Sumoylation of Topoisomerase I Is Involved in Its Partitioning between Nucleoli and Nucleoplasm and Its Clearing from Nucleoli in Response to Camptothecin
2002; Elsevier BV; Volume: 277; Issue: 42 Linguagem: Inglês
10.1074/jbc.m200388200
ISSN1083-351X
AutoresPrasad Rallabhandi, Keiko Hashimoto, Yin Yuan Mo, William T. Beck, Prasun Moitra, Peter D’Arpa,
Tópico(s)Biochemical and Molecular Research
ResumoPrevious studies identified a small fraction of putatively sumoylated topoisomerase I (TOP1) under basal conditions (∼1%), and anticancer camptothecins that trap the TOP1-DNA covalent intermediate markedly increase the sumoylation of TOP1 (≤10%). To study the role of the sumoylation of TOP1, we mutated sites on green fluorescent protein (GFP)-TOP1 corresponding to the consensus sequence for protein sumoylation (ΨKXE, where Ψ is a hydrophobic residue) and assayed the mutants for basal and camptothecin-induced sumoylation. Only one of the eight mutants, K117R, located in the highly charged NH2-terminal region, showed a substantial reduction (∼5-fold) in basal and camptothecin-induced sumoylation; thus, Lys-117 appears to be the major sumoylation site. A triple mutant having the ΨKXE sequences flanking K117R additionally mutated (K103R/K117R/K153R) showed little if any sumoylation, but was degraded like wild-type GFP-TOP1 during camptothecin treatment. However, K103R/K117R/K153R-GFP-TOP1 was markedly concentrated within nucleoli, depleted from the remainder of nucleus, and failed to be cleared from nucleoli in response to camptothecin treatment. These data are consistent with a model wherein basal transient sumoylation of the NH2-terminal, highly charged, disordered region prevents TOP1 binding to sites in nucleoli, thus driving it to bind in the nucleoplasm; and camptothecin treatment, which increases TOP1 sumoylation, further shifts the binding resulting in delocalization of TOP1 from nucleoli to nucleoplasm. Previous studies identified a small fraction of putatively sumoylated topoisomerase I (TOP1) under basal conditions (∼1%), and anticancer camptothecins that trap the TOP1-DNA covalent intermediate markedly increase the sumoylation of TOP1 (≤10%). To study the role of the sumoylation of TOP1, we mutated sites on green fluorescent protein (GFP)-TOP1 corresponding to the consensus sequence for protein sumoylation (ΨKXE, where Ψ is a hydrophobic residue) and assayed the mutants for basal and camptothecin-induced sumoylation. Only one of the eight mutants, K117R, located in the highly charged NH2-terminal region, showed a substantial reduction (∼5-fold) in basal and camptothecin-induced sumoylation; thus, Lys-117 appears to be the major sumoylation site. A triple mutant having the ΨKXE sequences flanking K117R additionally mutated (K103R/K117R/K153R) showed little if any sumoylation, but was degraded like wild-type GFP-TOP1 during camptothecin treatment. However, K103R/K117R/K153R-GFP-TOP1 was markedly concentrated within nucleoli, depleted from the remainder of nucleus, and failed to be cleared from nucleoli in response to camptothecin treatment. These data are consistent with a model wherein basal transient sumoylation of the NH2-terminal, highly charged, disordered region prevents TOP1 binding to sites in nucleoli, thus driving it to bind in the nucleoplasm; and camptothecin treatment, which increases TOP1 sumoylation, further shifts the binding resulting in delocalization of TOP1 from nucleoli to nucleoplasm. topoisomerase I Chinese hamster ovary human embryonic kidney green fluorescent protein camptothecin wild-type Topoisomerase I (TOP1)1is the molecular target of the camptothecin (CPT) class of cancer chemotherapeutics. CPT stabilizes the covalent TOP1-DNA intermediate by inhibiting the religation step of the DNA cleavage-religation mechanism. Previous studies have demonstrated that treatment of cells with CPT causes TOP1 to be hypersumoylated, ubiquitinated, and destroyed in proportion to the quantity of the covalent TOP1-DNA intermediate (1Beidler D.R. Cheng Y.C. Mol. Pharmacol. 