Expression of SUMO-2/3 Induced Senescence through p53- and pRB-mediated Pathways
2006; Elsevier BV; Volume: 281; Issue: 47 Linguagem: Inglês
10.1074/jbc.m608236200
ISSN1083-351X
AutoresTianwei Li, Rasa Santockyte, Rong‐Fong Shen, Ephrem Tekle, Guanghui Wang, David C.H. Yang, P Boon Chock,
Tópico(s)interferon and immune responses
ResumoThree highly homologous small ubiquitin-related modifier (SUMO) proteins have been identified in mammals. Modifications of proteins by SUMO-1 have been shown to regulate transcription, nucleocytoplasmic transport, protein stability, and protein-protein interactions. Relative to SUMO-1, little is known about the functions of SUMO-2 or SUMO-3 (referred to as SUMO-2/3). Here, stable cell lines overexpressing processed forms of SUMO-2/3 (SUMO-2/3GG) as well as their non-conjugatable derivatives, SUMO-2/3ΔGG, were established. Cells overexpressing SUMO-2/3GG showed a premature senescence phenotype as revealed by cellular morphology and senescence-associated galactosidase activity. The senescence pathway protein p21 was up-regulated in cells overexpressing SUMO-2/3GG. In contrast, cells overexpressing non-conjugatable forms of SUMO-2/3ΔGG showed neither an apparent senescent phenotype nor elevated p21. Both p53 and pRB were found to be modified by SUMO-2/3. Site-directed mutagenesis studies showed that Lys-386 of p53, the SUMO-1 modification site, is also the modification site for SUMO-2/3. In addition, H2O2 treatment of untransfected cells caused an increase in p53 sumoylation by SUMO-2/3, whereas that by SUMO-1 remained unchanged. Moreover, knocking down tumor suppressor proteins p53 or pRB using small interfering RNA significantly alleviated the premature senescence phenotypes in SUMO-2/3GG overexpressing cells. Together, our results reveal that p53 and pRB can be sumoylated by SUMO-2/3 in vivo, and such modification of p53 and pRB may play roles in premature senescence and stress response. Three highly homologous small ubiquitin-related modifier (SUMO) proteins have been identified in mammals. Modifications of proteins by SUMO-1 have been shown to regulate transcription, nucleocytoplasmic transport, protein stability, and protein-protein interactions. Relative to SUMO-1, little is known about the functions of SUMO-2 or SUMO-3 (referred to as SUMO-2/3). Here, stable cell lines overexpressing processed forms of SUMO-2/3 (SUMO-2/3GG) as well as their non-conjugatable derivatives, SUMO-2/3ΔGG, were established. Cells overexpressing SUMO-2/3GG showed a premature senescence phenotype as revealed by cellular morphology and senescence-associated galactosidase activity. The senescence pathway protein p21 was up-regulated in cells overexpressing SUMO-2/3GG. In contrast, cells overexpressing non-conjugatable forms of SUMO-2/3ΔGG showed neither an apparent senescent phenotype nor elevated p21. Both p53 and pRB were found to be modified by SUMO-2/3. Site-directed mutagenesis studies showed that Lys-386 of p53, the SUMO-1 modification site, is also the modification site for SUMO-2/3. In addition, H2O2 treatment of untransfected cells caused an increase in p53 sumoylation by SUMO-2/3, whereas that by SUMO-1 remained unchanged. Moreover, knocking down tumor suppressor proteins p53 or pRB using small interfering RNA significantly alleviated the premature senescence phenotypes in SUMO-2/3GG overexpressing cells. Together, our results reveal that p53 and pRB can be sumoylated by SUMO-2/3 in vivo, and such modification of p53 and pRB may play roles in premature senescence and stress response. The amino acid sequence of the small ubiquitin-modifier (SUMO) 2The abbreviations used are: SUMO, small ubiquitin-related modifier; pRB, retinoblastoma tumor suppressor protein; siRNA, small interfering RNA; HEK, human embryonic kidney; HA, hemagglutinin; MEF, mouse embryonic fibroblast; Ni-NTA, nickel-nitrilotriacetic acid; SA-β-Gal, senescence-associated β-galactosidase; E1, E1 is ubiquitin-activating enzyme; E3, ubiquitin-protein isopeptide ligase. 