Filamin A negatively regulates the transcriptional activity of p73alpha in the cytoplasm.
The transcription regulator p73alpha is structurally different from p53 in that it possesses a unique C-terminal domain, which has been implicated in transcriptional repression. To dissect the mechanism of repression by this domain, we performed a yeast two-hybrid screen of a HeLa cDNA library using residues 487-636 of p73alpha as bait and isolated a cDNA clone encoding the C-terminal portion (residues 2210-2647) of filamin A, a 280-kDa actin-binding protein. Additional yeast two-hybrid assays indicated that filamin A specifically interacts with the p73alpha C-terminus, which is lacking in p53 and p73beta. The interaction was confirmed by GST pull-down assays in vitro and by immunoprecipitation analysis in vivo. Immunofluorescence microscopy revealed that p73alpha remained in the cytoplasm in A7 melanoma cells stably expressing filamin A, whereas it was localized in the nucleus of filamin A-deficient M2 cells. Deletion of the C-terminus of p73alpha (residues 487-636) resulted in nuclear localization in both cell types. Consistent with our interaction data, transient co-expression of filamin A resulted in the down-regulation of p73alpha, but not of p53, transcriptional activity on various p53-responsive promoters. Taken together, our data suggest that p73alpha is sequestered in the cytoplasm by filamin A, thereby inhibiting its transcriptional activity.
Kim
Eun-Joo
EJ
Department of Bioscience & Biotechnology, Sejong University, 98 Kunja-dong, Kwangjin-gu, Seoul 143-747, Republic of Korea.
Park
Jong-Sup
JS
Um
Soo-Jong
SJ
D003285:Contractile Proteins / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D064448:Filamins; D005786:Gene Expression Regulation; D006801:Humans; D008545:Melanoma / Q000235:genetics / Q000378:metabolism*; D008840:Microfilament Proteins / Q000378:metabolism*; D009687:Nuclear Proteins / Q000378:metabolism*; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D000072160:Tumor Protein p73; D025521:Tumor Suppressor Proteins / Q000378:metabolism*