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.