2The abbreviations used are: SUMO, small ubiquitin-related modifier; pRB, retinoblastoma tumor suppressor protein; siRNA, small interfering RNA; HEK, human embryonic kidney; HA, hemagglutinin; MEF, mouse embryonic fibroblast; Ni-NTA, nickel-nitrilotriacetic acid; SA-β-Gal, senescence-associated β-galactosidase; E1, E1 is ubiquitin-activating enzyme; E3, ubiquitin-protein isopeptide ligase. is only 18% identical to that of ubiquitin, but its three-dimensional structure is very similar to ubiquitin. SUMO is attached to its target proteins by an enzymic mechanism that is analogous to the ubiquitinylation pathway (1Gill G. Genes Dev. 2004; 18: 2046-2059Crossref PubMed Scopus (618) Google Scholar, 2Schwartz D.C. Hochstrasser M. Trends Biochem. Sci. 2003; 28: 321-328Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar, 3Yeh E.T. Gong L. Kamitani T. Gene. 2000; 248: 1-14Crossref PubMed Scopus (411) Google Scholar). The mammalian SUMO family consists of SUMO-1, -2, and -3. Human SUMO-1 (SMT3C) exhibits 46% sequence identity with SUMO-2 (SMT3A) and SUMO-3 (SMT3B), whereas SUMO-2 and SUMO-3 are 96% identical in their processed forms; thus SUMO-2 and SUMO-3 are referred as SUMO-2/3 (4Johnson S.E. Annu. Rev. Biochem. 2004; 73: 355-382Crossref PubMed Scopus (1369) Google Scholar). SUMO-1 has been found to covalently modify >70 target proteins. Many of them are important regulatory proteins, such as p53, IκBα, c-Jun, promyelocytic leukemia protein (PML), and proliferating cell nuclear antigen (PCNA) (5Ulrich H.D. Trends Cell Biol. 2005; 15: 525-532Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar, 6Seeler J.S. Dejean A. Nat. Rev. Mol. Cell Biol. 2003; 4: 690-699Crossref PubMed Scopus (575) Google Scholar). The functions of SUMO-1 conjugation are target-specific and highly diverse. Sumoylation may be involved in the regulation of transcription, nucleocytoplasmic transport, DNA repair, protein stability, and chromosome separation (1Gill G. Genes Dev. 2004; 18: 2046-2059Crossref PubMed Scopus (618) Google Scholar, 2Schwartz D.C. Hochstrasser M. Trends Biochem. Sci. 2003; 28: 321-328Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar). Unlike SUMO-1, SUMO-2/3 can form polysumoylation chains due to their sequences containing the intrinsic SUMO consensus sequence ψKXE, where ψ stands for a large hydrophobic amino acid residue (7Tatham M.H. Jaffray E. Vaughan O.A. Desterro J.M. Botting C.H. Naismith J.H. Hay R.T. J. Biol. Chem. 2001; 276: 35368-35374Abstract Full Text Full Text PDF PubMed Scopus (634) Google Scholar). Another important difference between SUMO-2/3 and SUMO-1 conjugation pathways in mammalian cells is that the majority of SUMO-1 exists in the conjugated forms, whereas SUMO-2/3 exist primarily as free forms and readily conjugate to substrates under certain stresses (8Saitoh H. Hinchey J. J. Biol. Chem. 2000; 275: 6252-6258Abstract Full Text Full Text PDF PubMed Scopus (680) Google Scholar). In contrast to the large number of identified SUMO-1 target proteins, only a few SUMO-2/3-modified proteins have been reported. The physiological functions of SUMO-2/3 are not well understood.Cellular premature senescence is a program triggered by cells in response to various types of stresses, including DNA damage, oxidative stress, and oncogene activation (9Campisi J. Exp. Gerontol. 