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1 Department of Biological Sciences, Graduate School of Sciences, University of Tokyo, 7-8-1, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
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ICORP Project JST, 3-8-1, Komaba, Meguro-ku, Tokyo 153-8902, Japan
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Department of Life Sciences (Biology), Graduate School of Arts and Sciences, University of Tokyo, 3-8-1, Komaba, Meguro-ku, Tokyo 153-8902, Japan
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Department of Biochemistry and Second Department of Surgery, Faculty of Medicine, Hiroshima University, Minami-ku, Hiroshima 734-8551, Japan
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Introduction |
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The first mammalian protein shown to be sumoylated was RanGAP1, a GTPase-activating protein which plays an important role in the transport of proteins across the nuclear pore complex. Sumoylation facilitates binding of RanGAP1 to the nuclear pore, causing a change in the intracellular localization of target proteins (Bischoff et al. 1995; Matunis et al. 1996; Mahajan et al. 1997). Both I
B
and Mdm2, which function as suppressors of the transcriptional activity of NF-B and ubiquitin-protein ligase, respectively, can be sumoylated. Sumoylation stabilizes these proteins by preventing ubiquitination (Desterro et al. 1998; Buschmann et al. 2000). In addition, sumoylation of the tumour suppressor p53 reportedly enhances its transcriptional activity (Gostissa et al. 1999; Rodriguez et al. 1999). In yeast, lack of SUMO conjugation causes arrest of the cell cycle before anaphase (Seufert et al. 1995), suggesting an important role for sumoylation in cell cycle regulation.
The conjugation of SUMO is reversible. Several enzymes that remove SUMO from their substrates have been identified as SUMO-specific proteases in yeast and in mammals. These enzymes catalyse both the deconjugation of SUMO (desumoylation), and the hydrolysis of the SUMO C-terminal region. For example, in yeast, ubiquitin-like protease-1 (Ulp-1) catalyses processing of the C-terminal sequence of the Smt3 precursor (-GGATY) to its mature form (-GG), and deconjugates Smt3 from the lysine 
-amino group of target proteins (Li & Hochstrasser 1999). Several SUMO-specific proteases have been identified in mammals, including SENP1, SENP2/Axam, SUSP1, SMT3IP1, SMT3IP2/Axam2 and SuPr1, and others have been predicted to function as such, on the basis of DNA sequence homology (Gong et al. 2000; Kim et al. 2000; Yeh et al. 2000; Best et al. 2002; Nishida et al. 2000, 2001; Kadoya et al. 2002). The C-terminal domains of SUMO-specific proteases are highly conserved and function as catalytic domains, whereas the N-terminal regions display poor homology (Yeh et al. 2000). SENP1, Axam, SMT3IP1, and SuPr1 are predominantly nuclear proteins (Gong et al. 2000; Nishida et al. 2000; Best et al. 2002; Kadoya et al. 2002), whereas SUSP1 and SMT3IP2/Axam2 are mainly cytoplasmic (Kim et al. 2000; Nishida et al. 2001). Therefore, each SUMO-specific protease is thought to target a different protein. The SUMO-specific protease Axam (Axin associating molecule) has been reported to interact with Axin, which is a scaffold protein for Wnt signalling (Ikeda et al. 1998; Kishida et al. 1998; Kikuchi 1999; Kadoya et al. 2000). In mammalian cells, over-expression of Axam inhibits Wnt signalling by promoting ß-catenin degradation and this inhibition requires both the Axin-binding domain and the SUMO-specific protease catalytic domain (Kadoya et al. 2000, 2002). Together, these results suggested a relationship between Wnt signalling and sumoylation.
