• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of embojLink to Publisher's site
EMBO J. Oct 15, 1996; 15(20): 5606–5615.
PMCID: PMC452305

Regulation of WT1 by phosphorylation: inhibition of DNA binding, alteration of transcriptional activity and cellular translocation.

Abstract

Phosphorylation is one of the major post-translational mechanisms by which the activity of transcription factors is regulated. We have investigated the role of phosphorylation in the regulation of nucleic acid binding activity and the nuclear translocation of WT1. Two recombinant WT1 proteins containing the DNA binding domain with or without a three amino acid (KTS) insertion (WT1ZF + KTS and WT1ZF - KTS) were strongly phosphorylated by protein kinase A (PKA) and protein kinase C (PKC) in vitro. Both PKA and PKC phosphorylation inhibited the ability of WT1ZF + KTS or WT1ZF - KTS to bind to a sequence derived from the WT1 promoter region in gel mobility shift assays. The binding of WT1ZF - KTS to an EGR1 consensus binding site was also inhibited by prior PKA and PKC phosphorylation. We also demonstrate the RNA binding activity of WT1, but this was not altered by phosphorylation. PKA activation by dibutyryl cAMP in WT1-transfected cells resulted in the reversal of WT1 suppression of a reporter construct. Although WT1 protein is predominantly localized to the nucleus, this expression pattern is altered upon PKA activation, resulting in the cytoplasmic retention of WT1. Accordingly, phosphorylation may play a role in modulating the transcriptional regulatory activity of WT1 through interference with nuclear translocation, as well as by inhibition of WT1 DNA binding.

Full text

Full text is available as a scanned copy of the original print version. Get a printable copy (PDF file) of the complete article (2.5M), or click on a page image below to browse page by page. Links to PubMed are also available for Selected References.

