TY - JOUR

T1 - Changes in intracellular cAMP reported by a Redistribution assay using a cAMP-dependent protein kinase-green fluorescent protein chimera

AU - Almholt, Kasper

AU - Tullin, Søren

AU - Skyggebjerg, Ole

AU - Scudder, Kurt

AU - Thastrup, Ole

AU - Terry, Robert

PY - 2004

Y1 - 2004

N2 - We report on a novel method to monitor changes in intracellular cAMP concentration ([cAMP]i) within intact living cells using a chimeric fusion of the catalytic subunit of cAMP-dependent protein kinase to green fluorescent protein (PKAcat-GFP). In stably transfected unstimulated fibroblasts, fusion protein fluorescence is highly concentrated in aggregates throughout the cytoplasm and absent in the nucleus. Elevation of [cAMP]i disperses GFP fluorescence from the cytoplasmic aggregates within minutes. Spot-photobleach measurements show that the rate of exchange of GFP-labeled catalytic subunits at these aggregates increases in proportion to [cAMP]i. For any given stimulus, the response curve for dispersal of GFP fluorescence from aggregates agrees closely with the increase in total [cAMP]i as measured by standard in vitro methods (SPA). The redistribution of fluorescence is completely reversible: reduction of [cAMP]i results in return of fluorescence to the cytoplasmic aggregates. Consistent behaviour of PKAcat-GFP is seen in different cell backgrounds. We demonstrate that PKA Redistribution assays are suitable for measurement of changes in [cAMP]i brought about by both Gs- and Gi-protein-coupled receptor stimulation as well as by inhibition of cAMP phosphodiesterases.

AB - We report on a novel method to monitor changes in intracellular cAMP concentration ([cAMP]i) within intact living cells using a chimeric fusion of the catalytic subunit of cAMP-dependent protein kinase to green fluorescent protein (PKAcat-GFP). In stably transfected unstimulated fibroblasts, fusion protein fluorescence is highly concentrated in aggregates throughout the cytoplasm and absent in the nucleus. Elevation of [cAMP]i disperses GFP fluorescence from the cytoplasmic aggregates within minutes. Spot-photobleach measurements show that the rate of exchange of GFP-labeled catalytic subunits at these aggregates increases in proportion to [cAMP]i. For any given stimulus, the response curve for dispersal of GFP fluorescence from aggregates agrees closely with the increase in total [cAMP]i as measured by standard in vitro methods (SPA). The redistribution of fluorescence is completely reversible: reduction of [cAMP]i results in return of fluorescence to the cytoplasmic aggregates. Consistent behaviour of PKAcat-GFP is seen in different cell backgrounds. We demonstrate that PKA Redistribution assays are suitable for measurement of changes in [cAMP]i brought about by both Gs- and Gi-protein-coupled receptor stimulation as well as by inhibition of cAMP phosphodiesterases.

KW - 3',5'-Cyclic-AMP Phosphodiesterases

KW - Animals

KW - CHO Cells

KW - Cell Nucleus

KW - Cells, Cultured

KW - Cricetinae

KW - Cricetulus

KW - Cyclic AMP

KW - Cyclic AMP-Dependent Protein Kinases

KW - Cytoplasm

KW - Enzyme Activation

KW - Green Fluorescent Proteins

KW - HeLa Cells

KW - Humans

KW - Microscopy, Fluorescence

KW - Recombinant Fusion Proteins

U2 - 10.1016/j.cellsig.2004.01.006

DO - 10.1016/j.cellsig.2004.01.006

M3 - Journal article

C2 - 15157670

VL - 16

SP - 907

EP - 920

JO - Cellular Signalling

JF - Cellular Signalling

SN - 0898-6568

IS - 8

ER -