2003; 38: 5-11Crossref PubMed Scopus (171) Google Scholar, 10Campisi J. Cell. 2005; 120: 513-522Abstract Full Text Full Text PDF PubMed Scopus (1786) Google Scholar). Cells entering senescence undergo permanent cell cycle arrest with a set of functional and morphological changes. The cellular signals activated by the stresses are funneled to p53 and pRB, which determine whether cells enter senescence (10Campisi J. Cell. 2005; 120: 513-522Abstract Full Text Full Text PDF PubMed Scopus (1786) Google Scholar, 11Okada H. Mak T.W. Nat. Rev. Cancer. 2004; 4: 592-603Crossref PubMed Scopus (860) Google Scholar). Accordingly, most genes found to regulate senescence (i.e. p21 and p16INK4a) can be placed upstream or downstream of p53 or pRB in the signaling pathways (12Ben-Porath I. Weinberg R.A. Int. J. Biochem. Cell Biol. 2004; 37: 961-976Crossref PubMed Scopus (789) Google Scholar). p53 can be modified with SUMO-1 at lysine 386, and the sumoylation of p53 enhances its transcriptional activity (13Gostissa M. Hengstermann A. Fogal V. Sandy P. Schwarz S.E. Scheffner M. EMBO J. 1999; 18: 6462-6471Crossref PubMed Scopus (437) Google Scholar, 14Rodriguez M.S. Desterro J.M. Lain S. Midgley C.A. Lane D.P. Hay R.T. EMBO J. 1999; 18: 6455-6461Crossref PubMed Scopus (558) Google Scholar, 15Müller S. Berger M. Lehembre F. Seeler J.S. Haupt Y. Dejean A. J. Biol. Chem. 2000; 275: 13321-13329Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar). Most recently, pRB was also found to be modified by SUMO-1, and its activity was regulated by sumoylation (16Ledl A. Schmidt D. Muller S. Oncogene. 2005; 24: 3810-3818Crossref PubMed Scopus (87) Google Scholar). Because p53 and pRB as well as SUMO-2/3 (but not SUMO-1) are involved in cellular stress responses, it is of interest to determine whether SUMO-2/3 can modify p53 and pRB as a possible regulatory mechanism in cellular senescence.To further investigate the physiological functions of SUMO-2/3, we developed stable HEK293 Tet-On cell lines overexpressing Myc-His-SUMO-2/3GG and Myc-His-SUMO-2/3ΔGG. Our results reveal that p53 and pRB are modified by SUMO-2/3, and premature cellular senescence also occurs in cells overexpressing SUMO-2/3. Furthermore, H2O2 induced p53 modification by SUMO-2/3 in untransfected HEK293 cells. These findings provide new insights into the potential regulatory role of sumoylation in cellular senescence in response to oxidative stress.EXPERIMENTAL PROCEDURESAntibodies, Plasmids, and Mutagenesis—The monoclonal anti-Myc (9E10), anti-SUMO-1, anti-RanGAP1, anti-p53 (DO-1) antibodies, agarose-conjugated anti-Myc, and anti-p53 antibodies were purchased from Santa Cruz Biotechnology, monoclonal anti-HA and anti-β-actin antibodies from Sigma, monoclonal anti-p21 and anti-pRB antibodies from Upstate Biotechnology, and polyclonal anti-SUMO-2/3 antibody from Abcam. The cDNAs encoding the processed forms of SUMO-1 (1-97), SUMO-2 (1-92), and SUMO-3 (1-93) with Gly-Gly at their C termini were amplified by PCR. His6 tag sequences immediately upstream of the start codons of the SUMO-1/2/3 sequences were designed in the amplifying primers. The PCR-amplified cDNAs were inserted into the pTRE2hyg2-Myc vector (Clontech) as NheI/ClaI fragments to generate pTRE2hyg2-Myc-His-SUMO-1/2/3GG plasmids. The pTRE2hyg2-Myc-His-SUMO-2/3ΔGG plasmids harboring non-conjugatable SUMO-2 (1-90) and SUMO-3 (1-91) sequences, lacking the C-terminal Gly-Gly, were similarly constructed. The wild-type p53 cDNA was amplified by PCR and inserted into the pTRE2pur-HA (Clontech) as a MluI/ClaI fragment to generate the pTRE2pur-HA-p53WT plasmid. The pTRE2pur-HA-p53K386R (where Lys-386 is replaced by Arg) plasmid was constructed as described above using a 3′ primer specifically designed with the corresponding mutation. The p53 response reporter plasmids pRGC-Luc and pG13-Luc, were generous gifts from Dr. Seung J. Baek (University of Tennessee) and Dr. Ronald T. Hay (University of St. Andrews), respectively.Cell Culture, Transfection, and Cell Lines—The stable HEK293 Tet-On or mouse embryonic fibroblast (MEF) 3T3 Tet-Off cell lines overexpressing Myc-His-SUMO-1/2/3GG and Myc-His-SUMO-2/3ΔGG were established using the protocol previously described (17Li T. Evdokimov E. Shen R.F. Chao C.C. Tekle E. Wang T. Stadtman E.R. Yang D.C. Chock P.B. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 8551-8556Crossref PubMed Scopus (147) Google Scholar). FuGENE6 (Roche Applied Science) was used for transient transfection.Purification of His6-tagged SUMO Substrates—The His6-tagged proteins were purified under denaturing conditions using Ni-NTA-agarose according to the manufacturer's instructions (Qiagen) with some modifications. Briefly, after induction with 2 μg/ml doxycycline for 48 h, 1 × 108 cells were lysed in lysis buffer A (6 m guanidinium-HCl, pH 8.0, 100 mm Na2HPO4/NaH2PO4, 10 mm Tris-HCl, 10 mm imidazole, 10 mm β-mercaptoethanol, 20 mm N-ethylmaleimide, and 20 mm iodoacetamide). The clarified cell lysates were incubated with Ni-NTA-agarose beads for 1 h at 4 °C. The beads were washed twice for 2 min each time with 0.5 ml of each of the following buffers: lysis buffer A, washing buffer A (8 m urea, pH 8.0, 100 mm Na2HPO4/NaH2PO4, 10 mm Tris-HCl, 10 mm imidazole, and 20 mm N-ethylmaleimide), and washing buffer B (8 m urea, pH 6.3, 100 mm Na2HPO4/NaH2PO4, 10 mm Tris-HCl, 10 mm imidazole, 20 mm N-ethylmaleimide, and 0.2% Triton X-100). For Western blotting, the His6-tagged proteins were eluted by boiling Ni-NTA-agarose beads in 2× NuPAGE sample buffer containing 250 mm imidazole. Proteins were resolved by 4-12% NuPAGE gels (Invitrogen) and probed with specific antibodies.Immunoprecipitation and Immunoblotting—For immunoprecipitation, cells were lysed in lysis buffer B (50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 1% Nonidet P-40, 5 mm EDTA, 20 mm N-ethylmaleimide, 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml pepstatin, 20 μg/ml leupeptin, and 10 μg/ml aprotinin) to obtain whole cell extracts. Immunoprecipitation and immunoblotting were carried out as described previously (17Li T. Evdokimov E. Shen R.F. Chao C.C. Tekle E. Wang T. Stadtman E.R. Yang D.C. Chock P.B. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 8551-8556Crossref PubMed Scopus (147) Google Scholar).Two-dimensional Gel Electrophoresis—The whole cell extracts from stable cell lines overexpressing SUMO-1/2/3GG were analyzed by two-dimensional gel electrophoresis as described previously (17Li T. Evdokimov E. Shen R.F. Chao C.C. Tekle E. Wang T. Stadtman E.R. Yang D.C. Chock P.B. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 8551-8556Crossref PubMed Scopus (147) Google Scholar).p53 Transcriptional Transactivation Activity Assay—For the pRGC-Luc and pG13-Luc p53 response reporter plasmids, transfection was used to assess the transcriptional activity of p53 in vivo. The luciferase activity was determined with the Luciferase reporter assay kit (BioVision) using a Turner Design luminometer (Promega). The values obtained were normalized with protein concentration in each sample.Senescence-associated Galactosidase Assays—The senescence-associated expression of β-galactosidase (SA-β-Gal) activity was monitored using a senescence detection kit (BioVision).RNA Interference—The human p53 