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[Cancer Research 64, 7846-7851, November 1, 2004]
© 2004 American Association for Cancer Research

Regular Articles

SUMO-1 Modification of the Wilms’ Tumor Suppressor WT1

Gromoslaw A. Smolen1, Maria T. Vassileva2, Julie Wells1, Michael J. Matunis2 and Daniel A. Haber1

1 Massachusetts General Hospital Cancer Center and Harvard Medical School, Charlestown, Massachusetts; and 2 Department of Biochemistry and Molecular Biology, Bloomberg School of Hygiene and Public Health, Johns Hopkins University, Baltimore, Maryland


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
SUMO-1 conjugation modulates numerous cellular functions, including the subnuclear localization of its target proteins. The WT1 tumor suppressor encodes a four-zinc finger protein with distinct splicing isoforms. WT1(–KTS), encoding uninterrupted zinc fingers, functions as a transcription factor and has a diffusely nuclear distribution; WT1(+KTS), with an insertion of three amino acids (KTS) between zinc fingers three and four, localizes to discrete nuclear speckles, the function of which is unknown. Because the SUMO-1 E2-conjugating enzyme, Ubc9, interacts with WT1, we tested whether sumoylation modulates the cellular localization of WT1. We find here that both WT1 isoforms are directly sumoylated on lysine residues 73 and 177. Although RNA interference-mediated Ubc9 depletion effectively suppresses WT1 nuclear speckles, a SUMO-1–deficient WT1(+KTS)(K73, 177R) double mutant retains localization to speckles. Thus, direct sumoylation of WT1 is not responsible for its cellular localization, and other sumoylated proteins may target WT1 to these nuclear structures. Identification of other components of WT1-associated speckles is likely to provide clues to their function.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Wilms’ tumor is the most common pediatric kidney cancer, affecting 1/10,000 children. The first Wilms’ tumor susceptibility gene, WT1, encodes a four-zinc finger transcription factor with a specific pattern of expression in renal precursors (1 , 2) . Although about 10% of sporadic Wilms’ tumors harbor somatic inactivation of WT1, consistent with its characterization as a tumor suppressor, WT1-null mice demonstrate failure of both renal and gonadal differentiation (3) , pointing to an essential role in organogenesis. The WT1 locus gives rise to a number of isoforms resulting from alternative splicing events. The best characterized variation results from differential use of a splice donor site between exons 9 and 10, leading to insertion of three amino acids, lysine-threonine-serine (KTS), between zinc fingers three and four. A cumulative body of evidence clearly points to distinct cellular functions for WT1(–KTS) and WT1(+KTS) (4) .

Most functional studies of WT1 have focused on the WT1(–KTS) isoform, which only constitutes about 20% of the protein but which mediates potent transcription activation. WT1(–KTS) activates transcription of multiple genes, some of which are involved in cell cycle progression, as well as gonadal and renal cell differentiation (5, 6, 7, 8) . Specific inactivation of the WT1(–KTS) isoform leads to disruption of kidney architecture and failure of gonadal differentiation (9) . These abnormalities are similar to but less severe than those of WT1–null mice (3) , pointing to the potential contribution of the second major splice variant, WT1(+KTS).

WT1(+KTS) is the predominant cellular isoform, accounting for about 80% of WT1 transcripts, although its functional properties remain unknown. The (+KTS) insertion appears to disrupt DNA binding, and transcriptional regulation of target genes by WT1(+KTS) remains to be documented. The ratio between the (+KTS) and (–KTS) isoforms is critical for proper development in humans, because heterozygous individuals harboring a splice donor site mutation decreasing the (+KTS) to (–KTS) ratio display severe renal and gonadal defects (Frazier syndrome; ref. 10 ). WT1(+KTS) protein has been postulated to be involved in some aspect of pre-mRNA processing, based on its distinctive "speckled" subnuclear localization, along with snRNPs (11) , as well as its binding to a component of the splicing machinery, U2AF65 (12) . However, WT1(+KTS) does not colocalize with other key components of the splicing machinery such as SC35 (13) , and a specific effect of WT1(+KTS) on pre-mRNA processing has not been identified. Specific inactivation of WT1(+KTS) in the mouse leads to kidney developmental defects that are similar to those of WT1(–KTS)–null mice (9) . In contrast, gonadal differentiation proceeds to the stage of sex determination, with WT1(+KTS)–null XY animals displaying complete sex reversal, unlike WT(–KTS)–null mice, which fail to develop an undifferentiated gonad.