1995; 47: 907-914PubMed Google Scholar, 2Desai S.D. Liu L.F. Vazquez-Abad D. D'Arpa P. J. Biol. Chem. 1997; 272: 24159-24164Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar, 3Desai S.D., Li, T.K. Rodriguez-Bauman A. Rubin E.H. Liu L.F. Cancer Res. 2001; 61: 5926-5932PubMed Google Scholar). TOP1 is also known to delocalize from nucleoli following CPT treatment (4Danks M.K. Garrett K.E. Marion R.C. Whipple D.O. Cancer Res. 1996; 56: 1664-1673PubMed Google Scholar, 5Buckwalter C.A. Lin A.H. Tanizawa A. Pommier Y.G. Cheng Y.C. Kaufmann S.H. Cancer Res. 1996; 56: 1674-1681PubMed Google Scholar, 6Wadkins R.M. Danks M.K. Horowitz L. Baker S.D. Exp. Cell Res. 1998; 241: 332-339Crossref PubMed Scopus (13) Google Scholar), which has been suggested to involve sumoylation (7Mo Y.Y., Yu, Y. Shen Z. Beck W.T. J. Biol. Chem. 2002; 277: 2958-2964Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). TOP1 sumoylation increases rapidly after CPT treatment and has been hypothesized to be related to the ubiquitination, proteasome-dependent degradation, or the delocalization of TOP1 from nucleoli (1Beidler D.R. Cheng Y.C. Mol. Pharmacol. 1995; 47: 907-914PubMed Google Scholar, 2Desai S.D. Liu L.F. Vazquez-Abad D. D'Arpa P. J. Biol. Chem. 1997; 272: 24159-24164Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar, 3Desai S.D., Li, T.K. Rodriguez-Bauman A. Rubin E.H. Liu L.F. Cancer Res. 2001; 61: 5926-5932PubMed Google Scholar, 7Mo Y.Y., Yu, Y. Shen Z. Beck W.T. J. Biol. Chem. 2002; 277: 2958-2964Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar).Sumoylation has been shown to alter the cellular distribution, activity, or stability of a number of proteins (8Rodriguez 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, 9Muller 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, 10Buschmann T. Fuchs S.Y. Lee C.G. Pan Z.Q. Ronai Z. Cell. 2000; 101: 753-762Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar, 11Duprez E. Saurin A.J. Desterro J.M. Lallemand-Breitenbach V. Howe K. Boddy M.N. Solomon E. de The H. Hay R.T. Freemont P.S. J. Cell Sci. 1999; 112: 381-393Crossref PubMed Google Scholar, 12Matunis M.J., Wu, J. Blobel G. J. Cell Biol. 1998; 140: 499-509Crossref PubMed Scopus (376) Google Scholar, 13Rangasamy D. Woytek K. Khan S.A. Wilson V.G. J. Biol. Chem. 2000; 275: 37999-38004Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 14Sternsdorf T. Jensen K. Reich B. Will H. J. Biol. Chem. 1999; 274: 12555-12566Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar, 15Wilson V.G. Rangasamy D. Exp. Cell Res. 2001; 271: 57-65Crossref PubMed Scopus (86) Google Scholar, 16Bies J. Markus J. Wolff L. J. Biol. Chem. 2002; 277: 8999-9009Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). In the case of TOP1, a putative low steady state level of sumoylation of TOP1 (<1%) has been observed in the absence of CPT treatment (1Beidler D.R. Cheng Y.C. Mol. Pharmacol. 1995; 47: 907-914PubMed Google Scholar, 2Desai S.D. Liu L.F. Vazquez-Abad D. D'Arpa P. J. Biol. Chem. 1997; 272: 24159-24164Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar, 3Desai S.D., Li, T.K. Rodriguez-Bauman A. Rubin E.H. Liu L.F. Cancer Res. 2001; 61: 5926-5932PubMed Google Scholar), which suggests that sumoylation of TOP1 may be important for regulating its activity, localization, or abundance under basal conditions.To explore the role of TOP1 sumoylation under basal conditions and in response to camptothecins, we used site-directed mutagenesis of sumoylation consensus sequences to identify sumoylation sites on TOP1. The major sumoylation site was identified to be Lys-117 in the amino-terminal region, as only this mutant (K117R) showed a reduction in both basal and CPT-induced sumoylation. A triple mutant with two sumoylation site mutations besides K117R (K103R/K117R/K153R) showed an even greater reduction in CPT-induced sumoylation. This triple mutant almost totally lacks sumoylation, although it was degraded similarly to wild-type GFP-TOP1 (WT-GFP-TOP1). Surprisingly, a striking difference in the localization of K103R/K117R/K153R-GFP-TOP1 was observed in comparison to WT-GFP-TOP1; K103R/K117R/K153R-GFP-TOP1 was much more highly concentrated in nucleoli, was less abundant in the remainder of the nucleus, and failed to delocalize from nucleoli upon CPT treatment.DISCUSSIONSumoylation of TOP1 and TOP2 increases within minutes after treatment of cells with drugs that specifically target the respective enzymes (2Desai S.D. Liu L.F. Vazquez-Abad D. D'Arpa P. J. Biol. Chem. 1997; 272: 24159-24164Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar, 20Mao Y. Sun M. Desai S.D. Liu L.F. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4046-4051Crossref PubMed Scopus (181) Google Scholar, 21Mao Y. Desai S.D. Liu L.F. J. Biol. Chem. 2000; 275: 26066-26073Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, 22Mao Y. Desai S.D. Ting C.Y. Hwang J. Liu L.F. J. Biol. Chem. 2001; 276: 40652-40658Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). Basal sumoylation of endogenous TOP1 (i.e. without CPT treatment) has been observed in all the cell types we have analyzed and mainly consists of singly sumoylated TOP1, although a lesser abundance of doubly and triply sumoylated TOP1 is sometimes visible (e.g. see Fig. 3 A, compare WT (−) and (+) CPT; only mono- and di-sumoylated TOP1 are visible without CPT treatment). CPT treatment induces the sumoylation of TOP1 manyfold, and we and others have observed TOP1 with as many as six SUMO conjugates (2Desai S.D. Liu L.F. Vazquez-Abad D. D'Arpa P. J. Biol. Chem. 1997; 272: 24159-24164Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar, 21Mao Y. Desai S.D. Liu L.F. J. Biol. Chem. 2000; 275: 26066-26073Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar).For both basal and CPT-induced sumoylation, the monomer is the most abundant, the dimer is less abundant, and so on with the abundance of multiply sumoylated forms being inversely proportional to the number of sumoylations (Fig. 3 and see Refs. 2Desai S.D. Liu L.F. Vazquez-Abad D. D'Arpa P. J. Biol. Chem. 1997; 272: 24159-24164Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar and 21Mao Y. Desai S.D. Liu L.F. J. Biol. Chem. 2000; 275: 26066-26073Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). Here we have shown that within the highly charged amino-terminal region of TOP1, a conservative mutation of Lys-117 to Arg prevented the majority of both basal and CPT-induced sumoylation. However, residual sumoylations were observed on K117R-GFP-TOP1. To identify the sites of the residual sumoylations, the lysines of KXE sites flanking Lys-117 were additionally mutated. These double mutants, K103R/K117R-GFP-TOP1 and K117R/K153R-GFP-TOP1, exhibited significant residual sumoylation, and their partitioning between nucleoli and nucleoplasm was similar to WT-GFP-TOP1 and K117R-GFP-TOP1; they were also cleared from nucleoli during CPT treatment like WT-GFP-TOP1 and K117R-GFP-TOP1 (data not shown). Only the triple mutant, K103R/K117R/K153R-GFP-TOP1, which showed very little sumoylation, was found to be concentrated in nucleoli and depleted from nucleoplasm. None of the single mutants, except K117R, nor any of the double mutants having wild-type Lys-117 showed a detectable reduction in sumoylation. These results suggest that Lys-117 is the major sumoylation site on TOP1, and that the sumoylation of the sites flanking Lys-117 is sufficient to enable TOP1 to be cleared from nucleoli in response to CPT and to prevent the accumulation of TOP1 in nucleoli under basal conditions.Mutation of only a single lysine (Lys-117) eliminated the majority of sumoylated TOP1 despite the fact that up to six SUMO molecules have been seen attached to TOP1 (2Desai S.D. Liu L.F. Vazquez-Abad D. D'Arpa P. J. Biol. Chem. 1997; 272: 24159-24164Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar, 20Mao Y. Sun M. Desai S.D. Liu L.F. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4046-4051Crossref PubMed Scopus (181) Google Scholar). These observations can be explained by more than one model. First, the KXE sites may be differentially favored for sumoylation and de-sumoylation. If Lys-117 is the most favored sumoylation site with respect to its steady state sumoylation, it could account for the majority of mono-sumoylated TOP1. One of the sumoylation sites flanking Lys-117, either Lys-103 or Lys-153 may be the next favored; thus, sumoylation of Lys-117 plus Lys-103 or sumoylation of Lys-117 plus Lys-153 may account for the majority of di-sumoylated TOP1. Because mono-sumoylated TOP1 is severalfold more abundant than di-sumoylated TOP1, reduced sumoylation may only be observable when Lys-117 is mutated. This model of multiple mono-sumoylations predicts that the abundance of mono-sumoylated K117R-GFP-TOP1 might be roughly similar to di-sumoylated WT-GFP-TOP1. Our data are roughly consistent with this model (see Fig. 3,A and B). Our data do not exclude the possibility of a SUMO chain attached at Lys-117. SUMO chains consisting of SUMO-2/3 or SUMO-1 have been observed in vitro (23Tatham 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, 24Kirsh O. Seeler J.S. Pichler A. Gast A. Muller S. Miska E. Mathieu M. Harel-Bellan A. Kouzarides T. Melchior F. Dejean A. EMBO J. 2002; 21: 2682-2691Crossref PubMed Scopus (263) Google Scholar), and a low abundance of apparently di-sumoylated HDAC4 has been observed in cells co-transfected with HDAC4 and SUMO-2 (23Tatham 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). For consistency with the chain model, it would be necessary to postulate that a chain forms on a less favorable lysine when Lys-117 is mutated to explain the multiple sumoylations still visible on K117R-GFP-TOP1. Currently, the multiple mono-sumoylation model appears to be most consistent with our experimental data and with the fact that single SUMO conjugates on four closely spaced (IVL)KXE sites have been mapped on Cdc3 (25Johnson E.S. Blobel G. J. Cell Biol. 1999; 147: 981-994Crossref PubMed Scopus (325) Google Scholar). Notably, using antibodies specific to SUMO-1 or SUMO-2/3, we and others have observed SUMO-2/3 and SUMO-1 conjugated to TOP1 (20Mao Y. Sun M. Desai S.D. Liu L.F. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4046-4051Crossref PubMed Scopus (181) Google Scholar,21Mao Y. Desai S.D. Liu L.F. J. Biol. Chem. 2000; 275: 26066-26073Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). 3K. Hashimoto and P. D'Arpa, unpublished data. The close juxtaposition of multiple sumoylation sites on TOP1 (Lys-103, Lys-117, Lys-153) is similar to that found on the Saccharomyces cerevisiae septins Cdc3 (Lys-4, Lys-11, Lys-30, Lys-63) and Shs/Sep7 (Lys-426, Lys-437) (25Johnson E.S. Blobel G. J. Cell Biol. 1999; 147: 981-994Crossref PubMed Scopus (325) Google Scholar), the cytomegalovirus protein IE2 (Lys-175, Lys-180) (26Hofmann H. Floss S. Stamminger T. J. Virol. 2000; 74: 2510-2524Crossref PubMed Scopus (147) Google Scholar), HDAC1 (Lys-444, Lys-476) (27David G. Neptune M.A. DePinho R.A. J. Biol. Chem. 2002; 277: 23658-23663Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar), and GRIP1 (Lys-731, Lys-788) (28Kotaja N. Karvonen U. Janne O.A. Palvimo J.J. J. Biol. Chem. 2002; 277: 30283-30288Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar) (see Table I). Multiple closely spaced sumoylation sites may be a redundancy mechanism for ensuring sumoylation. In the case of TOP1, the closely spaced sumoylation sites may ensure sumoylation of the amino terminus, which is largely disordered (29Stewart L. Ireton G.C. Champoux J.J. J. Biol. Chem. 1996; 271: 7602-7608Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar) and in cells may dynamically switch among conformational forms.Sumoylation and ubiquitination sites in close proximity or involving the same lysine have been previously reported and suggested to serve a co-regulatory function. For example, sumoylation of the ubiquitin-protein isopeptide ligase Mdm2 appears to modulate its self-ubiquitination and destruction and thus the ubiquitination and proteasome-dependent degradation of p53 (10Buschmann T. Fuchs S.Y. Lee C.G. Pan Z.Q. Ronai Z. Cell. 2000; 101: 753-762Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar). In the case of