title: Identifications of Putative PKA Substrates with Quantitative Phosphoproteomics and Primary-Sequence-Based Scoring.
abstract: Protein kinase A (PKA or cAMP-dependent protein kinase) is a serine/threonine kinase that plays essential roles in the regulation of proliferation, differentiation, and apoptosis. To better understand the functions of PKA, it is necessary to elucidate the direct interplay between PKA and their substrates in living human cells. To identify kinase target substrates in a high-throughput manner, we first quantified the change of phosphoproteome in the cells of which PKA activity was perturbed by drug stimulations. LC-MS/MS analyses identified 2755 and 3191 phosphopeptides from experiments with activator or inhibitor of PKA. To exclude potential indirect targets of PKA, we built a computational model to characterize the kinase sequence specificity toward the substrate target site based on known kinase-substrate relationships. Finally, by combining the sequence recognition model with the quantitative changes in phosphorylation measured in the two drug perturbation experiments, we identified 29 reliable candidates of PKA targeting residues in living cells including 8 previously known substrates. Moreover, 18 of these sites were confirmed to be site-specifically phosphorylated in vitro. Altogether this study proposed a confident list of PKA substrate candidates, expanding our knowledge of PKA signaling network.
authors.affiliation: Graduate School of Pharmaceutical Sciences, Kyoto University , Sakyo-ku, Kyoto 606-8501, Japan.|European Bioinformatics Institute , European Molecular Biology Laboratory, Wellcome Genome Campus, Hinxton, Cambridge CB10 1SD, United Kingdom.
authors.forename: Haruna
authors.initials: H
authors.lastname: Imamura
authors.affiliation: European Bioinformatics Institute , European Molecular Biology Laboratory, Wellcome Genome Campus, Hinxton, Cambridge CB10 1SD, United Kingdom.
authors.forename: Omar
authors.initials: O
authors.lastname: Wagih
authors.affiliation: Graduate School of Pharmaceutical Sciences, Kyoto University , Sakyo-ku, Kyoto 606-8501, Japan.
authors.forename: Tomoya
authors.initials: T
authors.lastname: Niinae
authors.affiliation: Graduate School of Pharmaceutical Sciences, Kyoto University , Sakyo-ku, Kyoto 606-8501, Japan.
authors.forename: Naoyuki
authors.initials: N
authors.lastname: Sugiyama
authors.affiliation: European Bioinformatics Institute , European Molecular Biology Laboratory, Wellcome Genome Campus, Hinxton, Cambridge CB10 1SD, United Kingdom.
authors.forename: Pedro
authors.initials: P
authors.lastname: Beltrao
authors.affiliation: Graduate School of Pharmaceutical Sciences, Kyoto University , Sakyo-ku, Kyoto 606-8501, Japan.
authors.forename: Yasushi
authors.identifier: 0000-0001-7714-203X
authors.initials: Y
authors.lastname: Ishihama
mesh_terms: D000595:Amino Acid Sequence / Q000235:genetics; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D057166:High-Throughput Screening Assays; D006801:Humans; D010748:Phosphopeptides / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000235:genetics; D015398:Signal Transduction / Q000235:genetics; D013379:Substrate Specificity; D053719:Tandem Mass Spectrometry
chemical_list: D010748:Phosphopeptides; D017346:Protein Serine-Threonine Kinases; D017868:Cyclic AMP-Dependent Protein Kinases