Molecular basis for isoform-specific targeting of PKA

cAMP-dependent protein kinase (PKA) mediates physiological responses through phosphorylation of protein substrates. Two major types of PKA (I and II) are distinguished by inhibitory, regulatory (R) subunits, which are similar in domain organization, but functionally non-redundant. Here we examine the molecular basis for targeting RI and RII- isoforms of PKA to two dual-specific A Kinase Anchoring Proteins (AKAPs), D-AKAP1 and D-AKAP2. Although D-AKAP1 has multiple splice variants, the A Kinase Binding (AKB) domain was localized to residues 317-425 in the conserved core and, based on surface plasmon resonance, binds tightly to RI? and RII? (21 nM and 3nM). H/D exchange coupled with mass spectrometry showed the AKB domain was fully solvent-exposed, but RI and RII binding showed protection along a putative, ampipathic helix, defining a common interaction site for both isoforms. To define the molecular determinants for RII targeting, we solved the crystal structure of the N-terminal dimerization/docking (D/D) domain of RII? bound to a 21-residue peptide of D- AKAP2. This structure revealed a novel targeting mechanism that requires the ordering of one flexible N-terminal tail of RII?, upon AKAP binding. The two anti-parallel chains of the D/D domain form a stable hydrophobic core and the flexible tail is recruited to bolster the hydrophobic docking site. The D-AKAP2 peptide presents a hydrophobic ridge, enriched with branched side chains for docking. To further define isoform-diversity, I characterized the N- terminal disulfide bonds of RI?, a unique feature of RI- subunits. Mutagenesis showed the disulfide bonds were not required for dimerization or AKAP binding. Cys37 and Tyr19, however, were essential for AKAP binding. Tyr19 likely contributes to dimer packing and promoting disulfide bonding. Analysis of the NMR structures for RI? D/D domain suggests that RI may employ a mechanism similar to RII, but recruitment of the N-terminus may be regulated by the geometry and dynamics surrounding the disulfide bond. I also crystallized RI? D/D domain free and bound to the D- AKAP2 peptide used for the RII? structure. The crystals diffracted to 2.0 Å and we are now completing the phasing. These structures will elucidate how the flexible N- terminus contributes to RI targetin