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• W2042862271 abstract "SUMO-1 is a small ubiquitin-like protein that can be covalently conjugated to other proteins. A family of proteases catalyzes deconjugation of SUMO-1-containing species. Members of this family also process newly synthesized SUMO-1 into its conjugatable form. To understand these enzymes better, we have examined the localization and behavior of the human SUMO-1 protease SENP2. Here we have shown that SENP2 associates with the nuclear face of nuclear pores and that this association requires protein sequences near the N terminus of SENP2. We have also shown that SENP2 binds to Nup153, a nucleoporin that is localized to the nucleoplasmic face of the pore. Nup153 binding requires the same domain of SENP2 that mediates its targeting in vivo. Removal of the Nup153-interacting region of SENP2 results in a significant change in the spectrum of SUMO-1 conjugates within the cell. Our results suggest that association with the pore plays an important negative role in the regulation of SENP2, perhaps by restricting its activity to a subset of the conjugated proteins within the nucleus. SUMO-1 is a small ubiquitin-like protein that can be covalently conjugated to other proteins. A family of proteases catalyzes deconjugation of SUMO-1-containing species. Members of this family also process newly synthesized SUMO-1 into its conjugatable form. To understand these enzymes better, we have examined the localization and behavior of the human SUMO-1 protease SENP2. Here we have shown that SENP2 associates with the nuclear face of nuclear pores and that this association requires protein sequences near the N terminus of SENP2. We have also shown that SENP2 binds to Nup153, a nucleoporin that is localized to the nucleoplasmic face of the pore. Nup153 binding requires the same domain of SENP2 that mediates its targeting in vivo. Removal of the Nup153-interacting region of SENP2 results in a significant change in the spectrum of SUMO-1 conjugates within the cell. Our results suggest that association with the pore plays an important negative role in the regulation of SENP2, perhaps by restricting its activity to a subset of the conjugated proteins within the nucleus. SUMO-1 1The abbreviations used are: SUMOsmall ubiquitin-like modifierGFPgreen fluorescence proteinEGFPenhanced GFPGSTglutathione S-transferaseHIV-1human immunodeficiency virus, type 1AxamAxin associating moleculeAPCadenomatous polyposis coli1The abbreviations used are: SUMOsmall ubiquitin-like modifierGFPgreen fluorescence proteinEGFPenhanced GFPGSTglutathione S-transferaseHIV-1human immunodeficiency virus, type 1AxamAxin associating moleculeAPCadenomatous polyposis coli is a ubiquitin-like protein that can be covalently conjugated to other proteins through an isopeptide linkage in a manner similar to ubiquitin (1Melchior F. Annu. Rev. Cell Dev. Biol. 2000; 16: 591-626Crossref PubMed Scopus (646) Google Scholar). The SUMO-1 conjugation pathway utilizes proteins that both show sequence similarity to analogous enzymes in the ubiquitin pathway and utilize similar biochemical mechanisms (1Melchior F. Annu. Rev. Cell Dev. Biol. 2000; 16: 591-626Crossref PubMed Scopus (646) Google Scholar). A large and growing number of SUMO-1 conjugation substrates have been reported in vertebrates (1Melchior F. Annu. Rev. Cell Dev. Biol. 2000; 16: 591-626Crossref PubMed Scopus (646) Google Scholar). Notably, the profile of SUMO-1-conjugated proteins changes substantially in response to altered cellular conditions (see Ref. 2Azuma Y. Tan S.H. Cavenagh M.M. Ainsztein A.M. Saitoh H. Dasso M. FASEB J. 2001; 15: 1825-1827Crossref PubMed Scopus (45) Google Scholar), suggesting that there are mechanisms to control the specificity of conjugation and/or deconjugation of SUMO-1 differentially between distinct substrates. small ubiquitin-like modifier green fluorescence protein enhanced GFP glutathione S-transferase human immunodeficiency virus, type 1 Axin associating molecule adenomatous polyposis coli small ubiquitin-like modifier green fluorescence protein enhanced GFP glutathione S-transferase human immunodeficiency virus, type 1 Axin associating molecule adenomatous polyposis coli Unlike enzymes of the SUMO-1 conjugation pathway, enzymes involved in SUMO processing and deconjugation are not closely related by sequence to their ubiquitin counterparts. Rather, known SUMO proteases share sequence homology in their catalytic domains, which is more nearly conserved to viral proteases (3Li S.J. Hochstrasser M. Nature. 