Functional Heterogeneity of Small Ubiquitin-related Protein Modifiers SUMO-1 versus SUMO-2/3
2000; Elsevier BV; Volume: 275; Issue: 9 Linguagem: Inglês
10.1074/jbc.275.9.6252
ISSN1083-351X
AutoresHisato Saitoh, Joseph Hinchey,
Tópico(s)interferon and immune responses
ResumoPost-translational modification marked by the covalent attachment of the ubiquitin-like protein SUMO-1/SMT3C has been implicated in a wide variety of cellular processes. Recently, two cDNAs encoding proteins related to SUMO-1 have been identified in human and mouse. The functions and regulation of these proteins, known as SUMO-2/SMT3A and SUMO-3/SMT3B, remain largely uncharacterized. We describe herein quantitative and qualitative distinctions between SUMO-1 and SUMO-2/3 in vertebrate cells. Much of this was accomplished through the application of an antibody that recognizes SUMO-2 and -3, but not SUMO-1. This antibody detected multiple SUMO-2/3-modified proteins and revealed that, together, SUMO-2 and -3 constitute a greater percentage of total cellular protein modification than does SUMO-1. Intriguingly, we found that there was a large pool of free, non-conjugated SUMO-2/3 and that the conjugation of SUMO-2/3 to high molecular mass proteins was induced when the cells were subjected to protein-damaging stimuli such as acute temperature fluctuation. In addition, we demonstrated that SUMO-2/3 conjugated poorly, if at all, to a major SUMO-1 substrate, the Ran GTPase-activating protein RanGAP1. Together, these results support the concept of important distinctions between the SUMO-2/3 and SUMO-1 conjugation pathways and suggest a role for SUMO-2/3 in the cellular responses to environmental stress. Post-translational modification marked by the covalent attachment of the ubiquitin-like protein SUMO-1/SMT3C has been implicated in a wide variety of cellular processes. Recently, two cDNAs encoding proteins related to SUMO-1 have been identified in human and mouse. The functions and regulation of these proteins, known as SUMO-2/SMT3A and SUMO-3/SMT3B, remain largely uncharacterized. We describe herein quantitative and qualitative distinctions between SUMO-1 and SUMO-2/3 in vertebrate cells. Much of this was accomplished through the application of an antibody that recognizes SUMO-2 and -3, but not SUMO-1. This antibody detected multiple SUMO-2/3-modified proteins and revealed that, together, SUMO-2 and -3 constitute a greater percentage of total cellular protein modification than does SUMO-1. Intriguingly, we found that there was a large pool of free, non-conjugated SUMO-2/3 and that the conjugation of SUMO-2/3 to high molecular mass proteins was induced when the cells were subjected to protein-damaging stimuli such as acute temperature fluctuation. In addition, we demonstrated that SUMO-2/3 conjugated poorly, if at all, to a major SUMO-1 substrate, the Ran GTPase-activating protein RanGAP1. Together, these results support the concept of important distinctions between the SUMO-2/3 and SUMO-1 conjugation pathways and suggest a role for SUMO-2/3 in the cellular responses to environmental stress. nuclear pore complex glutathione S-transferase green fluorescent protein phosphate-buffered saline polyacrylamide gel electrophoresis nuclear domain 10 SUMO-1 is a highly conserved, smallubiquitin-related modifier that has been shown to be covalently conjugated to a variety of cellular proteins (1.Matunis M.J. Coutavas E. Blobel G. J. Cell Biol. 1996; 135: 1457-1470Crossref PubMed Scopus (949) Google Scholar, 2.Johnson E.S. Schwienhorst I. Dohmen R.J. Blobel G. EMBO J. 1997; 15: 5509-5519Crossref Scopus (440) Google Scholar, 3.Kamitani T. Nguyen H.P. Yeh E.T. J. Biol. Chem. 1997; 272: 14001-14004Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 