Isoform-specific PKA dynamics revealed by dye-triggered aggregation and DAKAP1α-mediated localization in living cells

The tetracysteine sequence YRECCPGCCMWR fused to the N terminus of green fluorescent protein (GFP) self-aggregates upon biarsenical labeling in living cells or in vitro. Such dye-triggered aggregates form temperature-dependent morphologies and are dispersed by photobleaching. Fusion of the biarsenical aggregating GFP to the regulatory (R) or catalytic (C) subunit of PKA traps intact holoenzyme in compact fluorescent puncta upon biarsenical labeling. Contrary to the classical model of PKA activation, elevated cAMP does not allow Rl alpha and C alpha to diffuse far apart unless the pseudosubstrate inhibitor PKI or locally concentrated substrate is coexpressed. However, Rll alpha releases Ca upon elevated cAMP alone, dependent on autophosphorylation of the Rlla inhibitory domain. DAKAPI a overexpression induced R and C outer mitochondrial colocalization and showed similar regulation. Overall, effective separation of type I PKA is substrate dependent, whereas type II PKA dissociation relies on autophosphorylation.