ENGINEERING FIREFLY LUCIFERASE AS AN INDICATOR OF CYCLIC AMP-DEPENDENT PROTEIN-KINASE IN LIVING CELLS

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.