AKIP1 Enhances NF-κB-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-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 AKIP1⅐p65 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.NF-B is an inducible transcription factor for the expression of wide variety of genes involved in immunoinflammatory responses, cell proliferation, and survival, thus playing crucial roles in the pathogenesis of many diseases including cancer, leukemia, and autoimmune diseases (1-4). NF-B exists as either a heterodimer or a homodimer, among which the p65/ p50 is the most ubiquitous heterodimer. In resting cells, NF-B dimers are sequestered in the cytoplasm through association with inhibitory proteins IBs (5). Upon treatment with NF-B inducers such as phorbol 12-myristate 13-acetate (PMA) 2 or pro-inflammatory cytokines, IB is phosphorylated and degraded through the ubiquitin/proteasome pathway, which eventually leads to the nuclear translocation of NF-B and binding to the B site of target genes (6, 7).It has been established that the phosphorylation of p65 is important for the transcriptional activity of NF-B (8 -12). The phosphorylation of p65 by the PKA catalytic subunit dramatically enhances NF-B transcriptional activity by recruiting histone acetyltransferase CBP/p300 (13-15). PKA, existing predominantly in the cytoplasm as an inactive tetramer holoenzyme in resting cells, is composed of two catalytic subunits and a homodimer of two regulatory subunits that can dissociate upon activation by cAMP (16 -20). In resting cells, PKAc is involved in the IB⅐NF-B complex present in the cytoplasm, a...

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“…A nuclear protein, A-kinase-interacting protein 1 (AKIP1), binds both to PKA (27) and p65 (28). This interaction has been suggested to enhance PKA-mediated transcriptional activity of p65 by promoting its nuclear retention and phosphorylation at S276, which potentially increases recruitment of transcriptional coactivators such as p300 to p65 (28).…”

“…This interaction has been suggested to enhance PKA-mediated transcriptional activity of p65 by promoting its nuclear retention and phosphorylation at S276, which potentially increases recruitment of transcriptional coactivators such as p300 to p65 (28). Given the constitutive activation of NF-κB and PKA signaling in Cry1 −/− ;Cry2 −/− cells (Figs.…”