siRNA, human pRB siRNA, or control siRNA (Santa Cruz Biotechnology) was transfected into SUMO-2GG or SUMO-3GG stable cells using siRNA transfection reagent (Santa Cruz Biotechnology) according to the manufacturer's instructions.RESULTSEnhancement of SUMO-2/3 Conjugations in Cells Expressing SUMO-2/3—Unlike SUMO-1, SUMO-2/3 are present mainly in their free forms instead of conjugated forms under normal conditions, and the conjugation of SUMO-2/3 increases significantly after certain stress conditions, such as heat shock and oxidative stress (8Saitoh H. Hinchey J. J. Biol. Chem. 2000; 275: 6252-6258Abstract Full Text Full Text PDF PubMed Scopus (680) Google Scholar). Stable cell lines overexpressing the processed forms of SUMO-2/3 (Myc-His-SUMO-2/3GG) and their non-conjugatable mutants (Myc-His-SUMO-2/3ΔGG) were established to analyze the consequences of the increased levels of SUMO-2/3 conjugation during stresses. As shown in Fig. 1, Myc-His-SUMO-2/3GG (abbreviated as SUMO-2/3GG) and Myc-His-SUMO-2/3ΔGG (abbreviated as SUMO-2/3ΔGG) were successfully expressed in the respective cell lines. A large number of high molecular weight sumoylated proteins and unconjugated SUMO-2/3 was detected by probing with either anti-Myc or anti-SUMO-2/3 antibody in cells expressing SUMO-2/3GG. The expression level of SUMO-2GG was apparently higher than that of SUMO-3GG, correlating with the higher level of sumoylated proteins observed by anti-SUMO-2/3 antibody in SUMO-2GG cells (Fig. 1). However, only unconjugated SUMO-2/3 were detected by anti-Myc antibody, and the level of endogenous sumoylated high molecular weight proteins detected by anti-SUMO-2/3 antibody is relatively low in cells expressing SUMO-2/3ΔGG (Fig. 1). This observation indicates that epitope-tagged SUMO-2/3GG are functional in cells, and high molecular weight proteins revealed by Western blotting are sumoylated proteins.The Patterns of Proteins Modified by SUMO-2 and SUMO-3 Are Similar but Different from That of SUMO-1—As shown in Fig. 2A, the majority of SUMO-1 is conjugated to the target proteins, whereas appreciable amounts of SUMO-2/3 are present in free forms. SUMO-1 modification apparently did not respond to H2O2 treatment; in contrast, SUMO-2/3 conjugates increased significantly after cells were treated with H2O2 (Fig. 2A, lanes 2). These results are consistent with a previous report (8Saitoh H. Hinchey J. J. Biol. Chem. 2000; 275: 6252-6258Abstract Full Text Full Text PDF PubMed Scopus (680) Google Scholar). Furthermore, Western blot analysis using anti-SUMO-2/3 antibody revealed that RanGAP1, the most abundant SUMO-1 substrate, was modified by SUMO-2/3. This sumoylation of RanGAP1 was further confirmed by immunoprecipitation with anti-RanGAP1 antibody (Fig. 2A, lanes 3). The global patterns of SUMO target proteins by different SUMO paralogues were further characterized by two-dimensional gel electrophoresis. As shown in Fig. 2B, the overall sumoylation patterns between SUMO-2 and SUMO-3 are apparently similar, indicating that SUMO-2 and SUMO-3 may target the same proteins with similar intensity. In contrast, SUMO-1 modified many neutral proteins and fewer high molecular weight proteins. These results indicate that SUMO-1 and SUMO-2/3 have different substrate preferences that may function specifically under distinct conditions.FIGURE 2The sumoylation patterns of SUMO-1/2/3 in HEK293 cells. A, 10 μg of the whole cell lysate of HEK293 cells (lanes 1), 10 μg of the whole cell lysate of HEK293 cells treated with 5 μm H2O2 for 20 min at 37 °C (lanes 2), and anti-RanGAP1 immunoprecipitate from 200 μg of the whole cell lysate of HEK293 cells (lanes 3) were resolved using NuPAGE gels and probed with anti-SUMO-1 or anti-SUMO-2/3 antibody, respectively. The two dark bands below the SUMO-1-modified RanGAP1 (lanes 3) are the heavy and light chains of anti-RanGAP antibody used in immunoprecipitation. B, 10 μg of the whole cell lysates from HEK293 Tet-On cells stably expressing Myc-His-tagged SUMO-1GG, SUMO-2GG, or SUMO-3GG were resolved using 7-cm two-dimensional gel electrophoresis and probed with anti-Myc antibody. Arrows indicate sumoylated RanGAP1.