Stress-induced Inactivation of the c-Myb Transcription Factor through Conjugation of SUMO-2/3 Proteins
2006; Elsevier BV; Volume: 281; Issue: 52 Linguagem: Inglês
10.1074/jbc.m609404200
ISSN1083-351X
AutoresMarek Šramko, Ján Markus, Juraj Kabát, Linda Wolff, Juraj Bies,
Tópico(s)Cell death mechanisms and regulation
ResumoPost-translational modifications, such as phosphorylation, acetylation, ubiquitination, and SUMOylation, play an important role in regulation of the stability and the transcriptional activity of c-Myb. Conjugation of small ubiquitin-like modifier type 1 (SUMO-1) to lysines in the negative regulatory domain strongly suppresses its transcriptional activity. Here we report conjugation of two other members of the SUMO protein family, SUMO-2 and SUMO-3, and provide evidence that this post-translational modification negatively affects transcriptional activity of c-Myb. Conjugation of SUMO-2/3 proteins is strongly enhanced by several different cellular stresses and occurs primarily on two lysines, Lys523 and Lys499. These lysines are in the negative regulatory domain of c-Myb and also serve as acceptor sites for SUMO-1. Stress-induced SUMO-2/3 conjugation is very rapid and independent of activation of stress-activated protein kinases of the SAPK and JNK families. PIAS-3 protein was identified as a new c-Myb-specific SUMO-E3 ligase that both catalyzes conjugation of SUMO-2/3 proteins to c-Myb and exerts a negative effect on c-Myb-induced reporter gene activation. Interestingly, co-expression of a SPRING finger mutant of PIAS-3 significantly suppresses SUMOylation of c-Myb under stress. These results argue that PIAS-3 SUMO-E3 ligase plays a critical role in stress-induced conjugation of SUMO-2/3 to c-Myb. We also detected stress-induced conjugation of SUMO-2/3 to c-Myb in hematopoietic cells at the levels of endogenously expressed proteins. Furthermore, according to the negative role of SUMO conjugation on c-Myb capacity, we have observed rapid stress-induced down-regulation of the targets genes c-myc and bcl-2 of c-Myb. Our findings demonstrate that SUMO-2/3 proteins conjugate to c-Myb and negatively regulate its activity in cells under stress. Post-translational modifications, such as phosphorylation, acetylation, ubiquitination, and SUMOylation, play an important role in regulation of the stability and the transcriptional activity of c-Myb. Conjugation of small ubiquitin-like modifier type 1 (SUMO-1) to lysines in the negative regulatory domain strongly suppresses its transcriptional activity. Here we report conjugation of two other members of the SUMO protein family, SUMO-2 and SUMO-3, and provide evidence that this post-translational modification negatively affects transcriptional activity of c-Myb. Conjugation of SUMO-2/3 proteins is strongly enhanced by several different cellular stresses and occurs primarily on two lysines, Lys523 and Lys499. These lysines are in the negative regulatory domain of c-Myb and also serve as acceptor sites for SUMO-1. Stress-induced SUMO-2/3 conjugation is very rapid and independent of activation of stress-activated protein kinases of the SAPK and JNK families. PIAS-3 protein was identified as a new c-Myb-specific SUMO-E3 ligase that both catalyzes conjugation of SUMO-2/3 proteins to c-Myb and exerts a negative effect on c-Myb-induced reporter gene activation. Interestingly, co-expression of a SPRING finger mutant of PIAS-3 significantly suppresses SUMOylation of c-Myb under stress. These results argue that PIAS-3 SUMO-E3 ligase plays a critical role in stress-induced conjugation of SUMO-2/3 to c-Myb. We also detected stress-induced conjugation of SUMO-2/3 to c-Myb in hematopoietic cells at the levels of endogenously expressed proteins. Furthermore, according to the negative role of SUMO conjugation on c-Myb capacity, we have observed rapid stress-induced down-regulation of the targets genes c-myc and bcl-2 of c-Myb. Our findings demonstrate that SUMO-2/3 proteins conjugate to c-Myb and negatively regulate its activity in cells under stress. c-Myb is a DNA-binding transcription factor that plays a major role in the development of erythroid, myeloid, and lymphoid lineages of definitive hematopoiesis. The c-myb gene is abundantly expressed in immature proliferating hematopoietic progenitor cells but not in mature non-proliferating cells (1Gonda T.J. Metcalf D. Nature. 