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 proper cellular function. Liquid-liquid phase separation (LLPS) of the ubiquitous PKA regulatory subunit RIα promotes cAMP compartmentation and signaling specificity. However, the molecular determinants of RIα LLPS remain unclear. Here, we reveal that two separate dimerization interfaces, combined with the cAMP-induced unleashing of the PKA catalytic subunit (PKA-C) from the pseudosubstrate inhibitory sequence, drive RIα condensate formation in the cytosol of mammalian cells, which is antagonized by docking to A-kinase anchoring proteins. Strikingly, we find that the RIα pseudosubstrate region is critically involved in forming a non-canonical R:C complex, which recruits active PKA-C to RIα condensates to maintain low basal PKA activity in the cytosol. Our results suggest that RIα 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.

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 RIα was recently identified as a major driver of cAMP compartmentation and signaling specificity. However, the molecular determinants of RIα 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 RIα condensate formation in cytosol, which is antagonized by docking to A-kinase anchoring proteins. Strikingly, we find that the RIα 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 RIα condensates. Our results suggest that RIα 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.

Measuring Spatiotemporal cAMP Dynamics Within an Endogenous Signaling Compartment Using FluoSTEP-ICUE.

cAMP is a ubiquitous second messenger involved in the regulation of diverse cellular processes. Spatiotemporal regulation of cAMP through compartmentalization within various subcellular microdomains is essential to ensure specific signaling. In the following protocol, we describe a method for directly visualizing signaling dynamics within cAMP microdomains using fluorescent sensors targeted to endogenous proteins (FluoSTEPs). Instead of overexpressing a biosensor-tagged protein of interest to target a microdomain, FluoSTEP Indicator of cAMP using Epac (FluoSTEP-ICUE) utilizes spontaneously complementing split GFP and CRISPR-Cas9 genome editing to localize a FRET-based cAMP biosensor to an endogenously expressed protein of interest. Utilizing this approach, FluoSTEP-ICUE can be used to measure cAMP levels within endogenous signaling compartments, thus providing a powerful tool for studying the spatiotemporal regulation of cAMP signaling.