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Overexpressing Processed Forms of SUMO-2/3-induced Senescence—Interestingly, cells overexpressing processed forms of SUMO-2/3 (SUMO-2/3GG) were found to grow slowly. Furthermore, many enlarged and flattened cells, a morphology commonly observed in senescent cells, were observed in the cells overexpressing SUMO-2/3GG (Fig. 3). Such a phenotype was not observed in cells overexpressing non-conjugatable forms of SUMO-2/3 (SUMO-2/3ΔGG). The SA-β-Gal activity was measured to confirm whether cells exhibiting the senescent morphology were indeed in senescence. As shown in Fig. 3, many senescent cells, stained blue due to SA-β-Gal activity, were found among cells overexpressing SUMO-2/3GG, but hardly any were found among cells overexpressing SUMO-2/3ΔGG. These results suggest that elevated cellular SUMO-2/3 modification levels in cells expressing SUMO-2/3GG may interfere with cell growth by inducing senescence. This unexpected observation with the immortal HEK293 cells was further confirmed in MEF 3T3 cells, a cell line typically used in senescence study. As shown in Fig. 3, overexpression of SUMO-2/3 also caused these cells to become enlarged and flattened, exhibiting SA-β-Gal activity.FIGURE 3The SA-β-Gal assay for processed and non-conjugatable SUMO-2/3 stable expression cells. The SA-β-Gal activity of HEK293 Tet-On cells or MEF 3T3 Tet-Off cells stably expressing Myc-His-SUMO-2/3GG or Myc-His-SUMO-2/3ΔGG was detected using a senescence detection kit (BioVision). The arrows indicate enlarged and flattened cells.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Knocking Down p53 or pRB Significantly Alleviated Senescence Phenotype—The tumor suppressor proteins p53 and pRB are two major regulatory proteins known to mediate premature cellular senescence. We wondered whether knocking down these proteins would alleviate the effects of overexpressing SUMO-2/3GG-induced senescence. As shown in Fig. 4A, when cells overexpressing SUMO-2/3GG were transfected with either p53 siRNA or pRB siRNA for 48 h, cellular p53 or pRB protein level was reduced to about one-third of that observed in cells transfected with a control siRNA consisting of a non-targeting 20-25-nt RNA sequence. Furthermore, the senescence-associated β-galactosidase activity was significantly reduced in p53 or pRB knock-down cells (Fig. 4B). These results indicate that the SUMO-2/3-induced premature cellular senescence is dependent on both p53 and pRB pathways.FIGURE 4Knocking down p53 or pRB in SUMO-2/3GG stable expression cells. A, HEK293 cells stably expressing SUMO-2/3GG were transfected with human p53 siRNA, pRB siRNA, or control (Contl) siRNA and cultured for 48 h. Whole cell extracts were resolved using NuPAGE gels and probed with anti-pRB, anti-p53, or anti-β-actin, respectively. β-actin was used to show equal loading of whole cell extracts. B, the SA-β-Gal activities of siRNA-transfected cells were assayed using a senescence detection kit (BioVision).View Large Image Figure ViewerDownload Hi-res image Download (PPT)p53 and pRB Are Modified by SUMO-2/3—The His6-tagged sumoylated proteins from