SUMO-1 Modification of the Wilms' Tumor Suppressor WT1

[CANCER RESEARCH 64, 7846–7851, November 1, 2004] SUMO-1 Modification of the Wilms’ Tumor Suppressor WT1

Gromoslaw A. Smolen,1 Maria T. Vassileva,2 Julie Wells,1 Michael J. Matunis,2 and Daniel A. Haber1 1Massachusetts General Hospital Cancer Center and Harvard Medical School, Charlestown, Massachusetts; and 2Department of Biochemistry and Molecular Biology, Bloomberg School of Hygiene and Public Health, Johns Hopkins University, Baltimore, Maryland

ABSTRACT remain unknown. The (ϩKTS) insertion appears to disrupt DNA binding, and transcriptional regulation of target genes by SUMO-1 conjugation modulates numerous cellular functions, including WT1(ϩKTS) remains to be documented. The ratio between the the subnuclear localization of its target proteins. The WT1 tumor sup- (ϩKTS) and (ϪKTS) isoforms is critical for proper development in pressor encodes a four-zinc finger protein with distinct splicing isoforms. WT1(؊KTS), encoding uninterrupted zinc fingers, functions as a tran- humans, because heterozygous individuals harboring a splice donor ϩ Ϫ scription factor and has a diffusely nuclear distribution; WT1(؉KTS), site mutation decreasing the ( KTS) to ( KTS) ratio display severe with an insertion of three amino acids (KTS) between zinc fingers three renal and gonadal defects (Frazier syndrome; ref. 10). WT1(ϩKTS) and four, localizes to discrete nuclear speckles, the function of which is protein has been postulated to be involved in some aspect of pre- unknown. Because the SUMO-1 E2-conjugating enzyme, Ubc9, interacts mRNA processing, based on its distinctive “speckled” subnuclear with WT1, we tested whether sumoylation modulates the cellular local- localization, along with snRNPs (11), as well as its binding to a ization of WT1. We find here that both WT1 isoforms are directly component of the splicing machinery, U2AF65 (12). However, sumoylated on lysine residues 73 and 177. Although RNA interference- WT1(ϩKTS) does not colocalize with other key components of the mediated Ubc9 depletion effectively suppresses WT1 nuclear speckles, a splicing machinery such as SC35 (13), and a specific effect of -SUMO-1–deficient WT1(؉KTS)(K73, 177R) double mutant retains local WT1(ϩKTS) on pre-mRNA processing has not been identified. Spe- ization to speckles. Thus, direct sumoylation of WT1 is not responsible for ϩ its cellular localization, and other sumoylated proteins may target WT1 to cific inactivation of WT1( KTS) in the mouse leads to kidney Ϫ these nuclear structures. Identification of other components of WT1- developmental defects that are similar to those of WT1( KTS)–null associated speckles is likely to provide clues to their function. mice (9). In contrast, gonadal differentiation proceeds to the stage of sex determination, with WT1(ϩKTS)–null XY animals displaying complete sex reversal, unlike WT(ϪKTS)–null mice, which fail to INTRODUCTION develop an undifferentiated gonad. The importance of subnuclear localization to the distinct properties Wilms’ tumor is the most common pediatric kidney cancer, affect- of WT1 isoforms is highlighted by the observation that Wilms’ ing 1/10,000 children. The first Wilms’ tumor susceptibility gene, tumor–derived mutations altering the DNA-binding domain lead to WT1, encodes a four-zinc finger transcription factor with a specific dramatic enhancement of nuclear bodies (13). As such, the compo- pattern of expression in renal precursors (1, 2). Although about 10% nents of these nuclear structures and the mechanisms by which WT1 of sporadic Wilms’ tumors harbor somatic inactivation of WT1, variants are targeted to these bodies may provide insight into their consistent with its characterization as a tumor suppressor, WT1-null functional properties. mice demonstrate failure