AKAP Signaling Islands: Venues for Precision Pharmacology

Abstract

Regulatory enzymes often have different roles in distinct subcellular compartments. Yet, most drugs indiscriminately saturate the cell. Thus, subcellular drug-delivery holds promise as a means to reduce off-target pharmacological effects. A-kinase anchoring proteins (AKAPs) sequester combinations of signaling enzymes within subcellular microdomains. Targeting drugs to these 'signaling islands' offers an opportunity for more precise delivery of therapeutics. Here, we review mechanisms that bestow protein kinase A (PKA) versatility inside the cell, appraise recent advances in exploiting AKAPs as platforms for precision pharmacology, and explore the impact of methodological innovations on AKAP research.

Keywords: drug targeting; protein kinase anchoring; scaffold proteins; subcellular compartmentalization.

Figure 1.. Active PKA Is Spatially Constrained.

(A) Structural model of an AKAP18-bound type II PKA holoenzyme. An RIIα (green) dimer is bound to two catalytic subunits (blue) and anchored to AKAP18 (gold) (adapted, with permission, from [19]). (B) Native mass spectrometry of RII+C+AKAP79 (297–427) complexes. In the presence of excess cAMP, the predominant species identified all involve PKAc still bound to R subunits. (C) Förster resonance energy transfer (FRET) measurements from cells expressing RII–CFP and PKAc–YFP. The β-adrenergic agonist isoproterenol was administered to all conditions. Supraphysiological accumulation of cAMP is induced by treatment with the PDE3 inhibitor rolipram. (D) Upon delivery of the agonist (cAMP stimulation) under physiological conditions (green), a minimal loss of FRET indicates that the PKA holoenzyme remains largely intact. By contrast, upon supraphysiologic accumulation of cAMP (grey) loss of FRET signal indicates pronounced dissociation of the R:C interface. (B–D adapted , with permission, from [20]). Abbreviations: AKAP, A-kinase anchoring protein; C, catalytic subunit; PKA, protein kinase A; PKAc, PKA catalytic subunit; R, receptor; stim, stimulation.

Figure 2.. AKAPs Recruit PKA to Distinct Subcellular Locations to Expand Enzyme Versatility.

(A) immunofluorescent labeling of mitochondrial D-AKAP1 (cyan) and proximity ligation detection of D-AKAP1–PKA signaling islands (magenta). (B) AKAP signaling islands are found in distinct signaling compartments throughout the cell. Each AKAP localizes to a specific subcellular domain and associates with unique groupings of signaling molecules. Abbreviations: AKAP, A-kinase anchoring protein; PKA, protein kinase A.

Figure 3.. Identifying AKAPs in Cells and Tissues.

(A) RII overlay shows binding of RII probe to AKAP helices after SDS-PAGE and immobilization on a membrane. The left lane was preincubated with the AKAP disruptor peptide Ht31 prior to RII incubation. Arrows indicate subsequently identified AKAPs in the right lane. (Images adapted from [76]). (B) Diagram depicting a proximity biotinylation experiment using an AKAP as the bait molecule fused to the promiscuous biotin ligase miniTurbo. Once labeled, the proteins are isolated and subjected to denaturing wash conditions. (C) Mass spectrometry identification and pathway analysis can yield protein interaction networks and candidate cell processes. Network depicted represents proteins that associate significantly more with a nuclear AKAP18 isoform versus a cytosolic isoform. Blue nodes indicate proteins involved in RNA splicing (Figure adapted from [42]). Abbreviations: AKAP, A-kinase anchoring protein; PKA, protein kinase A; R, receptor.