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FRET biosensors reveal AKAP‐mediated shaping of subcellular PKA activity and a novel mode of Ca 2+ /PKA crosstalk
Author(s) -
Schott Micah,
Grove Bryon
Publication year - 2015
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.29.1_supplement.558.5
Subject(s) - forskolin , crosstalk , protein kinase a , cytosol , microbiology and biotechnology , chemistry , biophysics , intracellular , kinase , biochemistry , enzyme , receptor , biology , physics , optics
A‐Kinase Anchoring Proteins (AKAPs) serve as scaffolds for cAMP‐dependent Protein Kinase (PKA) and other signaling enzymes within specific subcellular compartments. However, some AKAPs are not static anchors, but form dynamic signaling complexes that can traffic to different subcellular compartments when triggered. Gravin (AKAP12) is a multivalent AKAP that anchors PKA and other enzymes to the plasma membrane but redistributes to the cytosol upon intracellular Ca 2+ elevation. Through the redistribution of gravin, we postulate that Ca 2+ may regulate the interaction of PKA with downstream substrates, thus representing a novel crosstalk mechanism between Ca 2+ and PKA‐dependent pathways. To assess this, we measured the impact of gravin‐V5/His expression on compartmentalized PKA activity using the PKA FRET biosensor AKAR3. In cultured AN3 CA cells, which lack endogenous gravin, expression of gravin‐V5/His increased forskolin‐stimulated PKA activity in AKAR3 constructs targeted to the plasma membrane compared to control cells lacking gravin or expressing a ΔPKA‐gravin construct missing the PKA‐binding domain. In contrast, gravin‐V5/His expression decreased forskolin‐stimulated PKA activity in cytosolic AKAR3 constructs. A mutant gravin construct with inhibited membrane localization had no effect on membrane and cytosolic PKA activity. Finally, pretreatment with the calcium‐elevating agent thapsigargin caused the redistribution of gravin and prevented the gravin‐mediated increase in plasma membrane PKA activity following forskolin or isoproterenol treatment. These results reveal that Ca 2+ alters receptor‐mediated PKA activity through gravin redistribution and supports the hypothesis that gravin mediates crosstalk between Ca 2+ and PKA‐dependent pathways.