of both renal and gonadal differentiation (3), The small, ubiquitin-related protein SUMO-1 is highly conserved pointing to an essential role in organogenesis. The WT1 locus gives from yeast to humans (14) and has been associated with subnuclear rise to a number of isoforms resulting from alternative splicing events. localization of many cellular proteins. Like ubiquitination, sumoyla- The best characterized variation results from differential use of a tion leads to attachment of SUMO-1 to target proteins through the splice donor site between exons 9 and 10, leading to insertion of three ⑀ -NH2 group of lysine residues, using a cascade of E1, E2, and E3 amino acids, lysine-threonine-serine (KTS), between zinc fingers enzymes. In vitro, E1 and E2 enzymes (Aos1/Uba2 and Ubc9 in three and four. A cumulative body of evidence clearly points to humans) are sufficient to mediate sumoylation of a number of sub- Ϫ ϩ distinct cellular functions for WT1( KTS) and WT1( KTS) (4). strates. The recognition of substrates is achieved by Ubc9, which Ϫ Most functional studies of WT1 have focused on the WT1( KTS) interacts with proteins containing a consensus site, ⌿KXE, where ⌿ isoform, which only constitutes about 20% of the protein but which represents any hydrophobic residue and K is the particular lysine of Ϫ mediates potent transcription activation. WT1( KTS) activates tran- the target protein conjugated to SUMO-1 (15). In vivo, however, scription of multiple genes, some of which are involved in cell cycle several families of E3 enzymes are thought to contribute to substrate progression, as well as gonadal and renal cell differentiation (5–8). selection and specificity (16–19). Ϫ Specific inactivation of the WT1( KTS) isoform leads to disruption Unlike ubiquitination, sumoylation does not target proteins for of kidney architecture and failure of gonadal differentiation (9). These degradation, but it modulates a wide range of protein functions. abnormalities are similar to but less severe than those of WT1–null SUMO-1 is an important determinant of protein localization, required mice (3), pointing to the potential contribution of the second major for the speckled nuclear distribution of the proteins PML, TEL, and ϩ splice variant, WT1( KTS). HIPK2 (20–22). In addition, SUMO-1 has been shown to modulate ϩ WT1( KTS) is the predominant cellular isoform, accounting for the activity of the transcription factors p53, androgen receptor, and about 80% of WT1 transcripts, although its functional properties c-Jun (23–26). Although these are its most commonly reported functions, sumoylation can also exert an effect on nuclear transport, Received 4/28/04; revised 8/11/04; accepted 8/30/04. protein turnover, cell signaling, and other aspects of cellular homeo- Grant support: National Cancer Institute grant CA58596 (D. Haber) and NIH grant R01 GM60980-01 (M. Matunis). stasis (27). The costs of publication of this article were defrayed in part by the payment of page WT1 has been reported to interact physically with Ubc9 in yeast charges. This article must therefore be hereby marked advertisement in accordance with two-hybrid, glutathione S-transferase–pull down, as well as in vivo 18 U.S.C. Section 1734 solely to indicate this fact. Note: M. Vassileva and J. Wells contributed equally to this work. coimmunoprecipitation assays (28), suggesting that WT1 could be Requests for reprints: Daniel A. Haber, MGH Cancer Center, Building 149, 13th a potential substrate for SUMO-1 conjugation. However, the func- Street, Charlestown, MA 02129. Phone: 617-726-7805; Fax: 617-724-6919; E-mail: [email protected]. tional consequences of Ubc9 interaction have not been defined, nor ©2004 American Association for Cancer Research. is it known whether sumoylation is required for the distinctive 7846

