To determine whether this minimal peptide could disrupt the interaction of PKAc and AKIP 1A, HA-tagged 1-29 PKAc peptide was co-transfected into HeLa cells with GST-AKIP 1A. PKAc has been shown to trigger the activation and translocation of the p65 component of NF-kB. Afatinib msds However, the mechanism of translocation is unknown and was believed to be energy dependent. Furthermore, though the authors showed several years ago that phosphorylation by PKAc was a key event in p65 activation and translocation, the substrate for this reaction is still unknown. Our data shows that AKIP1 is the missing link regulating both translocation and activation. We first demonstrate that AKIP 1A, the catalytic subunit of PKA, and the p65 subunit of NF-kB can form a cytosolic complex. This complex could be modulated through activators of PKA, overexpression of AKIP 1A, and overexpression of a peptide that blocks the interaction of PKAc and AKIP 1A. Second, we show that both PKAc and AKIP1 regulate the rate at which NF-kB translocates into the nucleus in response to stimulus. TNFa mediated translocation of p65 into the nucleus of HeLa cells was greatly enhanced either by siRNA knock down of PKAc or over-expression of AKIP1. Third, we provide further evidence showing that the phosphorylation of p65 by PKAc is essential in its translocation. Expression of either AKIP 1A or concomitantly with CAT 1-29 resulted in a constitutive localization of p65 in the nucleus, indicating that AKIP1 regulates the rate at which p65 enters the nucleus. There was, however, no effect of AKIP 1A or CAT 1-29 on the rate of IkB degradation, suggesting the canonical NF-kB nuclear translocation pathway remained intact. IkBa was demonstrated to be present in this complex, suggesting that the pool of AKIP1 bound to PKAc and p65 Nutlin-3 overlaps with the total population of cytosolic NF-kB. Based on mRNA levels and protein expression data, the levels of endogenous AKIP 1A are significantly lower than p65 or PKAc. The observation that AKIP1 isoforms are limiting in the complex formation suggests that under normal cellular conditions, every AKIP1 protein will be associated with both a PKAc and p65. Therefore, when AKIP1 is no longer limiting, p65 nuclear translocation is altered. Each one of these salient features is addressed in detail below. The two other groups that worked on AKIP1 showed either the enhancement or abrogation of the transcriptional activity of p65 upon binding AKIP1. Though both groups have used similar stimuli and cell lines, they obtained opposite results especially with respect to the effects on the endogenous protein. Our studies found that the cell lines used had very little AKIP1. In human cell lines, the problem is further compounded by the presence of splice variants and thus the results obtained could be misleading.