Biochemical characterization and cellular imaging of a novel, membrane permeable fluorescent cAMP analog.
BACKGROUND
A novel fluorescent cAMP analog (8-[Pharos-575]- adenosine-3', 5'-cyclic monophosphate) was characterized with respect to its spectral properties, its ability to bind to and activate three main isoenzymes of the cAMP-dependent protein kinase (PKA-Ialpha, PKA-IIalpha, PKA-IIbeta) in vitro, its stability towards phosphodiesterase and its ability to permeate into cultured eukaryotic cells using resonance energy transfer based indicators, and conventional fluorescence imaging.


RESULTS
The Pharos fluorophore is characterized by a Stokes shift of 42 nm with an absorption maximum at 575 nm and the emission peaking at 617 nm. The quantum yield is 30%. Incubation of the compound to RIIalpha and RIIbeta subunits increases the amplitude of excitation and absorption maxima significantly; no major change was observed with RIalpha. In vitro binding of the compound to RIalpha subunit and activation of the PKA-Ialpha holoenzyme was essentially equivalent to cAMP; RII subunits bound the fluorescent analog up to ten times less efficiently, resulting in about two times reduced apparent activation constants of the holoenzymes compared to cAMP. The cellular uptake of the fluorescent analog was investigated by cAMP indicators. It was estimated that about 7 muM of the fluorescent cAMP analog is available to the indicator after one hour of incubation and that about 600 muM of the compound had to be added to intact cells to half-maximally dissociate a PKA type IIalpha sensor.


CONCLUSIONS
The novel analog combines good membrane permeability- comparable to 8-Br-cAMP - with superior spectral properties of a modern, red-shifted fluorophore. GFP-tagged regulatory subunits of PKA and the analog co-localized. Furthermore, it is a potent, PDE-resistant activator of PKA-I and -II, suitable for in vitro applications and spatial distribution evaluations in living cells.
Moll
Daniela
D
University of Kassel, Department of Biochemistry, Heinrich-Plett-Strasse 40, 34132 Kassel, Germany. daniela.moll@uni-kassel.de
Prinz
Anke
A
Brendel
Cornelia M
CM
Berrera
Marco
M
Guske
Katrin
K
Zaccolo
Manuela
M
Genieser
Hans-Gottfried
HG
Herberg
Friedrich W
FW
D000818:Animals; D019556:COS Cells; D002463:Cell Membrane Permeability; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D000242:Cyclic AMP / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D004735:Energy Transfer; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes / Q000737:chemistry* / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000378:metabolism; D011494:Protein Kinases / Q000737:chemistry / Q000378:metabolism; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence
C532148:8-(Pharos-575)-adenosine-3',5'-cyclic monophosphate; D005456:Fluorescent Dyes; D021122:Protein Subunits; D049452:Green Fluorescent Proteins; D000242:Cyclic AMP; D011494:Protein Kinases