1999; 398: 246-251Crossref PubMed Scopus (597) Google Scholar). Two SUMO proteases have been described in budding yeast, Ulp1p (3Li S.J. Hochstrasser M. Nature. 1999; 398: 246-251Crossref PubMed Scopus (597) Google Scholar) and Ulp2p/Smt4p (4Strunnikov A.V. Aravind L. Koonin E.V. Genetics. 2001; 158: 95-107Crossref PubMed Google Scholar, 5Li S.J. Hochstrasser M. Mol. Cell. Biol. 2000; 20: 2367-2377Crossref PubMed Scopus (308) Google Scholar). Ulp1p is concentrated near the nuclear periphery (5Li S.J. Hochstrasser M. Mol. Cell. Biol. 2000; 20: 2367-2377Crossref PubMed Scopus (308) Google Scholar) and interacts with nuclear pore components in two-hybrid assays (6Takahashi Y. Mizoi J. Toh E.A. Kikuchi Y. J. Biochem. (Tokyo). 2000; 128: 723-725Crossref PubMed Scopus (68) Google Scholar), while Ulp2p is localized throughout the nucleus (5Li S.J. Hochstrasser M. Mol. Cell. Biol. 2000; 20: 2367-2377Crossref PubMed Scopus (308) Google Scholar). ULP1 is an essential gene, and temperature-sensitive (ts) Ulp1p mutants arrest at the G2/M transition of the cell cycle. Ulp2 is not essential, but it is required for normal meiotic development, for regulation of spindle checkpoint arrest, and for chromatin condensation of rDNA during mitosis (4Strunnikov A.V. Aravind L. Koonin E.V. Genetics. 2001; 158: 95-107Crossref PubMed Google Scholar, 5Li S.J. Hochstrasser M. Mol. Cell. Biol. 2000; 20: 2367-2377Crossref PubMed Scopus (308) Google Scholar). Interestingly, these proteins do not appear to act in a simple complementary manner, since ulp1-ts/ulp2-null double mutants grow better than ulp2 single mutants under a variety of conditions (5Li S.J. Hochstrasser M. Mol. Cell. Biol. 2000; 20: 2367-2377Crossref PubMed Scopus (308) Google Scholar). In mammals, data base searches find at least seven members of the SUMO protease family (7Yeh E.T. Gong L. Kamitani T. Gene. 2000; 248: 1-14Crossref PubMed Scopus (410) Google Scholar), some of which have now been confirmed to act as SUMO proteases in vitro (8Gong L. Millas S. Maul G.G. Yeh E.T. J. Biol. Chem. 2000; 275: 3355-3359Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar, 9Kim K.I. Baek S.H. Jeon Y.J. Nishimori S. Suzuki T. Uchida S. Shimbara N. Saitoh H. Tanaka K. Chung C.H. J. Biol. Chem. 2000; 275: 14102-14106Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 10Nishida T. Tanaka H. Yasuda H. Eur. J. Biochem. 2000; 267: 6423-6427Crossref PubMed Scopus (141) Google Scholar, 11Nishida T. Kaneko F. Kitagawa M. Yasuda H. J. Biol. Chem. 2001; 276: 39060-39066Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). Outside of their conserved catalytic domain, these proteases possess non-conserved N-terminal extensions of varying lengths and relatively short non-conserved C-terminal sequences. SENP2 was discovered both through its homology to other SUMO proteases (7Yeh E.T. Gong L. Kamitani T. Gene. 2000; 248: 1-14Crossref PubMed Scopus (410) Google Scholar) and its interactions with murine Axin, a regulator of the Wnt signaling pathway (12Kadoya T. Kishida S. Fukui A. Hinoi T. Michiue T. Asashima M. Kikuchi A. J. Biol. Chem. 2000; 275: 37030-37037Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). When overexpressed in tissue culture cells or under in vitro conditions, the murine SENP2 homologue (Smt3IP2) cleaves conjugates of SUMO-1, SUMO-2, and SUMO-3 (11Nishida T. Kaneko F. Kitagawa M. Yasuda H. J. Biol. Chem. 2001; 276: 39060-39066Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). Here we have shown that full-length human SENP2 associates with nuclear pores in a manner similar to Ulp1 in yeast. This association occurs exclusively with the nuclear face of the pore and requires sequences near the N terminus of SENP2. We have also shown that SENP2 binds specifically to Nup153, a nucleoporin localized to the nucleoplasmic face of the nuclear pore and that this association requires the same domain of SENP2 that mediates its targeting in vivo. Remarkably, a mutant SENP2 protein that is unable to bind Nup153 is significantly more effective in promoting deconjugation of SUMO-1-conjugated species, indicating that localization of SENP2 to the nuclear pore plays an important role in spatially restricting the activity of this enzyme. Affinity-purified