1984; 310: 249-251Crossref PubMed Scopus (416) Google Scholar, 2Sitzmann J. Noben-Trauth K. Klempnauer K.H. Oncogene. 1995; 11: 2273-2279PubMed Google Scholar). The function of c-Myb as a regulator of hematopoiesis is achieved through transcriptional regulation of genes intimately involved in cellular processes such as proliferation, differentiation, and apoptosis (3Oh I.H. Reddy E.P. Oncogene. 1999; 18: 3017-3033Crossref PubMed Scopus (428) Google Scholar, 4Wolff L. Crit. Rev. Oncog. 1996; 7: 245-260Crossref PubMed Scopus (62) Google Scholar). Its critical role in establishment of the definitive hematopoietic system has been demonstrated in experiments with targeted disruption of the c-myb gene, where c-myb null mutant mouse embryos die around day 15 in utero due to severe anemia (5Mucenski M.L. McLain K. Kier A.B. Swerdlow S.H. Schreiner C.M. Miller T.A. Pietryga D.W. Scott Jr., W.J. Potter S.S. Cell. 1991; 65: 677-689Abstract Full Text PDF PubMed Scopus (904) Google Scholar). Originally, c-myb was identified as a transforming gene of two avian viruses, avian myeloblastosis virus and avian erythroleukemia virus E26 (6Lipsick J.S. Wang D.M. Oncogene. 1999; 18: 3047-3055Crossref PubMed Scopus (101) Google Scholar). c-myb can also be oncogenically activated following integration of replication-competent retroviruses into the c-myb locus in animal models (4Wolff L. Crit. Rev. Oncog. 1996; 7: 245-260Crossref PubMed Scopus (62) Google Scholar). Oncogenic forms of c-Myb protein are almost invariably accompanied with truncations of the amino and carboxyl termini resulting in removal of structural regions that negatively regulate c-Myb activity (4Wolff L. Crit. Rev. Oncog. 1996; 7: 245-260Crossref PubMed Scopus (62) Google Scholar). The c-Myb protein is composed of three functional domains: an amino-terminal DNA-binding domain, a central transactivation domain, and a carboxyl-terminal negative regulatory domain. The DNA-binding domain in the NH2-terminal region of c-Myb consists of three imperfect tandem repeats and binds to DNA through the consensus sequence 5′-PyAAC(G/T)G-3′ (7Biedenkapp H. Borgmeyer U. Sippel A.E. Klempnauer K.H. Nature. 1988; 335: 835-837Crossref PubMed Scopus (504) Google Scholar, 8Howe K.M. Reakes C.F. Watson R.J. EMBO J. 1990; 9: 161-169Crossref PubMed Scopus (132) Google Scholar, 9Weston K. Bishop J.M. 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Neoplasma. 2000; 47: 212-218PubMed Google Scholar, 22Kanei-Ishii C. Ninomiya-Tsuji J. Tanikawa J. Nomura T. Ishitani T. Kishida S. Kokura K. Kurahashi T. Ichikawa-Iwata E. Kim Y. Matsumoto K. Ishii S. Genes Dev. 2004; 18: 816-829Crossref PubMed Scopus (153) Google Scholar, 23Corradini F. Cesi V. Bartella V. Pani E. Bussolari R. Candini O. Calabretta B. J. Biol. Chem. 2005; 280: 30254-30262Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar), and conjugation of SUMO-1 (small ubiquitin-like modifier type 1) (24Bies J. Markus J. Wolff L. J. Biol. Chem. 2002; 277: 8999-9009Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar, 25Dahle O. Andersen T.O. Nordgard O. Matre V. Del Sal G. Gabrielson O.S. Eur. J. Biochem. 2003; 270: 1338-13348Crossref PubMed Scopus (62) Google Scholar) are crucial for modulation of the transactivation activity of c-Myb. SUMOylation is a reversible process that regulates the function of target proteins in a manner akin to phosphorylation (26Gill G. Curr. Opin. Genet. Dev. 2005; 15: 536-541Crossref PubMed Scopus (426) Google Scholar, 27Hay R.T. Mol. Cell. 2005; 18: 1-12Abstract Full Text Full Text PDF PubMed Scopus (1360) Google Scholar). The functional consequences of SUMOylation are not completely understood but this modification (unlike ubiquitination) does not directly involve targeting for proteasomal degradation. Rather it causes changes in protein-protein interactions, subnuclear localization, and conformational changes (26Gill G. Curr. Opin. Genet. Dev. 2005; 15: 536-541Crossref PubMed Scopus (426) Google Scholar, 27Hay R.T. Mol. Cell. 2005; 18: 1-12Abstract Full Text Full Text PDF PubMed Scopus (1360) Google Scholar). Three different isoforms of SUMO (SUMO-1, -2, -3) are present in mammals. SUMO-2 and SUMO-3 are 97% identical to each other and about 66% homologous to SUMO-1 (28Saitoh H. Hinchey J. J. Biol. Chem. 2000; 275: 6252-6258Abstract Full Text Full Text PDF PubMed Scopus (713) Google Scholar). At present, it is not entirely clear whether SUMO-1 and SUMO-2/3 play similar roles within cells. It was shown that oxidative stress, mild heat stress, or genotoxic stress (UV irradiation) cause a dramatic increase in the amount of SUMO-2/3 incorporated into high molecular weight complexes within cells (28Saitoh H. Hinchey J. J. Biol. Chem. 2000; 275: 6252-6258Abstract Full Text Full Text PDF PubMed Scopus (713) Google Scholar). However, the identity of these target proteins that are modified by conjugation of SUMO-2/3 in response to different stresses largely remains to be identified. Whereas there is a growing list of proteins modified with SUMO-1, only a few substrates for SUMO-2/3 modifications are validated in vivo. Here we show that Lys523 and Lys499, located in NRD of c-Myb, are modified with SUMO-2/3 in vivo. This modification is greatly enhanced by several different stresses such as heat stress, osmotic stress, and metabolic stress and to a lesser extent by genotoxic stress. Two SUMO-E3 ligases, PIASy and PIAS3, enhance conjugation of SUMO-2/3 proteins to c-Myb in a qualitatively different way. In hematopoietic cells under stress, SUMO-2/3 conjugation to c-Myb at the endogenous protein levels demonstrates the physiological relevance of this post-translational modification in regulation of c-Myb activity. Plasmid Constructs—Constructs encoding wild-type c-Myb (c-MybWT) and mutant forms (c-MybK499R, c-MybK523R, and c-Myb2K/R) were described previously (24Bies J. Markus J. Wolff L. J. Biol. Chem. 2002; 277: 8999-9009Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). Plasmids pCMV-T7-mPIASy (29Sachdev S. Bruhn L. Sieber H. Pichler A. Melchior F. Grosschedl R. Genes Dev. 2001; 15: 3088-3103Crossref PubMed Scopus (470) Google Scholar) and pCMV-FLAG-mPIAS3 (30Chung C.D. Liao J. Liu B. Rao X. Jay P. Berta P. Shuai K. Science. 1997; 278: 1803-1805Crossref PubMed Scopus (817) Google Scholar) were kindly provided by R. Grosschedl (University of Munich, Munich, Germany) and K. Shuai (University of California, Los Angeles, CA), respectively. The SPRING finger mutant of mPIAS3, where Cys334 was substituted with Ser, FLAG-PIAS3(Mut), was generated by site-directed mutagenesis using the QuikChange™ site-directed mutagenesis kit (Stratagene). The base pair substitution was confirmed by dideoxy sequencing using the BigDye™ Terminator version 1.0 Ready Reaction Cycle sequencing kit (Applied Biosystems). Plasmids encoding the c-MybS528A (pRmb3SVS528A) and c-MybS528D (pRmb3SV.9) mutants of c-Myb (31Aziz N. Miglarese M.R. Hendrickson R.C. Shabanowitz J. Sturgill T.W. Hunt D.F. Bender T.P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 6429-6433Crossref PubMed Scopus (74) Google