The importance of subnuclear localization to the distinct properties of WT1 isoforms is highlighted by the observation that Wilms’ tumor–derived mutations altering the DNA-binding domain lead to dramatic enhancement of nuclear bodies (13) . As such, the components of these nuclear structures and the mechanisms by which WT1 variants are targeted to these bodies may provide insight into their functional properties.

The small, ubiquitin-related protein SUMO-1 is highly conserved from yeast to humans (14) and has been associated with subnuclear localization of many cellular proteins. Like ubiquitination, sumoylation leads to attachment of SUMO-1 to target proteins through the {epsilon}-NH2 group of lysine residues, using a cascade of E1, E2, and E3 enzymes. In vitro, E1 and E2 enzymes (Aos1/Uba2 and Ubc9 in humans) are sufficient to mediate sumoylation of a number of substrates. The recognition of substrates is achieved by Ubc9, which interacts with proteins containing a consensus site, {Psi}KXE, where {Psi} represents any hydrophobic residue and K is the particular lysine of the target protein conjugated to SUMO-1 (15) . In vivo, however, several families of E3 enzymes are thought to contribute to substrate selection and specificity (16, 17, 18, 19) .

Unlike ubiquitination, sumoylation does not target proteins for degradation, but it modulates a wide range of protein functions. SUMO-1 is an important determinant of protein localization, required for the speckled nuclear distribution of the proteins PML, TEL, and HIPK2 (20, 21, 22) . In addition, SUMO-1 has been shown to modulate the activity of the transcription factors p53, androgen receptor, and c-Jun (23, 24, 25, 26) . Although these are its most commonly reported functions, sumoylation can also exert an effect on nuclear transport, protein turnover, cell signaling, and other aspects of cellular homeostasis (27) .

WT1 has been reported to interact physically with Ubc9 in yeast two-hybrid, glutathione S-transferase–pull down, as well as in vivo coimmunoprecipitation assays (28) , suggesting that WT1 could be a potential substrate for SUMO-1 conjugation. However, the functional consequences of Ubc9 interaction have not been defined, nor is it known whether sumoylation is required for the distinctive subnuclear localization of different WT1 isoforms. Here, we show that sumoylation is indeed required for the speckled distribution of WT1. Surprisingly, although WT1 itself is directly sumoylated, disruption of WT1 sumoylation does not affect its nuclear localization, indicating that other sumoylated WT1-binding partners are likely to target WT1 to these nuclear structures.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Plasmid Constructs.
Cytomegalovirus- and Rous sarcoma virus–driven WT1 constructs were as described previously (29 , 30) . K73R and K177R mutations were introduced using Quickchange kit (Stratagene, La Jolla, CA). Podocalyxin and Amphiregulin luciferase reporter constructs were as described previously (8 , 31) . All new plasmid constructs were verified by sequencing.

Cell Culture.
Human osteosarcoma U2OS cells and human cervical adenocarcinoma HeLa cells were maintained in Dulbecco’s modified medium supplemented with 10% fetal calf serum. Inducible cell lines (RSTEM and U2OS) were maintained in the above medium with an additional 1 µg/mL tetracycline. All U2OS cells were cultured at 5% CO2 at 37°C, whereas all RSTEM cells were kept at 5% CO2 at 32°C.

In vitro SUMO-1 Conjugation Assay.
cDNAs encoding full-length WT1 proteins were transcribed and translated in rabbit reticulocyte lysate in the presence of [35S]methionine according to the manufacturer’s instructions (Promega, Madison, WI). In vitro SUMO-1 modification reaction was initiated by adding 8 µL of the conjugation mix [20 mmol/L HEPES (pH 7.3), 110 mmol/L potassium acetate, 2 mmol/L magnesium acetate, 1 mmol/L dithiothreitol, 0.5 µmol/L recombinant Aos1/Uba2, 1 µmol/L recombinant Ubc9, 3 µmol/L recombinant SUMO-1, 1 mmol/L ATP, 10 mmol/L phosphocreatine, 40 units/mL creatine phosphokinase, and 1.2 µg/mL inositol pyrophosphatase] to 2 µL of WT1-translated protein. After 1 hour at 37°C, the reactions were stopped by the addition of an equal volume of SDS sample buffer and analyzed by SDS-PAGE followed by autoradiography.

