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J. Biol. Chem., Vol. 283, Issue 12, 7834-7843, March 21, 2008

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AKIP1 Enhances NF-B-dependent Gene Expression by Promoting the Nuclear Retention and Phosphorylation of p65^*

From the Department of Molecular and Cellular Biology, Nagoya City University Graduate School of Medical Sciences, Nagoya 467-8601, Japan

In this study, we have identified protein kinase A-interacting protein 1 (AKIP1) as a binding partner of NF-B p65 subunit, and AKIP1 enhances the NF-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·p65 complex and enhanced the transcriptional activity of NF-B by phosphorylating p65. In a transient luciferase assay, AKIP1 cotransfection efficiently increased the transcriptional activity of NF-B induced by phorbol 12-myristate 13-acetate (PMA). When AKIP1 was knocked down by RNA interference, the PMA-mediated NF-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 IB and NF-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-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-B signaling and suggest a novel mechanism by which AKIP1 augments the transcriptional competence of NF-B.

Received for publication, December 18, 2007

^* This work was supported in part by grants-in-aid from the Ministry of Health, Labor and Welfare and the Ministry of Education, Culture, Sports, Science, and Technology of Japan, and the Japanese Health Sciences Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya, Aichi 467-8601, Japan. Tel.: 81-52-853-8205; Fax: 81-52-859-1235; E-mail: tokamoto{at}med.nagoya-cu.ac.jp.

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