subnuclear localization of different WT1 isoforms. Here, we show units/mL creatine phosphokinase, and 1.2 ␮g/mL inositol pyrophosphatase] to that sumoylation is indeed required for the speckled distribution of 2 ␮L of WT1-translated protein. After 1 hour at 37°C, the reactions were WT1. Surprisingly, although WT1 itself is directly sumoylated, stopped by the addition of an equal volume of SDS sample buffer and analyzed disruption of WT1 sumoylation does not affect its nuclear local- by SDS-PAGE followed by autoradiography. ization, indicating that other sumoylated WT1-binding partners are Immunoprecipitations and Western Blotting. U2OS cells were plated at ϫ 5 likely to target WT1 to these nuclear structures. a density of 7 10 per 10-cm dish 1 day before transfection. Transient transfection was carried out with 6 ␮g of plasmid DNA per dish using Fugene 6 transfection reagent (Roche, Indianapolis, IN). Expression of transfected MATERIALS AND METHODS genes was analyzed 24 hours post-transfection. Cells were washed with PBS and disrupted with cold lysis buffer [150 mmol/L NaCl, 1% NP40, 50 mmol/L Plasmid Constructs. Cytomegalovirus- and Rous sarcoma virus–driven Tris (pH 8.0), 1 mmol/L EDTA, 20 mmol/L N-ethylmaleimide, and 1ϫ WT1 constructs were as described previously (29, 30). K73R and K177R protease inhibitor mixture (Complete EDTA-free; Roche)]. Cell lysates were mutations were introduced using Quickchange kit (Stratagene, La Jolla, CA). sonicated for 10 seconds and cleared by centrifugation at 14,000 rpm for 20 Podocalyxin and Amphiregulin luciferase reporter constructs were as described minutes at 4°C. Approximately 500 ␮g of protein lysate was used per IP previously (8, 31). All new plasmid constructs were verified by sequencing. Cell Culture. Human osteosarcoma U2OS cells and human cervical ade- reaction. Lysates were precleared with protein A agarose beads for 2 hours at ␮ nocarcinoma HeLa cells were maintained in Dulbecco’s modified medium 4°C. One g of antibody was added to the precleared lysates for 2 hours at supplemented with 10% fetal calf serum. Inducible cell lines (RSTEM and 4°C, followed by the addition of fresh protein A agarose beads and incubation U2OS) were maintained in the above medium with an additional 1 ␮g/mL at 4°C overnight. Beads were washed three times with 1 mL of cold lysis buffer. Bound proteins were eluted with 2ϫ SDS sample buffer and loaded tetracycline. All U2OS cells were cultured at 5% CO2 at 37°C, whereas all onto 10% SDS-PAGE gel (ReadyGel; Bio-Rad, Cambridge, MA). For immu- RSTEM cells were kept at 5% CO2 at 32°C. In vitro SUMO-1 Conjugation Assay. cDNAs encoding full-length WT1 noblotting analysis, proteins were transferred onto Immobilon polyvinylidene proteins were transcribed and translated in rabbit reticulocyte lysate in the difluoride membrane (Millipore, Bedford, MA) and visualized with Western presence of [35S]methionine according to the manufacturer’s instructions (Pro- Lightning Plus chemiluminescence kit (Perkin-Elmer, Boston, MA). mega, Madison, WI). In vitro SUMO-1 modification reaction was initiated by Antibodies. Anti-SUMO-1 antibodies used were D-11 (Santa Cruz Bio- adding 8 ␮L of the conjugation mix [20 mmol/L HEPES (pH 7.3), 110 mmol/L technology, Santa Cruz, CA) and 21C7 (Zymed, South San Francisco, CA). potassium acetate, 2 mmol/L magnesium acetate, 1 mmol/L dithiothreitol, 0.5 Anti-WT1 antibodies used were C-19 (Santa Cruz Biotechnology) and C8 (29). ␮mol/L recombinant Aos1/Uba2, 1 ␮mol/L recombinant Ubc9, 3 ␮mol/L Anti-SC35 antibody used was from Sigma (St. Louis, MO; S4045). Anti-Ubc9 recombinant SUMO-1, 1 mmol/L ATP, 10 mmol/L phosphocreatine, 40 antibody was from BD Biosciences (San Diego, CA; 610748).