Immunoprecipitations and Western Blotting.
U2OS cells were plated at a density of 7 x 105 per 10-cm dish 1 day before transfection. Transient transfection was carried out with 6 µg of plasmid DNA per dish using Fugene 6 transfection reagent (Roche, Indianapolis, IN). Expression of transfected 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 Tris (pH 8.0), 1 mmol/L EDTA, 20 mmol/L N-ethylmaleimide, and 1x protease inhibitor mixture (Complete EDTA-free; Roche)]. Cell lysates were sonicated for 10 seconds and cleared by centrifugation at 14,000 rpm for 20 minutes at 4°C. Approximately 500 µg of protein lysate was used per IP reaction. Lysates were precleared with protein A agarose beads for 2 hours at 4°C. One µg of antibody was added to the precleared lysates for 2 hours at 4°C, followed by the addition of fresh protein A agarose beads and incubation at 4°C overnight. Beads were washed three times with 1 mL of cold lysis buffer. Bound proteins were eluted with 2x SDS sample buffer and loaded onto 10% SDS-PAGE gel (ReadyGel; Bio-Rad, Cambridge, MA). For immunoblotting analysis, proteins were transferred onto Immobilon polyvinylidene difluoride membrane (Millipore, Bedford, MA) and visualized with Western Lightning Plus chemiluminescence kit (Perkin-Elmer, Boston, MA).

Antibodies.
Anti-SUMO-1 antibodies used were D-11 (Santa Cruz Biotechnology, Santa Cruz, CA) and 21C7 (Zymed, South San Francisco, CA). Anti-WT1 antibodies used were C-19 (Santa Cruz Biotechnology) and C8 (29) . Anti-SC35 antibody used was from Sigma (St. Louis, MO; S4045). Anti-Ubc9 antibody was from BD Biosciences (San Diego, CA; 610748).

Immunofluorescence Analysis.
Sixty percent confluent U2OS cell were grown on coverslips in 12-well dishes. Transfection of 0.5 µg 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 withdrawal.

Reverse Transcription-Polymerase Chain Reaction Analysis.
Reverse transcription reactions were standardized by adjusting the total RNA input to 1 µg. The following primers were used: Hs.Ubc9f, 5'-GGCACGATGAACCTCATGAACTGG-3'; Hs.Ubc9r, 5'-GCCTCTGCTTGAGCTGGGTCTTGG-3'; Hs.GAPDHf, 5'-ACCACAGTCCATGCCATCAC-3'; Hs.GAPDHr, 5'-TCCACCACCCTGTTGCTGTA-3'; Hs.WT1f, 5'-TCAGGATGTGCGACGTGTGCCTGGAGTAGC-3'; and Hs.WT1r, 5'-GTGATGGCGGACTAATTCATCTGACCGGGC-3'.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Ubc9 Is Required for Formation of WT1 Nuclear Speckles.
To determine whether sumoylation is required for the subnuclear localization of WT1 variants, we first targeted the essential SUMO-1–conjugating enzyme, Ubc9, using RNA interference. In these experiments, we made use of U2OS cells with tetracycline-regulated expression of WT1(delZ), a mutation that deletes the zinc finger domain of WT1 and gives rise to clear and readily quantifiable nuclear speckles. This mutation has been reported in patients with Denys Drash syndrome, associated with dominant-negative WT1 mutations. Using RNA interference, we successfully knocked down the levels of Ubc9 mRNA (Fig. 1A)Citation as well as Ubc9 protein (Fig. 1B)Citation . Prolonged exposure of cells to the Ubc9-targeting silencing duplexes resulted in some cell death, however, we are able to assess the WT1 nuclear speckles in morphologically normal cells at an earlier time point. At 48 hours after Ubc9 siRNA treatment, WT1(delZ) expression became diffusely nuclear (Fig. 1C)Citation . This effect was specific, because the morphology of speckles associated with the splicing factor SC-35 was not affected (Fig. 1C)Citation . Thus, Ubc9 is required for WT1 nuclear speckle formation, suggesting a role for sumoylation.



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  		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.