The Wnt signalling pathway is conserved from worms to humans and is essential for normal cellular function. Wnt signalling is involved in many developmental processes, including axis identity, tissue morphogenesis, determination of cell fate, patterning of the central nervous system (CNS) and convergent extension movement (Cadigan & Nusse 1997; Patapoutian & Reichardt 2000; Tada et al. 2002). Some Wnt signalling components are known as oncogenes or tumour suppressor genes and defects in the Wnt signalling pathway result in oncogenesis (Polakis 2000). In the canonical Wnt pathway (Wnt/ß-catenin pathway), secreted Wnt ligands form complexes with receptors of the Frizzled family (Bhanot et al. 1996; Yang-Snyder et al. 1996). In the cytoplasm, the signal is transduced through Dvl, which in turn prevents phosphorylation of ß-catenin by GSK3ß in a multiprotein complex containing Axin, GSK3ß and adenomatous polyposis coli (APC) protein (Noordermeer et al. 1994; Itoh et al. 1998; Kishida et al. 1998, 1999). Unphosphorylated ß-catenin escapes recognition by ß-TrCP, a component of an E3 ubiquitin ligase, and therefore can translocate to the nucleus, where it associates with transcription factors such as TCF and LEF to enhance their transcriptional activity (Molenaar et al. 1996; Hart et al. 1999).
Recently, some Wnt signalling molecules have been shown to be sumoylated. One of them is Axin, which acts as a scaffold protein in the canonical Wnt signalling. Mutants of Axin that cannot be sumoylated fail to activate JNK, while effectively down-regulating ß-catenin (Rui et al. 2002). LEF1 and Tcf4 are transcription factors activated by Wnt signalling, and are also sumoylated. Interestingly, sumoylation of LEF1 inhibits its transcription activity, while sumoylation of Tcf4 promotes it (Sachdev et al. 2001; Yamamoto et al. 2003). These results suggest that sumoylation directly regulates Wnt signal transduction.
In this study, we identified a novel SUMO-specific protease in Xenopus laevis, which we named XSENP1. XSENP1 displays amino acid conservation with other SUMO-specific protease family members, and demonstrates SUMO-specific protease activity in vitro. In Xenopus embryos, dorsal expression of XSENP1 causes head defects and inhibits expression of Xnr3, a Wnt signalling target gene. Furthermore, coexpression with other Wnt signalling components suggested that XSENP1 inhibits the Wnt signalling pathway downstream of ß-catenin and upstream of siamois.
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Results |
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To investigate the roles of sumoylation in Xenopus laevis, we aimed to isolate a Xenopus SUMO-specific protease. A search of the DNA Data Bank of Japan (DDBJ) revealed a Xenopus EST sequence that was highly homologous to the C-terminal region of mammalian SUMO-specific proteases. The 200 C-terminal amino acids of SUMO-specific proteases contain the catalytic domains, which are highly conserved. We denoted this region the SPH (SUMO-specific protease homology) domain in this paper. We designed primers for the Xenopus EST clone, and amplified the EST fragment using PCR. Using this fragment as a probe, we screened a stage-28 Xenopus embryo cDNA library by plaque hybridization. Positive clones were isolated and sequenced. We identified two cDNA clones encoding predicted proteins of 618 and 616 amino acids, respectively. Of the known SUMO-specific proteases, the identified clones were most homologous to hSENP1 (Fig. 1B), therefore we named them XSENP1a and XSENP1b (XSENP1s). XSENP1a was 89% identical to XSENP1b and the SPH domains of XSENP1a and XSENP1b were 83% and 85% identical, respectively, to the domain of hSENP1. Regions outside the SPH domain did not show high sequence homology with hSENP1. Full-length XSENP1a and XSENP1b had 52% and 53% identity, respectively, with hSENP1 (Fig. 1C).
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To investigate the temporal pattern of XSENP1s expression, we carried out RT-PCR with XSENP1a- or XSENP1b-specific primers. The mRNAs of XSENP1a and XSENP1b existed maternally, their expression decreased at the gastrula stage, and then increased again after the neural stage. The temporal expression patterns of XSENP1a and XSENP1b were very similar (Fig. 2A). To investigate the spatial expression pattern of XSENP1s, we performed whole-mount in situ hybridization with a DIG-labelled RNA probe for XSENP1s. At the gastrula stage, XSENP1s mRNA expression was not detected (Fig. 2Ba,b). During the neurula stage, XSENP1s transcripts were detected weakly in neural tissues including the anterior neural plate region (Fig. 2Bc–f). In 2-day-old tadpoles (Stage 31), XSENP1s transcripts were restricted to the anterior neural structures such as the eye vesicle (Fig. 2Bg,h), and levels of expression were increased in 3-day-old tadpoles (Stage 41). Signal was detected in most of the head region and weak signal was observed in the presumptive spinal cord region (Fig. 2Bi,j).