IκBα, sumoylation may compete with ubiquitination for the same acceptor site lysine (18Desterro J.M. Rodriguez M.S. Hay R.T. Mol. Cell. 1998; 2: 233-239Abstract Full Text Full Text PDF PubMed Scopus (905) Google Scholar). These examples prompted us to test the hypothesis that the sumoylation of TOP1 might somehow regulate its proteasome-dependent degradation. However, we did not detect a difference between the degradation of transfected WT- and K103R/K117R/K153R-GFP-TOP1 (Fig. 4 and Table III). We did find that the degradation of both WT- and K103R/K117R/K153R-GFP-TOP1 mimicked the degradation of the endogenous TOP1 in the cell lines tested. In conclusion, a near complete lack of sumoylation of TOP1 had no effect on the CPT-induced degradation of TOP1.Sumoylation has been shown to alter the cellular distribution of several proteins (11Duprez E. Saurin A.J. Desterro J.M. Lallemand-Breitenbach V. Howe K. Boddy M.N. Solomon E. de The H. Hay R.T. Freemont P.S. J. Cell Sci. 1999; 112: 381-393Crossref PubMed Google Scholar, 12Matunis M.J., Wu, J. Blobel G. J. Cell Biol. 1998; 140: 499-509Crossref PubMed Scopus (376) Google Scholar, 13Rangasamy D. Woytek K. Khan S.A. Wilson V.G. J. Biol. Chem. 2000; 275: 37999-38004Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 15Wilson V.G. Rangasamy D. Exp. Cell Res. 2001; 271: 57-65Crossref PubMed Scopus (86) Google Scholar, 30Mahajan R. Gerace L. Melchior F. J. Cell Biol. 1998; 140: 259-270Crossref PubMed Scopus (236) Google Scholar). In every cell type we have tested (at least 10), we identified putative sumoylated forms of endogenous TOP1 under basal conditions (i.e. in the absence of CPT treatment), albeit at markedly lower abundance than after CPT treatment. We also observed basal sumoylation on WT-GFP-TOP1, but these forms were significantly reduced on K117R-GFP-TOP1 and K103R/K117R/K153R-GFP-TOP1, indicating that a small fraction of TOP1 is normally sumoylated in the absence of CPT. Suggesting a role for basally sumoylated TOP1, WT-GFP-TOP1 and K103R/K117R/K153R-GFP-TOP1 distribute differently within the nucleus. WT-GFP-TOP1 distributes to both nucleoplasm and nucleoli similar to endogenous TOP1 immunostaining; in contrast, the K103R/K117R/K153R-GFP-TOP1 concentrates in nucleoli and is depleted from the nucleoplasm (Fig. 5,A and B). Interestingly, the single mutant K117R-GFP-TOP1 and the double mutants K103R/K117R-GFP-TOP1 and K117R/K153R-GFP-TOP1 localized similarly to WT-GFP-TOP1, which may be the consequence of their residual sumoylation. This result suggests that basally sumoylated TOP1, whose steady state levels are only ∼1%, plays a role in partitioning TOP1 between nucleoli and the nucleoplasm.TOP1 is well known to be cleared from nucleoli after CPT treatment (4Danks M.K. Garrett K.E. Marion R.C. Whipple D.O. Cancer Res. 1996; 56: 1664-1673PubMed Google Scholar, 5Buckwalter C.A. Lin A.H. Tanizawa A. Pommier Y.G. Cheng Y.C. Kaufmann S.H. Cancer Res. 1996; 56: 1674-1681PubMed Google Scholar, 6Wadkins R.M. Danks M.K. Horowitz L. Baker S.D. Exp. Cell Res. 1998; 241: 332-339Crossref PubMed Scopus (13) Google Scholar). We found that TOP1 clears from nucleoli during CPT treatment in the presence of proteasome inhibitors, suggesting that relocalization to the nucleoplasm rather than degradation of nucleolar TOP1 is responsible for the clearing (data not shown). Recent studies have shown that CPT-induced nucleolar clearing of TOP1 correlated with changes in total protein sumoylation and TOP1 sumoylation, which were experimentally varied by heat shock or the expression of wild-type or dominant negative Ubc9 (7Mo Y.Y., Yu, Y. Shen Z. Beck W.T. J. Biol. Chem. 2002; 277: 2958-2964Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Here we have shown that WT-GFP-TOP1 is cleared from nucleoli within 5 min of CPT treatment, and its distribution within the nucleoplasm remained unchanged up to the longest CPT treatment time (60 min). After CPT treatment, WT-GFP-TOP1 localized to perinucleolar structures as have been previously observed (31Christensen M.O. Barthelmes H.U. Feineis