antibodies against Xenopus Nup153 were a kind gift from K. Ullman (Huntsman Cancer Institute, Salt Lake City). Affinity-purified antibodies against Xenopus Nup98 were a kind gift from M. Powers (Emory University, Atlanta). The mouse monoclonal antibody mAb414 against nucleoporins, mouse monoclonal antibody against RanGAP1, and anti-V5 antibody were purchased from BAbCO (Richmond, CA), Zymed Laboratories, Inc. (South San Francisco, CA), and Strategene (La Jolla, CA) respectively. Mouse monoclonal anti-FLAG antibody and rabbit polyclonal antibody against the green fluorescence protein (GFP) were purchased from CLONTECH, Inc. (Palo Alto, CA). Rhodamine- and Alexa 488-conjugated secondary antibodies were purchased from Molecular Probes, Inc. (Eugene, OR). The SENP2 cDNA was amplified from the human universal QUICK-CloneTM cDNA (CLONTECH). The sequence of the encoded protein was identical to human cDNAs that have been reported by other laboratories (GenBankTMaccession numbers NM021627 and AF151697). The encoded protein has previously been designated as SENP2, and we will use this nomenclature throughout this report. We fused the SENP2 coding sequence in-frame to the 3′-end of the GFP coding sequence by insertion between the EcoRI and SalI sites of pEGFP-C2 (CLONTECH). Similarly, we prepared a vector encoding a version of SENP2 with an N-terminal FLAG tag by insertion of the SENP2 coding region between EcoRI and SalI sites of pCMV-Tag2B (Strategene). Truncation mutants were generated by PCR using Pfu DNA polymerase (Strategene) and subcloned into the same vectors. The SENP2 truncation mutants were also subcloned into pGEX4T-1 between EcoRI and SalI for expression of glutathione S-transferase (GST) fusion proteins in bacteria. Expression of the recombinant GST·SENP2 fusion proteins was induced with 0.05 mm isopropyl-1-thio-β-d-galactopyranoside at room temperature for 4 h and purified according to the manufacturer's instructions (Amersham Biosciences). A cDNA encoding the mature form of SUMO-1 (amino acids 1–97) was subcloned into pcDNA4/HisMax C (Invitrogen) between the BamHI and NotI restriction sites, allowing expression with six histidine and Xpress epitope tags at its N terminus. A human RanGAP1 cDNA was amplified from the pET-RanGAP1 plasmid kindly provided by Volker Gerke (University of Muenster, Muenster, Germany). The cDNA was cloned into the pcDNA3.1/V5-His-TOPO TA cloning vector (Invitrogen), allowing its expression with the V5 epitope and six histidine tags at its C terminus. A form of RanGAP1 that cannot be modified by SUMO-1 was generated by PCR using Pfu DNA polymerase to produce a point mutation that substituted lysine 524 with an arginine residue. COS7 and HeLa cells were cultured in Dulbecco's modified Eagle's medium (Invitrogen) containing 10% fetal bovine serum, 100 μg/ml penicillin, 100 μg/ml streptomycin, and 2 mm glutamine (Biofluids, Rockville, MD). In all cases, the cells were transfected using Effectene transfection reagent (Qiagene Inc., Valencia, CA). To measure the effect of SENP2 expression on the overall profile of conjugated substrates (Fig. 5A), COS7 cells were grown in 6-well plates and transfected with vectors expressing His-Xpress-tagged SUMO-1 and EGFP·SENP2, as indicated. To measure the effect of SENP2 expression specifically on RanGAP1 conjugation (Fig. 5B), COS7 cells were transfected with vectors expressing EGFP or EGFP·SENP2, as well as vectors expressing V5-tagged wild type or unconjugatable mutant human RanGAP1. In Fig. 5, A and B, the cells were washed twice with cold phosphate-buffered saline 24 h after transfection and suspended in 125 μl of boiling 2× SDS sample buffer, followed by brief sonication. The samples were analyzed by Western blotting as described below. HeLa cells were grown for immunofluorescence on glass coverslips. In all figures except Fig. 1B, the cells were fixed for 5 min with 4% formaldehyde plus 2% sucrose in KB buffer (10 mmTris-Cl, pH 7.7, 150 mm NaCl, 0.1% bovine serum albumin) and permeabilized with 0.1% Triton X-100 in KB buffer. In Fig. 1B, HeLa cells were fixed with 3% formaldehyde for 20 min at room temperature, and cells were alternatively permeabilized with Triton X-100 as described above or permeabilized with 0.004% digitonin at 4 °C for 15 min. In all cases, the coverslips were incubated for 1 h at room temperature in primary and secondary antibodies that had been