4.Mahajan R. Delphin C. Guan T. Gerace L. Melchior F. Cell. 1997; 88: 97-107Abstract Full Text Full Text PDF PubMed Scopus (1002) Google Scholar). Like ubiquitin, SUMO-1 is believed to form an isopeptide bond between the carboxyl terminus of SUMO-1 and a lysine side chain(s) of the target protein (2.Johnson E.S. Schwienhorst I. Dohmen R.J. Blobel G. EMBO J. 1997; 15: 5509-5519Crossref Scopus (440) Google Scholar, 3.Kamitani T. Nguyen H.P. Yeh E.T. J. Biol. Chem. 1997; 272: 14001-14004Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 5.Mahajan R. Gerace L. Melchior F. J. Cell Biol. 1998; 140: 259-270Crossref PubMed Scopus (236) Google Scholar). The conjugation of SUMO-1 to cellular proteins has been implicated in multiple vital cellular processes, including nuclear transport, cell cycle control, oncogenesis, inflammation, and the response to virus infection (6.Johnson P.R. Hochstrasser M. Trends Cell Biol. 1997; 7: 408-413Abstract Full Text PDF PubMed Scopus (69) Google Scholar, 7.Saitoh H. Pu R.T. Dasso M. Trends Biochem. Sci. 1997; 22: 374-376Abstract Full Text PDF PubMed Scopus (125) Google Scholar, 8.Hodges M. Tissot C. Freemont P. Curr. Biol. 1998; 8: 749-752Abstract Full Text Full Text PDF PubMed Google Scholar). It has been proposed that SUMO-1 conjugation may function antagonistically to ubiquitin conjugation (9.Desterro J.M. Rodriguez M.S. Hay R.T. Mol. Cell. 1998; 2: 233-239Abstract Full Text Full Text PDF PubMed Scopus (905) Google Scholar) and/or that SUMO-1 conjugation may regulate the target protein's interaction with other cellular components (5.Mahajan R. Gerace L. Melchior F. J. Cell Biol. 1998; 140: 259-270Crossref PubMed Scopus (236) Google Scholar,10.Matunis M.J. Wu J. Blobel G. J. Cell Biol. 1998; 140: 499-509Crossref PubMed Scopus (376) Google Scholar, 11.Muller S. Matunis M.J. Dejean A. EMBO J. 1998; 17: 61-70Crossref PubMed Scopus (577) Google Scholar).Among known SUMO-1 substrates in vertebrate species, the Ran GTPase-activating protein RanGAP1 is the most abundant and best characterized. It is a highly conserved protein that enhances GTP hydrolysis on Ran, a Ras-related small nuclear GTP-binding protein required for nucleocytoplasmic trafficking (12.Bischoff F.R. Krebber H. Kemp F.T. Hermes I. Ponstingl H. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1749-1753Crossref PubMed Scopus (220) Google Scholar, 13.Melchior F. Gerace L. Trends Cell Biol. 1998; 8: 175-179Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). It has been demonstrated both in vitro and in vivo that a single lysine residue at position 526 in the C terminus of mouse RanGAP1 is modified by SUMO-1 (5.Mahajan R. Gerace L. Melchior F. J. Cell Biol. 1998; 140: 259-270Crossref PubMed Scopus (236) Google Scholar, 10.Matunis M.J. Wu J. Blobel G. J. Cell Biol. 1998; 140: 499-509Crossref PubMed Scopus (376) Google Scholar). A large fraction of SUMO-modified RanGAP1 appears to be tightly associated with the nuclear envelope via interaction with RanBP2/Nup358, a component of cytoplasmic filaments in the nuclear pore complex (NPC)1 (1.Matunis M.J. Coutavas E. Blobel G. J. Cell Biol. 1996; 135: 1457-1470Crossref PubMed Scopus (949) Google Scholar, 4.Mahajan R. Delphin C. Guan T. Gerace L. Melchior F. Cell. 1997; 88: 97-107Abstract Full Text Full Text PDF PubMed Scopus (1002) Google Scholar, 14.Saitoh H. Cooke C.A. Burgess W.H. Earnshaw W.C. Dasso M. Mol. Biol. Cell. 1996; 7: 1319-1334Crossref PubMed Scopus (51) Google Scholar, 15.Saitoh H. Pu R. Cavenagh M. Dasso M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3736-3741Crossref PubMed Scopus (163) Google Scholar). Unmodified RanGAP1 is present in the cytoplasm, suggesting that the conjugation of a ubiquitin-like moiety at the C-terminal domain may target