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Previous reports have shown that SUMO-specific proteases such as Ulp1, hSUSP1, and Axam possess two enzymatic activities. One is an isopeptidase activity that deconjugates SUMO from the lysine -amino group of the target protein and the other is a hydrolase activity that cleaves the C-terminal Gly-Gly residues from immature SUMO-1 to produce the mature form (Li & Hochstrasser 1999; Kim et al. 2000; Kadoya et al. 2002). Because XSENP1s contain predicted SPH domains, they were expected to possess the same two enzymatic activities as the known SUMO-specific proteases. We therefore measured the desumoylation and hydrolase activity of full-length XSENP1s and three different mutants of XSENP1a: (i) C-XSENP1a: SPH-domain of XSENP1a; (ii) N-XSENP1a: N-terminal and central region of XSENP1a; and (iii) XSENP1aC602S a cysteine to serine substitution at residue 602 (Fig. 3A). This residue is one of the three conserved amino acids (His, Asp, and Cys) that are required for the protein's catalytic activity.
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Dorsal expression of XSENP1s or C-XSENP1a mRNA in Xenopus embryos causes head defects and suppresses expression of Xnr3 without degradation of ß-catenin
To analyse the role of XSENP1s in vivo, we injected full-length and mutant XSENP1s mRNA into Xenopus embryos. Embryos injected with XSENP1s or C-XSENP1a dorsally showed head defects, while embryos injected with N-XSENP1a or XSENP1aC602S showed insignificant effect (Fig. 4A). As above, we showed that XSENP1s possess activities similar to that shown by the known SUMO-specific proteases and that the C-terminal region of XSENP1s is required for these activities (Fig. 3). These two results suggested that dorsally expressed XSENP1s cause head defects and that SUMO-specific protease activity is required for this effect. In contrast, ventral expression of XSENP1s and mutants did not cause head defects (data not shown). It is therefore likely that the head defect induced by expression of the XSENP1s was due to the inhibition of a dorsal event in Xenopus embryos. In Xenopus laevis, ectopic expression of negative regulators of the canonical Wnt signalling pathway in the dorsal marginal zone inhibits axis formation and causes a ventralization phenotype. The embryo phenotypes seen after dorsal injection of the XSENP1s or C-XSENP1a mRNAs were very similar to those caused by dorsal expression of negative regulators such as GSK3ß, Axin, Duplin, IDAX and Axam (He et al. 1995; Zeng et al. 1997; Kadoya et al. 2000; Sakamoto et al. 2000; Hino et al. 2001). XSENP1 and Axam possess the activities common to SUMO-specific proteases, so XSENP1 might inhibit the Wnt signalling pathway in the same way as Axam. To confirm whether dorsal expression of XSENP1s led to the ventralization phenotype via negative regulation of Wnt signalling, we performed RT-PCR to determine the expression levels of Xnr3, a direct target gene in the pathway (McKendry et al. 1997). We found that Xnr3 was indeed down-regulated, suggesting that dorsal expression of XSENP1s mRNA caused ventralization by suppression of the canonical Wnt signalling pathway (Fig. 4B). Destabilization of ß-catenin by Axam requires both binding to Axin and SUMO-specific protease activity (Kadoya et al. 2000, 2002). The N-terminal regions of the XSENP1s show low amino acid sequence homology to Axam. Thus, it was predicted that the XSENP1s would not be able to degrade ß-catenin. To examine the effect of XSENP1s on the stabilization of ß-catenin, XSENP1s were co-injected with myc-ß-catenin and Western blotting was performed. When GSK-3ß was co-injected into the dorsal marginal zone of 4-cell stage embryos, exogenous ß-catenin was destabilized (Fig. 4C, lanes 2 and 5). In contrast, co-injection with XSENP1a or XSENP1b caused almost no degradation of ß-catenin (Fig. 4C, lanes 2–4). This result demonstrates that XSENP1s cannot degrade ß-catenin, suggesting that XSENP1s have a different function to Axam.