Fig. 1. Requirement of Ubc9 for WT1 speckles. A, semiquantitative reverse transcription-PCR analysis of endogenous Ubc9 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; control) mRNAs after transient transfection of U2OS cells with Ubc9 siRNA, a nonspecific duplex, or no treatment (mock). B, anti-Ubc9 immunoblotting of U2OS cells transiently transfected with Ubc9 siRNA, a nonspecific duplex, or no treatment (mock). A nonspecific band (NS) from the anti-Ubc9 immunoblot was used to show equal protein loading in each lane. C, anti-WT1 and anti-SC-35 immunofluorescence analysis of U2OS cells treated with the duplexes indicated (red channel). Cell viability was assessed using a vital dye 5-chloromethylfluorescein diacetate (green channel). Overlay of the two channels in shown under merged. Representative nuclei from each transfection are shown. 7847

Immunofluorescence Analysis. Sixty percent confluent U2OS cell were grown on coverslips in 12-well dishes. Transfection of 0.5 ␮g DNA per well was performed using Fugene 6 transfection reagent. CellTracker Green 5-chloromethylfluorescein diacetate was used according to the manufacturer’s instructions (Molecular Probes, Eugene, OR). Staining and detection was done as described previously (13). RNA Interference. Short interfering RNAs (siRNA) targeting Ubc9 were a custom SMARTpool mixture from Dharmacon (Lafayette, CO). U2OS cells with tetracycline regulatable expression of WT1(delZ) were used (UZ11). Cells were seeded in 12-well plates on coverslips in the presence of tetracycline. The next day, tetracycline was withdrawn, and the cells were transfected with the duplexes (final concentration, 100 nmol/L) using Oligofectamine reagent. Cells were retransfected 24 hours later and fixed for immunofluorescence analyses 48 hours after the initial transfection and tetracycline with- drawal.

Fig. 3. Mapping of the sumoylation site on WT1. A, schematic representation of WT1 showing the two lysines targeted for sumoylation at the NH2 terminus and the KTS alternative splice in the zinc finger domain. The Ubc9 consensus sites are aligned with the gray box indicating SUMO-1-modified lysine residues. B, anti-WT1 immunoblotting of anti-SUMO-1 immunoprecipitates from U2OS cells transiently transfected with empty expression vector (pCDNA3.1-) or with constructs encoding wild-type or the indicated mutants of WT1(ϪKTS). Ten percent of cell lysate was immunoblotted with anti-WT1 antibody to demonstrate the expression levels. Mr markers are indicated on the left, whereas the position of WT1-SUMO-1 conjugates is indicated on the right of the panel.

Reverse Transcription-Polymerase Chain Reaction Analysis. Reverse transcription reactions were standardized by adjusting the total RNA input to 1 ␮g. The following primers were used: Hs.Ubc9f, 5Ј-GGCACGATGAA- CCTCATGAACTGG-3Ј; Hs.Ubc9r, 5Ј-GCCTCTGCTTGAGCTGGGTCTT- GG-3Ј; Hs.GAPDHf, 5Ј-ACCACAGTCCATGCCATCAC-3Ј; Hs.GAPDHr, 5Ј-TCCACCACCCTGTTGCTGTA-3Ј; Hs.WT1f, 5Ј-TCAGGATGTGCGA- CGTGTGCCTGGAGTAGC-3Ј; and Hs.WT1r, 5Ј-GTGATGGCGGACTAA- TTCATCTGACCGGGC-3Ј.

