Transcriptional activation by wild-type but not transforming mutants of the p53 anti-oncogene

Abstract

The protein encoded by the wild-type p53 proto-oncogene has been shown to suppress transformation, whereas certain mutations that alter p53 become transformation competent. Fusion proteins between p53 and the GAL4 DNA binding domain were made to anchor p53 to a DNA target sequence and to allow measurement of transcriptional activation of a reporter plasmid. The wild-type p53 stimulated transcription in this assay, but two transforming mutations in p53 were unable to act as transcriptional activators. Therefore, p53 can activate transcription, and transformation-activating mutations result in a loss of function of the p53 protein. The inability of the p53 mutant proteins to activate transcription may enable them to be transformation competent.

Fig. 1

The p53-GAL4 fusion proteins specifically activate transcription of CAT. (A) A diagrammatic representation of the different plasmids used. The effector plasmids contain the NH₂-terminal amino acids of p53 and the sequence coding for the GAL4 DNA binding domain; the reporter plasmid contains CAT coding sequences driven by the SV40 promoter. Two individual in-frame p53-GAL4 fusion proteins were made that contain the p53 transactivating domain and the GAL4 DNA binding domain. The p53-GAL4 plasmids consist of the Harvey murine sarcoma virus (H-MSV)–long terminal repeat, wild-type p53 coding sequences from amino acid 1–343 (fusion 1) or amino acids 1–330 (fusion 2) dispersed by p53 introns 2 and 3 and fused to GAL4 sequences (amino acids 4–147). The DNA construct labeled “p53 truncated” contains the H-MSV LTR and encodes only p53 amino acids 1–343. All p53 clones retain the nuclear localization signal of p53. GAL4 (amino acids 4–147) encodes only the GAL4 DNA binding domain. GAL4 (amino acids 4–881) encodes the entire GAL4 protein consisting of both the DNA binding domain and the transactivating domain (31). The GAL4 plasmids also use the H-MSV LTR enhancer-promoter. All regions of fusion were sequenced to ensure that the protein sequences remained in-frame. The CAT reporter plasmid used contains 23bp of the SV40 enhancer, the SV40 21-bp repeats, and the TATA box and CAT sequences in the bluescript vector (27). The oligonucleotide 5′-CTAGACGGAAGACTCTCCTCCGT-3′, which contains the GAL4 recognition sequence bounded by Xba I linkers, was inserted at the Xba I site ofthe CAT plasmid,155 bp upstream of the start of transcription (32). The reporter, CAT, contains either 0,2, or 4 DNA recognition sequences for the GAL4 binding domain. (B) The activator and reporter plasmids (10 μg of each) were cotransfected with a plasmid containing the β-galactosidase gene (5 μg) with calcium phosphate precipitation into HeLa cells essentially as described (33). A β-galactosidase plasmid was used to monitor and normalize for transfection efficiency. The activity ofCAT is measured by the conversion of [¹⁴C]chloramphenicol (lower dot) to acetyl and diacetyl chloramphenicol, respectively (34). Effector A, none; effector B, GAL4 (amino acids 4–147); effector C, GAL4; effector D, truncated p53; effector E, p53-GAL4 (fusion 1); and effector F, p53-GAL4 (fusion 2).

Fig. 2

Mutations within the p53-activating domain of the p53-GAL4 fusion protein eliminate transcriptional activity. Mutant 1 (A)and mutant 2 (B)sequences were fused to GAL4 (amino acids 4–147) sequences as in Fig. 1, fusion 1. Plasmids were separately transfected into HeLa cells with the CAT target plasmid and β-galactosidase marker gene as in Fig. 1. In (A) effector A, none; effector B, GAL4 (amino acids 4–147); effector C, wild-type p53-GAL4; effector D, MI p53-GAL4;and effector E,GAL4.

Fig. 3

In vitro translation of p53-GAL4 fusion proteins and GAL4 proteins. To ensure that the appropriate proteins were produced, we made RNA in vitro and translated it into protein with rabbit reticulocyte lysate and a mixture of ³⁵S-labeled Cys and ³⁵S-labeled Met according to manufacturer’s instructions. On the left is a 7.5% acrylamide gel with the higher molecular mass proteins. On the right is a 12% acrylamide gel with the wild-type p53 protein product and the small GAL4 DNA binding domain protein. Size markers are shown in kilodaltons on both sides of the figure. Lane A, wild-type p53; lane B, p53-GAL4 (fusion 1); lane C, p53-GAL4 (fusion 2); lane D, p53-GAL4 (Ml); lane E, p53-GAL4 (M2); and lane F, GAL4. Lane a, wild-type p53; and lane b, GAL4 (amino acids 4–147).

Fig. 4

Immunoprecipitation of p53-GAL4 fusion proteins. Plasmids encoding wild-type p53-GAL4 (Fl), p53-GAL4 mutant 1 (Ml), p53-GAL4 mutant 2 (M2), or vector alone (–) were cotransfected with a plasmid containing the β-galactosidase gene into COS cells with DEAE dextran. The β-galactosidase plasmid was used to monitor transfection efficiency. Transfected cells were labeled with a mixture of ³⁵S-labeled Cys and ³⁵S-labeled Met. Immunoprecipitations were performed as described with p53 monoclonal antibody PAb248 and equal amounts of trichloroacetic acid–precipitable counts (28). Also shown are in vitro–translated wild-type p53- GAL4 fusion 1 (lane 5) and p53 mutant 1 immunoprecipitated from cells that overexpress this protein (lane 6). Molecular weight markers are shown in kilodaltons.