diluted in KB buffer with 2% normal horse serum. Hoechst 33258 was added to the secondary antibody incubation to stain the DNA. After each incubation, the cells were rinsed five times for 2 min in KB buffer with 2% normal horse serum. The coverslips were mounted in Vectashield (Vector Laboratories, Burlingame, CA). Images were captured on a Zeiss Axioskop microscope with a Hamamatsu Orca II digital CCD camera (Carl Zeiss, Inc., Thornwood, NY) and Openlab software (Improvision, Inc., Lexington, MA). Proteins were resolved in 4–20% SDS-PAGE gel and transferred to polyvinylidene difluoride membrane. The blots were blocked in 5% nonfat dry milk in phosphate-buffered saline (Biofluids) containing 0.15% Tween-20 (PBS-T) at room temperature for 1 h, incubated in primary and horseradish peroxidase-labeled secondary antibodies for 1 h each, thoroughly rinsed with PBS-T after each incubation, and detected using ECL Plus reagents (AmershamBiosciences). Xenopus interphase egg extracts were prepared exactly as described elsewhere (13Smythe C. Newport J.W. Methods Cell Biol. 1991; 35: 449-468Crossref PubMed Scopus (152) Google Scholar). 15 μg of purified recombinant GST·SENP2 fusion proteins were incubated with 15 μl of egg extract plus 500 μl of buffer B (20 mmTris-Cl, pH 8.0, 50 mm NaCl, 0.5 mmdithiothreitol, 2.5 mm MgCl2, 0.1% Triton X-100, 10% glycerol) for 5 h at 4 °C. 20 μl of glutathione-agarose beads that had previously been equilibrated with buffer B were added to the reaction and incubated overnight at 4 °C. The beads were washed four times with 1.5 ml of buffer B, and the bound proteins were eluted using 30 μl of SDS sample buffer. The products were subjected to Western blotting using the indicated antibodies. To better understand the function of individual SUMO proteases, we cloned the human homologue of SENP2 by PCR and performed experiments to confirm that a recombinant fragment of the human SENP2 protein (amino acids 178–590, containing the putative catalytic region) had activity in assays for SUMO-1 processing and isopeptide cleavage of bonds between SUMO-1 and RanGAP1 (data not shown). Our results were very similar to those previously described for the in vitro analysis of mouse SENP2 (11Nishida T. Kaneko F. Kitagawa M. Yasuda H. J. Biol. Chem. 2001; 276: 39060-39066Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). To pursue a better understanding of SENP2 in vivo, we subcloned the SENP2 open reading frame into plasmid vectors for the expression of fusion proteins encoding SENP2 tagged with either green fluorescent protein (EGFP·SENP2) or a FLAG epitope (FLAG-SENP2). To determine the localization of SENP2, we examined the localization of a fusion between GFP and the full SENP2 coding region (EGFP·SENP2) in HeLa cells. When expressed at low levels (Fig. 1A), this protein co-localized with the nuclear envelope, whereas EGFP alone was diffusely distributed. At higher levels of expression, we found the fusion protein not only at the nuclear envelope but also within inclusions in the nucleus (data not shown). EGFP·SENP2 distribution overlapped with immunofluorescent staining using the monoclonal antibody 414 (mAb414), which recognizes a family of FXFG-containing nuclear pore proteins (Fig. 1A). Notably, the distribution of eGFP·SENP2 was more restricted than mAb414 staining and appeared to be found primarily on the nuclear side of the nuclear envelope (Fig. 1A, lower panel). The FLAG-SENP2 localized similarly (data not shown), suggesting that the pattern of localization was independent of the fusion moiety. In addition, polyclonal rabbit antibodies directed against SENP2 showed strong staining of the nuclear envelope in immunofluorescence experiments (data not shown), suggesting that the endogenous SENP2 protein is also localized to the nuclear envelope. Together, these observations demonstrate unambiguously that SENP2 is associated with the nuclear envelope and strongly indicate that SENP2 is resident on the nuclear face of the nuclear pore. To test more directly whether SENP2 is restricted to the nuclear side of the pore, we examined EGFP·SENP2 accessibility to anti-GFP antibody staining under different permeabilization conditions (Fig. 1B). When cells were permeabilized with digitonin, which disrupts the plasma membrane but leaves the nuclear envelope intact (14Adam S.A. Marr R.S. Gerace L. J. Cell Biol. 1990; 111: 807-816Crossref