Imaging of cAMP‐dependent protein kinase activity in living neural cells using a novel fluorescent substrate

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Abstract
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.