RanGAP1 to the NPC (5.Mahajan R. Gerace L. Melchior F. J. Cell Biol. 1998; 140: 259-270Crossref PubMed Scopus (236) Google Scholar, 10.Matunis M.J. Wu J. Blobel G. J. Cell Biol. 1998; 140: 499-509Crossref PubMed Scopus (376) Google Scholar).Two cDNAs coding for proteins related to SUMO-1 have been isolated from human and mouse cDNA libraries and are referred to as SUMO-2/SMT3A and SUMO-3/SMT3B (6.Johnson P.R. Hochstrasser M. Trends Cell Biol. 1997; 7: 408-413Abstract Full Text PDF PubMed Scopus (69) Google Scholar, 16.Lapenta V. Chiurazzi P. van der Spek P. Pizzuti A. Hanaoka F. Brahe C. Genomics. 1997; 40: 362-366Crossref PubMed Scopus (102) Google Scholar, 17.Chen A. Mannen H. Li S.S. Biochem. Mol. Biol. Int. 1998; 46: 1161-1174PubMed Google Scholar). Both transcripts are expressed in a wide range of tissues and cell types, suggesting that their gene products play a role in some fundamental cellular processes. A comparison of the amino acid sequences of SUMO-1, -2, and -3 has revealed that SUMO-1 shares 48% identity with SUMO-2 and 46% identity with SUMO-3. Since SUMO-2 and -3 share 95% identity, it is reasonable to group SUMO-2 and -3 into a subfamily distinct from SUMO-1. It has been previously reported that both SUMO-2 and -3 could be transferred to other proteins in a pattern similar to that of SUMO-1 when SUMO-2/3 was transiently overexpressed in mammalian culture cells (18.Kamitani T. Nguyen H.P. Kito K. Fukuda-Kamitani T. Yeh E.T. J. Biol. Chem. 1998; 273: 3117-3120Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar, 19.Kamitani T. Kito K. Nguyen H.P. Fukuda-Kamitani T. Yeh E.T. J. Biol. Chem. 1998; 273: 11349-11353Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). This result suggested that SUMO-2/3 might function in a capacity similar to that of SUMO-1. Very little is known, however, about the specific function of SUMO-2/3 and mechanistic differences that could support cellular distinctions between two SUMO subfamilies.To gain a better understanding of the role of SUMO-2/3 and the differential regulation of SUMO-2/3 versus SUMO-1 conjugation in vertebrate cells, we have begun to characterize SUMO-2/3 at the protein level in vivo. We describe herein the development of an antibody specific to SUMO-2 and -3. Using this antibody and a complementary SUMO-1-specific antibody, we found that RanGAP1 was preferentially modified by SUMO-1, but very poorly by SUMO-2/3, despite a higher concentration of SUMO-2/3 than SUMO-1 in intracellular pools. We also demonstrated that the SUMO-2/3 conjugation pathway could be up-regulated by several protein-damaging stimuli. This is the first evidence for the involvement of a ubiquitin-like protein modification in cellular stress responses. Based on these results, we propose that there is distinct regulation of SUMO-2/3 modification as compared with SUMO-1 modification and that the SUMO-2/3 pathway may constitute an element of the cellular response to environmental stress.DISCUSSIONOn the basis of sequence homology, the known smallubiquitin-related modifiers (SUMO) can be divided into two groups in vertebrates: one including human SUMO-1 and its interspecies homologs and the other comprising human SUMO-2, SUMO-3, and their counterparts in other species. Data base searches for proteins homologous to SUMO-2/3 revealed that SUMO-2/3 is highly conserved not only in vertebrate species, but also in insect species, suggesting a biologically significant role for the SUMO-2/3 conjugation pathway.Using a newly developed anti-SUMO-2/3 antibody, we demonstrated that SUMO-2/3- and SUMO-2/3-conjugated proteins are present in vertebrate cells. One obvious difference between SUMO-1 and -2/3 was the presence of a large pool of free or non-conjugated SUMO-2/3 in COS-7 