AKIP1 enhances NF-κB-dependent gene expression by promoting the nuclear retention and phosphorylation of p65
AKIP1 enhances NF-κB-dependent gene expression by promoting the nuclear retention and phosphorylation of p65
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DOI：
10.1074/jbc.m710285200
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发表时间：
2008-03-21
影响因子：
4.8
通讯作者：
Okamoto, Takashi
中科院分区：
文献类型：
--
作者：
Gao, Nan;Asamitsu, Kaori;Okamoto, Takashi
在本研究中，我们已经确定蛋白激酶A相互作用蛋白1（AKIP 1）作为NF-κ B B p65亚基的结合伴侣，并且AKIP 1增强NF-κ B介导的基因表达。AKIP 1是一种核蛋白，已知与PKA的催化亚基（PKAc）相互作用。我们通过酵母双杂交筛选，以p65的N端区域为诱饵鉴定了AKIP 1。通过谷胱甘肽S-转移酶pull-down实验和免疫沉淀-Western blotting实验证实了AKIP 1与p65之间的相互作用。我们发现PKAc存在于AKIP 1中心点p65复合物中，并通过磷酸化p65增强NF-κ B B的转录活性。在瞬时荧光素酶测定中，AKIP 1共转染有效地增加了由佛波酯12-肉豆蔻酸酯13-乙酸酯（PMA）诱导的NF-κ B B的转录活性。当AKIP 1被RNA干扰敲低时，PMA介导的NF-κ B依赖性基因表达被消除，表明AKIP 1的生理作用。我们发现PKAc通过与静息细胞中的I κ B α和NF-κ B结合而保持非活性形式，其被PMA诱导的信号传导激活并可使p65磷酸化。AKIP 1的过表达增加PKAc与p65的结合，并增强PKAc介导的p65在Ser-276的磷酸化。有趣的是，这种p65磷酸化促进了p65的核转位并增强了NF-κ B的转录。事实上，我们观察到AKIP 1与p65在细胞内共定位，并且似乎将p65保留在细胞核中。这些发现表明AKIP 1在NF-κ B信号传导中的积极作用，并提出了一种新的机制，AKIP 1通过该机制增强NF-κ B的转录能力。
In this study, we have identified protein kinase A-interacting protein 1 (AKIP1) as a binding partner of NF-kappa B p65 subunit, and AKIP1 enhances the NF-kappa B-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 center dot p65 complex and enhanced the transcriptional activity of NF-kappa B by phosphorylating p65. In a transient luciferase assay, AKIP1 cotransfection efficiently increased the transcriptional activity of NF-kappa B induced by phorbol 12-myristate 13-acetate (PMA). When AKIP1 was knocked down by RNA interference, the PMA-mediated NF-kappa B-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 I kappa B alpha and NF-kappa B 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-kappa B 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-kappa B signaling and suggest a novel mechanism by which AKIP1 augments the transcriptional competence of NF-kappa B.