Scholar) were kindly provided by T. Bender (University of Virginia, Charlottesville, VA). Plasmids encoding HA-SUMO-2 (pCMV-HA-SUMO-2-GG) and HA-SUMO-3 (pCMV-HA-SUMO-3-GG) and their K11R mutants (pCMV-HA-SUMO-2-K11R and pCMV-HA-SUMO-3-K11R) (32Tatham 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 (666) Google Scholar) were kindly provided by R. Hay (University of St. Andrews, St. Andrews, United Kingdom). Plasmid p5xMRE-A-luc was gift of B. Luscher (Medizinische Hochschule, Hannover, Germany). Plasmids pDsREd2-cMyb and p-EGFP-SUMO-2 were constructed by in-frame cloning of PCR-amplified murine c-Myb and SUMO-2 cDNAs into fluorescent expression vectors p-EGFP-C1 and pDsRed2-N1 (Clontech), respectively. Plasmid pEGFP-PML was kindly provided by H. Katano (National Institute of Infectious Diseases, Shinjuku, Tokyo, Japan). Cell Lines and Culture Conditions—SV40-transformed monkey kidney cells (COS-7; American Type Culture Collection) and murine erythroleukemia cells (DS19; a kind gift from S. Ruscetti, NCI-Frederick Cancer Research and Development Center, Frederick, MD) were cultured in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum (Sigma), 2 mm l-glutamine (Invitrogen), penicillin/streptomycin (100 μg/ml each) (Invitrogen). Transient Transfections—For transient transfections, COS-7 cells were plated at a density of 3 × 105 cells/100-mm tissue culture dish 1 day prior to transfection. Transfections were carried out with 2 μg of plasmid DNA/dish using the Effectene™ transfection reagent (Qiagen) according to the manufacturer's instructions. Expression of the transfected gene was analyzed 36 h post-transfection by Western immunoblotting. Western Immunoblotting—Cells were disrupted in ice-cold lysis buffer (20 mm Tris-HCl (pH 7.5), 50 mm NaCl, 0.5% Nonidet P-40, 0.5% SDS, and 0.5% sodium deoxycholate) supplemented with N-ethylmaleimide (10 mm, Sigma) and a mixture of protease inhibitors (Complete™, Roche Molecular Biochemicals). Cell lysates were sonicated for 20 s and clarified by centrifugation at 13,000 × g for 15 min at 4 °C. The c-Myb protein was immunoprecipitated with rabbit antiserum (24Bies J. Markus J. Wolff L. J. Biol. Chem. 2002; 277: 8999-9009Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar) and SUMO-2/3 proteins were immunoprecipitated with rabbit polyclonal anti-SUMO-2/3 antibody (33Azuma Y. Amaoutov A. Dasso M. J. Cell Biol. 2003; 163: 477-487Crossref PubMed Scopus (175) Google Scholar), kindly provided by Y. Azuma (NICHD, National Institutes of Health). Immunoprecipitated proteins or whole cell lysates were fractionated by SDS-PAGE on an 8% gel and electrophoretically transferred to a nitrocellulose membrane. Immunoblots were incubated with primary antibodies and visualized with SuperSignal West Pico chemiluminescent substrate (Pierce) in accordance with the manufacturer's instructions. Primary antibodies were anti-c-Myb monoclonal antibody (a kind gift of Eric Westin), anti-FLAG monoclonal antibody M2 (Sigma), and anti-HA monoclonal antibody (Covance), and anti-T7 monoclonal antibodies (Novagen). Confocal Laser Microscopy—COS-7 cells were seeded onto glass coverslips and cotransfected with constructs encoding green (pEGFP-SUMO-2 or pEGFP-PML) and red (pDsRed2-cMyb) fluorescent fusion proteins. Thirty-six hours after transfection coverslips were fixed in 2% paraformaldehyde in phosphate-buffered saline for 10 min, washed two times in phosphate-buffered saline, and mounted on microscopic slides using Fluoromount G (Southern Biotechnology). Confocal images were acquired with a Leica DMIRBE inverted microscope (Leica) equipped with a digital scanning head (Leica SP2 Confocal Microscope) using excitation wavelengths of 488 nm for GFP and 594 nm for Red2. The channels were recorded independently and pseudocolor images were generated and superimposed