Imaging of cAMP-dependent protein kinase activity in living neural cells using a novel fluorescent substrate.
In order to visualize the activity of the cAMP-dependent protein kinase (PKA) in living cells, we have constructed a new fluorescence PKA substrate by conjugating a fluorescence probe to a partial amino acid sequence of PKA regulatory domain II which contains a specific autophosphorylation site. The fluorescent peptide was cell-permeable and became phosphorylated when the intracellular cAMP concentration was increased, resulting in a decrease in its fluorescence intensity. In NG108-15 cells, PKA activity was localized to the cytosol around the nucleus. In cultured hippocampal neurons, addition of L-glutamate caused PKA activation associated with increase of the cellular cAMP.
Higashi
H
H
Mitsubishi Kasei Institute of Life Sciences, Machida, Tokyo, Japan. henry@libra.ls.m-kagaku.co.jp
Sato
K
K
Ohtake
A
A
Omori
A
A
Yoshida
S
S
Kudo
Y
Y
D000595:Amino Acid Sequence; D000818:Animals; D002463:Cell Membrane Permeability; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D003600:Cytosol / Q000201:enzymology; D004789:Enzyme Activation; D005456:Fluorescent Dyes / Q000378:metabolism*; D018698:Glutamic Acid / Q000494:pharmacology; D006624:Hippocampus / Q000166:cytology / Q000196:embryology; D007091:Image Processing, Computer-Assisted; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009474:Neurons / Q000201:enzymology*; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D051381:Rats; D014407:Tumor Cells, Cultured
D005456:Fluorescent Dyes; D010446:Peptide Fragments; D010768:Phosphoserine; D018698:Glutamic Acid; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases