Kinase-anchoring proteins and the intracellular targeting of cyclic adenosine monophosphate (cAMP)-dependent protein kinase
thesisposted on 15.12.2014, 10:35 by Alison Jane Davis
Kinase anchoring proteins (AKAPs) are a family of structurally diverse multidomain proteins classified solely by their ability to bind cAMP-dependent protein kinase/protein kinase A (PKA). We show that the prototypic AKAP, AKAP79, targets PKA to the plasma membrane. This subcellular localisation is mediated via interactions between basic regions on AKAP79 and acidic phospholipids in the plasma membrane. We show that while PKA and protein kinase C (PKC)-dependent phosphorylation of AKAP79 targeting residues has no effect on its subcellular localization, Gq-coupled receptor-driven depletion of the lipid anchor PtdIns(4,5)P2 causes release of AKAP79 from the membrane. Receptor-mediated regulation of AKAP membrane binding may be an important feedback mechanism controlling the degree of access that AKAP-bound enzymes have to membrane-associated substrates. Real-time confocal imaging failed to show significant redistribution of AKAP79 following Gq-coupled receptor activation, which may reflect only limited short-range movements.;In addition to regulated association with membrane, we also show through co-immunoprecipitation studies a direct interaction between AKAP79 and all three members of the inwardly rectifying potassium channel Kir2 family. Using a combination of deletion analysis and structural modelling we show that AKAP79 binds the Kir2 family via a novel binding motif located on the C terminus of the ion channel and not via a leucine zipper as has been reported for other AKAP-channel interactions. Interestingly, we also show using a PKA regulatory subunit overlay assays that this region of Kir2.1 is capable of isolating other, potentially novel, AKAPs from rat brain and heart homogenates.;Finally, we use whole-cell patch clamp analysis to demonstrate that cAMP-dependent modulation of Kir2.1 channel activity is significantly enhanced in the presence of AKAP79, thus confirming an important role for AKAP79 in targeting PKA to key channel phosphorylation sites.