using Imaris version 5.0.3 software (Bitplane). The acquired digital images were processed using Adobe Photoshop 6.0 software. Transactivation Assay—COS-7 cells were plated at a density of 1 × 104 cells/well in 12-well tissue culture plates and grown overnight prior to transfections. Transfections were carried out using the FuGENE 6 Transfection Reagent (Roche). Cells were cotransfected with 100 ng of the c-Myb-responsive reporter plasmid p5xMRE-A-luc (containing five copies of the mim-1A, Myb-responsive element (MRE) upstream of minimal herpes simplex virus thymidine kinase promoter and the firefly luciferase gene), 50 ng of pRL-tk-Renilla luciferase vector (Promega), and expression vectors encoding wild-type (cMybWT) or a mutant form of cMyb (cMyb2K/R) (100 ng of each). To assess the effect of PIAS3 expression on the transcriptional activity of c-Myb, increasing concentrations (1, 10, and 50 ng) of plasmid encoding murine PIAS3 was also transfected as indicated. The total amount of transfected DNA was adjusted to 500 ng with empty vector pcDNA3.1 (Invitrogen). Forty-eight hours post-transfection, cells were processed for both firefly and Renilla luciferase activities using the Dual Luciferase® reporter assay system (Promega) and Turner TD-20e luminometer (Turner Designs). Each transfection experiment was performed in triplicate and repeated two times. RNA Isolation and Northern Blot Analysis—Total RNA was prepared using the TRIzol reagent (Invitrogen). Samples containing 10 μg of total RNA were resolved on a 1.2% agarose gel containing 0.25 m formaldehyde in MOPS buffer system. Separated RNAs were capillary transferred onto nylon membrane (Nytran SuperCharge, Schleicher & Schuell) and UV cross-linked using a Stratalinker 1800 (Stratagene). Blots were hybridized with cDNA probes labeled by the random priming method using Ready-to-Go DNA Labeling Beads (Amersham Biosciences). Quantitative analysis was performed with a Storm 840 phosphorimager using ImageQuant 5.1 software (GE Healthcare). Post-translational Modification of c-Myb by Covalent Attachment of SUMO-2/3—It has been demonstrated that the negative regulatory domain of c-Myb can be modified by SUMO-1 (24Bies J. Markus J. Wolff L. J. Biol. Chem. 2002; 277: 8999-9009Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar, 25Dahle O. Andersen T.O. Nordgard O. Matre V. Del Sal G. Gabrielson O.S. Eur. J. Biochem. 2003; 270: 1338-13348Crossref PubMed Scopus (62) Google Scholar) within its so-called PEST/EVES motif, and this modification is important for the inhibitory function of this domain. Whereas many targets have no preferences for SUMO isoforms, others are preferentially modified by one isoform, for example, RanGAP1 with SUMO-1 (28Saitoh H. Hinchey J. J. Biol. Chem. 2000; 275: 6252-6258Abstract Full Text Full Text PDF PubMed Scopus (713) Google Scholar) or topoisomerase-II by SUMO-2/3 (34Azuma Y. Amaoutov A. Anan T. Dasso M. EMBO J. 2005; 24: 2172-2182Crossref PubMed Scopus (120) Google Scholar). To determine whether, in addition to SUMO-1, c-Myb is modified in vivo by SUMO-2/3, we analyzed transiently transfected COS-7 cells with plasmids encoding either wild-type c-Myb (c-MybWT), HA-tagged SUMO-2 (HA-SUMO-2), or both. Thirty-six hours post-transfection the cells were lysed, c-Myb was immunoprecipitated and analyzed by Western immunoblotting with anti-HA and anti-c-Myb monoclonal antibodies. As shown in Fig. 1A, anti-HA antibody recognized two HA-immunoreactive bands in the immunoprecipitates from cells transfected with both c-MybWT and HA-SUMO-2-encoding constructs, but not in cells transfected with either construct alone. Anti-c-Myb monoclonal antibodies were used to confirm that similar levels of c-Myb were expressed and immunoprecipitated from cells transfected with c-MybWT. Anti-c-Myb monoclonal