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

Sakamoto, Yoshimasa; Yoshida, Mitsuaki; Semba, Kentaro; Hunter, Tony

doi:10.1038/sj.onc.1201391

Cited by 52 publications

(35 citation statements)

References 35 publications

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“…Recently, PKA has been shown to activate MMP-9 in human ovarian epithelium (41) and we have confirmed this findings in human LEC. WT1 is regulated by PKA through phosphorylation of its serine residues (26,34,42) in agreement with our findings.…”

Section: Discussionsupporting

confidence: 82%

The Transcription Factor Wilms Tumor 1 Regulates Matrix Metalloproteinase-9 through a Nitric Oxide-Mediated Pathway

Marcet‐Palacios

Ulanova

Duta

et al. 2007

The Journal of Immunology

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Matrix metalloproteinase-9 (MMP-9) is released by human lung epithelial cells (LEC) in conditions such as asthma and chronic obstructive pulmonary disease and expression of MMP-9 correlates with the severity of these disorders. MMP-9 production has been reported to be regulated by a NO/soluble guanylate cyclase-dependent pathway. Transcriptional regulation of this enzyme, however, is poorly understood. Using phylogenetic analysis, we observed a highly conserved sequence in the 5′ flanking region of the MMP-9 gene containing binding sites for the transcription factor Wilms tumor 1 (WT1). We confirmed the presence of WT1 in human LEC and that treatment with TNF or a mixture containing LPS, PMA, and IFN-γ resulted in translocation of WT1 from the nucleus to the cytosol. This translocation coincided with increased expression of MMP-9 and could be blocked by inhibitors of the NO/soluble guanylate cyclase pathway. WT1 knockdown using small-interfering RNA up-regulated MMP-9 expression in the presence of the NO synthase inhibitor 1400W. Using either WT1 pulldown with probes for the conserved region of the MMP-9 promoter or chromatin immunoprecipitation, we confirmed WT1 binding to the MMP-9 promoter. These findings indicate WT1 is a repressor of MMP-9, regulated by a NO-mediated pathway in human LEC. To our knowledge, this is the first report of WT1 regulating MMP-9 expression. Further study is needed to determine whether clinical conditions exhibiting tissue remodeling, such as asthma and/or chronic obstructive pulmonary disease, demonstrate reduced levels of WT1 or its repressor activity.

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Section: Discussionsupporting

confidence: 82%

The Transcription Factor Wilms Tumor 1 Regulates Matrix Metalloproteinase-9 through a Nitric Oxide-Mediated Pathway

Marcet‐Palacios

Ulanova

Duta

et al. 2007

The Journal of Immunology

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“…An example of negative regulation of DNA binding by distal site phosphorylation has been described for the transcription factor c-myb, in which phosphorylation at sites ϳ50 residues from the DNA binding domain inhibit transcription factor docking (35). The DNA binding activity of the transcriptional repressor, Wilms' tumor gene product WT1, is similarly inhibited by phosphorylation within a distal zinc-finger region (34,36). Sometimes phosphorylation of transcription factors regulates transactivation function via post-binding mechanisms.…”

Section: Discussionmentioning

confidence: 99%

“…Thus, casein kinase 2 activity may provide an important regulatory signal for Tax-mediated activation of the NF-B pathway. This phosphorylation site lies within the zinc-finger domain of Tax and may represent a negative regulatory mechanism similar to that described for WT1 (36). Future studies will be aimed at uncovering the kinetic relationship of Tax post-translational modifications and Tax function.…”

Section: Discussionmentioning

confidence: 99%

Site-specific Phosphorylation Differentiates Active from Inactive Forms of the Human T-cell Leukemia Virus Type 1 Tax Oncoprotein

et al. 2006

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The human T-cell leukemia virus type 1 oncoprotein Tax is a phosphoprotein with a predominately nuclear subcellular localization that accomplishes multiple functions via protein-protein interactions. It has been proposed that regulation of this protein's pleiotropic functions may be accomplished through phosphorylation of specific amino acid residues. We have conducted a phosphoryl mapping of mammalian-expressed Tax protein using a combination of affinity purification, liquid chromatography tandem mass spectrometry, and site-directed substitution mutational analysis. We achieved physical coverage of 77% of the Tax sequence and identified four novel sites of phosphorylation at Thr-48, Thr-184, Thr-215, and Ser-336. Previously identified potential serine phosphorylation sites at Ser-10, Ser-77, and Ser-274 could not be confirmed by mass spectrometry. The functional significance of these novel phosphorylation events was evaluated by mutational analysis and subsequent evaluation for activity via both CREB and NF-B-responsive promoters. Our results demonstrate that phosphorylation at Thr-215 is associated with loss of both Tax functions, phosphorylation at Thr-48 was specifically deficient for activation via NF-B, and phosphorylation at Thr-184 and Ser-336 had no effect on these Tax functions. Semiquantitation of phosphopeptides revealed that the majority of Tax was phosphorylated at Thr-48, Thr-184, Thr-215, and Ser-336, whereas only a minor population of Tax was phosphorylated at either Ser-300 or Ser-301. These results suggest that both positive and negative phosphorylation signals result in the maintenance of a subfraction of Tax as "active" protein. Human T-cell leukemia virus type 1 (HTLV-1)2 is a human transforming retrovirus. Infection with HTLV-1 can give rise to adult T-cell leukemia and HTLV-1-associated myelopathy/ tropical spastic paraparesis as well as other subneoplastic conditions (1-5). Although cellular transformation can be achieved by expression of a single viral transactivating protein, Tax, the exact mechanism of transformation is not known (6). Tax is thought to induce genomic instability and, thus, to contribute to cellular transformation through interaction with cellular proteins involved in cell cycle control and the DNA damage repair response (7-11). In addition, Tax can activate or repress a variety of cellular genes predominately through the CREB (cAMP-response element-binding protein) and NF-B pathways (6, 12). Thus, uncovering the regulatory mechanism for controlling the various Tax activities is critical to understanding HTLV-1-mediated cellular transformation.There have been several structure-function studies of the Tax protein predominately utilizing molecular biology techniques. Specifically, with respect to the regulation of multiple Tax activities, it has been noted that mutation or deletion of individual domains results in a selective loss of function (13). Important domains for Tax function include a nuclear localization signal (13, 14), nuclear export signal (15, 16), activatio...

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“…Serine phosphorylation within the zinc finger domain of WT1 has been shown to interfere with nuclear localization and DNA binding (5,6). WT1 physically associates with other cellular proteins, including SF1 (7), p53 (8), and PAR4 (9).…”

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confidence: 99%

Identification of Connective Tissue Growth Factor as a Target of WT1 Transcriptional Regulation

Stanhope-Baker¹,

Williams

2000

Journal of Biological Chemistry

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The Wilms tumor suppressor WT1 has transcriptionactivating and -suppressing capabilities. WT1-responsive promoters have been described; however, in large part, it remains unclear which potential downstream genes are physiologically relevant and mediate the function of WT1 in tumorigenesis and development. To identify genes regulated by WT1 in vivo, we used a dominantnegative version of WT1 to modulate WT1 activity in a Wilms tumor cell line. Screening oligonucleotide arrays with RNA from these cells uncovered a number of genes whose expression was altered by abrogation of WT1 function. Several of the genes encode members of the CCN family of growth regulators. The promoter of one of these genes, connective tissue growth factor (CTGF), is suppressed by WT1 both in its endogenous location and in reporter constructs. WT1 regulation of CTGF expression is not mediated by previously identified WT1 recognition elements and may therefore involve a novel mechanism. Our results indicate that CTGF is a bona fide target of WT1 transcriptional suppression and likely plays a role in Wilms tumorigenesis and associated disease syndromes.Wilms tumor (pediatric nephroblastoma) is one of the most common solid tumors found in children. A subset (5-10%) of Wilms tumors is caused by mutations in the tumor suppressor gene, WT1 (reviewed in Refs. 1 and 2). In addition to its role in tumorigenesis, WT1 is also required for normal kidney and urogenital development. WT1 is expressed in a temporally and spatially restricted pattern in the developing kidney and urogenital structures, and WT1 knockout mice die before birth with both kidney and urogenital development blocked at an early stage (3). Based upon these findings, it has been suggested that WT1 is required for the expression of signaling molecules and receptors involved in the reciprocal inductive events of early kidney differentiation (reviewed in Ref. Although it is clear that WT1 plays an important role in both development and tumorigenesis, the mechanisms through which it functions in these processes remain poorly understood. WT1 has been implicated in such diverse pathways as RNA splicing, DNA replication, and apoptosis (reviewed in Refs. 2 and 4). The most well characterized function of WT1, however, is that of a transcription factor. The amino terminus of WT1 is rich in proline and glutamine, a feature characteristic of transactivation domains, and the carboxyl terminus contains four C2H2 zinc finger DNA-binding motifs and a nuclear localization signal sequence. In vivo, there are four major isoforms of WT1 generated by alternative splicing at two sites. Splicing of exon 5 removes 17 amino acids from the middle of the protein, and a second alternative splicing event removes three amino acids (KTS) from between the third and fourth zinc fingers of the protein. The four isoforms of WT1 are present in a constant ratio that is conserved among species, suggesting that they have non-overlapping functions.WT1 binds to DNA via its zinc finger motifs, but this binding is isoform-depen...

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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

Cited by 52 publications

References 35 publications

The Transcription Factor Wilms Tumor 1 Regulates Matrix Metalloproteinase-9 through a Nitric Oxide-Mediated Pathway

The Transcription Factor Wilms Tumor 1 Regulates Matrix Metalloproteinase-9 through a Nitric Oxide-Mediated Pathway

Site-specific Phosphorylation Differentiates Active from Inactive Forms of the Human T-cell Leukemia Virus Type 1 Tax Oncoprotein

Identification of Connective Tissue Growth Factor as a Target of WT1 Transcriptional Regulation

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