RESULTS

Ubc9 Is Required for Formation of WT1 Nuclear Speckles. To determine whether sumoylation is required for the subnuclear local- Fig. 2. SUMO-1 modification of WT1. A, SUMO-1 conjugation of in vitro translated ization of WT1 variants, we first targeted the essential SUMO-1– [35S]-labeled WT1 proteins after incubation with various combinations of SUMO-1 modification mix and SDS-PAGE followed by autoradiography. B, in vivo SUMO-1 conjugating enzyme, Ubc9, using RNA interference. In these exper- modification of WT1, demonstrated by immuprecipitation-Western analysis in U2OS cells iments, we made use of U2OS cells with tetracycline-regulated transiently transfected with empty expression vector (pCDNA3.1-) or with constructs expression of WT1(delZ), a mutation that deletes the zinc finger encoding WT1(ϪKTS) or WT1(ϩKTS). The order of antibodies used for immuprecipitation (IP) and immunoblotting (WB) is indicated above each panel. Ten percent of cell domain of WT1 and gives rise to clear and readily quantifiable nuclear lysate was immunoblotted with anti-WT1 antibody to measure the expression levels speckles. This mutation has been reported in patients with Denys (bottom panel). C, Immunoprecipitation-Western analysis in rat embryonic kidney Drash syndrome, associated with dominant-negative WT1 mutations. RSTEM cells in which a 2- to 3-fold induction of WT1 is mediated by a tetracycline- regulated promoter (ϩtet., off; Ϫtet, on). The order of antibodies used for immuprecipi- Using RNA interference, we successfully knocked down the levels of tation (IP) and immunoblotting (WB) is indicated above each panel. Ten percent of cell Ubc9 mRNA (Fig. 1A) as well as Ubc9 protein (Fig. 1B). Prolonged lysate was immunoblotted with anti-WT1 antibody to measure the expression levels. Mr markers are indicated on the left, whereas the position of WT1-SUMO-1 conjugates is exposure of cells to the Ubc9-targeting silencing duplexes resulted in indicated on the right of the panels. some cell death, however, we are able to assess the WT1 nuclear 7848

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 2004 American Association for Cancer Research. SUMOYLATION OF WT1 speckles in morphologically normal cells at an earlier time point. At Mapping of the Sumoylated Residues within WT1. Ubc9 di- 48 hours after Ubc9 siRNA treatment, WT1(delZ) expression became rectly interacts with a number of SUMO-1 substrates through a diffusely nuclear (Fig. 1C). This effect was specific, because the consensus motif ⌿KXE, where ⌿ is any hydrophobic residue. The morphology of speckles associated with the splicing factor SC-35 was lysine contained within such a consensus is the recipient of the not affected (Fig. 1C). Thus, Ubc9 is required for WT1 nuclear SUMO-1 modification. WT1 contains two such motifs, both in the speckle formation, suggesting a role for sumoylation. NH2 terminus and centered around lysine 73 and lysine 177 (Fig. 3A). WT1 Is Modified by SUMO-1 In vitro and In vivo. To investi- We therefore introduced K73R and K177R point mutations, either gate whether WT1 is itself modified by SUMO-1, we first used a individually or in combination, into expression constructs and tested standard in vitro modification assay, involving incubation of 35S- their ability to be sumoylated in vivo. Lysine 73 was the major site of labeled in vitro translated WT1, purified recombinant SUMO-1, Ubc9 SUMO-1 modification, because the K73R mutant displayed a signif- (E2 activity), and SAE1/2 heterodimer (E1 activity). Both major icant reduction in the amount of immunoprecipitated conjugates, splice forms of WT1, (ϩKTS) and (ϪKTS), were modified by whereas the K177R mutant had a small effect (Fig. 3B). Virtual