SUMO-2/3GG and SUMO-2/3ΔGG overexpressing cells were purified using Ni-NTA-agarose and probed with anti-RanGAP1, anti-p53, or anti-pRB antibodies, respectively. As shown in Fig. 5, protein bands corresponding to the sizes of the monosumoylated RanGAP1, p53, and pRB were observed in cells expressing SUMO-2/3GG but not in those expressing SUMO-2/3ΔGG. These results indicate that RanGAP1, p53, and pRB can be modified by SUMO-2/3. Interestingly, some slower migrated bands corresponding to polysumoylated p53 were also found, implying that p53 might be polysumoylated by SUMO-2/3.FIGURE 5The modification of p53 and pRB by SUMO-2/3. After 48 h of incubation with 2 μg/ml doxycycline, whole cell extracts from HEK293 cells stably expressing SUMO-2/3GG or SUMO-2/3 ΔGG were purified using Ni-NTA-agarose beads and probed with anti-RanGAP1, anti-p53, or anti-pRB as indicated. The solid arrows indicate the band corresponding to the size of monosumoylated target proteins. The open arrow indicates the bands corresponding to the sizes of polysumoylated proteins.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The Sumoylation Site of p53 by SUMO-2/3 Has Been Identified to be Lys-386—To determine whether SUMO-2/3 also conjugates to p53 at Lys-386, HA-tagged wild-type p53 (p53WT) and HA-tagged mutant p53 (p53K386R) were transiently expressed in cells stably overexpressing SUMO-2/3GG (13Gostissa M. Hengstermann A. Fogal V. Sandy P. Schwarz S.E. Scheffner M. EMBO J. 1999; 18: 6462-6471Crossref PubMed Scopus (437) Google Scholar, 14Rodriguez M.S. Desterro J.M. Lain S. Midgley C.A. Lane D.P. Hay R.T. EMBO J. 1999; 18: 6455-6461Crossref PubMed Scopus (558) Google Scholar, 15Müller S. Berger M. Lehembre F. Seeler J.S. Haupt Y. Dejean A. J. Biol. Chem. 2000; 275: 13321-13329Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar). When His6-tagged SUMO-2/3-conjugated proteins were isolated using Ni-NTA-agarose and analyzed by Western blot with anti-HA antibody, only p53 WT (but not p53K386R) was found to co-purify with His6-tagged SUMO-2/3-conjugated proteins (Fig. 6A). These results indicate that SUMO-2/3 and SUMO-1 share the same conjugating site, Lys-386, in p53. However, no polysumoylated p53 was observed, likely because of the limited expression level of HA-tagged p53.FIGURE 6Modification of p53 by SUMO-2/3 at lysine 386 and its response to H2O2. A, HEK293 cells stably expressing SUMO-2/3GG were transfected with pTRE2pur-HA-p53WT or pTRE2pur-HA-p53K386R plasmid. His-tagged SUMO-2/3-conjugated proteins were purified using Ni-NTA-agarose beads and probed with anti-HA antibody. The whole cell extracts (WCE) were used to show equal loading. B, HEK293 cells were incubated with 5 μm H2O2 for 20 min at 37 °C, and the whole cell lysates were immunoprecipitated with anti-p53 antibody. The precipitates were probed with anti-SUMO-1 or anti-SUMO-2/3 antibody. The arrows indicate the band corresponding to the size of monosumoylated p53.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Oxidative Stress Stimulated p53 Sumoylation by SUMO-2/3 but Not SUMO-1—It has been reported that sumoylation by SUMO-2/3 (but not SUMO-1) responds to a number of stress conditions, e.g. heat shock and oxidative stress (8Saitoh H. Hinchey J. J. Biol. Chem. 2000; 275: 6252-6258Abstract Full Text Full Text PDF PubMed Scopus (680) Google Scholar). As shown in Fig. 6B, when untransfected HEK293 cells were treated with 5 μm H2O2 for 20 min at 37 °C, the SUMO-2/3-modified p53 was elevated from an undetectable level to a clearly visible level in Western analysis monitored by anti-SUMO-2/3 antibody. However, no polysumoylated p53 was detected under these