 
WT1 Is Modified by SUMO-1 In vitro and In vivo.
To investigate whether WT1 is itself modified by SUMO-1, we first used a standard in vitro modification assay, involving incubation of 35S-labeled in vitro translated WT1, purified recombinant SUMO-1, Ubc9 (E2 activity), and SAE1/2 heterodimer (E1 activity). Both major splice forms of WT1, (+KTS) and (–KTS), were modified by SUMO-1 to a similar extent (Fig. 2A)Citation . The observed Mr shift was present only when all of the components described above were included in the modification reaction. To confirm that WT1 is modified in vivo, we transiently transfected human osteosarcoma cells U2OS with both of the WT1 splice forms and used coimmunoprecipitation to demonstrate the presence of WT1–SUMO-1 conjugates. Immunoprecipitation using anti-WT1 antibody followed by immunoblotting analysis with anti-SUMO-1 antibody revealed two major bands, suggesting that WT1 may be modified by either one or two molecules of SUMO-1 (Fig. 2B)Citation . Similar results were observed in the reciprocal experiment, using anti-SUMO-1 immunoprecipitation, followed by anti-WT1 immunoblotting analysis. To demonstrate that WT1 is modified by SUMO-1 in a more physiologic setting, we used embryonic rat kidney RSTEM cells, which display detectable levels of endogenous WT1 and retain their in vivo differentiation potential (32) . RSTEM cells have been engineered to express either WT1(–KTS) or WT1(+KTS) under the control of a tetracycline-repressible promoter (8) , resulting in a modest 2- to 3-fold induction of WT1 expression over the endogenous levels (Fig. 2C)Citation . Immunoprecipitation and immunoblotting analyses in RSTEM cells clearly show the presence of WT1(–KTS)–SUMO-1 (Fig. 2C)Citation as well as WT1(+KTS)–SUMO-1 conjugates (data not shown) after WT1 induction. Significantly, the conjugates were also observed for endogenous levels of WT1 when these cells were grown in the presence of tetracycline. Collectively, these findings demonstrate that both WT1(–KTS) and WT1(+KTS) are substrates for SUMO-1 conjugation.



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  		Fig. 2. SUMO-1 modification of WT1. A, SUMO-1 conjugation of in vitro translated [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 modification of WT1, demonstrated by immuprecipitation-Western analysis in U2OS cells transiently transfected with empty expression vector (pCDNA3.1-) or with constructs encoding WT1(–KTS) or WT1(+KTS). The order of antibodies used for immuprecipitation (IP) and immunoblotting (WB) is indicated above each panel. Ten percent of cell lysate was immunoblotted with anti-WT1 antibody to measure the expression levels (bottom panel). C, Immunoprecipitation-Western analysis in rat embryonic kidney RSTEM cells in which a 2- to 3-fold induction of WT1 is mediated by a tetracycline-regulated promoter (+tet., off; –tet, on). The order of antibodies used for immuprecipitation (IP) and immunoblotting (WB) is indicated above each panel. Ten percent of cell 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 indicated on the right of the panels.

 
Mapping of the Sumoylated Residues within WT1.
Ubc9 directly interacts with a number of SUMO-1 substrates through a consensus motif {Psi}KXE, where {Psi} is any hydrophobic residue. The lysine contained within such a consensus is the recipient of the SUMO-1 modification. WT1 contains two such motifs, both in the NH2 terminus and centered around lysine 73 and lysine 177 (Fig. 3A)Citation . We therefore introduced K73R and K177R point mutations, either individually or in combination, into expression constructs and tested their ability to be sumoylated in vivo. Lysine 73 was the major site of SUMO-1 modification, because the K73R mutant displayed a significant reduction in the amount of immunoprecipitated conjugates, whereas the K177R mutant had a small effect (Fig. 3B)Citation . Virtual absence of WT1–SUMO-1 conjugates was observed when the mutations were combined in the (K73, 177R) double mutant. Similar findings were observed for both WT1(–KTS) and WT1(+KTS) isoforms (data not shown). Therefore, WT1 is modified in vivo on lysine 73 and lysine 177 residues, with lysine 73 being the major site of sumoylation. WT1(K73, 177R) double mutant construct allowed us to test the functional consequences of sumoylation on the characteristic subnuclear speckling by WT1(+KTS).



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  		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(–KTS). 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.

 
Effect of Sumoylation on WT1(+KTS) Nuclear Localization.
To determine whether SUMO-1 conjugation of WT1 is directly responsible for its localization to nuclear speckles, we transiently tranfected U2OS cells with wild-type or lysine-to-arginine WT1 mutants and examined their nuclear localization using immunofluorescence microscopy (Fig. 4)Citation . Remarkably, abrogation of SUMO-1 conjugation had no effect on the nuclear distribution of WT1 variants, including the WT1(+KTS) physiologic splicing variant, and the WT1(delZ) mutant. Thus, although sumoylation is required for the appropriate nuclear localization of these variants, as demonstrated by Ubc9 RNAi experiments, this effect does not require direct SUMO-1 conjugation to WT1 itself.



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  		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 (+/–KTS) and zinc finger-deleted WT1(delZ). Wild-type WT1(–KTS) is expressed diffusely in the nucleus, WT1(+KTS) 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.