SUMO-1 to a similar extent (Fig. 2A). The observed Mr shift was absence of WT1–SUMO-1 conjugates was observed when the muta- present only when all of the components described above were in- tions were combined in the (K73, 177R) double mutant. Similar cluded in the modification reaction. To confirm that WT1 is modified findings were observed for both WT1(ϪKTS) and WT1(ϩKTS) in vivo, we transiently transfected human osteosarcoma cells U2OS isoforms (data not shown). Therefore, WT1 is modified in vivo on with both of the WT1 splice forms and used coimmunoprecipitation to lysine 73 and lysine 177 residues, with lysine 73 being the major site demonstrate the presence of WT1–SUMO-1 conjugates. Immunopre- of sumoylation. WT1(K73, 177R) double mutant construct allowed us cipitation using anti-WT1 antibody followed by immunoblotting anal- to test the functional consequences of sumoylation on the character- ysis with anti-SUMO-1 antibody revealed two major bands, suggest- istic subnuclear speckling by WT1(ϩKTS). .ing that WT1 may be modified by either one or two molecules of Effect of Sumoylation on WT1(؉KTS) Nuclear Localization SUMO-1 (Fig. 2B). Similar results were observed in the reciprocal To determine whether SUMO-1 conjugation of WT1 is directly re- experiment, using anti-SUMO-1 immunoprecipitation, followed by sponsible for its localization to nuclear speckles, we transiently tran- anti-WT1 immunoblotting analysis. To demonstrate that WT1 is mod- fected U2OS cells with wild-type or lysine-to-arginine WT1 mutants ified by SUMO-1 in a more physiologic setting, we used embryonic and examined their nuclear localization using immunofluorescence rat kidney RSTEM cells, which display detectable levels of endoge- microscopy (Fig. 4). Remarkably, abrogation of SUMO-1 conjugation nous WT1 and retain their in vivo differentiation potential (32). had no effect on the nuclear distribution of WT1 variants, including RSTEM cells have been engineered to express either WT1(ϪKTS) or the WT1(ϩKTS) physiologic splicing variant, and the WT1(delZ) WT1(ϩKTS) under the control of a tetracycline-repressible promoter mutant. Thus, although sumoylation is required for the appropriate (8), resulting in a modest 2- to 3-fold induction of WT1 expression nuclear localization of these variants, as demonstrated by Ubc9 RNAi over the endogenous levels (Fig. 2C). Immunoprecipitation and im- experiments, this effect does not require direct SUMO-1 conjugation munoblotting analyses in RSTEM cells clearly show the presence of to WT1 itself. WT1(ϪKTS)–SUMO-1 (Fig. 2C) as well as WT1(ϩKTS)–SUMO-1 WT1 protein is known to self-associate (33, 34), and it may there- conjugates (data not shown) after WT1 induction. Significantly, the fore be possible for low levels of endogenous wild-type WT1 in conjugates were also observed for endogenous levels of WT1 when U2OS cells to recruit the transfected WT1 into preexisting nuclear these cells were grown in the presence of tetracycline. Collectively, structures. To eliminate this possibility, we identified a human cell these findings demonstrate that both WT1(ϪKTS) and WT1(ϩKTS) line, HeLa, with no expression of WT1 detectable by reverse tran- are substrates for SUMO-1 conjugation. scription-PCR (Fig. 5A) and analyzed the nuclear distribution of

Fig. 4. Nuclear localization of WT1 sumoylation mutants. Anti-WT1 immunofluorescence analysis of U2OS cells transfected with the indicated constructs. Each mutation (K73R, K177R, and the double mutant) was introduced into the physiologic isoforms of WT1 (ϩ/ϪKTS) and zinc finger- deleted WT1(delZ). Wild-type WT1(ϪKTS) is expressed diffusely in the nucleus, WT1(ϩKTS) forms small speckles, whereas WT1(delZ) is expressed in larger discrete speckles. None of these patterns is disrupted by mutations of WT1 sumoylation sites. Representative nuclei for each construct are displayed.

7849

WT1 has been reported to interact with Ubc9, suggesting that it could be a SUMO-1 substrate (28). Using a variety of in vitro and in vivo methods, we have shown here that WT1 is indeed a substrate for SUMO-1 conjugation. In the in vitro system, the addition of Aos1/Uba2 (E1 enzyme) and Ubc9 (E2 enzyme) was sufficient for recognition of WT1 as a substrate and conjugation of a single SUMO-1 molecule onto WT1. However, in vivo, we observed two major immunoprecipitated bands, suggesting that two SUMO-1 molecules may be conjugated to WT1. Consistent with this result, WT1 has two motifs that conform to the consensus sequence (⌿KXE) recognized by Ubc9. Enhanced modification of WT1 in vivo might reflect the presence of factors not present in our in vitro system, such as E3 proteins. In U2OS cells, WT1 is a robust SUMO-1 substrate, as abundant WT1–SUMO-1 conjugates were observed with overexpression of WT1 alone, without the need for coexpression with components of the SUMO-1 conjugation pathway. Gross overexpression of WT1 is not required for sumoylation, because this was also observed in RSTEM cells, derived from the undifferentiated mesenchyme of embryonic rat kidney (32). Although baseline sumoylation of WT1 was observed in these cells, induction of modest amounts of WT1 from a regulated promoter led to a significant enhancement. Strong sumoylation of newly expressed WT1 is of particular interest as expression of WT1 in a developing embryo is strongly induced at the mesenchymal-to-epithelial transition (4).

