Molecular Determinants and Signaling Effects of PKA RIα Phase Separation

Spatiotemporal regulation of intracellular signaling molecules, such as the 3,5-cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA), ensures the specific execution of various cellular functions. Liquid-liquid phase separation (LLPS) of the ubiquitously expressed PKA regulatory subunit RI was recently identified as a major driver of cAMP compartmentation and signaling specificity. However, the molecular determinants of RI LLPS remain unclear. Here, we reveal that two separate dimerization interfaces combined with the cAMP-induced release of the PKA catalytic subunit (PKA-C) from the pseudosubstrate inhibitory sequence are required to drive RI condensate formation in cytosol, which is antagonized by docking to A-kinase anchoring proteins. Strikingly, we find that the RI pseudosubstrate region is critically involved in the formation of a non-canonical R:C complex, which serves to maintain low basal PKA activity in the cytosol by enabling the recruitment of active PKA-C to RI condensates. Our results suggest that RI LLPS not only facilitates cAMP compartmentation but also spatially restrains active PKA-C, thus highlighting the functional versatility of biomolecular condensates in driving signaling specificity.