Centrosomal P4.1-associated protein is a new member of transcriptional coactivators for nuclear factor-kappaB.
Nuclear factor-kappaB (NF-kappaB) is a transcription factor important for various cellular events such as inflammation, immune response, proliferation, and apoptosis. In this study, we performed a yeast two-hybrid screening using the N-terminal domain of the p65 subunit (RelA) of NF-kappaB as bait and isolated centrosomal P4.1-associated protein (CPAP) as a candidate for a RelA-associating partner. Glutathione S-transferase pull-down assays and co-immunoprecipitation experiments followed by Western blotting also showed association of CPAP with RelA. When overexpressed, CPAP enhanced NF-kappaB-dependent transcription induced by tumor necrosis factor-alpha (TNFalpha). Reduction of the protein level of endogenous CPAP by RNA interference resulted in decreased activation of NF-kappaB by TNFalpha. After treatment with TNFalpha, a portion of CPAP was observed to accumulate in the nucleus, although CPAP was found primarily in the cytoplasm without any stimulation. Moreover, CPAP was observed in a complex recruited to the transcriptional promoter region containing the NF-kappaB-binding motif. One hybrid assay showed that CPAP has the potential to activate gene expression when tethered to the transcriptional promoter. These data suggest that CPAP functions as a coactivator of NF-kappaB-mediated transcription. Since a physiological interaction between CPAP and the coactivator p300/CREB-binding protein was also observed and synergistic activation of NF-kappaB-mediated transcription was achieved by these proteins, CPAP-dependent transcriptional activation is likely to include p300/CREB-binding protein.
Koyanagi
Michiyo
M
Department of Viral Oncology, Institute for Virus Research, Kyoto University, Sakyo-ku, Kyoto 606-8507, Japan.
Hijikata
Makoto
M
Watashi
Koichi
K
Masui
Osamu
O
Shimotohno
Kunitada
K
D020816:Amino Acid Motifs; D001665:Binding Sites; D015153:Blotting, Western; D002460:Cell Line; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism; D018385:Centrosome / Q000648:ultrastructure*; D003593:Cytoplasm / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D008156:Luciferases / Q000378:metabolism; D008869:Microtubule-Associated Proteins / Q000378:metabolism* / Q000502:physiology*; D008957:Models, Genetic; D016328:NF-kappa B / Q000737:chemistry / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D034622:RNA Interference; D011994:Recombinant Proteins / Q000737:chemistry; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D051996:Transcription Factor RelA; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D020798:Two-Hybrid System Techniques
C414735:CENPJ protein, human; D008869:Microtubule-Associated Proteins; D016328:NF-kappa B; D011994:Recombinant Proteins; D051996:Transcription Factor RelA; D014409:Tumor Necrosis Factor-alpha; D004247:DNA; D008156:Luciferases; D005982:Glutathione Transferase