Physical and Functional Interaction Between WT1 and P53 Proteins SHYAMALA MAHESWARAN*, SEON PARK*, AMY BERNARD*, JENNIFER F
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Proc. Natl. Acad. Sci. USA Vol. 90, pp. 5100-5104, June 1993 Genetics Physical and functional interaction between WT1 and p53 proteins SHYAMALA MAHESWARAN*, SEON PARK*, AMY BERNARD*, JENNIFER F. MoRRISt, FRANK J. RAUSCHER IIlt, DAVID E. HILLY, AND DANIEL A. HABER*§ *Massachusetts General Hospital Cancer Center and Harvard Medical School, Charlestown, MA 02129; tThe Wistar Institute, Philadelphia, PA 19104; and tOncogene Science, Inc., Cambridge, MA 02139 Communicated by Kurt J. Isselbacher, February 22, 1993 ABSTRACT WTI is a tumor-suppressor gene expressed in rabbit polyclonal antibodies directed against overlapping the developing kidney, whose inactivation leads to the devel- N-terminal peptides, aa 1-173 and 85-173, respectively (9); opment of Wilms tumor, a pediatric kidney cancer. WTI DG10 is a monoclonal antibody generated against human encodes a transcription factor which binds to the EGRI con- WT1 synthesized in Escherichia coli. For immunoprecipita- sensus sequence, mediating transcriptional repression. We now tions, cells were labeled with [35S]methionine and extracted demonstrate that p53, the product of a tumor-suppressor gene with either ELB buffer (50 mM Hepes, pH 7.0/250 mM with ubiquitous expression, physically associates with WT1 in NaCl/0.5 mM EDTA/0.1% Nonidet P-40) or RIPA buffer (10 transfected cells. The interaction between WT1 and p53 mod- mM Tris, pH 7.4/150 mM NaCl, 1% Triton X-100/1% sodium deoxycholate/0.1% SDS). For sequential immunoprecipita- ulates their ability to transactivate their respective targets. In tions, cellular lysates were extracted with ELB buffer and the absence of p53, WT1 acts as a potent transcriptional immunoprecipitated with the first antibody. The immune activator of the early growth response gene 1 (EGRI) site, complex was then dissociated in RIPA buffer and the re- rather than a transcriptional repressor. In contrast, WT1 leased proteins were immunoprecipitated with the second exerts a cooperative effect on p53, enhancing its ability to antibody. Peptide maps ofimmunoprecipitated WT1 proteins transactivate the muscle creatine kinase promoter. were generated using various concentrations ofStaphylococ- cus aureus V8 protease (10). For immunoprecipitation/ Wilms tumor has been linked to the inactivation of the WTI Western analysis, anti-p53 antibodies were covalently tumor-suppressor gene at the 11p13 chromosomal locus crosslinked to protein A-Sepharose (10), and after immuno- (reviewed in ref. 1). WTI encodes a developmentally regu- precipitation, proteins were released from the antibody by lated transcription factor of 52-54 kDa. The C terminus incubation with 2% SDS/50 mM Tris, pH 6.8, at room contains four zinc fingers which confer binding specificity to temperature for 5 min. Western blot analysis was performed the EGRI DNA consensus (2), while the N terminus mediates according to the ECL protocol (Amersham), using antibody transcriptional repression in transient transfection assays (3, WT-6F1 (9) at a dilution of 1:2000. To crosslink proteins in 4). A complex pattern of alternative splicing leads to distinct cells, cultures were incubated with 3 mM dithiobis(succin- WTI gene products (5). Alternative splice II, which encodes imidyl propionate) (DSP, Pierce; 100-mg/ml stock in dimeth- three amino acids [Lys-Thr-Ser (KTS)], interrupts the spac- yl sulfoxide) for 30 min, DSP was inactivated with 30 mM ing between the third and fourth zinc fingers, altering the ammonium acetate (pH 7.0), and protein was extracted. For DNA-binding specificity of WT1 protein (2, 6). WTI muta- two-dimensional gel electrophoresis, proteins were cross- tions detected in Wilms tumor specimens have also provided linked with DSP, immunoprecipitated, and resolved by SDS/ reagents to dissect the functional properties of WT1 protein. 3-10% PAGE. The lanes were excised, the crosslinking was Of particular interest is a dominant negative mutation, reversed by incubation in 5% 2-mercaptoethanol/10%o glyc- WTAR (7), which encodes an in-frame deletion of the third erol/2.3% SDS, 62.5 mM Tris, pH 6.8, and the proteins were zinc finger and demonstrates oncogenic potential in baby rat electrophoresed in the second dimension. For gel filtration, kidney (BRK) cell transformation assays (8). Disruption of ELB extracts from unlabeled ceils were resolved on a Su- the DNA-binding domain by the WTAR mutation suggests perose 12 FPLC column (Pharmacia); fractions were con- that its dominant effect may result from interactions with centrated by ethanol precipitation and analyzed by Western other cellular proteins. blotting. Using stable BRK cell lines immortalized by transfection Chloramphenicol Acetyltransferase (CAT) Assays. Cultures with the adenovirus EIA gene along with WTI, we demon- were transfected by calcium phosphate/DNA precipitation strate the presence of a complex containing WT1 and p53 with expression constructs under the control ofthe cytomeg- proteins. This complex is also observed in BRK cells ex- alovirus promoter, and the total amount