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Bickmore WA, Oghene K, Little MH, Seawright A, van Heyningen V, Hastie ND. Modulation of DNA binding specificity by alternative splicing of the Wilms tumor wt1 gene transcript. Science. 1992 Jul 10;257(5067):235–237. [PubMed]
  • Boulikas T. Putative nuclear localization signals (NLS) in protein transcription factors. J Cell Biochem. 1994 May;55(1):32–58. [PubMed]
  • Buckler AJ, Pelletier J, Haber DA, Glaser T, Housman DE. Isolation, characterization, and expression of the murine Wilms' tumor gene (WT1) during kidney development. Mol Cell Biol. 1991 Mar;11(3):1707–1712. [PMC free article] [PubMed]
  • Burd CG, Dreyfuss G. Conserved structures and diversity of functions of RNA-binding proteins. Science. 1994 Jul 29;265(5172):615–621. [PubMed]
  • Call KM, Glaser T, Ito CY, Buckler AJ, Pelletier J, Haber DA, Rose EA, Kral A, Yeger H, Lewis WH, et al. Isolation and characterization of a zinc finger polypeptide gene at the human chromosome 11 Wilms' tumor locus. Cell. 1990 Feb 9;60(3):509–520. [PubMed]
  • Campbell CE, Huang A, Gurney AL, Kessler PM, Hewitt JA, Williams BR. Antisense transcripts and protein binding motifs within the Wilms tumour (WT1) locus. Oncogene. 1994 Feb;9(2):583–595. [PubMed]
  • Chakraborty T, Brennan T, Olson E. Differential trans-activation of a muscle-specific enhancer by myogenic helix-loop-helix proteins is separable from DNA binding. J Biol Chem. 1991 Feb 15;266(5):2878–2882. [PubMed]
  • Coppes MJ, Campbell CE, Williams BR. The role of WT1 in Wilms tumorigenesis. FASEB J. 1993 Jul;7(10):886–895. [PubMed]
  • Darnell JE, Jr, Kerr IM, Stark GR. Jak-STAT pathways and transcriptional activation in response to IFNs and other extracellular signaling proteins. Science. 1994 Jun 3;264(5164):1415–1421. [PubMed]
  • Delarue F, Virone A, Hagege J, Lacave R, Peraldi MN, Adida C, Rondeau E, Feunteun J, Sraer JD. Stable cell line of T-SV40 immortalized human glomerular visceral epithelial cells. Kidney Int. 1991 Nov;40(5):906–912. [PubMed]
  • Dey BR, Sukhatme VP, Roberts AB, Sporn MB, Rauscher FJ, 3rd, Kim SJ. Repression of the transforming growth factor-beta 1 gene by the Wilms' tumor suppressor WT1 gene product. Mol Endocrinol. 1994 May;8(5):595–602. [PubMed]
  • Drummond IA, Madden SL, Rohwer-Nutter P, Bell GI, Sukhatme VP, Rauscher FJ., 3rd Repression of the insulin-like growth factor II gene by the Wilms tumor suppressor WT1. Science. 1992 Jul 31;257(5070):674–678. [PubMed]
  • Eccles MR, Grubb G, Ogawa O, Szeto J, Reeve AE. Cloning of novel Wilms tumor gene (WT1) cDNAs; evidence for antisense transcription of WT1. Oncogene. 1994 Jul;9(7):2059–2063. [PubMed]
  • Englert C, Vidal M, Maheswaran S, Ge Y, Ezzell RM, Isselbacher KJ, Haber DA. Truncated WT1 mutants alter the subnuclear localization of the wild-type protein. Proc Natl Acad Sci U S A. 1995 Dec 19;92(26):11960–11964. [PMC free article] [PubMed]
  • Gashler AL, Bonthron DT, Madden SL, Rauscher FJ, 3rd, Collins T, Sukhatme VP. Human platelet-derived growth factor A chain is transcriptionally repressed by the Wilms tumor suppressor WT1. Proc Natl Acad Sci U S A. 1992 Nov 15;89(22):10984–10988. [PMC free article] [PubMed]
  • Gessler M, Poustka A, Cavenee W, Neve RL, Orkin SH, Bruns GA. Homozygous deletion in Wilms tumours of a zinc-finger gene identified by chromosome jumping. Nature. 1990 Feb 22;343(6260):774–778. [PubMed]
  • Goodyer P, Dehbi M, Torban E, Bruening W, Pelletier J. Repression of the retinoic acid receptor-alpha gene by the Wilms' tumor suppressor gene product, wt1. Oncogene. 1995 Mar 16;10(6):1125–1129. [PubMed]
  • Gorski K, Carneiro M, Schibler U. Tissue-specific in vitro transcription from the mouse albumin promoter. Cell. 1986 Dec 5;47(5):767–776. [PubMed]
  • Haber DA, Sohn RL, Buckler AJ, Pelletier J, Call KM, Housman DE. Alternative splicing and genomic structure of the Wilms tumor gene WT1. Proc Natl Acad Sci U S A. 1991 Nov 1;88(21):9618–9622. [PMC free article] [PubMed]
