Engineering a bioluminescent indicator for cyclic AMP-dependent protein kinase.
cDNA coding for the luciferase in the firefly Photinus pyralis was amplified in vitro to generate cyclic AMP-dependent protein kinase phosphorylation sites. The DNA was transcribed and translated to generate light-emitting protein. A valine at position 217 was mutated to arginine to generate a site RRFS and the heptapeptide kemptide, the phosphorylation site of the porcine pyruvate kinase, was added at the N- or C-terminus of the luciferase. The proteins carrying phosphorylation sites were characterized for their specific activity, pI, effect of pH on the colour of the light emitted and effect of the catalytic subunit of protein kinase A in the presence of ATP. Only one of the recombinant proteins (RRFS) was significantly different from wild-type luciferase. The RRFS mutant had a lower specific activity, lower pH optimum, emitted greener light at low pH and when phosphorylated it decreased its activity by up to 80%. This latter effect was reversed by phosphatase. This recombinant protein is a good candidate to measure for the first time cyclic AMP-dependent phosphorylation in live cells.
Sala-Newby
G B
GB
Department of Medical Biochemistry, University of Wales College of Medicine, Cardiff, U.K.
Campbell
A K
AK
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D007202:Indicators and Reagents; D008156:Luciferases / Q000737:chemistry*; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000235:genetics; D010766:Phosphorylation; D016133:Polymerase Chain Reaction; D015202:Protein Engineering; D011494:Protein Kinases / Q000235:genetics*; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics
D007202:Indicators and Reagents; D009842:Oligopeptides; D011994:Recombinant Proteins; C037642:kemptide; D008156:Luciferases; D011494:Protein Kinases