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To examine how XSENP1s suppress Wnt signalling, we co-injected XSENP1a mRNA ventrally with positive regulators such as XDvl, Xß-catenin, Xß-cateninSA (constitutively active form of ß-catenin), and with siamois, a target gene of Wnt signalling (DeMarais & Moon 1992; Sokol et al. 1995; Carnac et al. 1996; Yost et al. 1996). In Xenopus, it is known that ventral expression of positive regulators of the Wnt pathway induces secondary axis formation (Moon & Kimelman 1998). If ectopic axis induction by positive regulators of Wnt signalling can be inhibited by co-injection with XSENP1, the positive regulators would be assumed to function upstream of XSENP1 in the pathway. Consistent with the result shown in Fig. 4C, when Dvl, ß-catenin, and ß-cateninSA were co-injected ventrally with XSENP1a mRNA, secondary axis formation was suppressed (Fig. 5Ac–h). Therefore, XSENP1a is thought to function downstream of Dvl and ß-catenin. Furthermore, XSENP1a did not inhibit axis induction by ectopic siamois expression, and siamois was able to prevent the defects in head formation induced by the dorsal expression of XSENP1a (Fig. 5Ai,j and Fig. 5B). Taken together, our results suggest that XSENP1 functions downstream of ß-catenin and upstream of siamois in the canonical Wnt signalling pathway.
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Discussion |
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We report here that XSENP1, a novel member of the SUMO-specific protease family, can induce defects in head formation in Xenopus laevis and that this effect could result from inhibition of Wnt signal transduction. XSENP1 possesses all amino acids that are conserved among other SUMO-specific proteases, and shows the highest homology with hSENP1. The SPH (SUMO-specific protease family homology) domains of XSENP1 and hSENP1 are highly identical, although the N-terminal and central regions show little similarity. The SPH domains of SUMO-specific proteases are highly conserved among family members and function as catalytic domains, while the N-terminal regions are thought to regulate cellular localization and confer substrate specificity (Yeh et al. 2000). Therefore, although XSENP1 shows the greatest homology with hSENP1, the two proteins may display quite different substrate specificities. The differences in amino acid sequences are most likely due to the divergence between species, although the possibility that other orthologous genes of hSENP1 exist in Xenopus laevis cannot be excluded. However, there is some partial amino acid sequence homology in the central regions of XSENP1 and hSENP1. If XSENP1 and hSENP1 do target the same substrate, these amino acid sequences may be important for determination of target specificity. The coding sequence of XSENP1a has 91% similarity to XSENP1b, and the 3'-UTR of XSENP1a is approximately 70% similar to that of XSENP1b. Although these two proteins show very similar patterns of expression and functions, we do not believe that XSENP1a and XSENP1b are allelic variants.
Protease activities of XSENP1 and inhibition of head organization
The C-terminal region of SUMO-specific proteases (approximately 200 amino acids, termed the SPH domain in this paper) functions as the catalytic domain and is highly conserved among SUMO-specific protease family members. Consistent with this, the desumoylation activity of XSENP1 requires the SPH domain. Furthermore, dorsal expression of XSENP1s causes head defects and this effect also requires the SPH domain. Axam functions as a SUMO-specific protease and can inhibit Wnt signalling via the activity of its C-terminal region (Kadoya et al. 2002). We therefore predicted that the head defect induced by XSENP1 expression was a result of Wnt signalling inhibition. In fact, our results showed that XSENP1 inhibits the expression of Xnr3, a direct target gene of Wnt signalling. In the case of Axam, the central region of the protein binds to Axin and this binding is required for down-regulation of ß-catenin (Kadoya et al. 2000, 2002). The SPH domain of XSENP1 shows high homology with the corresponding region of Axam, while the central regions show little homology. Therefore, XSENP1 probably does not bind to Axin and may inhibit the canonical Wnt signalling pathway by a different mechanism from Axam. Western blot analysis to investigate the effect of XSENP1 expression on the stabilization of ß-catenin showed that XSENP1s did not promote ß-catenin degradation, which supports our proposal that XSENP1 plays a different role from Axam in the canonical Wnt pathway. Despite screening Xenopus cDNA and genomic libraries, we were unable to identify a Xenopus orthologue of Axam/SENP2. Axam/SENP2 may not be expressed in Xenopus, or may be present only at very low levels.
