Gao Nan, Asamitsu Kaori, Hibi Yurina, Ueno Takaharu, Okamoto Takashi
Department of Molecular and Cellular Biology, Nagoya City University Graduate School of Medical Sciences, Nagoya 467-8601, Japan.
J Biol Chem. 2008 Mar 21;283(12):7834-43. doi: 10.1074/jbc.M710285200. Epub 2008 Jan 4.
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.
在本研究中，我们已确定蛋白激酶A相互作用蛋白1（AKIP1）为核因子-κB p65亚基的结合伴侣，且AKIP1可增强核因子-κB介导的基因表达。AKIP1是一种核蛋白，已知其与蛋白激酶A（PKA）的催化亚基（PKAc）相互作用。我们以p65的N端区域为诱饵，通过酵母双杂交筛选鉴定出了AKIP1。AKIP1与p65之间的相互作用在体外通过谷胱甘肽S-转移酶下拉试验以及在体内通过免疫沉淀-蛋白质印迹试验得以证实。我们发现PKAc存在于AKIP1-p65复合物中，并通过使p65磷酸化来增强核因子-κB的转录活性。在瞬时荧光素酶试验中，共转染AKIP1可有效增强佛波酯12-肉豆蔻酸酯13-乙酸酯（PMA）诱导的核因子-κB的转录活性。当通过RNA干扰敲低AKIP1时，PMA介导的核因子-κB依赖性基因表达被消除，这表明了AKIP1的生理作用。我们发现，在静息细胞中通过与IκBα和核因子-κB结合而维持无活性形式的PKAc，可被PMA诱导的信号激活并使p65磷酸化。AKIP1的过表达增加了PKAc与p65的结合，并增强了PKAc介导的p65第276位丝氨酸的磷酸化。有趣的是，这种p65磷酸化促进了p65的核转位并增强了核因子-κB的转录。事实上，我们观察到AKIP1与细胞内的p65共定位，并且似乎将p65保留在细胞核中。这些发现表明AKIP1在核因子-κB信号传导中发挥积极作用，并提示了一种AKIP1增强核因子-κB转录能力的新机制。