PubMed Scopus (763) Google Scholar), we observed no staining with anti-GFP antibodies. However, it was clear that the fusion protein was still present because the GFP emission was observed on the nuclear envelope. By contrast, permeabilization with a detergent that disrupts the nuclear envelope (Triton X-100) allowed staining, indicating that the fusion protein could be recognized by anti-eGFP antibodies. These observations show that SENP2 is localized to the nucleoplasmic side of the nuclear envelope. We wished to determine which sequences within SENP2 are required for its correct targeting to the nuclear envelope. To do this, we made a series of deletions in the SENP2 fusion protein, encompassing both the N and C termini (Fig. 2A). Deletion of as few as 30 amino acids from the N terminus of SENP2 disrupted its association to the pore (Fig. 2B). By contrast, fusion proteins that were extensively deleted from the C terminus, including one that retained only 70 amino acids of the N terminus of SENP2 (EGFP·SENP2-(1–70)), were able to localize correctly to the nuclear envelope (Fig. 3). A similar deletion analysis using FLAG-SENP2 fusion proteins provided essentially identical results (data not shown), confirming that this finding was independent of the fusion epitope used. These observations show that the sequences within the N-terminal 70 amino acids of SENP2 are both necessary and sufficient for its correct localization at the nuclear pore.Figure 3Sequences at the N terminus of SENP2 are sufficient for localization to the nuclear pore. EGFP fusion proteins with the indicated segments of SENP2 were transfected into HeLa cells. Localization of the fusion proteins was visualized by eGFP fluorescence (left column, green). DNA stained with Hoechst 33258 dye is shown in the center column(blue), and merged images are shown in the right column. Similar results were observed using indirect immunofluorescence with anti-FLAG antibodies against FLAG-tagged SENP2 proteins.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To test whether SENP2 binds to nuclear pore proteins, we utilized interphase Xenopus egg extracts (13Smythe C. Newport J.W. Methods Cell Biol. 1991; 35: 449-468Crossref PubMed Scopus (152) Google Scholar) as a source of unassembled, soluble nuclear pore components. We incubated the egg extracts with recombinant GST fusion proteins that encoded different regions of the SENP2 protein. After incubation, we purified the fusion proteins by affinity chromatography and examined whether the samples specifically retained extract proteins that could be recognized in Western blot assays by mAb414 (Fig. 4, upper panel). Fusion proteins encoding the N terminus of SENP2 (e.g.GST·SENP2-(1–170) and GST·SENP2-(1–70)) specifically retained a mAb414-reactive band that migrated with a mobility corresponding to 180 kDa on gel electrophoresis. Previous characterization of mAb414-reactive proteins in Xenopus egg extracts (15Meier E. Miller B.R. Forbes D.J. J. Cell Biol. 1995; 129: 1459-1472Crossref PubMed Scopus (74) Google Scholar) suggested that the 180-kDa band was likely to be the nucleoporin Nup153 (16Sukegawa J. Blobel G. Cell. 1993; 72: 29-38Abstract Full Text PDF PubMed Scopus (242) Google Scholar). To confirm this identification, we subjected the same samples to Western blotting using antibodies against the Xenopus Nup153 (17Ullman K.S. Shah S. Powers M.A. Forbes D.J. Mol. Biol. Cell. 1999; 10: 649-664Crossref PubMed Scopus (110) Google Scholar). This analysis showed that Nup153 associated with GST·SENP2-(1–170) and GST·SENP2-(1–70) (Fig. 4, lanes 5 and 6) but did not associate with GST or with fusion proteins containing any other region of SENP2 (Fig. 4). Several additional observations suggested that the retention of Nup153 was highly specific. First, no other mAb414-reactive bands were specifically retained in association with GST or any of the GST fusion proteins. Second, Western blots using antibodies directed against a GLFG nucleoporin associated with the nucleoplasmic side of the pore (18Powers M.A. Forbes D.J. Dahlberg J.E. Lund E. J. Cell Biol. 1997; 136: 241-250Crossref PubMed Scopus (179) Google Scholar), Nup98, did not recognize any proteins associated with SENP2 (Fig. 4, lower panel). Taken together, our findings strongly suggest that SENP2 interacts with Nup153 in Xenopus egg extracts. This finding is consistent with the localization of SENP2 determined in Figs. 1 and 2, because Nup153 has been demonstrated to be a component of the nuclear basket in vertebrates (19Pante N. Bastos R. McMorrow I. Burke B. Aebi U. J. Cell Bio. l. 1994; 126: 603-617Crossref PubMed Scopus (171) Google Scholar). The specificity of these interactions was independently confirmed by directed two-hybrid analysis in which strong interactions were observed between Nup153 and either full-length SENP2 or the N-terminal domain of SENP2 (data not shown). By contrast, no specific interactions were observed between Nup153 and SENP2 sequences outside of the first 100 amino acids. To determine whether SENP2 localization has any role in regulation of its activity, we transfected COS7 cells with wild type EGFP·SENP2, a mutant lacking the N-terminal 70 amino acids of SENP2 (EGFP·SENP2-(71–590)), and a mutant in which a critical cysteine in the predicted active site of the enzyme was changed to serine (EGFP·SENP2-C/S). EGFP·SENP2-C/S correctly targeted to the nuclear pore in a manner that was indistinguishable from the wild type protein (data not shown). To monitor the pattern of SUMO-1 conjugation within the transfected cells, we simultaneously transfected with a lower concentration of a plasmid expressing tagged SUMO-1 protein (His-Xpress-SUMO-1) and assayed the conjugation of the tagged SUMO-1 by Western blot (Fig. 5). We observed that His-Xpress-SUMO-1 became conjugated to a variety of proteins when it was co-transfected with a vector expressing EGFP, most easily observed on Western blots as a high molecular weight smear. This spectrum was slightly attenuated when the EGFP expression vector was replaced with a vector expressing wild type SENP2. By contrast, when EGFP·SENP2-C/S was co-expressed with the tagged SUMO-1 it dramatically increased the level of SUMO-1 conjugation within transfected cells, indicating that it was not only enzymatically inactive but also functioned in a dominant fashion to disrupt deconjugation by endogenous SENP2. We do not currently know the mechanism whereby EGFP·SENP2-C/S inhibited the endogenous protein. Most remarkably, expression of EGFP·SENP2-(71–590) caused the loss of almost all conjugated forms of His-Xpress-SUMO-1, indicating that SENP2 was much more effective in deconjugation of nuclear proteins when it was no longer tethered to the nuclear pore. These results suggest that association with the pore may play an important negative role in the regulation of SENP2, restricting its activity to a subset of the conjugated proteins within the nucleus and that allowing the protein to access other locations within the nucleus promotes inappropriate deconjugation of SUMO-1 species, which are not normally substrates for this enzyme. Because SENP2 is resident at the nuclear pore, we also examined the capacity of overexpressed SENP2 to alter the conjugation status of RanGAP1 in vivo. RanGAP1 is localized in the cytosol, where SUMO-1 conjugation targets it to the nuclear pore through association with a large nucleoporin, RanBP2 (20Matunis M.J. Coutavas E. Blobel G. J. Cell Biol. 1996; 135: 1457-1470Crossref PubMed Scopus (948) Google Scholar, 21Mahajan R. Delphin C. Guan T. Gerace L. Melchior F. Cell. 1997; 88: 97-107Abstract Full Text Full Text PDF PubMed Scopus (999) Google Scholar). In this experiment, we co-transfected a plasmid expressing V5-tagged RanGAP1 with the plasmid expressing EGFP·SENP2 (Fig. 5B). As a control, we performed similar experiments with a mutant version of RanGAP1 lacking the single lysine residue that becomes modified by SUMO-1 conjugation (RanGAP1-K524R, Ref. 22Matunis M.J., Wu, J. Blobel G. J. Cell Biol. 1998; 140: 499-509Crossref PubMed Scopus (376) Google Scholar). EGFP·SENP2 expression did not substantially alter the pattern of RanGAP1 modification. Moreover, under conditions of moderate or even massive EGFP·SENP2 overexpression, RanGAP1 staining at the nuclear pore was retained (data not shown), further arguing that its SUMO-1 modification status is not regulated by SENP2 despite the fact that RanGAP1 can be deconjugated from SUMO-1 in vitro by SENP2 (Ref. 11Nishida T. Kaneko F. Kitagawa M. Yasuda H. J. Biol. Chem. 2001; 276: 39060-39066Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, data not shown). These observations suggest that the primary substrate of SENP2 at the nuclear pore is unlikely to be RanGAP1. SUMO proteases share homology