S. Knudsen B.R. Andersen A.H. Boege F. Mielke C. J. Biol. Chem. 2002; 277: 15661-15665Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). In contrast, K103R/K117R/K153R-GFP-TOP1 failed to clear from the nucleoli even after 60 min of CPT treatment (Fig. 5, A andB). K103R/K117R/K153R-GFP-TOP1 also failed to clear from nucleoli following treatment with 5,6-dichlorobenzimidazole riboside or actinomycin D, which caused endogenous TOP1 and WT-GFP-TOP1 to rapidly clear from nucleoli (data not shown). Thus, these results suggest that sumoylation of TOP1 is required for its clearing from nucleoli during exposure to CPT, 5,6-dichlorobenzimidazole riboside, or actinomycin D.The major sumoylation sites were found on the amino-terminal region of TOP1. The amino-terminal region of TOP1 has several notable features indicating an important role in regulating the cellular localization of TOP1. First, it contains the nuclear localization signals. Second, it contains the nucleolin binding site (32Bharti A.K. Olson M.O. Kufe D.W. Rubin E.H. J. Biol. Chem. 1996; 271: 1993-1997Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar), and inSaccharomyces, the nucleolin ortholog, Nsr1p, physically interacts with yeast TOP1; and null mutants of NSR1 exhibited altered localization of TOP1 (33Edwards T.K. Saleem A. Shaman J.A. Dennis T. Gerigk C. Oliveros E. Gartenberg M.R. Rubin E.H. J. Biol. Chem. 2000; 275: 36181-36188Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). Third, the NH2 terminus ofDrosophila TOP1 was sufficient for recruitment to active sites of transcription (34Shaiu W.L. Hsieh T.S. Mol. Cell. Biol. 1998; 18: 4358-4367Crossref PubMed Google Scholar). Finally, the NH2-terminal region per se fused to GFP has been shown to concentrate in nucleoli and to clear from nucleoli in response to CPT (7Mo Y.Y., Yu, Y. Shen Z. Beck W.T. J. Biol. Chem. 2002; 277: 2958-2964Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 35Mao Y. Mehl I.R. Muller M.T. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1235-1240Crossref PubMed Scopus (39) Google Scholar) or ultraviolet light (35Mao Y. Mehl I.R. Muller M.T. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1235-1240Crossref PubMed Scopus (39) Google Scholar), which indicates that formation of the covalent TOP1-DNA intermediate is not required for nucleolar clearing. We have also found that CPT-induced clearing of WT-GFP-TOP1 was not changed after mutagenizing the active site tyrosine to phenylalanine (data not shown). Together these results suggest that the major reason TOP1 clears from nucleoli during CPT treatment is not a result of slower nucleoplasmic versus nucleolar mobility resulting from preferential nucleoplasmic formation of the TOP1-DNA covalent intermediate as recently suggested (31Christensen M.O. Barthelmes H.U. Feineis S. Knudsen B.R. Andersen A.H. Boege F. Mielke C. J. Biol. Chem. 2002; 277: 15661-15665Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar).WT-GFP-TOP1 was recently shown to move continuously between nucleoli and nucleoplasm (31Christensen M.O. Barthelmes H.U. Feineis S. Knudsen B.R. Andersen A.H. Boege F. Mielke C. J. Biol. Chem. 2002; 277: 15661-15665Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar) similar to other nucleolar proteins (36Phair R.D. Misteli T. Nature. 2000; 404: 604-609Crossref PubMed Scopus (953) Google Scholar, 37Chen D. Huang S. J. Cell Biol. 2001; 153: 169-176Crossref PubMed Scopus (267) Google Scholar, 38Dundr M. Misteli T. Biochem. J. 2001; 356: 297-310Crossref PubMed Scopus (329) Google Scholar). Basic stretches of amino acids have been identified as elements that may mediate nucleolar retention of proteins through interaction with polyanions (39van Eenennaam H. van der Heijden A. Janssen R.J. van Venrooij W.J. Pruijn G.J. Mol. Biol. Cell. 2001; 12: 3680-3689Crossref PubMed Scopus (22) Google Scholar), and in the case of TOP1, the 120 amino acids centered about Lys-117 are 35% Lys and Arg (28% Asp and Glu). It is possible that SUMO conjugates may disrupt this highly charged region, effectively dislodging