Abstract
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.ResultsAKAR2-AKAP12 can be expressed in mammalian cells, is fully functional, and reveals the spatial-temporal activation of AKAP12 undergoing phosphorylation by PKA in response to beta-adrenergic activation in human epidermoid carcinoma A431 cells.ConclusionThe dynamic phosphorylation of AKAP12 "biosensed" by AKAR2-AKAP12 reveals the scaffold in association with the cell membrane, undergoing rapid phosphorylation by PKA. The perinuclear, cytoplasmic accumulation of phosphorylated scaffold reflects the phosphorylated, PKA-activated form of AKAP12, which catalyzes the resensitization and recycling of desensitized, internalized G-protein-coupled receptors.
Highlights
The 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
It is the first time that a biosensor has been fused with a scaffold molecule in an attempt to yield a biosensor fusion protein designed to detect the phosphorylation and activation of a targeted PKA substrate, in this case AKAP12
The AKAR2AKAP12 displays full ability to function in resensitization and recycling of G protein-coupled receptors (GPCR) to the cell membrane, once they have undergone activation/desensitization
Summary
The 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. AKAPs participate dynamically in such large, macromolecular signaling complexes that can include protein kinases and phosphoprotein phosphatases, and phosphodiesterases (PDE), One of the major obstacles in understanding the precise functions of signaling molecules like AKAP scaffolds is the inability to ascertain the spatial-temporal dynamics of the molecules during activation of a signaling pathway [11]. The advent of powerful "biosensors" that can report on changes in function as a result of activation of a kinase (e.g., AKAR/AKAR2 reporting phosphorylation events catalyzed by PKA) provide a novel avenue with which to study of the spatial-temporal signaling of PKA [12]. We characterize a fusion construct that retains all the functional capability of the AKAP12, but can, by virtue of its AKAR2 moiety, report as a biosensor on the phosphorylation of AKAR2-AKAP12 in living cells
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