cells. This fraction of SUMO-2/3 appears to be readily available for conjugation reactions since its conjugation can be induced by cellular stresses such as acute heat elevation, oxidative stress, and ethanol addition, which are known to cause the accumulation of damaged proteins in the cells and to induce several stress-responsive kinase cascades (23.Kyriakis J.M. Avruch J. J. Biol. Chem. 1996; 271: 24313-24316Abstract Full Text Full Text PDF PubMed Scopus (1024) Google Scholar, 24.Meriin A.B. Yaglom J.A. Gabai V.L. Mosser D.D. Zon L. Sherman M.Y. Mol. Cell. Biol. 1999; 19: 2547-2555Crossref PubMed Scopus (225) Google Scholar). Recently, we found that treatment by the peptide aldehyde protease inhibitor MG-132, which has been reported to induce protein damage (25.Lee D.H. Goldberg A.L. Trends Cell Biol. 1998; 8: 397-403Abstract Full Text Full Text PDF PubMed Scopus (1233) Google Scholar), also effectively up-regulates the conjugation of SUMO-2/3 to a high molecular mass protein fraction (data not shown). We are in the process of investigating proteins specifically modified by SUMO-2/3 in a stress-responsive manner and the fate of such SUMO-2/3-modified proteins in stressed cells.It has been reported that the ubiquitin pathway responds to a wide variety of protein-damaging and environmental stresses (26.Wilkinson K. Annu. Rev. Nutr. 1995; 15: 161-189Crossref PubMed Scopus (133) Google Scholar). Intriguingly, heat shock (27.Carlson N. Rogers S. Rechsteiner M. J. Cell Biol. 1987; 104: 547-555Crossref PubMed Scopus (97) Google Scholar, 28.Parag H.A. Raboy B. Kulka R.G. EMBO J. 1987; 6: 55-61Crossref PubMed Scopus (168) Google Scholar) and oxidative (29.Shang F. Gong X. Taylor A. J. Biol. Chem. 1997; 272: 23086-23093Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar) stresses have been known to induce the nearly quantitative conversion of free ubiquitin to the conjugated pool, reminiscent of the stress-induced SUMO-2/3 conjugation described in this study. Given that ubiquitin conjugation to damaged cellular proteins induces rapid degradation of the conjugates by 26 S proteasomes, the similar mode of stress response in the SUMO-2/3 pathway might represent a novel mechanism by which damaged proteins are identified and processed. For instance, SUMO-2/3 conjugation might stabilize the target protein by protecting against ubiquitin conjugation and the subsequent rapid proteasomal degradation. Recent observations suggest that IκBα, an inhibitor for the NF-κB transcription factor, can be conjugated with either ubiquitin or SUMO-1 on Lys21 (9.Desterro J.M. Rodriguez M.S. Hay R.T. Mol. Cell. 1998; 2: 233-239Abstract Full Text Full Text PDF PubMed Scopus (905) Google Scholar). IκBα conjugated with SUMO-1 remains stable, but ubiquitin conjugates are subjected to rapid proteasomal degradation. Since modifications of IκBα with SUMO and ubiquitin are mutually exclusive, it is likely that these modifications functionally antagonize each other. Alternatively, SUMO-2/3 conjugation might enhance the denaturation process during stress and augment the ubiquitin-dependent degradation of such SUMO-2/3-conjugated, denatured/damaged protein by 26 S proteasomes. Conjugation by either SUMO-1 or SUMO-2/3, however, has not yet been reported to increase protein instability.Whatever its role, our results suggest that the SUMO-2/3 conjugation pathway represents a new class of acute and reversible stress response. The rapid kinetics of SUMO-2/3 conjugation and deconjugation reveal highly dynamic features of the regulation of the SUMO-2/3 conjugation pathway in the context of environmental stress. A complete understanding of the SUMO-2/3 conjugation pathway in relation to environmental stress will require further information not only on