TOP1 from binding sites in nucleoli and enabling a large part to continuously partition into the nucleoplasm under basal conditions. CPT treatment may cause an equilibrium shift toward lesser nucleolar retention and greater nucleoplasmic localization because of its known effect of increasing TOP1 sumoylation.Whether cancer cells can exploit the sumoylation-dependent partitioning of TOP1 to evade the cytotoxic effects of CPT requires further investigation. Topoisomerase I (TOP1)1is the molecular target of the camptothecin (CPT) class of cancer chemotherapeutics. CPT stabilizes the covalent TOP1-DNA intermediate by inhibiting the religation step of the DNA cleavage-religation mechanism. Previous studies have demonstrated that treatment of cells with CPT causes TOP1 to be hypersumoylated, ubiquitinated, and destroyed in proportion to the quantity of the covalent TOP1-DNA intermediate (1Beidler D.R. Cheng Y.C. Mol. Pharmacol. 1995; 47: 907-914PubMed Google Scholar, 2Desai S.D. Liu L.F. Vazquez-Abad D. D'Arpa P. J. Biol. Chem. 1997; 272: 24159-24164Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar, 3Desai S.D., Li, T.K. Rodriguez-Bauman A. Rubin E.H. Liu L.F. Cancer Res. 2001; 61: 5926-5932PubMed Google Scholar). TOP1 is also known to delocalize from nucleoli following CPT treatment (4Danks M.K. Garrett K.E. Marion R.C. Whipple D.O. Cancer Res. 1996; 56: 1664-1673PubMed Google Scholar, 5Buckwalter C.A. Lin A.H. Tanizawa A. Pommier Y.G. Cheng Y.C. Kaufmann S.H. Cancer Res. 1996; 56: 1674-1681PubMed Google Scholar, 6Wadkins R.M. Danks M.K. Horowitz L. Baker S.D. Exp. Cell Res. 1998; 241: 332-339Crossref PubMed Scopus (13) Google Scholar), which has been suggested to involve sumoylation (7Mo Y.Y., Yu, Y. Shen Z. Beck W.T. J. Biol. Chem. 2002; 277: 2958-2964Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). TOP1 sumoylation increases rapidly after CPT treatment and has been hypothesized to be related to the ubiquitination, proteasome-dependent degradation, or the delocalization of TOP1 from nucleoli (1Beidler D.R. Cheng Y.C. Mol. Pharmacol. 1995; 47: 907-914PubMed Google Scholar, 2Desai S.D. Liu L.F. Vazquez-Abad D. D'Arpa P. J. Biol. Chem. 1997; 272: 24159-24164Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar, 3Desai S.D., Li, T.K. Rodriguez-Bauman A. Rubin E.H. Liu L.F. Cancer Res. 2001; 61: 5926-5932PubMed Google Scholar, 7Mo Y.Y., Yu, Y. Shen Z. Beck W.T. J. Biol. Chem. 2002; 277: 2958-2964Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Sumoylation has been shown to alter the cellular distribution, activity, or stability of a number of proteins (8Rodriguez 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, 9Muller S. Berger M. Lehembre F. Seeler J.S. Haupt Y. 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Biol. Chem. 2002; 277: 8999-9009Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). In the case of TOP1, a putative low steady state level of sumoylation of TOP1 (<1%) has been observed in the absence of CPT treatment (1Beidler D.R. Cheng Y.C. Mol. Pharmacol. 1995; 47: 907-914PubMed Google Scholar, 2Desai S.D. Liu L.F. Vazquez-Abad D. D'Arpa P. J. Biol. Chem. 1997; 272: 24159-24164Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar, 3Desai S.D., Li, T.K. Rodriguez-Bauman A. Rubin E.H. Liu L.F. Cancer Res. 2001; 61: 5926-5932PubMed Google Scholar), which suggests that sumoylation of TOP1 may be important for regulating its activity, localization, or abundance under basal conditions. To explore the role of TOP1 sumoylation under basal conditions and in response to camptothecins, we used site-directed mutagenesis of sumoylation consensus sequences to identify sumoylation sites on TOP1. The major sumoylation site was identified to be Lys-117 in the amino-terminal region, as only this mutant (K117R) showed a reduction in both basal and CPT-induced sumoylation. A triple mutant with two sumoylation site mutations besides K117R (K103R/K117R/K153R) showed an even greater reduction in CPT-induced sumoylation. This triple mutant
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