Expression of a chimeric, cGMP-sensitive regulatory subunit of the cAMP-dependent protein kinase type I alpha.
To study the fluctuations of cGMP in living cells through changes of energy transfer of dissociable fluorescence labeled subunits, we constructed a cGMP-sensitive probe by combining the N-terminus of the type I regulatory subunit of cAMP-dependent protein kinase (PKA) with the cGMP binding sites of cGMP-dependent protein kinase I alpha (PKG). This chimeric regulatory subunit retained PKA-like dimerization and PKG-compatible cGMP binding constants (Kd = 53 nM) for both binding sites. High affinity interaction with the PKA catalytic subunit was verified by Surface Plasmon Resonance (Kd = 3.15 nM). Additionally, the chimera inhibits the formation of wild-type holoenzyme with an apparent Ki of 1.05 nM. Furthermore, cGMP dissociated the mutant holoenzyme with an apparent activation constant of 146 nM. Thus, our construct provides all the requirements needed to investigate changes in intracellular cGMP concentrations.
Wild
N
N
Institut für Pharmakologie und Toxikologie, Technische Universität München, Germany.
Herberg
F W
FW
Hofmann
F
F
Dostmann
W R
WR
D001483:Base Sequence; D001665:Binding Sites; D002850:Chromatography, Gel; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D006152:Cyclic GMP / Q000378:metabolism / Q000494:pharmacology*; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011993:Recombinant Fusion Proteins; D012045:Regulatory Sequences, Nucleic Acid
D046911:Macromolecular Substances; D011993:Recombinant Fusion Proteins; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D006152:Cyclic GMP