the SUMO-2/3 conjugation substrate(s), but also on the regulation of the SUMO-2/3 conjugation/deconjugation enzymes during environmental stress.Strikingly, the SUMO-1 and -2/3 modification pathways can be clearly distinguished by their differential modification of RanGAP1, which is a major SUMO-1 substrate, but is very poorly modified by SUMO-2/3. According to previously published reports, there is no indication of any ubiquitin-related polypeptide modification of RanGAP1 purified from mammalian cells and X. laevis egg extracts except SUMO-1 (1.Matunis M.J. Coutavas E. Blobel G. J. Cell Biol. 1996; 135: 1457-1470Crossref PubMed Scopus (949) Google Scholar,5.Mahajan R. Gerace L. Melchior F. J. Cell Biol. 1998; 140: 259-270Crossref PubMed Scopus (236) Google Scholar, 14.Saitoh H. Cooke C.A. Burgess W.H. Earnshaw W.C. Dasso M. Mol. Biol. Cell. 1996; 7: 1319-1334Crossref PubMed Scopus (51) Google Scholar). These investigations of endogenous RanGAP1 support the hypothesis of selective modification of RanGAP1 by SUMO-1 in vivo. Others studies have shown, however, that RanGAP1 is efficiently modified by SUMO-2 and -3 as well as by SUMO-1 when cDNA carrying SUMO-2 or -3 is transiently overexpressed in mammalian cell culture (18.Kamitani T. Nguyen H.P. Kito K. Fukuda-Kamitani T. Yeh E.T. J. Biol. Chem. 1998; 273: 3117-3120Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar, 19.Kamitani T. Kito K. Nguyen H.P. Fukuda-Kamitani T. Yeh E.T. J. Biol. Chem. 1998; 273: 11349-11353Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). Since we identified the preferential modification of RanGAP1 by SUMO-1 not only in COS-7 cells, but also in other cells such as human HeLa, 293, SK-N-M, and NB4 and mouse NIH-3T3 and liver, a more reasonable explanation might be that overproduction of either SUMO-2 or -3 by transient transfection perturbs the specificity of the normal modification system.The mechanism that governs the specificity of modification between SUMO-1 and -2/3 is currently unexplained, although three possibilities appear feasible. First, there may be two parallel enzymatic cascades present in cells that independently facilitate the modification of proteins with either the SUMO-1 or -2/3 family, respectively. For example, there is evidence for the existence of two slightly different isoforms of human Ubc9p (30.Jiang W. Koltin Y. Mol. Gen. Genet. 1996; 251: 153-160PubMed Google Scholar) and three isoforms of mouse Ubc9p (31.Tsytsykova A.V. Tsitsikov E.N. Wright D.A. Futcher B. Geha R.S. Mol. Immunol. 1998; 35: 1057-1067Crossref PubMed Scopus (8) Google Scholar). Ubc9p is an enzyme whose activity has been demonstrated to be required for SUMO-1 conjugation to RanGAP1 (20.Saitoh H. Sparrow D.B. Shiomi T. Pu R.T. Nishimoto T. Mohun T.J. Dasso M. Curr. Biol. 1998; 8: 121-124Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 32.Lee G.W. Melchior F. Matunis M.J. Mahajan R. Tian Q. Anderson P. J. Biol. Chem. 1998; 273: 6503-6507Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). It will be interesting to compare these isoforms in terms of their specificity for SUMO-1 or -2/3. Second, similar to the ubiquitin conjugation system, one might predict the existence of a ubiquitin-protein isopeptide ligase-like activity that docks Ubc9p with RanGAP1 and determines the specificity of SUMO-1 conjugation to RanGAP1. One likely candidate for this putative activity is RanBP2/Nup358, as it has been shown to interact with both Ubc9p and SUMO-1-modified RanGAP1 (14.Saitoh H. Cooke C.A. Burgess W.H. Earnshaw W.C. Dasso M. Mol. Biol. Cell. 1996; 7: 1319-1334Crossref PubMed Scopus (51) Google Scholar, 15.Saitoh H. Pu R. Cavenagh M. Dasso M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3736-3741Crossref PubMed Scopus (163) Google Scholar). Third, SUMO-1 and -2/3 might both be conjugated to RanGAP1, followed by selective deconjugation of SUMO-2/3 from RanGAP1. This scenario would require SUMO-2/3-specific isopeptidase activity in the context of SUMO-1 modification of RanGAP1. The sulfhydryl proteases from yeast and bovine brain that exhibit SUMO-1-releasing activity from SUMO-1-conjugated RanGAP1 (31.Tsytsykova A.V. Tsitsikov E.N. Wright D.A. Futcher B. Geha R.S. Mol. Immunol. 