Identifications of protein kinase A substrates with quantitative phosphoproteomics and primary-sequence based scoring.(H-89)

Protein kinase A (PKA) is a serine/threonine kinase who plays essential roles in the complex signalling pathways. To better understand the functionalities, it is necessary to elucidate direct interplays between PKA and their substrates in living system. In order to screen them in a high-throughput manner, we firstly quantified the change of phosphoproteome in the cells of which PKA activity was perturbed by drag stimulations. LC-MS/MS analysis identified 2,755 and 3,191 phosphopeptides from either experiments with activator or inhibitor of PKA. To exclude indirect targets of PKA, we built the simple model to characterize the kinase sequential specificity to its substrate recognitions based on the known kinase-substrate relationships. Finally, by applying the metric to the sites of which phosphorylation were changed due to the drug stimulations, we identified 29 reliable candidates of PKA targeting residues in living cells including 8 known substrates. Moreover, 18 sites out of them were confirmed to be site-specifically phosphorylated in vitro. Altogether this study proposed confident list of PKA substrate and expanded the knowledge of PKA signalling network.