Abstract
Hormones can transmit signals through adenosine 3',5'-monophosphate (cAMP) to precise intracellular locations. The fidelity of these responses relies on the activation of localized protein kinase A (PKA) holoenzymes. Association of PKA regulatory type II (RII) subunits with A-kinase-anchoring proteins (AKAPs) confers location, and catalytic (C) subunits phosphorylate substrates. Single-particle electron microscopy demonstrated that AKAP79 constrains RII-C subassemblies within 150 to 250 angstroms of its targets. Native mass spectrometry established that these macromolecular assemblies incorporated stoichiometric amounts of cAMP. Chemical-biology- and live cell-imaging techniques revealed that catalytically active PKA holoenzymes remained intact within the cytoplasm. These findings indicate that the parameters of anchored PKA holoenzyme action are much more restricted than originally anticipated.