conditions. This observation is likely because of the presence of the relatively low abundance of modified p53. On the other hand, the level of SUMO-1-modified p53 exhibited no noticeable change following H2O2 treatment (Fig. 6B).Increase of p53 Transcriptional Activation by Modification with SUMO-2/3—To assess the functional changes of p53 in cells resulting from SUMO-2/3 modification, reporter gene constructs containing binding sites for p53 at the promoter regions were used to analyze the transcriptional activity of p53. When stable cell lines were transfected with the pRGC-Luc p53 reporter plasmid, the luciferase activity in cells expressing SUMO-2GG was found to be elevated ∼4-fold over those expressing SUMO-2ΔGG. Similarly, luciferase activity in cells expressing SUMO-3GG was ∼2-fold over those expressing SUMO-3ΔGG. In contrast, both SUMO-2ΔGG- and SUMO-3ΔGG-transfected cell lines showed similarly low levels of luciferase activity (Fig. 7). Similar results were also observed when pG13-Luc was used as the p53 reporter plasmid or when MEF 3T3 Tet-Off cells were co-transfected with pRGC-Luc, p53WT, and the four SUMO-2/3 constructs (data not shown). In addition, p21, which is a direct target of p53, was induced in cells overexpressing SUMO-2/3 but not in those overexpressing the non-conjugatable SUMO-2/3ΔGG (Fig. 1). These findings suggest that overexpressing SUMO-2/3GG elevates the p53 sumoylation level, which in turn stimulates p53 transcriptional activity.FIGURE 7The stimulation of p53 transcriptional activity by overexpressing SUMO-2/3. Shown are HEK293 Tet-On cells stably expressing SUMO-2ΔGG (1), SUMO-2GG (2), SUMO-3ΔGG (3), or SUMO-3GG (4) transfected with pRGC-luc, a p53-response reporter plasmid, 1 μg/well in a 12-well plate. Twenty-four hours after transfection, luciferase reporter activity was assayed, and the values obtained were normalized by protein concentration in each sample. Each value is the mean from three independent transfections with the error bar indicating S.D.View Large Image Figure ViewerDownload Hi-res image Download (PPT)DISCUSSIONConsidering the distinct aspects of the elevation of protein modification by SUMO-2/3 in response to biological stresses (8Saitoh H. Hinchey J. J. Biol. Chem. 2000; 275: 6252-6258Abstract Full Text Full Text PDF PubMed Scopus (680) Google Scholar), it is of interest to identify SUMO-2/3 target proteins and to investigate the consequences of these modifications. In this study, stable cell lines overexpressing SUMO-2/3GG were used to serve as models for evaluating the effects of the elevated level of SUMO-2/3 modification under stress conditions. As shown in the right panel of Fig. 1, the overall SUMO-2/3GG conjugation patterns, except for the elevation of conjugation levels, are similar to endogenous SUMO-2/3 conjugation patterns in cells expressing non-conjugatable SUMO-2/3ΔGG. These results indicate that our stable cell lines could serve as suitable models mimicking the stress-induced elevation of SUMO-2/3 conjugation. We showed clearly that RanGAP1, the most abundant substrate of SUMO-1, is also sumoylated by SUMO-2/3 at their endogenous levels in HEK293 cells. (Fig. 2A) and that overexpression of SUMO-2 or SUMO-3 significantly elevated the level of SUMO-2/3-modified RanGAP1 (Fig. 1). This finding is different from a previous report that SUMO-2/3 conjugated poorly, if at all, to RanGAP1 (8Saitoh H. Hinchey J. J. Biol. Chem. 2000; 275: 6252-6258Abstract Full Text Full Text PDF PubMed Scopus (680) Google Scholar). The discrepancy could result from the facts that COS-7 cells, which contain hig
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