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 resists
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.