in their catalytic domains but otherwise have widely divergent primary sequences in their N- and C-terminal domains. We have found that human SENP2 localizes to the nucleoplasmic face of nuclear pores through sequences in the N-terminal domain. We have further found that this domain of SENP2 interacts with Nup153, a nucleoporin on the nucleoplasmic face of the nuclear pore. Together, our observations suggest that at least one function of the divergent regions in SUMO proteases is their proper localization within the nucleus. Notably, elimination of the N-terminal domain of SENP2 not only allows it to relocalize to other parts of the nucleus but also increases its capacity to deconjugate SUMO-1-conjugated species within the cell. These observations suggest that targeting SENP2 to the nuclear pore is a mechanism to sequester it from SUMO-1-conjugated proteins in the nuclear interior. Alternatively, the N-terminal domain may have a role in negatively regulating SENP2 through additional mechanisms. Our findings suggest that interactions between Nup153 and SENP2 may be responsible for the localization of SENP2 at the nuclear pore (Fig. 4). Nup153 is associated with the basket structure on the nucleoplasmic side of the pore (19Pante N. Bastos R. McMorrow I. Burke B. Aebi U. J. Cell Bio. l. 1994; 126: 603-617Crossref PubMed Scopus (171) Google Scholar), and it has been implicated in multiple aspects of nuclear transport and pore structure (17Ullman K.S. Shah S. Powers M.A. Forbes D.J. Mol. Biol. Cell. 1999; 10: 649-664Crossref PubMed Scopus (110) Google Scholar, 23Walther T.C. Fornerod M. Pickersgill H. Goldberg M. Allen T.D. Mattaj I.W. EMBO J. 2001; 20: 5703-5714Crossref PubMed Scopus (164) Google Scholar). The association between Nup153 and SENP2 is therefore entirely consistent with immunofluorescence data showing that SENP2 was found only on the nucleoplasmic side of the nuclear envelope (Fig. 1). Ulp1p, a protease for budding yeast SUMO-1 (Smt3), is found at the nuclear pore (5Li S.J. Hochstrasser M. Mol. Cell. Biol. 2000; 20: 2367-2377Crossref PubMed Scopus (308) Google Scholar) and has been reported to bind Nup42p (6Takahashi Y. Mizoi J. Toh E.A. Kikuchi Y. J. Biochem. (Tokyo). 2000; 128: 723-725Crossref PubMed Scopus (68) Google Scholar). Like Nup153, Nup42p is an FG-repeat containing nucleoporin (24Vasu S.K. Forbes D.J. Curr. Opin. Cell Biol. 2001; 13: 363-375Crossref PubMed Scopus (217) Google Scholar). These observations may suggest a conserved role for SUMO-1 deconjugation in the regulation of pore activity. However, there are likely to be some differences between vertebrates and yeast in the details of this function because Nup42p is localized on the cytosolic face of the nuclear envelope in yeast (24Vasu S.K. Forbes D.J. Curr. Opin. Cell Biol. 2001; 13: 363-375Crossref PubMed Scopus (217) Google Scholar). Although SENP2 was localized to the nuclear pore, we have not yet found any role for SENP2 in nuclear trafficking. Overexpression of wild type or mutant versions of SENP2 did not significantly alter nuclear import or export of a GFP-labeled chimeric model substrate consisting of HIV-1 Rev and a hormone-inducible nuclear localization sequence (Rev-GR-GFP, Ref. 25Love D.C. Sweitzer T.D. Hanover J.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10608-10613Crossref PubMed Scopus (84) Google Scholar) (data not shown). Although such negative results do not exclude the possibility that some nuclear transport pathways are controlled by SENP2, they suggest that modulation of SENP2 activity does not grossly alter the role of Nup153 in pore structure or nuclear import. Interestingly, Pichler et al. (26Pichler A. Gast A. Seeler J.S. Dejean A. Melchior F. Cell. 2002; 108: 109-120Abstract Full Text Full Text PDF PubMed Scopus (631) Google Scholar) have demonstrated that RanBP2 is an E3 enzyme for SUMO-1 that can catalyze both its own hyperconjugation to SUMO-1 and the conjugation of Sp100 in vitro. Pichler et al. (26Pichler A. Gast A. Seeler J.S. Dejean A. Melchior F. Cell. 2002; 108: 109-120Abstract Full Text Full Text PDF PubMed Scopus (631) Google Scholar) have proposed a model wherein RanBP2 may couple nuclear import with the conjugation of a subset of SUMO-1 substrates (26Pichler A. Gast A. Seeler J.S. Dejean A. Melchior F. Cell. 2002; 108: 109-120Abstract Full Text Full Text PDF PubMed Scopus (631) Google Scholar). It would be