Engineering firefly luciferase as an indicator of cyclic AMP‐dependent protein kinase in living cells

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Abstract
A bioluminescent indicator for protein kinase A has been developed by mutating V217 in firefly (Photinus pyralis) luciferase to R, and the C‐terminal peroxisomal signal removed by PCR. The cDNA for normal and the RRFS mutant luciferase were inserted into pSV7d and expressed in COS‐7 cells. Transient expression in approximately 5% of cells was confirmed by extraction of active luciferase, light emission from cells in the presence of luciferin, and immuno‐localisation. The cyclic‐AMP analogue, 8‐(4‐chlorophenylthio)‐cyclic AMP caused a 5–10% decrease in light emission within 4 min in COS cells expressing the RRFS mutant, but not in cells expressing normal luciferase. This provides for the first time an indicator for detecting and quantifying protein kinase A activation in living cells.