PKA: Assembly of Dynamic Macromolecular Signaling Scaffolds

Although we have learned a great deal about the protein kinase superfamily from our structure/function studies of the free PKA catalytic (C) subunit and the free cAMP-bound regulatory (R) subunits, these studies did not allow us to understand how the C-subunit was actually inhibited nor did we understand at the molecular level how the holoenzymes were activated by cAMP. Four functionally non-redundant PKA R-subunits (RIα, RIβ, RIIα, and RIIβ) are assembled as a dimer. The N-terminal dimerization/docking (D/D) is followed by a flexible linker and two tandem cAMP binding domains. The D/D domain also serves as the docking site for A Kinase Anchoring Proteins (AKAPs) which function as scaffolds that target the PKA holoenzyme to specific sites in the cell in close proximity to selected substrates. Isoform diversity of the R-subunits is a major mechanism for achieving specificity in PKA signaling. We describe here, for the first time, how PKA is assembled into diverse tetrameric holoenzymes. This allows us to begin to fully appreciate the role of the R-subunits in creating novel tetramers that are then recruited in unique ways to supramolecular complexes where they are poised to phosphorylate nearby substrates. How these scaffolds are then assembled as part of polyvalent PKA signaling scaffolds is our next challenge. By showing how the RII subunit is anchored to a PDZ domain through a dual specific AKAP, DAKAP2, we are beginning to understand how larger scaffolds are assembled and anchored to the C-termini of ion transporters. We are also exploring PKA signaling scaffolds at the mitochondria.