title: Analysis of A-kinase anchoring protein (AKAP) interaction with protein kinase A (PKA) regulatory subunits: PKA isoform specificity in AKAP binding.
abstract: 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 RIIalpha very well (apparent K(D) values of 0.5 and 2 nM, respectively). Both proteins also bind RIIbeta quite well, but with three- to fourfold lower affinities than those observed versus RIIalpha. However, only S-AKAP84/D-AKAP1 interacts with RIalpha at a nanomolar affinity (apparent K(D) of 185 nM). In comparison, AKAP95 binds RIIalpha (apparent K(D) of 5.9 nM) with a tenfold higher affinity than RIIbeta and has no detectable binding to RIalpha. 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 RIIalpha and RIIbeta to all the AKAPs examined (EC(50)-values from 6 to 360 nM). Furthermore, RIalpha 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.
authors.affiliation: Ruhr-Universität Bochum, Bochum, 44801, Germany. W.Herberg@ruhr-uni-bochum.de
authors.forename: F W
authors.initials: FW
authors.lastname: Herberg
authors.forename: A
authors.initials: A
authors.lastname: Maleszka
authors.forename: T
authors.initials: T
authors.lastname: Eide
authors.forename: L
authors.initials: L
authors.lastname: Vossebein
authors.forename: K
authors.initials: K
authors.lastname: Tasken
mesh_terms: D054758:A Kinase Anchor Proteins; D048868:Adaptor Proteins, Signal Transducing; D001667:Binding, Competitive; D002352:Carrier Proteins / Q000378:metabolism*; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry* / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins; D047908:Intracellular Signaling Peptides and Proteins; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008565:Membrane Proteins / Q000378:metabolism*; D009687:Nuclear Proteins / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism; D010455:Peptides / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013379:Substrate Specificity; D020349:Surface Plasmon Resonance; D013816:Thermodynamics
chemical_list: D054758:A Kinase Anchor Proteins; C105137:AKAP1 protein, human; C487775:AKAP5 protein, human; C490117:AKAP8L protein, human; D048868:Adaptor Proteins, Signal Transducing; C486332:Akap1 protein, rat; C415703:Akap5 protein, rat; D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D036341:Intercellular Signaling Peptides and Proteins; D047908:Intracellular Signaling Peptides and Proteins; D007527:Isoenzymes; D008565:Membrane Proteins; D009687:Nuclear Proteins; D010446:Peptide Fragments; D010455:Peptides; D011993:Recombinant Fusion Proteins; C400087:S Ht31; C067297:8-aminohexylamino cAMP; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases