Phosphatases control the duration and range of cAMP/PKA microdomains

The spatiotemporal interplay between the second messenger cyclic AMP (cAMP) and its main effector, Protein Kinase A (PKA) is crucial for the pleotropic nature of this cascade. To maintain a high degree of specificity, the cAMP/PKA axis is organized into functional multiprotein complexes, called microdomains, precisely distributed within the cell. While the subcellular allocation of PKA is guaranteed by a family of tethers called A-Kinase-anchoring Proteins (AKAPs), the mechanisms underlying the efficient confinement of a microdomain’s functional effects are not fully understood. Here we used FRET-based sensors to detect cAMP levels and PKA-dependent phosphorylation within specific subcellular compartments and found that, while free cAMP is responsible for the activation of local PKA enzymes, the dephosphorylating actions of phosphatases dictate the duration of the microdomain’s effects. To test the range of action of PKA microdomains we used rigid aminoacidic nanorulers to distance our FRET sensors from their original location for 10 or 30 nm. Interestingly, we established that cAMP levels do not affect the spatial range of the microdomain while on the contrary, phosphatase activity acts as the main functional boundary for phosphorylated PKA targets. Our findings contribute to the design of a picture where two microdomain-forming events have distinct roles. Cyclic AMP elevations trigger the initial activation of subcellular PKA moieties, while the temporal and spatial extent of the PKA’s actions is regulated by phosphatases.