title: AKIP1 enhances NF-kappaB-dependent gene expression by promoting the nuclear retention and phosphorylation of p65.
abstract: In this study, we have identified protein kinase A-interacting protein 1 (AKIP1) as a binding partner of NF-kappaB p65 subunit, and AKIP1 enhances the NF-kappaB-mediated gene expression. AKIP1 is a nuclear protein and known to interact with the catalytic subunit of PKA (PKAc). We identified AKIP1 by a yeast two-hybrid screen using the N terminus region of p65 as bait. The interaction between AKIP1 and p65 was confirmed by glutathione S-transferase pull-down assay in vitro and immunoprecipitation-Western blotting assay in vivo. We found that the PKAc was present in the AKIP1.p65 complex and enhanced the transcriptional activity of NF-kappaB by phosphorylating p65. In a transient luciferase assay, AKIP1 cotransfection efficiently increased the transcriptional activity of NF-kappaB induced by phorbol 12-myristate 13-acetate (PMA). When AKIP1 was knocked down by RNA interference, the PMA-mediated NF-kappaB-dependent gene expression was abolished, indicating a physiological role of AKIP1. We found that PKAc, which is maintained in an inactive form by binding to IkappaBalpha and NF-kappaB in resting cells, was activated by PMA-induced signaling and could phosphorylate p65. Overexpression of AKIP1 increased the PKAc binding to p65 and enhanced the PKAc-mediated phosphorylation of p65 at Ser-276. Interestingly, this p65 phosphorylation promoted nuclear translocation of p65 and enhanced NF-kappaB transcription. In fact, we observed that AKIP1 colocalized with p65 within the cells and appeared to retain p65 in nucleus. These findings indicate a positive role of AKIP1 in NF-kappaB signaling and suggest a novel mechanism by which AKIP1 augments the transcriptional competence of NF-kappaB.
authors.affiliation: Department of Molecular and Cellular Biology, Nagoya City University Graduate School of Medical Sciences, Nagoya 467-8601, Japan.
authors.forename: Nan
authors.initials: N
authors.lastname: Gao
authors.forename: Kaori
authors.initials: K
authors.lastname: Asamitsu
authors.forename: Yurina
authors.initials: Y
authors.lastname: Hibi
authors.forename: Takaharu
authors.initials: T
authors.lastname: Ueno
authors.forename: Takashi
authors.initials: T
authors.lastname: Okamoto
mesh_terms: D021581:Active Transport, Cell Nucleus / Q000187:drug effects / Q000502:physiology; D048868:Adaptor Proteins, Signal Transducing; D002273:Carcinogens / Q000494:pharmacology; D002467:Cell Nucleus / Q000235:genetics / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology*; D006367:HeLa Cells; D006801:Humans; D046912:Multiprotein Complexes / Q000235:genetics / Q000378:metabolism*; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism*; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D017434:Protein Structure, Tertiary / Q000502:physiology; D034622:RNA Interference; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D051996:Transcription Factor RelA / Q000235:genetics / Q000378:metabolism*; D020798:Two-Hybrid System Techniques
chemical_list: C471621:AKIP1 protein, human; D048868:Adaptor Proteins, Signal Transducing; D002273:Carcinogens; D046912:Multiprotein Complexes; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; C498143:RELA protein, human; D051996:Transcription Factor RelA; D017868:Cyclic AMP-Dependent Protein Kinases; D013755:Tetradecanoylphorbol Acetate