Defining sites of interaction on PKAc with AKIP 1A.
A) Fifteen-residue peptides covering the catalytic domain (amino acids 1-38) of PKAc were staggered by one amino acid, spotted onto a membrane, and probed with AKIP1 splice variants proteins. Binding was detected with anti-AKIP1 antibody. The blot showed differential binding to the splice variants with the most intense binding to AKIP 1A, the full length protein. The residues in orange are weak binding and in red the strongest binding peptides. B) The amino terminal helix of PKAc is highlighted with exposed amino acids facing out towards the AKIP 1A binding surface: these amino acids may be important for the interaction with AKIP 1. C) HEK 293 cells were transfected with GST-AKIP 1A in the presence of the 1-29 PKAc disruptor peptide. Scrambled 1-29 peptide and a proline mutant at positions 20 and 22 to disrupt subsequent integrity of 1-29 helical structure served as controls. After 24 h, cells harvested and subjected to GST pull downs and immunoblotting showed that AKIP1/PKAc interaction was disrupted by the 1-29 peptide but not the two controls. D) Quantification of the amount of the relative amount of PKAc bound to GST-AKIP 1A. E) HeLa cells were transfected with Myc-AKIP 1A in the presence or absence of either CAT 1-29 or scrambled CAT 1-29. Cells were stimulated with 8-CPT-cAMP (50 µM) or TNFα (1 ng/ml) for 1 hour, fractionated to obtain nuclear enriched fractions and nuclear PKAc levels were measured by Western blot and quantified.