title: The rate of NF-κB nuclear translocation is regulated by PKA and A kinase interacting protein 1.
abstract: The mechanism of PKAc-dependent NF-κB activation and subsequent translocation into the nucleus is not well defined. Previously, we showed that A kinase interacting protein 1 (AKIP1) was important for binding and retaining PKAc in the nucleus. Since then, other groups have demonstrated that AKIP1 binds the p65 subunit of NF-κB and regulates its transcriptional activity through the phosphorylation at Ser 276 by PKAc. However, little is known about the formation and activation of the PKAc/AKIP1/p65 complex and the rate at which it enters the nucleus. Initially, we found that the AKIP1 isoform (AKIP 1A) simultaneously binds PKAc and p65 in resting and serum starved cells. Using peptide arrays, we refined the region of AKIP 1A binding on PKAc and mapped the non-overlapping regions on AKIP 1A where PKAc and p65 bind. A peptide to the amino-terminus of PKAc (CAT 1-29) was generated to specifically disrupt the interaction between AKIP 1A and PKAc to study nuclear import of the complex. The rate of p65 nuclear translocation was monitored in the presence or absence of overexpressed AKIP 1A and/or (CAT 1-29). Enhanced nuclear translocation of p65 was observed in the presence of overexpressed AKIP1 and/or CAT 1-29 in cells stimulated with TNFα, and this correlated with decreased phosphorylation of serine 276. To determine whether PKAc phosphorylation of p65 in the cytosol regulated nuclear translocation, serine 276 was mutated to alanine or aspartic acid. Accelerated nuclear accumulation of p65 was observed in the alanine mutant, while the aspartic acid mutation displayed slowed nuclear translocation kinetics. In addition, enhanced nuclear translocation of p65 was observed when PKAc was knocked-down by siRNA. Taken together, these results suggest that AKIP 1A acts to scaffold PKAc to NF-κB in the cytosol by protecting the phosphorylation site and thereby regulating the rate of nuclear translocation of p65.
authors.affiliation: Department of Pediatrics, University of California San Diego, La Jolla, California, United States of America.
authors.forename: Charles C
authors.initials: CC
authors.lastname: King
authors.forename: Mira
authors.initials: M
authors.lastname: Sastri
authors.forename: Philip
authors.initials: P
authors.lastname: Chang
authors.forename: Juniper
authors.initials: J
authors.lastname: Pennypacker
authors.forename: Susan S
authors.initials: SS
authors.lastname: Taylor
mesh_terms: D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D016895:Culture Media, Serum-Free; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D006801:Humans; D016328:NF-kappa B / Q000378:metabolism*; D010766:Phosphorylation; D010770:Phosphotransferases / Q000378:metabolism*; D011485:Protein Binding; D021381:Protein Transport
chemical_list: D016895:Culture Media, Serum-Free; D016328:NF-kappa B; D010770:Phosphotransferases; D017868:Cyclic AMP-Dependent Protein Kinases