 
WT1 protein is known to self-associate (33 , 34) , and it may therefore be possible for low levels of endogenous wild-type WT1 in U2OS cells to recruit the transfected WT1 into preexisting nuclear structures. To eliminate this possibility, we identified a human cell line, HeLa, with no expression of WT1 detectable by reverse transcription-PCR (Fig. 5A)Citation and analyzed the nuclear distribution of transfected WT1 lysine-to-arginine mutants (Fig. 5B)Citation . As in U2OS cells, transfected HeLa cells showed no significant differences between wild-type and mutant WT1 constructs. Hence, dimerization with endogenous WT1 is unlikely to explain the persistent nuclear localization of lysine-to-arginine WT1 mutants, pointing to other sumoylated WT1-interacting proteins that may target WT1 to these nuclear structures.



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  		Fig. 5. Nuclear localization of WT1 sumoylation mutants in cells not expressing endogenous WT1. A, reverse transcription-PCR expression analysis of WT1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; control) mRNAs in U2OS and HeLa cells, showing the lack of WT1 expression in HeLa cells. B, anti-WT1 immunofluorescence analysis of HeLa cells transfected with the indicated constructs. Two mutations, K73R and K177R, were both introduced into the physiologic isoforms of WT1 (+/–KTS) and zinc finger-deleted WT1(delZ). Wild-type WT1(–KTS) is expressed diffusely in the nucleus, WT1(+KTS) 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.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
WT1 plays an important role in both tumorigenesis and normal genito-urinary development, yet regulatory mechanisms that modulate its function are not well understood. These appear to include a very specific spatial and temporal pattern of expression in the developing embryo, a complex pattern of pre-mRNA splicing, potential protein interacting partners, and finally, posttranslational modifications (4) . In this report, we present evidence that WT1 is modified by SUMO-1 and analyze the functional consequences of this modification.

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 ({Psi}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, 34, 35, 36) . Although we have defined the consequence of Ubc9 binding to WT1, the consequences of WT1 sumoylation on other molecular interactions 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(–KTS) in the activation of target gene transcription is well documented. However, we were unable to show any SUMO-1–dependent effect on WT1-mediated transcriptional activation of two well-characterized WT1 target genes, Amphiregulin and Podocalyxin (data not shown). Therefore, we focused our analysis on the WT1(+KTS) isoform. Assessing the consequences of WT1(+KTS) sumoylation is difficult, because there are no defined assays for the function of this isoform; thus, its 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, including the naturally occurring chromosomal translocation product EWS-WT1 and synthetic deletion constructs (4) . Despite these observations, our analysis of (K73, 177R) mutants revealed no significant differences in the distribution of WT1 isoforms, indicating that WT1(+KTS) nuclear distribution is not dependent on direct sumoylation of the NH2 terminus. The Ubc9 dependence of WT1 localization argues that the SUMO-1 conjugation pathway is necessary for speckle formation, raising the possibility that sumoylation of a separate, WT1-interacting protein is itself critical for the formation of WT1 speckles.

An emerging theme relevant to protein subnuclear localization is the central role played by speckle "organizing" proteins. The arginine/serine-rich domain present within a family of pre-mRNA splicing factors is necessary and sufficient for targeting other associated proteins, which themselves lack a targeting motif, to these nuclear speckles (37 , 38) . The effect of sumoylation itself has been best studied for the PML protein, which is targeted to nuclear bodies. In PML–/– cells, transfected nonsumoylateable PML cannot form nuclear bodies (39 , 40) , whereas expression of wild-type PML reconstitutes the nuclear bodies and recruits resident proteins Daxx and Sp100. In PML+/+ cells, nonsumoylateable PML is correctly targeted to nuclear bodies, presumably through interaction with the wild-type endogenous PML protein. Although our data indicate that WT1 dimerization does not explain the persistent nuclear speckling of nonsumoylateable WT1 contructs, other proteins within these structures may be sumoylated and recruit WT1. Additional molecular definition of proteins that make up the WT1(+KTS) nuclear speckles will elucidate the role of this WT1 isoform in cancer as well as normal organogenesis.


   FOOTNOTES
 
Grant support: National Cancer Institute grant CA58596 (D. Haber) and NIH grant R01 GM60980-01 (M. Matunis).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: M. Vassileva and J. Wells contributed equally to this work.

Requests for reprints: Daniel A. Haber, MGH Cancer Center, Building 149, 13th Street, Charlestown, MA 02129. Phone: 617-726-7805; Fax: 617-724-6919; E-mail: Haber{at}helix.mgh.harvard.edu

Received 4/28/04. Revised 8/11/04. Accepted 8/30/04.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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