antibodies also detected a SUMO-2-modified form migrating ∼20 kDa above the nonmodified form of c-MybWT (Fig. 1A). Transfection efficiency was evaluated by Western immunoblotting of total cellular proteins. Anti-HA antibody detected a ladder of bands representing the HA-SUMO-2-conjugated cellular proteins only in cells transfected with HA-SUMO-2 (Fig. 1B). Anti-c-Myb monoclonal antibodies detected the c-MybWT protein (75-kDa form) only in cells transfected with the c-MybWT construct (Fig. 1B). Identical results were obtained with HA-tagged SUMO-3 constructs (data not shown). Both proteins SUMO-2 and-3 are virtually identical (in the processed form they differ only in 3 amino acids) and presently no antibodies are available that would distinguish between these two isoforms. All the figures show representative results that were obtained with the HA-SUMO-2 construct, even though all experiments in COS-7 cells were performed with both SUMO-2 and SUMO-3 with virtually identical results. These experiments clearly show that SUMO-2/3 proteins efficiently modify c-Myb. We also observed that, at least in COS-7 cells, SUMO-2/3 have a higher affinity toward c-Myb than SUMO-1. Transfection of c-MybWT and equimolar amounts of either HA-SUMO-2 or HA-SUMO-3 leads to conjugation of SUMO-2/3, where 20-40% of c-Myb is detected in the SUMOylated form, whereas transfected c-MybWT and SUMO-1 leads to only 5-10% of SUMOylated c-Myb (Refs. 24Bies J. Markus J. Wolff L. J. Biol. Chem. 2002; 277: 8999-9009Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar and 25Dahle O. Andersen T.O. Nordgard O. Matre V. Del Sal G. Gabrielson O.S. Eur. J. Biochem. 2003; 270: 1338-13348Crossref PubMed Scopus (62) Google Scholar and data not shown). Previously we have shown that SUMO-1 protein conjugates to two lysines, Lys499 and Lys523, located in the negative regulatory domain of c-Myb. Sequences around both Lys499 and Lys523 conform perfectly to the minimal SUMO modification consensus sequence ΨKXE (where Ψ is a hydrophobic amino acid and X any amino acid). Interestingly, we also observed that the single mutation K523R completely abolished modification of c-Myb with SUMO-1, suggesting that SUMOylation of c-Myb is an interdependent process, where modification of Lys523 is required for modification of the second Lys499 to occur. To determine whether the same lysines are sites of SUMO-2/3 conjugation, we cotransfected COS-7 cells with HA-SUMO-2/3 and wild-type c-Myb, as well as c-Myb constructs in which Lys499 (cMybK499R), Lys523 (cMybK523R), or both (cMyb2K/R) were changed to arginines. Lysates from transfected cells were immunoprecipitated with anti-c-Myb polyclonal antiserum and analyzed by immunoblotting using anti-HA and anti-c-Myb antibodies (Fig. 1C). These results demonstrate that Lys499 and Lys523 are major modification sites for SUMO-2/3 proteins, with an obvious preference for modification of Lys523 with SUMO-2/3. The single mutation K523R strongly inhibited modification of c-Myb with SUMO-2/3 at both sites. Thus, conjugation of SUMO-2/3 is ordered, where the first modification of c-Myb with SUMO-2/3 takes place almost exclusively at Lys523, and is necessary for the attachment of the second molecule to Lys499. As expected, mutation of both sites completely abolished modification of c-Myb with SUMO-2/3. Thus, Lys499 and Lys523 are the two major sites in the NRD of c-Myb modified with SUMO-2/3. Subnuclear Co-localization of c-Myb with SUMO-2—Next, we examined the co-localizations of c-Myb with SUMO-2/3 in COS-7 cells. COS-7 cells were transfected with constructs encoding the fusion fluorescent proteins EGFP-SUMO2 and DsRed2-cMyb, and analyzed the cells by confocal microscopy. DsRed2-cMyb was detected in the nucleus in the form of bright nuclear speckles, as well as in a fine granular form. GFP-SUMO-2 