attractive to speculate that SENP2 might have a role in transport-linked deconjugation of the same subset of SUMO-1-conjugated proteins. Notably, translocation through the nuclear pore is not essential for their efficient conjugation of Sp100 by RanBP2 (26Pichler A. Gast A. Seeler J.S. Dejean A. Melchior F. Cell. 2002; 108: 109-120Abstract Full Text Full Text PDF PubMed Scopus (631) Google Scholar). Furthermore, nonconjugatable forms of Sp100 show no defects in nuclear localization in vivo(27Sternsdorf T. Jensen K. Reich B. Will H. J. Biol. Chem. 1999; 274: 12555-12566Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar), suggesting that SUMO-1 modification cannot be essential for its nuclear import. These observations further indicate that pore-associated SUMO-1 conjugation and deconjugation activities are unlikely to be involved in nuclear transport per se. An alternative function might be the specific marking of newly imported proteins, perhaps to direct their localization after nuclear entry or to regulate their activity before their assembly into macromolecular complexes within the nucleus (28Senger B. Simos G. Bischoff F.R. Podtelejnikov A. Mann M. Hurt E. EMBO J. 1998; 17: 2196-2207Crossref PubMed Scopus (157) Google Scholar). SENP2 is closely related to a rat SUMO-1 protease, Axam, which has been reported as an Axin-binding protein (11Nishida T. Kaneko F. Kitagawa M. Yasuda H. J. Biol. Chem. 2001; 276: 39060-39066Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 12Kadoya T. Kishida S. Fukui A. Hinoi T. Michiue T. Asashima M. Kikuchi A. J. Biol. Chem. 2000; 275: 37030-37037Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Axam antagonizes the binding of Dvl-1 to Axin and suppresses GSK-3β-dependent phosphorylation in the Axin complex, thereby enhancing β-catenin degradation (12Kadoya T. Kishida S. Fukui A. Hinoi T. Michiue T. Asashima M. Kikuchi A. J. Biol. Chem. 2000; 275: 37030-37037Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Enhancement of β-catenin degradation does not require SUMO protease activity (11Nishida T. Kaneko F. Kitagawa M. Yasuda H. J. Biol. Chem. 2001; 276: 39060-39066Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar), suggesting that SENP2 may have multiple, independent functions. Earlier reports on Axam also indicated that Axam and SENP2 (SMT3IP2) are localized to the cytosol (11Nishida T. Kaneko F. Kitagawa M. Yasuda H. J. Biol. Chem. 2001; 276: 39060-39066Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 12Kadoya T. Kishida S. Fukui A. Hinoi T. Michiue T. Asashima M. Kikuchi A. J. Biol. Chem. 2000; 275: 37030-37037Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). We believe that the difference between these observations and ours was that both reports visualized the localization of N-terminal-truncated forms of Axam or SENP2, missing 72 or 42 amino acids, respectively. We suspect that expression levels and cell type-specific differences may also contribute to the differences between our observations and those reports, since we did not observe localization of truncated forms of SENP2 in the cytosol. We cannot currently explain the relationship between the APC/β-catenin pathway and SENP2, but it will be of interest to examine whether SENP2 or other SUMO-1 pathway enzymes regulate nuclear translocation or subnuclear localization of any of the proteins of the APC/β-catenin pathway. In summary, we have shown that full-length SENP2 localizes to the nuclear face of the nuclear pore. This localization is likely to be achieved through interaction with Nup153. Appropriate localization to the pore plays an important role for the correct regulation of SENP2 because its mislocalization leads to the inappropriate deconjugation of many SUMO-1-conjugated species. It will be of interest in the future to determine which SUMO-1-conjugated species are normally the targets of SENP2 activity. We thank Jomon Joseph and Tadashi Anan for help in generating constructs expressing tagged human RanGAP1 and SUMO-1. We thank Shyh-Han Tan for help with confocal microscopy. Finally, we thank Alexei Arnaoutouv, Yoshiaki Azuma, Byrn Booth Quimby, and Shyh-Han Tan for critical reading of this report." @default.
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• W2042862271 title "Association of the Human SUMO-1 Protease SENP2 with the Nuclear Pore" @default.
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