Functional characterization of the interacting domains of the positive coactivator PC4 with the transcription factor AP-2alpha.
The transcriptional positive cofactor 4 (PC4) physically interacts with the transcription factor, activator protein-2 (AP-2) alpha, and overexpression of PC4 results in a relief of the AP-2 transcriptional self-interference, which is induced by high levels of AP-2alpha expression. PC4 was initially described as a DNA-binding protein that enhances the activator-dependent transcription of class II genes in vitro, but it was later shown that PC4 could also act as a potent repressor of transcription on specific DNA structures such as single-stranded (ss) DNA, DNA ends and heteroduplex DNA. To further explore the functional domains of PC4 and its ssDNA-binding effect in the interaction with AP-2alpha and on AP-2 transcriptional activity, we investigated the C-terminal domain of PC4 (PC4-CTD) and several PC4 mutants in which the ssDNA binding function was interrupted. We found that the C-terminal domain of PC4 physically interacts with AP-2alpha and retains the function of full-length protein in relieving transcription self-interference of AP-2. A point-mutated form of PC4 within the C-terminal domain beta-ridge, PC4 W89A, or a triple mutant in the beta2-beta3 loop of PC4, F77A/K78G/K80G, inactivate the ability of PC4 to bind AP-2alpha and to relieve the transcription self-interference of AP-2alpha. In addition, point-mutated forms of AP-2alpha within the activation domain (AD) that inactivate AP-2 transcription activity also lose their self-interference function. Our data suggest that the C-terminal domain of the transcription cofactor PC4 is critical for AP-2alpha transcriptional interference that is mediated by the activation domain of AP-2alpha.
Program in Molecular Biology and Genetics, Barbara Ann Karmanos Cancer Institute, Wayne State University School of Medicine, 110 East Warren Avenue, Detroit, MI 48201, USA.
Li
L
Zhong
Yingqun
Y
Wang
Perry
P
Kannan
Michael A
MA
Tainsky
D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D015153:Blotting, Western; D045744:Cell Line, Tumor; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D006801:Humans; D017874:Immediate-Early Proteins; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D009154:Mutation; D011233:Precipitin Tests; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D050656:Transcription Factor AP-2; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic; D014162:Transfection
D004268:DNA-Binding Proteins; C490043:IFRD2 protein, human; D017874:Immediate-Early Proteins; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; C493673:TFAP2A protein, human; D015534:Trans-Activators; D050656:Transcription Factor AP-2; D014157:Transcription Factors; D015500:Chloramphenicol O-Acetyltransferase; D005982:Glutathione Transferase