  • Harrington MA, Konicek B, Song A, Xia XL, Fredericks WJ, Rauscher FJ., 3rd Inhibition of colony-stimulating factor-1 promoter activity by the product of the Wilms' tumor locus. J Biol Chem. 1993 Oct 5;268(28):21271–21275. [PubMed]
  • Hewitt SM, Hamada S, McDonnell TJ, Rauscher FJ, 3rd, Saunders GF. Regulation of the proto-oncogenes bcl-2 and c-myc by the Wilms' tumor suppressor gene WT1. Cancer Res. 1995 Nov 15;55(22):5386–5389. [PubMed]
  • Huang A, Campbell CE, Bonetta L, McAndrews-Hill MS, Chilton-MacNeill S, Coppes MJ, Law DJ, Feinberg AP, Yeger H, Williams BR. Tissue, developmental, and tumor-specific expression of divergent transcripts in Wilms tumor. Science. 1990 Nov 16;250(4983):991–994. [PubMed]
  • Huang RP, Adamson ED. The phosphorylated forms of the transcription factor, Egr-1, bind to DNA more efficiently than non-phosphorylated. Biochem Biophys Res Commun. 1994 May 16;200(3):1271–1276. [PubMed]
  • Hunter T, Karin M. The regulation of transcription by phosphorylation. Cell. 1992 Aug 7;70(3):375–387. [PubMed]
  • Joseph LJ, Le Beau MM, Jamieson GA, Jr, Acharya S, Shows TB, Rowley JD, Sukhatme VP. Molecular cloning, sequencing, and mapping of EGR2, a human early growth response gene encoding a protein with "zinc-binding finger" structure. Proc Natl Acad Sci U S A. 1988 Oct;85(19):7164–7168. [PMC free article] [PubMed]
  • Kennedy D, Ramsdale T, Mattick J, Little M. An RNA recognition motif in Wilms' tumour protein (WT1) revealed by structural modelling. Nat Genet. 1996 Mar;12(3):329–331. [PubMed]
  • Lanoix J, Belhumeur P, Lussier M, Royal A, Bravo R, Skup D. Regulated expression of Krox-24 and other serum-responsive genes during differentiation of P19 embryonal carcinoma cells. Cell Growth Differ. 1991 Aug;2(8):391–399. [PubMed]
  • Larsson SH, Charlieu JP, Miyagawa K, Engelkamp D, Rassoulzadegan M, Ross A, Cuzin F, van Heyningen V, Hastie ND. Subnuclear localization of WT1 in splicing or transcription factor domains is regulated by alternative splicing. Cell. 1995 May 5;81(3):391–401. [PubMed]
  • Li L, Zhou J, James G, Heller-Harrison R, Czech MP, Olson EN. FGF inactivates myogenic helix-loop-helix proteins through phosphorylation of a conserved protein kinase C site in their DNA-binding domains. Cell. 1992 Dec 24;71(7):1181–1194. [PubMed]
  • MacGregor GR, Caskey CT. Construction of plasmids that express E. coli beta-galactosidase in mammalian cells. Nucleic Acids Res. 1989 Mar 25;17(6):2365–2365. [PMC free article] [PubMed]
  • Madden SL, Rauscher FJ., 3rd Positive and negative regulation of transcription and cell growth mediated by the EGR family of zinc-finger gene products. Ann N Y Acad Sci. 1993 Jun 11;684:75–84. [PubMed]
  • Madden SL, Cook DM, Morris JF, Gashler A, Sukhatme VP, Rauscher FJ., 3rd Transcriptional repression mediated by the WT1 Wilms tumor gene product. Science. 1991 Sep 27;253(5027):1550–1553. [PubMed]
  • Maheswaran S, Park S, Bernard A, Morris JF, Rauscher FJ, 3rd, Hill DE, Haber DA. Physical and functional interaction between WT1 and p53 proteins. Proc Natl Acad Sci U S A. 1993 Jun 1;90(11):5100–5104. [PMC free article] [PubMed]
  • Maheswaran S, Englert C, Bennett P, Heinrich G, Haber DA. The WT1 gene product stabilizes p53 and inhibits p53-mediated apoptosis. Genes Dev. 1995 Sep 1;9(17):2143–2156. [PubMed]
  • Mosialos G, Hamer P, Capobianco AJ, Laursen RA, Gilmore TD. A protein kinase-A recognition sequence is structurally linked to transformation by p59v-rel and cytoplasmic retention of p68c-rel. Mol Cell Biol. 1991 Dec;11(12):5867–5877. [PMC free article] [PubMed]
  • Müller HJ, Skerka C, Bialonski A, Zipfel PF. Clone pAT 133 identifies a gene that encodes another human member of a class of growth factor-induced genes with almost identical zinc-finger domains. Proc Natl Acad Sci U S A. 1991 Nov 15;88(22):10079–10083. [PMC free article] [PubMed]
