Evaluation of transcriptional activity of p53 in individual living mammalian cells.
To estimate the transcriptional activity of p53 in individual living mammalian cells, we constructed the enhanced green fluorescent protein-red fluorescent protein (EGFP-DsRed) reporter system with the EGFP-p53 expression vector and the reporter plasmid, which carried a p53-dependent promoter. The expression level and transcriptional activity of EGFP-p53 were determined simultaneously by green and red fluorescence signals, respectively. In this system, we could target only the cells expressing p53 at endogenous levels, as observed in UV- or adriamycin-stimulated A549 cells. Using this system, we investigated the transcriptional activity of mutant p53s in tetramerization domain. Transcriptional activities were nearly abolished by seven mutations and significantly reduced in several mutant p53s. However, under overexpression conditions, the latter mutant p53s showed activity similar to that observed in wild-type p53. These results indicated the importance of physiological concentration for p53 proteins in cells so as to analyze their activities. Fluorescence intensity distribution analysis indicated that the mutant p53s lacking transcriptional activity presented as monomer forms in the cellular extract. In most of the mutant p53s, the decrease in transcriptional activity correlated with an increase in the fraction of monomers. This reporter system can be used for estimating the transcriptional activity of mutant p53s without contribution of the cells overexpressing p53.
Imagawa
Toshiaki
T
Laboratory of Biological Chemistry, Department of Chemistry, Faculty of Science, Hokkaido University, Sapporo 060-0810, Japan. toshi@sci.hokudai.ac.jp
Terai
Tomoko
T
Yamada
Yoshifumi
Y
Kamada
Rui
R
Sakaguchi
Kazuyasu
K
D049452:Green Fluorescent Proteins / Q000032:analysis; D006801:Humans; D008164:Luminescent Proteins; D009154:Mutation; D017434:Protein Structure, Tertiary; D014158:Transcription, Genetic; D016159:Tumor Suppressor Protein p53 / Q000032:analysis* / Q000737:chemistry / Q000502:physiology; D000097573:Red Fluorescent Protein
D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D016159:Tumor Suppressor Protein p53; D000097573:Red Fluorescent Protein; C485184:enhanced green fluorescent protein