cDNA cloning and its pronephros-specific expression of the Wilms' tumor suppressor gene, WT1, from Xenopus laevis

Semba, Kentaro; Saito-Ueno, Rika; Takayama, Gensuke; Kondo, Mariko

doi:10.1016/0378-1119(96)00143-6

Cited by 33 publications

(24 citation statements)

References 22 publications

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“…A variety of hormonal signals can activate PKA in vivo in principle leading to phosphorylation of WT1, but without knowing more about which signals regulate WT1 and the nature of its target genes it is hard to determine the exact eect of the Phe-365 mutation on WT1 function. WT1 cDNAs have been cloned from various vertebrates including human, mouse, rat, chicken, alligator, Xenopus laevis and zebra®sh (Call et al, 1990;Gessler et al, 1990;Kent et al, 1995;Semba et al, 1996;Sharma et al, 1992). All these vertebrate WT1 proteins have Ser residues corresponding to both Ser-365 and Ser-393.…”

Section: Discussionmentioning

confidence: 99%

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Inhibition of the DNA-binding and transcriptional repression activity of the Wilms' tumor gene product, WT1, by cAMP-dependent protein kinase-mediated phosphorylation of Ser-365 and Ser-393 in the zinc finger domain

et al. 1997

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The Wilms' tumor suppressor gene, WT1, encodes a transcription factor in the zinc ®nger family, which binds to GC-rich sequences and functions as a transcriptional activator or repressor. The WT1 protein plays a crucial role in urogenital development in mammals and its function is thought to be conserved during vertebrate evolution. Although accumulating evidence suggests that WT1 regulates a subset of genes including growth factor and growth factor receptor genes, little is known about regulators or signal cascades that could modulate the function of WT1. In this study, we show that the WT1 protein expressed exogenously in ®broblasts was phosphorylated in vivo, and that treatment with forskolin, which activates the cAMP-dependent protein kinase (PKA) in vivo, induced phosphorylation of additional sites in WT1. We identi®ed the forskolin-induced phosphorylation sites as Ser-365 and Ser-393, which lie in the zinc ®nger domain in zinc ®ngers 2 and 3, respectively. PKA phosphorylated WT1 at Ser-365 and Ser-393 in vitro, as well as at additional sites, and this phosphorylation abolished the DNA-binding activity of WT1 in vitro. Using WT1 mutants in which Ser-365 and Ser-393 were mutated to Ala individually and in combination, we showed that phosphorylation of these sites was critical for inhibition of DNA binding in vivo. Thus, coexpression of the PKA catalytic subunit with wild type WT1 reduced the level of WT1 DNA-binding activity detected in nuclear extracts, and decreased transcriptional repression activity in vivo. In contrast to wild type WT1, all of the phosphorylation site mutants retained signi®cant DNAbinding activity and repression activity in the presence of PKA. Analysis of the mutants showed that phosphorylation of Ser-365 and Ser-395 had additive inhibitory eects on WT1 DNA-binding in vivo and that phosphorylation at both sites was required for neutralization of repression activity. Therefore, we conclude that PKA modulates the activity of WT1 in vivo through phosphorylation of Ser-365 and Ser-393, which inhibits DNA binding. This in turn results in a decrease in WT1 transcriptional repression. Our ®ndings provide the ®rst evidence that the function of WT1 can be modulated by its phosphorylation in vivo.

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Section: Discussionmentioning

confidence: 99%

“…WT1 knockout mice exhibit a failure of kidney and gonad development (Kreidberg et al, 1993) con®rming that WT1 is required for urogenital development in mammals. Recent observations suggest that the function of WT1 in urogenital development has been conserved during vertebrate evolution (Kent et al, 1995;Semba et al, 1996).…”

Section: Introductionmentioning

confidence: 99%

Inhibition of the DNA-binding and transcriptional repression activity of the Wilms' tumor gene product, WT1, by cAMP-dependent protein kinase-mediated phosphorylation of Ser-365 and Ser-393 in the zinc finger domain

et al. 1997

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“…Of particular interest is alternative splicing of exon 9 that generates a three amino-acid insertion (lysine-threonine-serine, KTS) between the third and fourth zinc fingers. This alternative splicing is conserved during evolution (Kent et al, 1995;Semba et al, 1996), and WT1 lacking KTS (WT1ÀKTS) and WT1 containing KTS (WT1 þ KTS) have distinct biological functions during sex determination and nephron formation (Hammes et al, 2001). WT1 binds directly to 9-12 base GC-rich consensus sequences and regulates transcription (Rauscher et al, 1990;Drummond et al, 1994;Nakagama et al, 1995).…”

