FRET biosensors reveal AKAP-mediated shaping of subcellular PKA activity and a novel mode of Ca2+/PKA crosstalk

• Published in: Cellular signalling Vol. 28; no. 4; pp. 294 - 306
• Main Authors: Schott, Micah B., Gonowolo, Faith, Maliske, Benjamin, Grove, Bryon
• Format: Journal Article
• Language: English
• Published: Elsevier Inc 01.04.2016
• Subjects: A-kinase anchoring protein; AKAR3; Calcium; Gravin; Isoproterenol; Protein kinase A; SES; Protein kinase A; Calcium; AKAP; ECFP; cAMP; CFP; EGFP; Isoproterenol; CaM; AKAR3; Tg; FRET; WT; EYFP; β2AR; Fsk; PB1-3; [Ca2+]i; Iso; SSeCKS; IBMX; PDE4; PKA; PKC; RII; CB4; GPCR; kD; A-kinase anchoring protein; Gravin; NES; PM
• ISSN: 0898-6568; 1873-3913
• DOI: 10.1016/j.cellsig.2016.01.001

Scaffold proteins play a critical role in cellular homeostasis by anchoring signaling enzymes in close proximity to downstream effectors. In addition to anchoring static enzyme complexes, some scaffold proteins also form dynamic signalosomes that can traffic to different subcellular compartments upon stimulation. Gravin (AKAP12), a multivalent scaffold, anchors PKA and other enzymes to the plasma membrane under basal conditions, but upon [Ca2+]i elevation, is rapidly redistributed to the cytosol. Because gravin redistribution also impacts PKA localization, we postulate that gravin acts as a calcium “switch” that modulates PKA-substrate interactions at the plasma membrane, thus facilitating a novel crosstalk mechanism between Ca2+ and PKA-dependent pathways. To assess this, we measured the impact of gravin-V5/His expression on compartmentalized PKA activity using the FRET biosensor AKAR3 in cultured cells. Upon treatment with forskolin or isoproterenol, cells expressing gravin-V5/His showed elevated levels of plasma membrane PKA activity, but cytosolic PKA activity levels were reduced compared with control cells lacking gravin. This effect required both gravin interaction with PKA and localization at the plasma membrane. Pretreatment with calcium-elevating agents thapsigargin or ATP caused gravin redistribution away from the plasma membrane and prevented gravin from elevating PKA activity levels at the membrane. Importantly, this mode of Ca2+/PKA crosstalk was not observed in cells expressing a gravin mutant that resisted calcium-mediated redistribution from the cell periphery. These results reveal that gravin impacts subcellular PKA activity levels through the spatial targeting of PKA, and that calcium elevation modulates downstream β-adrenergic/PKA signaling through gravin redistribution, thus supporting the hypothesis that gravin mediates crosstalk between Ca2+ and PKA-dependent signaling pathways. Based on these results, AKAP localization dynamics may represent an important paradigm for the regulation of cellular signaling networks. •The FRET biosensor AKAR3 was used to investigate gravin's impact on PKA activity.•Gravin elevated plasma membrane PKA activity and reduced cytosolic PKA activity.•Gravin is redistributed away from the cell periphery upon Ca2+ elevation.•Ca2+ elevation reduced plasma membrane PKA activity through gravin redistribution.