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.
Sala-Newby
G
G
Department of Medical Biochemistry, University of Wales College of Medicine, Heath Park, Cardiff, UK.
Campbell
A K
AK
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000494:pharmacology; D004247:DNA; D005455:Fluorescent Antibody Technique; D007313:Insecta; D007700:Kinetics; D008156:Luciferases / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D015202:Protein Engineering; D011494:Protein Kinases / Q000032:analysis*; D013873:Thionucleotides / Q000494:pharmacology
D013873:Thionucleotides; C015929:8-((4-chlorophenyl)thio)cyclic-3',5'-AMP; D004247:DNA; D000242:Cyclic AMP; D008156:Luciferases; D011494:Protein Kinases