The expression pattern and predicated roles of XSENP1s
RT-PCR analysis indicated that XSENP1s mRNA exists maternally, so we predicted that XSENP1 plays a role prior to the midblastula transition (MBT). Dorsal injection with XSENP1 caused head defects and inhibited the canonical Wnt signalling pathway, thus we hypothesized that XSENP1 might play a role in the determination of dorso-ventral axis formation. However, loss-of-function analysis of XSENP1 by injection with morpholino anti-sense oligos directed against XSENP1a and XSENP1b showed little effect, contrary to our expectation (data not shown). This result suggests that XSENP1 is not involved in dorso-ventral axis formation. However, the lack of an effect following depletion of XSENP1, even though it is clear that XSENP1 functions in early development, may be due to one or more of the following reasons. First, maternal XSENP1 protein may exist in eggs, and if these levels are sufficient to function, the morpholino effect may be obscured. As mentioned, both XSENP1a and XSENP1b transcripts are maternally expressed. Second, the loss-of-function effect might not be observed when only XSENP1 is depleted. Perhaps another factor(s) is required to see a clear effect of the depletion of XSENP1. Finally, other SUMO-specific proteases may be involved in dorso-ventral axis formation instead of XSENP1. In fact, several mammalian SUMO-specific proteases have been isolated and some Xenopus EST sequences highly homologous to SUMO-specific proteases have been registered in DDBJ. For these reasons, we therefore cannot exclude the possibility that XSENP1 regulates dorso-ventral axis formation in early development via down-regulation of canonical Wnt signalling. A recent report suggested that sumoylation of Tcf4 promotes transcription activity in cultured cells (Yamamoto et al. 2003). It is therefore possible that sumoylation and desumoylation of Xenopus Tcf3 may be involved in the regulation of canonical Wnt signalling in early development.
Whole-mount in situ hybridization and RT-PCR showed that XSENP1s mRNA is localized to the central nervous system (CNS) region, including the head region after the tail-bud stage, and, at very low levels, at the gastrula and neural stage. Wnt signalling is crucial to CNS development and antero-posterior axis patterning after MBT (Pierce & Kimelman 1996; Itoh & Sokol 1997; Niehrs 1999; Heasman et al. 2000), thus XSENP1 may play a role in CNS development and/or maintenance by inhibition of the canonical Wnt signalling pathway. However, XSENP1s-pCS2 injection into the animal pole of 2-cell stage embryos caused no remarkable effects, which argues against a role for XSENP1s in CNS development and antero-posterior axis patterning.
The mechanism of inhibition of Wnt signalling by XSENP1
XSENP1 mRNA inhibits ectopic axis formation that is induced by Dvl, ß-catenin, and a constitutively active form of ß-catenin (ß-cateninSA) but not by siamois. Furthermore, XSENP1 does not promote ß-catenin degradation. These results suggested to us that XSENP1 inhibits the Wnt signalling pathway downstream of ß-catenin and upstream of siamois, for instance, by inhibiting transcription factors such as Tcf3/4 or LEF1. Consistent with this idea, a recent report shows that sumoylation activates the transcription activity of Tcf4 (Yamamoto et al. 2003). Tcf4 that has been sumoylated by PIASy (one of the SUMO-ligases) is activated and this promotes ß-catenin-dependent transcription. Axam can remove the SUMO molecule from Tcf4 and reduction of Tcf4 protein levels by RNA interference leads to an increase in sumoylated Tcf4 and subsequent activation of Tcf4. So, XSENP1 may suppress the Wnt signalling pathway by removing SUMO from sumoylated Tcf3/4 during Xenopus development. However, another study showed that sumoylation of LEF1 by PIASy repressed the transcriptional activity of LEF1 (Sachdev et al. 2001). This contradiction may be due to the different experimental systems used in each study and/or the differing functions of Tcf4 and LEF1. Complex regulation systems may mediate the sumoylation of Tcf3/4 and LEF1.