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confidence: 99%

Transcriptional activity of testis-determining factor SRY is modulated by the Wilms’ tumor 1 gene product, WT1

2003

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The Wilms' tumor 1 (WT1) and sex-determining region of the Y chromosome (SRY) genes are essential for development of the mammalian gonads and mutations in these genes are associated with gonadal dysgenesis in humans. The SRY gene encodes a transcription factor with one high-mobility group (HMG) box as a DNAbinding domain. WT1 encodes a transcription factor that contains four contiguous C 2 H 2 -type zinc-finger motifs as a DNA/RNA binding or protein-protein interaction domain. Here we report that WT1 binds to and acts synergistically with SRY to activate transcription from a promoter containing SRY-binding sites. This interaction is mediated by the WT1 zinc-finger domain and the SRY HMG box. WT1 mutants associated with Denys-Drash syndrome (DDS), which is characterized by Wilms' tumor, pseudohermaphroditism, and nephropathy, fail to interact with SRY. Wildtype WT1 is recruited to SRYbinding sites in an SRY-dependent manner, whereas DDS mutants are not recruited as efficiently. These results suggest that WT1 forms a complex with SRY to regulate transcription and that this WT1-SRY interaction is important in testis development.

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“…After sex determination, WT1 becomes specific to Sertoli cells in the testis, follicle cells in the ovary, and cells in the surface epithelium of both sexes (Pelletier et al, 1991;Mundlos et al, 1993). Using RNA expression studies, WT1 has been observed in the gonads of nonmammalian species, such as chickens (Kent et al, 1995), Xenopus (Semba et al, 1996), and alligators (Western et al, 2000). In T. Scripta, Northern blots of the urogenital ridge suggested that WT1 may have a role in gonad development in turtles.…”

Section: Discussion Wt1 Expression Pattern Suggests a Conserved Role mentioning

confidence: 99%

Colocalization of WT1 and cell proliferation reveals conserved mechanisms in temperature‐dependent sex determination

Schmahl

Yao

Pierucci-Alves

et al. 2003

Genesis

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SummaryDuring vertebrate development the gonad has two possible fates, the testis or the ovary. The choice between these fates is made by a variety of sex-determining mechanisms, from the sexdetermining gene on the Y chromosome (Sry) in mammals, to nongenetic temperature-dependent systems in many reptiles. Despite the differences in the mechanisms at the top of the sexdetermining cascade, the resulting morphology and many genes involved in early testis and ovarian development are common to most vertebrates, leading to the hypothesis that the underlying processes of sex determination are conserved. In this study, we examined the early steps of gonad development in the red-eared slider turtle (Trachemys scripta), a species that uses the temperature of egg incubation to determine sex. A dramatic increase in cell proliferation was observed in the male gonad during the earliest stages of sex determination. Using the localization of Wilms' Tumor suppressor 1 (WT1), we determined that this proliferation increase occurred in a population that contained pre-Sertoli cells. The proliferation of pre-Sertoli cells has been documented during sex determination in both mice and alligators, suggesting that proliferation of this cell type has an important role in vertebrate testis organogenesis and the determination of male fate.

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cDNA cloning and its pronephros-specific expression of the Wilms' tumor suppressor gene, WT1, from Xenopus laevis

Cited by 33 publications

References 22 publications

Inhibition of the DNA-binding and transcriptional repression activity of the Wilms' tumor gene product, WT1, by cAMP-dependent protein kinase-mediated phosphorylation of Ser-365 and Ser-393 in the zinc finger domain

Inhibition of the DNA-binding and transcriptional repression activity of the Wilms' tumor gene product, WT1, by cAMP-dependent protein kinase-mediated phosphorylation of Ser-365 and Ser-393 in the zinc finger domain

Transcriptional activity of testis-determining factor SRY is modulated by the Wilms’ tumor 1 gene product, WT1

Colocalization of WT1 and cell proliferation reveals conserved mechanisms in temperature‐dependent sex determination

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