Analysis of A-kinase anchoring protein (AKAP) interaction with protein kinase A (PKA) regulatory subunits: PKA isoform specificity in AKAP binding

Compartmentalization of cAMP-dependent protein kinase (PKA) is in part mediated by specialized protein motifs in the dimerization domain of the regulatory (R)-subunits of PKA that participate in protein-protein interactions with an amphipathic helix region in A-kinase anchoring proteins (AKAPs). In order to develop a molecular understanding of the subcellular distribution and specific functions of PKA isozymes mediated by association with AKAPs, it is of importance to determine the apparent binding constants of the R-subunit-AKAP interactions. Here, we present a novel approach using surface plasmon resonance (SPR) to examine directly the association and dissociation of AKAPs with all four R-subunit isoforms immobilized on a modified cAMP surface with a high level of accuracy. We show that both AKAP79 and S-AKAP84/D-AKAP1 bind RIIα very well (apparent K D values of 0.5 and 2 nM, respectively). Both proteins also bind RIIβ quite well, but with three- to fourfold lower affinities than those observed versus RIIα. However, only S-AKAP84/D-AKAP1 interacts with RIα at a nanomolar affinity (apparent K D of 185 nM). In comparison, AKAP95 binds RIIα (apparent K D of 5.9 nM) with a tenfold higher affinity than RIIβ and has no detectable binding to RIα. Surface competition assays with increasing concentrations of a competitor peptide covering amino acid residues 493 to 515 of the thyroid anchoring protein Ht31, demonstrated that Ht31, but not a proline-substituted peptide, Ht31-P, competed binding of RIIα and RIIβ to all the AKAPs examined ( EC 50-values from 6 to 360 nM). Furthermore, RIα interaction with S-AKAP84/D-AKAP1 was competed ( EC 50 355 nM) with the same peptide. Here we report for the first time an approach to determine apparent rate- and equilibria binding constants for the interaction of all PKA isoforms with any AKAP as well as a novel approach for characterizing peptide competitors that disrupt PKA-AKAP anchoring.