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.

A‐kinase anchoring proteins (AKAPs) play a critical role in signaling by targeting cAMP dependent signaling complexes to specific cellular sites. Gravin, a multivalent AKAP that targets PKA and possibly PDE4 to the cell membrane, undergoes redistribution to the cytosol in response to elevated [Ca2+]i suggesting it may play a role in cross‐talk between Ca2+ and cAMP dependent pathways. We have shown previously that gravin redistribution alters PKA activity in cytosolic and membrane regions, but the effect of gravin redistribution on cAMP levels in these compartments is unknown. To begin assessing this, we used a FRET based cAMP biosensor (lck‐EPAC2‐camps) targeted to the membrane to determine the effect of gravin expression on cAMP levels. Treatment of cells expressing the biosensor and gravin with IBMX showed a greater increase in cAMP levels compared to cells lacking gravin. In addition, cells expressing gravin and the biosensor, showed a significantly higher increase in cAMP when treated with rolipram (PDE4)/forskolin compared to cilostamide (PDE3)/forskolin. These experiments indicate that gravin expression affects cAMP levels at the membrane following PDE inhibition and sets the stage for determining the role of gravin redistribution in regulating cytosolic and membrane cAMP levels. EPAC2‐camps was a gift from Drs. V. Nikolaev and M. Lohse. Supported by NIH grants 1P30GM103329 and 1S100D016250‐01.

Scaffold proteins play a critical role in cellular homeostasis by positioning signaling molecules in proximity to downstream effectors. However, some scaffold proteins do not assemble static enzyme complexes, but form dynamic signalosomes that traffic to different subcellular compartments in response to stimuli. Gravin (AKAP12) ‐ a multivalent scaffold linked to cell cycle progression, cellular migration, β2‐adrenergic receptor regulation, and hippocampal learning and memory ‐ anchors PKA and other enzymes to the plasma membrane but is redistributed to the cytosol after [Ca2+]i elevation. We postulate that gravin redistribution represents a novel mechanism for cross‐talk between Ca2+‐dependent and cAMP‐dependent signaling pathways. To assess this, we measured the impact of gravin localization on compartmentalized PKA activity using the PKA FRET biosensor AKAR3. Expression of gravin in cells lacking endogenous gravin caused an increase in forskolin‐dependent PKA activity in AKAR3 constructs targeted to the plasma membrane when compared to control cells lacking gravin or expressing a gravin construct lacking the PKA‐binding domain. Expression of a gravin mutant with reduced membrane localization showed no increase in membrane PKA activity compared to control cells lacking gravin. Interestingly, gravin expression caused a decrease in forskolin‐dependent PKA activity in cytosolic AKAR3 constructs compared to control cells lacking gravin. These results confirm that gravin localization regulates PKA signaling and support the hypothesis that gravin mediates cross‐talk between Ca2+ and cAMP‐dependent signaling in functions such as in cell cycle progression, cellular migration, β2‐adrenergic receptor regulation, and hippocampal learning and memory. Based on these results, dynamics in scaffold protein localization likely represents an important paradigm for the regulation of cellular signaling networks. Grant Funding Source: NIH P30GM103329