Mutant p53 proteins have diverse intracellular abilities to oligomerize and activate transcription.
Accumulating evidence supports the hypothesis that tumor-suppressor p53 can act as a transcriptional activator. Insertion of high-affinity p53 DNA binding sites upstream of a promoter yields a p53-responsive vector. Chimeric proteins fusing p53 and the GAL4 DNA-binding domain demonstrate the presence of a transcriptional activating domain in the N-terminus of p53. GAL4-p53 chimeras constructed using naturally occurring p53 mutations at either codon 141 (Tyr-141) or 175 (His-175) of p53 had little ability to activate the reporter gene; in contrast, mutations at either codon 248 (Trp-248) or 273 (His-273) produced greater transcriptional activities than did wild-type p53. GAL4 chimeras can be used to analyse interactions between different domains of p53 and between different p53 alleles; a DNA binding site is defined, and a simple measurement can be made of function. We had expected that coexpression of GAL4 chimeras and p53 alleles would squelch transcriptional activation downstream of GAL binding sites. Surprisingly, coexpression of either p53 (Trp-248) or (His-273) with the GALA-p53 (wild-type, His-273, Trp-248, His-175, Tyr-141) effectors conferred an increase in transcriptional activation as compared with the effector alone. Oligomerization of p53 alleles with GAL4-p53 chimeras could underlie this effect, leading to an increase in transcription-activating motifs near the promoter. To test this possibility, we constructed a GAL4-p53 C-terminal chimera with p53 residues 160-393, lacking the transcriptional activating domain but retaining regions believed to be important in p53 oligomerization. Neither GAL4-p53 (C-terminus) nor p53 expression vectors were able to transactivate G5E1B-CAT alone. Both p53 (His-273) and (Trp-248) co-expressed with GAL4-p53 (C-terminus) were able to transactivate the G5E1B-CAT reporter gene; in contrast, p53 (Tyr-141) was not able to activate transcription. p53 (Tyr-141/His-273) behaved as a dominant negative mutant and inhibited the ability of the combination of p53 (His-273) and GAL4-p53 (C-terminus) to stimulate the reporter gene. Double immunoprecipitation by sequentially using GAL4 and p53 antibodies 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 oligomerization; some mutations, such as His-273 and Trp-248, participate in these functions.(ABSTRACT TRUNCATED AT 400 WORDS)
Department of Medicine, UCLA School of Medicine 90048.
C W
CW
Miller
A
A
Chumakov
J
J
Said
D L
DL
Chen
A
A
Aslo
H P
HP
Koeffler
D000483:Alleles; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D006801:Humans; D009154:Mutation; D009842:Oligopeptides / Q000378:metabolism; D011233:Precipitin Tests; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship; D015533:Transcriptional Activation; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000502:physiology*
D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; D016159:Tumor Suppressor Protein p53