AKAR2-AKAP12 fusion protein biosenses dynamic phosphorylation and localization of a GPCR-based scaffold

摘要

BackgroundThe cAMP-dependent protein kinase A (PKA) plays a pivotal role in virtually all cells, there being a multitude of important target molecules that are substrates for PKA in cell signaling. The spatial-temporal dynamics of PKA activation in living cells has been made accessible by the development of clever biosensors that yield a FRET signal in response to the phosphorylation by PKA. AKAR2 is genetically encoded fluorescent probe that acts as a biosensor for PKA activation. AKAP12 is a scaffold that docks PKA, G-protein-coupled receptors, cell membrane negatively-charged phospholipids, and catalyzes receptor resensitization and recycling. In the current work, the AKAR2 biosensor was fused to the N-terminus of AKAP12 to evaluate its ability to function and report on dynamic phosphorylation of the AKAP12 scaffold.