The two sites of SUMO-1 conjugation mapped to the NH2 terminus of WT1, which has been ascribed both transcriptional activation and repression functions in distinct cellular contexts. This may also reflect

the effects of proteins reported to interact with the NH2 terminus of WT1, including Ubc9, Hsp70, BASP1, and WT1 itself (28, 33–36). Although we have defined the consequence of Ubc9 binding to WT1, the consequences of WT1 sumoylation on other molecular interac- tions remain to be elucidated. Given the fact that both isoforms of WT1 are equally sumoylated, we analyzed the effects of this modification on characteristic functional properties of each splice form. The role of WT1(ϪKTS) in the activation of target gene transcription is well documented. However, Fig. 5. Nuclear localization of WT1 sumoylation mutants in cells not expressing we were unable to show any SUMO-1–dependent effect on WT1- endogenous WT1. A, reverse transcription-PCR expression analysis of WT1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; control) mRNAs in U2OS and HeLa mediated transcriptional activation of two well-characterized WT1 cells, showing the lack of WT1 expression in HeLa cells. B, anti-WT1 immunofluores- target genes, Amphiregulin and Podocalyxin (data not shown). There- cence analysis of HeLa cells transfected with the indicated constructs. Two mutations, ϩ K73R and K177R, were both introduced into the physiologic isoforms of WT1 (ϩ/ϪKTS) fore, we focused our analysis on the WT1( KTS) isoform. Assessing and zinc finger-deleted WT1(delZ). Wild-type WT1(ϪKTS) is expressed diffusely in the the consequences of WT1(ϩKTS) sumoylation is difficult, because nucleus, WT1(ϩKTS) forms small speckles, whereas WT1(delZ) is expressed in larger there are no defined assays for the function of this isoform; thus, its discrete speckles. None of these patterns is disrupted by mutations of WT1 sumoylation sites. Representative nuclei for each construct are displayed. distinct speckled nuclear distribution serves as a surrogate for its functional integrity. Several lines of evidence suggest that the domain

responsible for speckling resides within the NH2 terminus of WT1, transfected WT1 lysine-to-arginine mutants (Fig. 5B). As in U2OS including the naturally occurring chromosomal translocation product cells, transfected HeLa cells showed no significant differences be- EWS-WT1 and synthetic deletion constructs (4). Despite these obser- tween wild-type and mutant WT1 constructs. Hence, dimerization vations, our analysis of (K73, 177R) mutants revealed no significant with endogenous WT1 is unlikely to explain the persistent nuclear differences in the distribution of WT1 isoforms, indicating that localization of lysine-to-arginine WT1 mutants, pointing to other WT1(ϩKTS) nuclear distribution is not dependent on direct sumoy- sumoylated WT1-interacting proteins that may target WT1 to these lation of the NH2 terminus. The Ubc9 dependence of WT1 localiza- nuclear structures. tion argues that the SUMO-1 conjugation pathway is necessary for speckle formation, raising the possibility that sumoylation of a sepa- DISCUSSION rate, WT1-interacting protein is itself critical for the formation of WT1 speckles. WT1 plays an important role in both tumorigenesis and normal An emerging theme relevant to protein subnuclear localization is genito-urinary development, yet regulatory mechanisms that modulate the central role played by speckle “organizing” proteins. The arginine/ its function are not well understood. These appear to include a very serine-rich domain present within a family of pre-mRNA splicing specific spatial and temporal pattern of expression in the developing factors is necessary and sufficient for targeting other associated pro- embryo, a complex pattern of pre-mRNA splicing, potential protein teins, which themselves lack a targeting motif, to these nuclear speck- interacting partners, and finally, posttranslational modifications (4). In les (37, 38). The effect of sumoylation itself has been best studied for this report, we present evidence that WT1 is modified by SUMO-1 the PML protein, which is targeted to nuclear bodies. In PMLϪ/Ϫ and analyze the functional consequences of this modification. cells, transfected nonsumoylateable PML cannot form nuclear bodies 7850

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