1998; 35: 1057-1067Crossref PubMed Scopus (8) Google Scholar, 33.Suzuki T. Ichiyama A. Saitoh H. Kawakami T. Omata M. Chung C.H. Kimura M. Shimbara N. Tanaka K. J. Biol. Chem. 1999; 274: 31131-31134Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar) and the previously described deconjugation activity associated with the NPC fraction (1.Matunis M.J. Coutavas E. Blobel G. J. Cell Biol. 1996; 135: 1457-1470Crossref PubMed Scopus (949) Google Scholar) represent likely candidates for the SUMO-2/3 isopeptidase, but preferential SUMO-2/3 deconjugation activity has not yet been identified.Prolonged exposure of SUMO-2/3 immunoblots revealed multiple discrete minor bands in the range of 100 kDa (Fig. 5). Most of these immunoreactive proteins were absent in the immunoblot probed with anti-SUMO-1 antibody, suggesting that a number of cellular proteins may become selectively modified by SUMO-2/3 as opposed to SUMO-1. However, one might point out that some of the high molecular mass bands (>120 kDa) were similar in size to the high molecular mass bands that appeared in the immunoblot probed with anti-SUMO-1 antibody. Although the identities of these proteins remain to be established, some might represent common substrates for SUMO-1 and -2/3 (i.e. mixed conjugation products including both SUMO-1 and -2/3). This observation argues for the possibility of cross-talk or convergence of the SUMO-1 and -2/3 modification pathways, at least with regard to some common substrates.Nuclear foci detected by anti-SUMO-1 antibody have been colocalized with the nuclear matrix-associated, large multiprotein complex known as nuclear domain 10 (ND10), nuclear bodies, or promyelocytic leukemia protein oncogenic domains (11.Muller S. Matunis M.J. Dejean A. EMBO J. 1998; 17: 61-70Crossref PubMed Scopus (577) Google Scholar, 34.Sternsdorf T. Jensen K. Will H. J. Cell Biol. 1997; 139: 1621-1634Crossref PubMed Scopus (289) Google Scholar, 35.Everett R.D. Freemont P. Saitoh H. Dasso M. Orr A. Kathoria M. Parkinson J. J. Virol. 1998; 72: 6581-6591Crossref PubMed Google Scholar, 36.Ishov A.M. Sotnikov A.G. Negorev D. Vladimirova O.V. Neff N. Kamitani T. Yeh E.T.H. Strauss J.F. Maul G.G. J. Cell Biol. 1999; 147: 221-233Crossref PubMed Scopus (677) Google Scholar). The functions of ND10 are not well understood, but size, number, and composition appear to be regulated throughout the cell cycle and to respond to environmental stress, interferon, and infection of several DNA viruses. We found that the nuclear foci detected by anti-SUMO-2/3 antibody appeared to represent staining patterns exhibited in common with SUMO-1 (Fig.5 B). Currently, we observed that As2O3, which has been reported to affect the assembly/maintenance of ND10 (11.Muller S. Matunis M.J. Dejean A. EMBO J. 1998; 17: 61-70Crossref PubMed Scopus (577) Google Scholar), induced the accumulation of both SUMO-1 and -2/3 in the nuclear punctate structures (data not shown). These results suggest that SUMO-1 and -2/3 are components of ND10 and imply involvement of the SUMO-2/3 pathway in the assembly or function of ND10.The data presented thus far indicate that although there is functional heterogeneity between the SUMO-1 and -2/3 subfamily members, it is currently unknown whether further functional heterogeneity might be present