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Engineering firefly luciferase as an indicator of cyclic AMP‐dependent protein kinase in living cells
Author(s) -
Sala-Newby Graciela,
Campbell Anthony K.
Publication year - 1992
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(92)80776-d
Subject(s) - luciferase , bioluminescence , luciferin , protein kinase a , mutant , microbiology and biotechnology , kinase , complementary dna , biology , chemistry , biochemistry , transfection , gene
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.