDM2, and expression of viral oncoproteins, including HPV16 E6, adenovirus E1A, and SV40 large T antigen (32Hollstein M. Hainaut P. Massively regulated genes. the example of Tp53.J. Pathol. 2010; 220: 164-173Crossref PubMed Scopus (108) Google Scholar), can all lead to p53 inactivation. Although several ubiquitin E3 ligases have been shown to regulate p53 levels (33Brooks C.L. Gu W. p53 ubiquitination. Mdm2 and beyond.Mol. Cell. 2006; 21: 307-315Abstract Full Text Full Text PDF PubMed Scopus (690) Google Scholar), the genetically modified mouse without Mdm2 has unequivocally demonstrated the centrality of Mdm2 in regulating the p53 protein level (34Toledo F. Wahl G.M. Regulating the p53 pathway. In vitro hypotheses, in vivo veritas.Nat. Rev. Cancer. 2006; 6: 909-923Crossref PubMed Scopus (1041) Google Scholar). As described earlier, we found that SKI can enhance the MDM2-mediated p53 decrease (Fig. 1, A and C); therefore, we examined whether SKI can positively regulate MDM2-mediated ubiquitination of p53. As shown in Fig. 2A, although expression of MDM2 alone enhanced the ubiquitination of p53, co-expression of SKI dramatically increased the p53 ubiquitination shown both by the ubiquitin in the p53 immunoprecipitation as well as p53 in the immunoprecipitation of ubiquitin (supplemental Fig. 2). These results suggest that SKI can enhance MDM2-mediated p53 ubiquitination and thus proteasomal degradation, which likely results in a decreased p53 level. It has been shown that a mutant SKI with an insertion of four amino acids, alanine, arginine, proline, and glycine (ARPG) replacing aspartic acid at position 181, inactivated the transforming function of SKI in chicken embryo fibroblast cells (35Colmenares C. Teumer J.K. Stavnezer E. Transformation-defective v-ski induces MyoD and myogenin expression but not myotube formation.Mol. Cell. Biol. 1991; 11: 1167-1170Crossref PubMed Scopus (50) Google Scholar), and we therefore tested whether the transforming capacity of SKI is correlated with its ability to enhance ubiquitination of p53. As shown in Fig. 2B, lane 4, expression of the SKI mutant ARPG, at a comparable level of wild type SKI protein, did not enhance, if reduced, p53 ubiquitination, suggesting that the transforming ability of SKI is tightly associated with its interaction with MDM2 to result in p53 ubiquitination. In addition, we also tested whether SKI at a physiological level can regulate MDM2-mediated p53 ubiquitination. As shown in Fig. 2C, siRNA-mediated reduction of endogenous SKI protein also reduced p53 ubiquitination conferred upon by MDM2 expression. These results suggest that SKI at both normal and overexpression levels is capable of enhancing MDM2-mediated ubiquitination of p53, and this activity is tightly associated with the transforming function of SKI. The ability of SKI to enhance MDM2-mediated ubiquitination of p53 suggests several possible modes of action of SKI in the regulation of p53. It is possible that SKI directly regulates p53's propensity of being ubiquitinated by MDM2 followed by proteosomal degradation. It is also possible that SKI can positively enhance the activity of MDM2 through direct physical interaction with MDM2; alternatively, SKI can regulate MDM2 through post-translational modification without direct physical interaction. We established that in an overexpression system, SKI could physically interact with MDM2 (supplemental Fig. 3). However, repeated attempts in a variety of cell types failed to demonstrate this physical interaction at endogenous expression levels. Nevertheless, we can clearly demonstrate that overexpression of SKI increases the abundance of MDM2, consistent with its ability to decrease the p53 level (Fig. 3, A, and middle panel of B). Conversely, a depletion of SKI by siRNA in HepG2 cells can decrease endogenous MDM2 levels (Fig. 3B, right panel), supporting the view that SKI can positively regulate the abundance of MDM2. Moreover, the increased level of MDM2 by SKI is likely a result of increased stability of MDM2 as shown in Fig. 3C, whereas the SKI mutant ARPG fails to increase the stability of MDM2 (Fig. 3C, right panel). These results suggest that SKI can functionally enhance MDM2, likely its protein level or activity, yet it does not do so through high affinity or direct physical interaction with MDM2. 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• W2021818279 created "2016-06-24" @default.
• W2021818279 creator A5029912396 @default.
• W2021818279 creator A5040974046 @default.
• W2021818279 creator A5051269814 @default.
• W2021818279 date "2012-04-01" @default.
• W2021818279 modified "2023-10-12" @default.
• W2021818279 title "Overexpression of SKI Oncoprotein Leads to p53 Degradation through Regulation of MDM2 Protein Sumoylation" @default.
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