MUTANT-P53 PROTEINS HAVE DIVERSE INTRACELLULAR ABILITIES TO OLIGOMERIZE AND ACTIVATE TRANSCRIPTION

Accumulating evidence supports the hypothesis that tumor-suppressor p5 3 can act as a transcriptional activator. Insertion of high-affinity p 53 DNA binding sites up-stream of a promoter yields a p53-responsive v ector. Chimeric proteins fusing p53 and the GAL4 DNA-binding domain de monstrate the presence of a transcriptional activating domain in the N -terminus of p53. GAL4-p53 chimeras constructed using naturally occurr ing p53 mutations at either codon 141 (Tyr-141) or 175 (His-175) of p5 3 had little ability to activate the reporter gene; in contrast, mutat ions at either codon 248 (Trp-248) or 273 (His-273) produced greater t ranscriptional activities than did wild-type p53. GAL4 chimeras can be used to analyse interactions between different domains of p53 and bet ween different p53 alleles; a DNA binding site is defined, and a simpl e measurement can be made of function. We had expected that coexpressi on of GAL4 chimeras and p53 alleles would squelch transcriptional acti vation downstream of GAL binding sites. Surprisingly, coexpression of either p53 (Trp-248) or (His-273) with the GALA-p53 (wild-type, His-27 3, Trp-248, His-175, Tyr-141) effectors conferred an increase in trans criptional activation as compared with the effector alone. Oligomeriza tion of p53 alleles with GAL4-p53 chimeras could underlie this effect, leading to an increase in transcription-activating motifs near the pr omoter. To test this possibility, we constructed a GAL4- p53 C-termina l chimera with p53 residues 160-393, lacking the transcriptional activ ating domain but retaining regions believed to be important in p53 oli gomerization. Neither GAL4-p53 (C-terminus) nor p53 expression vectors were able to transactivate G5E1B-CAT alone. Both p53 (His-273) and (T rp-248) co-expressed with GAL4-p53 (C-terminus) were able to transacti vate the G5E1B-CAT reporter gene; in contrast, p53 (Tyr-141) was not a ble to activate transcription. p53 (Tyr-141/His-273) behaved as a domi nant negative mutant and inhibited the ability of the combination of p 53 (His-273) and GAL4-p53 (C-terminus) to stimulate the reporter gene. Double immunoprecipitation by sequentially using GAL4 and p53 antibod ies showed that p53 (His-273) and (Tyr-141/ His-273), but not p53 (Tyr -141), can efficiently oligomerize in vivo to the C-terminal region of p53. Transcriptional activating function of p53 may be modulated by o ligomerization; some mutations, such as His-273 and Trp-248, participa te in these functions. The p53 proteins with mutations at Tyr-141 and His-175 have lost their abilities to transactivate and oligomerize eff iciently in vivo, perhaps resulting in loss of their tumor-suppressor activities. In contrast, mutations in the region of codons 248 and 273 result in p53 proteins with potent abilities to transactivate and oli gomerize in vivo, suggesting that these proteins in conjunction with l oss of the normal allele of p53 may be able to behave in a similar way to recessive oncogenes.