An important key to understanding the function of XSENP1 will be to identify its actual target molecule(s). Many substrates for sumoylation have been identified and the consensus target amino acid sequence for sumoylation is ‘
KxE/D’, where is a hydrophobic residue and x is any amino acid (Muller et al. 2001). This consensus sequence is found in several Wnt signalling players including XLEF1, XTcf3/4, XAxin, XAxil, Xß-catenin, XAPC, XNLK, and XTAB1, although there is no evidence that these proteins are actually sumoylated. As described above, one candidate substrate for XSENP1 is XTcf3/4. XSENP1s can remove SUMO-1 from sumoylated Tcf4 in vitro, and Tcf4 functions downstream of ß-catenin and upstream of siamois in the canonical Wnt signalling pathway. However, it may difficult to determine the specific target of XSENP1 in vivo, because sumoylation probably occurs in multiple processes and sites, and there are already a multitude of reported proteins that can be sumoylated or desumoylated.
In this study, we demonstrated that XSENP1, a member of the SUMO-specific protease family, can inhibit normal head organization when it is expressed dorsally, and that this inhibition is caused by negative regulation of the canonical Wnt signalling pathway. Previous studies have shown the importance of sumoylation in the regulation of essential cellular events such as cell-cycling, development and oncogenesis. Understanding the mysterious post-transcriptional modification system underlying sumoylation and desumoylation may thereby help in the understanding of many other complex biological mechanisms.
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Experimental procedures |
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Eggs of Xenopus laevis were obtained following injection of 300 IU of human chorionic gonadotropin (Gestron, Denka Seiyaku) into Xenopus laevis. Fertilized embryos were cultured in 10% Steinberg's solution and staged according to Nieuwkoop & Faber (1956).
Identification of XSENP1a and XSENP1b cDNA clones
The EST sequence (BI350328 [GenBank] ) that was highly homologous to the C-terminus of rAxam and hSENP1 was identified from the Xenopus oocyte cDNA library and was amplified using the PCR primers, 5'-GGACCCTAACCTGCAAGACGAG-3' and 5'-GATGAAGGATCTCCCAAACCATTCTC-3'. Using this clone as a probe, a stage-28 Xenopus cDNA library was screened by the plaque hybridization method and positive clones were isolated and sequenced.
Estimation of evolutionary relationship
A sequence of about 200 amino acids from the C-terminal region of XSENP1a and XSENP1b was aligned with SUMO-specific protease family members using the GeneWorks evolutionary relationship algorithm.
RT-PCR assays
Total RNA was extracted from Xenopus embryos using ISOGEN (Nippon Gene). First-strand cDNAs were synthesized from 1 µg of the total RNA with oligo(dT) primers, using the reverse transcription enzyme, superscript‘ II (Invitrogen). PCR was performed with the following primers: XSENP1a, 5'-GATTTCATAGTTTCTCTAGTCGTATTT-3' and 5'-CAGGAAACTGAAAGTTGAAGGC-3'; XSENP1b, 5'-AACAAACAACACAGGCAATTTCGG-3' and 5'-GTAGTCGGCTTGCTGGAAACTG-3'; XODC, 5'-GTCAATGATGGAGTGTATGGATC-3' and 5'-TCCATTCCGCTCTCCTGAGCAC-3'; Xnr3, 5'-CTTCTGCACTAGATTCTG-3' and 5'-CAGCTTCTGGCCAAGACT -3'
Whole-mount in situ hybridization
Whole-mount in situ hybridization was performed as previously described (Harland 1991). Briefly, digoxigenin (DIG)-labelled anti-sense mRNA probes were transcribed from XSENP1a- and XSENP1b-pBluescript II SK (XSENP1a and XSENP1b inserted in pBluescript II SK) using T7 RNA polymerase, following linearization with PstI (XSENP1a) or SpeI (XSENP1b). Albino embryos were fixed with MEMFA. The embryos were hybridized with the DIG-labelled probes, followed by anti-DIG antibody labelling. Embryos were stained using BM purple (Roche).
