Genetically encoded FRET probe for PKC activity based on pleckstrin.
We developed a probe for investigating protein kinase C (PKC) activity in living cells. The probe is based on a fragment of pleckstrin enclosed by two FRET-capable fluorophores. PKC activity modulation was reliably followed by FRET change in vitro and in vivo. The probe responds quickly to PKC activation by phorbol ester. FRET changes were reversible when PKC inhibitors were administered. Stimulation of cellular signaling pathways using histamine or bradykinin triggered PKC in a physiologically relevant way.
Schleifenbaum
Andreas
A
European Molecular Biology Institute, Meyerhofstrasse 1, 69117 Heidelberg, Germany.
Stier
Gunter
G
Gasch
Alexander
A
Sattler
Michael
M
Schultz
Carsten
C
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001798:Blood Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001920:Bradykinin / Q000494:pharmacology; D045744:Cell Line, Tumor; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006632:Histamine / Q000494:pharmacology; D006801:Humans; D008164:Luminescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D009447:Neuroblastoma; D009474:Neurons / Q000187:drug effects / Q000201:enzymology; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010750:Phosphoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011487:Protein Conformation; D011493:Protein Kinase C / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D001426:Bacterial Proteins; D001798:Blood Proteins; D008164:Luminescent Proteins; D010446:Peptide Fragments; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; C039077:platelet protein P47; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins; D006632:Histamine; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D001920:Bradykinin