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

AKAR2-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.The 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.The discovery of a class of scaffold proteins that harbor a binding site for the regulatory subunits (i.e., RI/RII) of cyclic AMP-dependent protein kinase A (PKA, A-kinase) was seminal in our understanding of the roles of these A-Kinase Anchoring Proteins (AKAPs), in many facets of cellular signaling [1]. AKAPs not only dock PKA, but also can act as molecular "tool boxes" that capable of docking protein kinases other than PKA (including protein kinase C, PKC, and the Src-family tyrosine kinases), phosphoprotein phosphatases (such as protein phosphatase-2B), and adaptor molecules [2]. AKAPs participate dynamically in such large, macromolecular signaling complexes that can include not only protein kinases and phosphoprotein phosphatases, but also phosphodiesterases (PDE), adaptor molecules (like Grb2), ion channels, and members of the superfamily of G protein-coupled receptors (GPCR) [3]. Two AKAPs, AKAP12 (as known as AKAP250, gravin, and SSECKS) and AKAP5 associate with the beta-adrenergic receptors and have been the focus of intense research [2-6]. Human epidermoid carcinoma A431 cells express AKAP12 and a full complement of the prototypic GPCR, the beta2-adrenergic receptor [7-10]. A431 cells have been well-characterized with respect to PKA-based cell signaling [7-10] and were adopted for the current studies.One of the major obstac