The importance of this site has recently been studied through mutational analysis. Mutation of serine 276 to alanine is embryonic lethal despite translocation to the nucleus and interaction with DNA, demonstrating the regulatory importance of this site. Two recent studies identified AKIP1 as a p65 binding protein. In the first study, the 1B isoform of AKIP1, upon neddylation, was found to bind p65, and recruit histone deacetylase, SirT1, to the CPI-613 complex resulting in transcriptional repression. The second study identified p65 as an AKIP 1A binding partner through a yeast two-hybrid screen. AKIP 1A was recruited to a nuclear complex that contained p65 and PKAc. The net result of this complex was an increase in NF-kB dependent transcription. In this study, we wanted to examine the effect of PKAc and AKIP1 on the rate of translocation of NF-kB into the nucleus. We provide evidence that AKIP1 acts as a molecular scaffold that simultaneously binds PKAc and p65 in the cytosol and disruption of this complex alters the rate at which NF-kB enters the nucleus. Our data also suggests that phosphorylation of p65 by PKAc is abrogated by AKIP1 in the cytosol and this allows p65 to translocate faster into the nucleus. PKAc CX-4945 PKC inhibitor preferentially interacts with the AKIP 1A isoform, and the PKAc binding site was previously mapped to the amino terminus. To further map the interaction site on PKAc, we performed a peptide walk to determine specific amino acids required for PKAc to bind to the three AKIP1 splice variants. Specifically, 15-mer peptides derived from the amino terminal 38 amino acids of PKAc were spotted onto a membrane and overlaid with in vitro translated AKIP 1A, AKIP 1B, or AKIP 1C proteins as previously described. The three AKIP1 isoforms specifically bound clusters of sequences, shown in Fig. 4A, with the sequence of the spotted peptide shown to the right of the peptide spot. The complete peptide walk of AKIP 1A over the amino terminus of PKAc and the identified binding sites are shown in Figure S1. Exposed lysines at either end of the peptide appear to enhance AKIP1 binding to PKAc. To further define the optimal binding regions and critical amino acids within this region, peptides containing amino acids 11�C30 were systematically shortened by one amino acid from the amino and/or carboxy terminal and tested for AKIP 1A binding. A peptide composed of amino acids 15�C29 was identified as a minimal binding region. Figure 4B indicates the exposed amino acids on the solvent exposed surface of the N-terminal helix of PKAc that are thought to be important for interaction with AKIP 1A. Previously, AKIP1 was found to enhance translocation of PKAc into the nucleus of HeLa cells stimulated with forskolin.