Isoform Specific Differences in Binding of a Dual-Specificity A-Kinase Anchoring Protein to Type I and Type II Regulatory Subunits of PKA

Dual-specificity AKAPs bind to type I (RI) and type II (RII) regulatory subunits of cAMP-dependent protein kinase A (PKA), potentially recruiting distinct cAMP responsive holoenzymes to a given intracellular location. To understand the molecular basis for this “dual” functionality, we have examined the pH-dependence, the salt-dependence, and the kinetics of binding of the A-kinase binding (AKB) domain of D-AKAP2 to the regulatory subunit isoforms of PKA. Using fluorescence anisotropy, we have found that a 27-residue peptide corresponding to the AKB domain of D-AKAP2 bound 25-fold more tightly to RIIα than to RIα. The higher affinity for RIIα was the result of a slower off-rate as determined by surface plasmon resonance. The high-affinity interaction for RIα and RIIα was pH-independent from pH 7.4 to 5.0. At pH 4.0, both isoforms had a reduction in binding affinity. Additionally, binding of the AKB domain to RIα was independent of solution ionic strength, whereas RIIα had an increased binding affinity at higher ionic strength. This suggests that the relative energetic contribution of the charge stabilization is different for the two isoforms. This prediction was confirmed by mutagenesis in which acidic mutations, primarily of E10 and D23, in the AKB domain affected binding to RIα but not to RIIα. These isoform-specific differences provide a foundation for developing isoform-specific peptide inhibitors of PKA anchoring by dual-specificity AKAPs, which can be used to evaluate the physiological significance of dual-specificity modes of PKA anchoring.