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

Abstract

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.

Protein kinase A‐mediated phosphorylation of naked cuticle homolog 2 stimulates cell‐surface delivery of transforming growth factor‐α for epidermal growth factor receptor transactivation

“…AKAP12 was a logical candidate to examine because it was the only AKAP detected in a comprehensive proteomic analysis of NKD2‐associated vesicles . Interaction of AKAP12 and PKA has been reported previously . In Figure A, we show that NKD2 and AKAP12 interact by reciprocal co‐IP in HEK293 cells transiently expressing untagged NKD2 and AKAP12‐EGFP.…”

“…29 Interaction of AKAP12 and PKA has been reported previously. [30][31][32] In Figure 5A, we show that NKD2 and AKAP12 interact by reciprocal co-IP in HEK293 cells transiently expressing untagged NKD2 and AKAP12-EGFP. We also show that the two proteins colocalize in cytoplasmic puncta and at the plasma membrane in MDCK cells transiently expressing NKD2-EGFP and AKAP12-mCherry ( Figure 5B).…”

Potential for therapeutic targeting of AKAP signaling complexes in nervous system disorders

“…These mechanisms include the rapid redistribution of Gravin-PKA to intracellular membrane compartments following stimulation of PKC (Lin et al, 1996; Wassler et al, 2001; Yan et al, 2009; Schott & Grove, 2013) or in response to increases in intracellular Ca 2+ that activate Ca 2+ -CaM. The interaction between Ca 2+ -CaM and gravin occurs at the CB4 putative CaM binding site and is thought to play a key role in Ca 2+ -CaM-dependent redistribution (Schott & Grove, 2013; Schott et al, 2016). Mechanisms such as these that direct the signaling complex to intracellular compartments may serve to limit PKA activity at the cell membrane.…”

cAMP: a multifaceted modulator of immune synapse assembly and T cell activation

“…AKAPs are the mainstay of PKA-mediated regulation, as they are responsible for bringing PKA in close proximity to specific substrates among the plethora that coexist within the cell [80][81][82]. In T lymphocytes, AKAPs are responsible for the dynamics of PKA during IS assembly.…”