Assessment of the hydrolase activity of XSENP1s and XSENP1a mutants
A total of 1.5 µg of GST-SUMO-1-Myc was incubated with 0.5 µg of GST-XSENP1a, GST-XSENP1b, GST-N-XSENP1a, GST-C-XSENP1a or GST-XSENP1aC602S in 50 µL of reaction mixture (100 mM Tris/HCl [pH 8.0], 2 mM DTT, 1 mM EDTA, and 5% glycerol) at 30 °C for 3 h. After incubation, the mixtures were resolved by SDS-polyacrylamide gel electrophoresis (PAGE) and probed with the anti-SUMO-1 (GMP1) antibody (Zymed Laboratories Inc.).
Analyses of the desumoylation activity of XSENP1s and XSENP1a mutants
HA-SUMO-1 was expressed with Flag-PIASy and Tcf4 in 293 cells (60-mm-diameter dish). Cells were then lysed in 200 µL of RIPA buffer (10 mM Na-phosphate buffer pH 7.2, 150 mM NaCl, 1% Na-deoxycholate, 1% Triton X-100, and 0.1% SDS) containing 1 µg/mL aprotinin, 1 µg/mL leupeptin, 10 mM phenylmethylsulphonyl fluoride, 1 mM NaF, 0.4 mM Na-orthovanadate, and 10 mM N-ethylmaleimide. The lysates were immunoprecipitated with the anti-Tcf4 antibody (Upstate Biotechnology Inc.), and then the immunoprecipitates were incubated further with 0.5 µg of GST-XSENP1a, GST-XSENP1b, GST-N-XSENP1a, GST-C-XSENP1a, or GST-XSENP1aC602S in 30 µL of reaction mixture (50 mM Tris/HCl [pH 7.5], 1 mM DTT, and 150 mM NaCl) for 3 h at 30 °C. After incubation, the mixtures were subjected to SDS-PAGE and probed with the anti-HA (16B12) antibody (Covance Inc.). LipofectAMINE 2000 (Life Technologies Inc.) was used to transfect the 293 cells.
RNA injection
XSENP1s cDNAs were cloned into pCS2+. Constructs linearized by the appropriate restriction enzymes were used as templates. Capped mRNAs were produced using an mMessage mMachine kit (Ambion). mRNA of other Wnt components used in this paper was synthesized in the same way. Fertilized Xenopus eggs were dejellied with 100% Steinberg's solution containing 4.5%L-cysteine hydrochloride monohydrate and cultured in 100% Steinberg's solution. Four- to 8-cell stage embryos were injected with the capped mRNAs into the dorsal or ventral marginal zone in 4% Ficoll. After injection, embryos were cultured for 3 days in 10% Steinberg's solution and observed or cultured for about 8 h (until stage 10) in 10% Steinberg's solution and dissolved in ISOGEN (Nippon Gene) for RNA extraction.
Analyses of destabilization of ß-catenin
Five Xenopus embryos injected with mRNA into the dorsal marginal zone were lysed in 200 µL of SDS page buffer (124 mM Tris/HCl [pH 6.8], 20% Glycerol, 4% SDS, 2% 2-mercaptoethanol, 0.001% Bromophenol Blue, 0.5% NONIDET P-40) at Stage 9. The mixtures were subjected to SDS-PAGE and probed with the anti-myc antibody (CALBIOCHEM) or anti--tubulin antibody (SIGMA).
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Acknowledgements |
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Footnotes |
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* Correspondence: E-mail: asashi{at}bio.c.u-tokyo.ac.jp
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References |
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Received: 14 February 2004
Accepted: 12 May 2003
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