protein had a more homogenous distribution in the nucleus than c-Myb, but overall there was a strong co-localization of DsRed2-cMyb and EGFP-SUMO-2 (Fig. 2A; superimposition of confocal images). It was shown previously that c-Myb associates with PML in vivo in very bright dot-like structures that represent PML (ND10) nuclear bodies (35Dahle O. Bakke O. Gabrielson O.S. Exp. Cell Res. 2004; 297: 118-126Crossref PubMed Scopus (17) Google Scholar). We also observed co-localization of c-Myb and PML in COS-7 cells when these cells were cotransfected with HA-SUMO-2, and fluorescent fusion proteins encoding DsRed2-cMyb and EGFP-PML (Fig. 2B). However, the majority of DsRed2-cMyb protein was localized outside of GFP-PML-positive nuclear bodies. Thus, modification of c-Myb with SUMO-2/3 proteins seems to occur in subnuclear compartments that may differ from PML (ND10) nuclear bodies. Stress Induces Conjugation of SUMO-2/3 Proteins to c-Myb— It was shown previously that SUMO-2/3 proteins are more abundant in their free form than SUMO-1 in COS-7 cells. Additionally, Saitoh and Hinchey (28Saitoh H. Hinchey J. J. Biol. Chem. 2000; 275: 6252-6258Abstract Full Text Full Text PDF PubMed Scopus (713) Google Scholar) reported that pools of free SUMO-2/3 decrease rapidly when cells are exposed to heat, ethanol, or hydrogen peroxide. Therefore, SUMO-2/-3 modification of cellular proteins may be involved in the cellular response to environmental stresses. To investigate whether conjugation of SUMO-2/3 to c-Myb is modulated by stress, COS-7 cells transfected with HA-SUMO-2/3 and wild-type c-Myb were subjected to different forms of stress and analyzed by immunoprecipitation and Western immunoblotting. As shown in Fig. 3A, the amount of SUMO-2/3 conjugated to c-Myb dramatically increased after exposure of cells to heat stress (43 °C for 30 min), osmotic stress (0.7 m NaCl for 30 min), and also metabolic stress (ethanol 7% for 30 min). In contrast, genotoxic stress (UV irradiation) increased conjugation only mildly (Fig. 3B). With the environmental stress, we observed, not only was there a dramatic increase in modification of two major SUMOylation sites Lys523 and Lys499, but also several slower migrating species that correspond to c-Myb modified with more than two molecules of SUMO-2/3 (Fig. 3A). SUMO-2 and SUMO-3 contain the consensus SUMO modification site (ΨKXE) in their N-terminal regions and it is absent in the sequence of SUMO-1. These sites have been shown to be utilized by SUMO-E1-activating and SUMO-E2-conjugating enzymes to form polymeric chains of SUMO-2/3 on protein substrates in vitro, and SUMO-2/3 chains have also been detected in vivo (32Tatham 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 (666) Google Scholar). Thus, although all SUMO species share the same conjugation machinery, modification by SUMO-1 and SUMO-2/-3 may have distinct functional consequences. We decided to explore the possibility that the slower migrating bands detected in cells under stress (Fig. 3A) are actually c-Myb species modified by the conjugation of poly-SUMO-2/3 chains. Mutation K11R in SUMO-2/3 proteins (HA-SUMO-2/3-K11R) destroys the ability of the proteins to create polymeric chains in vitro and in vivo (32Tatham 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 (666) Google Scholar). COS-7 cells were transfected with c-Myb and either wild-type or mutated forms (K11R) of SUMO-2/3 proteins. Thirty-six hours post-transfection cells were subjected to heat stress, lysed, and c-Myb protein was immunoprecipitated. Immunoprecipitates were subjected to Western immunoblotting analysis with anti-HA and anti-cMyb antibodies. Heat stress strongly induced SUMOylation of c-Myb and none of the SUMO-2/3-modified c-Myb forms were affected by cot
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