in the SUMO-2 versus SUMO-3 pathways. It will be important in the future to generate a monoclonal antibody that distinguishes between SUMO-2 and -3 subfamily members, but this is likely to be complicated by the high homology between SUMO-2 and -3.In summary, we have generated specific antibodies to SUMO-2/3 and have found that a number of proteins exist in SUMO-2/3-conjugated forms. The combination of this antibody and anti-SUMO-1 antibody revealed several differences between the SUMO-1 and -2/3 modification pathways. First, SUMO-2/3 is present in greater abundance than SUMO-1, and there is a large pool of non-conjugated SUMO-2/3 in COS-7 cells. Remarkably, this pool of free SUMO-2/3 was promptly activated and incorporated into high molecular mass proteins by protein-damaging stresses such as heat shock. Second, RanGAP1 is preferentially modified by SUMO-1, but very poorly by SUMO-2/3 in vivo, and SUMO-1-modified RanGAP1 appears to localize more abundantly at the NPC/nuclear envelope than the little SUMO-2/3-modified RanGAP1 that may be present, if any. Collectively, these results imply that regulatory and functional heterogeneity is present among SUMO-1, -2, and -3 modification systems and suggest a role for SUMO-2/3 in the response to environmental stress. SUMO-1 is a highly conserved, smallubiquitin-related modifier that has been shown to be covalently conjugated to a variety of cellular proteins (1.Matunis M.J. Coutavas E. Blobel G. J. Cell Biol. 1996; 135: 1457-1470Crossref PubMed Scopus (949) Google Scholar, 2.Johnson E.S. Schwienhorst I. Dohmen R.J. Blobel G. EMBO J. 1997; 15: 5509-5519Crossref Scopus (440) Google Scholar, 3.Kamitani T. Nguyen H.P. Yeh E.T. J. Biol. Chem. 1997; 272: 14001-14004Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 4.Mahajan R. Delphin C. Guan T. Gerace L. Melchior F. Cell. 1997; 88: 97-107Abstract Full Text Full Text PDF PubMed Scopus (1002) Google Scholar). Like ubiquitin, SUMO-1 is believed to form an isopeptide bond between the carboxyl terminus of SUMO-1 and a lysine side chain(s) of the target protein (2.Johnson E.S. Schwienhorst I. Dohmen R.J. Blobel G. EMBO J. 1997; 15: 5509-5519Crossref Scopus (440) Google Scholar, 3.Kamitani T. Nguyen H.P. Yeh E.T. J. Biol. Chem. 1997; 272: 14001-14004Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 5.Mahajan R. Gerace L. Melchior F. J. Cell Biol. 1998; 140: 259-270Crossref PubMed Scopus (236) Google Scholar). The conjugation of SUMO-1 to cellular proteins has been implicated in multiple vital cellular processes, including nuclear transport, cell cycle control, oncogenesis, inflammation, and the response to virus infection (6.Johnson P.R. Hochstrasser M. Trends Cell Biol. 1997; 7: 408-413Abstract Full Text PDF PubMed Scopus (69) Google Scholar, 7.Saitoh H. Pu R.T. Dasso M. Trends Biochem. Sci. 1997; 22: 374-376Abstract Full Text PDF PubMed Scopus (125) Google Scholar, 8.Hodges M. Tissot C. Freemont P. Curr. Biol. 1998; 8: 749-752Abstract Full Text Full Text PDF PubMed Google Scholar). It has been proposed that SUMO-1 conjugation may function antagonistically to ubiquitin conjugation (9.Desterro J.M. Rodriguez M.S. Hay R.T. Mol. Cell. 1998; 2: 233-239Abstract Full Text Full Text PDF PubMed Scopus (905) Google Scholar) and/or that SUMO-1 conjugation may regulate the target protein's interaction with other cellular components (5.Mahajan R. Gerace L. Melchior F. J. Cell Biol. 1998; 140: 259-270Crossref PubMed Scopus (236) Google Scholar,10.Matunis M.J. Wu J. Blobel G. J. Cell Biol. 1998; 140: 499-509Crossref PubMed Scopus (376) Google Scholar, 11.Muller S. Matunis M.J. Dejean A. EMBO J. 1998; 17: 61-70Crossref PubMed Scopus (577) Google Scholar). Among known SUMO-1 substrates in vertebrate species, the Ran GTPase-activating protein RanGAP1 is the most abundant and best characterized. It is a highly conserved protein that enhances GTP hydrolysis on Ran, a Ras-related small nuclear GTP-binding protein required for nucleocytoplasmic trafficking (12.Bischoff F.R. Krebber H. Kemp F.T. Hermes I. Ponstingl H. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1749-1753Crossref PubMed Scopus (220) Google Scholar, 13.Melchior F. Gerace L. Trends Cell Biol. 1998; 8: 175-179Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). It has been demonstrated both in vitro and in vivo that a single lysine residue at position 526 in the C terminus of mouse RanGAP1 is modified by SUMO-1 (5.Mahajan R. Gerace L. Melchior F. J. Cell Biol. 1998; 140: 259-270Crossref PubMed Scopus (236) Google Scholar, 10.Matunis M.J. Wu J. Blobel G. J. Cell Biol. 1998; 140: 499-509Crossref PubMed Scopus (376) Google Scholar). A large fraction of SUMO-modified RanGAP1 appears to be tightly associated with the nuclear envelope via interaction with RanBP2/Nup358, a component of cytoplasmic filaments in the nuclear pore complex (NPC)1 (1.Matunis M.J. Coutavas E. Blobel G. J. Cell Biol. 1996; 135: 1457-1470Crossref PubMed Scopus (949) Google Scholar, 4.Mahajan R. Delphin C. Guan T. Gerace L. Melchior F. Cell. 1997; 88: 97-107Abstract Full Text Full Text PDF PubMed Scopus (1002) Google Scholar, 14.Saitoh H. Cooke C.A. Burgess W.H. Earnshaw W.C. Dasso M. Mol. Biol. Cell. 1996; 7: 1319-1334Crossref PubMed Scopus (51) Google Scholar, 15.Saitoh H. Pu R. Cavenagh M. Dasso M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3736-3741Crossref PubMed Scopus (163) Google Scholar). Unmodified RanGAP1 is present in the cytoplasm, suggesting that the conjugation of a ubiquitin-like moiety at the C-terminal domain may target RanGAP1 to the NPC (5.Mahajan R. Gerace L. Melchior F. J. Cell Biol. 1998; 140: 259-270Crossref PubMed Scopus (236) Google Scholar, 10.Matunis M.J. Wu J. Blobel G. J. Cell Biol. 1998; 140: 499-509Crossref PubMed Scopus (376) Google Scholar). Two cDNAs coding for proteins related to SUMO-1 have been isolated from human and mouse cDNA libraries and are referred to as SUMO-2/SMT3A and SUMO-3/SMT3B (6.Johnson P.R. Hochstrasser M. Trends Cell Biol. 1997; 7: 408-413Abstract Full Text PDF PubMed Scopus (69) Google Scholar, 16.Lapenta V. Chiurazzi P. van der Spek P. Pizzuti A. Hanaoka F. Brahe C. Genomics. 1997; 40: 362-366Crossref PubMed Scopus (102) Google Scholar, 17.Chen A. Mannen H. Li S.S. Biochem. Mol. Biol. Int. 1998; 46: 1161-1174PubMed Google Scholar). Both transcripts are expressed in a wide range of tissues and cell types, suggesting that their gene products play a role in some fundamental cellular processes. A comparison of the amino acid sequences of SUMO-1, -2, and -3 has revealed that SUMO-1 shares 48% identity with SUMO-2 and 46% identity with SUMO-3. Since SUMO-2 and -3 share 95% identity, it is reasonable to group SUMO-2 and -3 into a subfamily distinct from SUMO-1. It has been previously reported that both SUMO-2 and -3 could be transferred to other proteins in a pattern similar to that of SUMO-1 when SUMO-2/3 was transiently overexpressed in mammalian culture cells (18.Kamitani T. Nguyen H.P. Kito K. Fukuda-Kamitani T. Yeh E.T. J. Biol. Chem. 1998; 273: 3117-3120Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar, 19.Kamitani T. Kito K. Nguyen H.P. Fukuda-Kamitani T. Yeh E.T. J. Biol. Chem. 1998; 273: 11349-11353Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). This result suggested that SUMO-2/3 might function in a capacity similar to that of SUMO-1. Very little is known, however, about the specific function of SUMO-2/3 and mechanistic differences that could support cellul
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