  • Murre C, McCaw PS, Vaessin H, Caudy M, Jan LY, Jan YN, Cabrera CV, Buskin JN, Hauschka SD, Lassar AB, et al. Interactions between heterologous helix-loop-helix proteins generate complexes that bind specifically to a common DNA sequence. Cell. 1989 Aug 11;58(3):537–544. [PubMed]
  • Patwardhan S, Gashler A, Siegel MG, Chang LC, Joseph LJ, Shows TB, Le Beau MM, Sukhatme VP. EGR3, a novel member of the Egr family of genes encoding immediate-early transcription factors. Oncogene. 1991 Jun;6(6):917–928. [PubMed]
  • Pavletich NP, Pabo CO. Zinc finger-DNA recognition: crystal structure of a Zif268-DNA complex at 2.1 A. Science. 1991 May 10;252(5007):809–817. [PubMed]
  • Pearson RB, Kemp BE. Protein kinase phosphorylation site sequences and consensus specificity motifs: tabulations. Methods Enzymol. 1991;200:62–81. [PubMed]
  • Rangnekar VM, Aplin AC, Sukhatme VP. The serum and TPA responsive promoter and intron-exon structure of EGR2, a human early growth response gene encoding a zinc finger protein. Nucleic Acids Res. 1990 May 11;18(9):2749–2757. [PMC free article] [PubMed]
  • Rauscher FJ, 3rd, Morris JF, Tournay OE, Cook DM, Curran T. Binding of the Wilms' tumor locus zinc finger protein to the EGR-1 consensus sequence. Science. 1990 Nov 30;250(4985):1259–1262. [PubMed]
  • Rihs HP, Jans DA, Fan H, Peters R. The rate of nuclear cytoplasmic protein transport is determined by the casein kinase II site flanking the nuclear localization sequence of the SV40 T-antigen. EMBO J. 1991 Mar;10(3):633–639. [PMC free article] [PubMed]
  • Rupprecht HD, Drummond IA, Madden SL, Rauscher FJ, 3rd, Sukhatme VP. The Wilms' tumor suppressor gene WT1 is negatively autoregulated. J Biol Chem. 1994 Feb 25;269(8):6198–6206. [PubMed]
  • Ryan G, Steele-Perkins V, Morris JF, Rauscher FJ, 3rd, Dressler GR. Repression of Pax-2 by WT1 during normal kidney development. Development. 1995 Mar;121(3):867–875. [PubMed]
  • Sharma PM, Bowman M, Yu BF, Sukumar S. A rodent model for Wilms tumors: embryonal kidney neoplasms induced by N-nitroso-N'-methylurea. Proc Natl Acad Sci U S A. 1994 Oct 11;91(21):9931–9935. [PMC free article] [PubMed]
  • Sleigh MJ. A nonchromatographic assay for expression of the chloramphenicol acetyltransferase gene in eucaryotic cells. Anal Biochem. 1986 Jul;156(1):251–256. [PubMed]
  • Sukhatme VP. Early transcriptional events in cell growth: the Egr family. J Am Soc Nephrol. 1990 Dec;1(6):859–866. [PubMed]
  • Sukhatme VP, Cao XM, Chang LC, Tsai-Morris CH, Stamenkovich D, Ferreira PC, Cohen DR, Edwards SA, Shows TB, Curran T, et al. A zinc finger-encoding gene coregulated with c-fos during growth and differentiation, and after cellular depolarization. Cell. 1988 Apr 8;53(1):37–43. [PubMed]
  • Wang ZY, Qiu QQ, Huang J, Gurrieri M, Deuel TF. Products of alternatively spliced transcripts of the Wilms' tumor suppressor gene, wt1, have altered DNA binding specificity and regulate transcription in different ways. Oncogene. 1995 Feb 2;10(3):415–422. [PubMed]
  • Werner H, Re GG, Drummond IA, Sukhatme VP, Rauscher FJ, 3rd, Sens DA, Garvin AJ, LeRoith D, Roberts CT., Jr Increased expression of the insulin-like growth factor I receptor gene, IGF1R, in Wilms tumor is correlated with modulation of IGF1R promoter activity by the WT1 Wilms tumor gene product. Proc Natl Acad Sci U S A. 1993 Jun 15;90(12):5828–5832. [PMC free article] [PubMed]
  • Yeger H, Cullinane C, Flenniken A, Chilton-MacNeil S, Campbell C, Huang A, Bonetta L, Coppes MJ, Thorner P, Williams BR. Coordinate expression of Wilms' tumor genes correlates with Wilms' tumor phenotypes. Cell Growth Differ. 1992 Dec;3(12):855–864. [PubMed]

Articles from The EMBO Journal are provided here courtesy of The European Molecular Biology Organization

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...