of promoter se- pressing mutant WTAR and a mutated p53 gene (codon 248), quence transfected into each dish was equalized by the as well as in specimens of sporadic Wilms tumor. A potential addition ofvector DNA. Transfection efficiencies were stan- functional interaction between WT1 and p53 is suggested by dardized by cotransfection of a human growth hormone transactivation assays using their respective target se- reporter construct, and all experiments were repeated at least quences. While WT1 enhances transcriptional activation by three times. CAT activity was quantitated by excising the p53, wild-type p53 appears to convert WT1 from a transcrip- appropriate sections ofthe TLC plates for scintillation count- tional activator to a transcriptional repressor. ing. MATERIALS AND METHODS RESULTS Coimmunoprecipitation of WT1 and p53 in Transfected Immunoprecipitations and Western Blot Analyses. Three BRK Cells. To study WT1-associated proteins in an appro- anti-WT1 antibodies were used: WT-6F1 and WT-91 are Abbreviations: BRK, baby rat kidney; DSP, dithiobis(succinimidyl The publication costs ofthis article were defrayed in part by page charge propionate); MCK, muscle creatine kinase; CAT, chloramphenicol payment. This article must therefore be hereby marked "advertisement" acetyltransferase. in accordance with 18 U.S.C. §1734 solely to indicate this fact. §To whom reprint requests should be addressed. 5100 Downloaded by guest on September 26, 2021 Genetics: Maheswaran et al. Proc. Natl. Acad. Sci. USA 90 (1993) 5101 a 1st Ab Nor.specifir aWf-91 priate kidney-derived cell type, we made use of EIA- immortalized BRK cell lines, stably transfected with con- N c\ C\j structs encoding either wild-type WT1 (clone A6) or mutant WTAR (clone 6.3) (8). Immunoprecipitation of WT1 from 2nd Ab C L BRK cells under nonionic detergent conditions (ELB buffer) ~ Q resulted in the coprecipitation of a protein comigrating with M p53, while immunoprecipitation of p53 coprecipitated a band kDa that comigrated with WT1. Coprecipitation of these proteins was reduced by extraction under more stringent conditions 66- (RIPA buffer), consistent with an unstable protein associa- .- 5 tion. To determine whether WT1 and p53 were in fact coimmunoprecipitated, we performed sequential immuno- precipitations with antibodies directed against these two proteins. We first used 6.3 cells, expressing transfected 45 - WTAR and a mutated endogenous p53 (with an Arg His substitution in codon 246 of the rat p53 gene, analogous to human codon 248). Radiolabeled, ELB-extracted lysates were immunoprecipitated with anti-WT1 antibody WT-91, a p53 a p53 and the immune complex was dissociated in RIPA buffer 1st Ab Nonspecific PAB 122 PAS 122 before immunoprecipitation with anti-p53 antibody PAb421 or PAb122. Fig. la shows the precipitation of a protein, to to .-Q released from the WT1 immune complex, that comigrated 9D- 2nd Ab a an C-1 with authentic p53. Substitution of nonspecific antibodies of a: 33 4 + the same isotype for either the anti-WT1 or the anti-p53 kDa antibody abolished precipitation of p53. In the reciprocal experiment, immunoprecipitation of extracts with anti-p53 antibody PAbl22, dissociation of the immune complex, and 66 - immunoprecipitation with either of two anti-WT1 antibodies (WT-91 and DG10) precipitated a protein that comigrated - WTAR --- with authentic WTAR (Fig. lb). Use of nonspecific antibod- 45 - ies or preincubation of DG10 with unlabeled vaccinia- produced WT1 protein abolished the immunoprecipitation of WTAR from the p53 immune complex (Fig. lb). Sequential- immunoprecipitation experiments also demonstrated the co- precipitation of p53 and wild-type WT1 from extracts of A6 cells. Partial V8 protease digestion of WT1 either directly C V8 (,u g) 1 5 50 1 5 immunoprecipitated from A6 cells or sequentially immuno- kDa precipitated with anti-p53 followed by anti-WT1 antibodies 45 - confirmed the presence of authentic WT1 within the p53 immune complex (Fig. lc). Immunoprecipitation/Western Blot Analysis of BRK Cells and Wilms Tumor Specimens. We used a second approach to 29 - demonstrate the association between WT1 and p53, consist- ing of immunoprecipitation with the first antibody, followed 21 - by Western blot analysis of the immunoprecipitate with the second antibody. Unlabeled ELB-extracted proteins were 14.3 -. - .: immunoprecipitated with anti-p53 antibody and analyzed by translated p53 or WT1. RIPA buffer was sufficiently stringent to dissociate the WT1/p53 complex without affecting the primary antigen-antibody interaction. (b) Immunoprecipitation with anti-p53 antibody followed by anti-WT1 antibodies. Radiolabeled proteins extracted with ELB buffer from 6.3 cells were first immunoprecip- itated with either PAb122 (lst Ab, lanes 3 and 4) or a nonspecific antibody of the same isotype (lst Ab, lanes 1 and 2). Immune WTI direct ip WTI Seq ip complexes were dissociated and reprecipitated with anti-WT-91 (2nd aWT DGIO ap53 PAbl22 Ab, lanes 2 and 4) or a 10-fold excess of nonspecific rabbit antibody Il (2nd Ab, lanes 1 and 3). WTAR directly immunoprecipitated from 6.3 aWTI DGI10 cells served as a marker (lane M). In a separate experiment, proteins FIG.