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Peptide microarrays for detailed, high-throughput substrate identification, kinetic characterization, and inhibition studies on protein kinase A
Authors:Riet Hilhorst  Liesbeth Houkes  Adriënne van den Berg  Rob Ruijtenbeek
Institution:PamGene International BV, 5200 BJ ’s-Hertogenbosch, The Netherlands
Abstract:A microarray-based mix-and-measure, nonradioactive multiplex method with real-time detection was used for substrate identification, assay development, assay optimisation, and kinetic characterization of protein kinase A (PKA). The peptide arrays included either up to 140 serine/threonine-containing peptides or a concentration series of a smaller number of peptides. In comparison with existing singleplex assays, data quality was high, variation in assay conditions and reagent consumption were reduced considerably, and assay development could be accelerated because phosphorylation kinetics were monitored simultaneously on 4, 12, or 96 arrays. PKA was shown to phosphorylate many peptides containing known PKA phosphorylation sites as well as some new substrates. The kinetic behavior of the enzyme and the mechanism of inhibition by AMP-PNP, staurosporin, and PKA inhibitor peptide on the peptide microarray correlated well with data from homogeneous assays. Using this multiplex setup, we showed that the kinetic parameters of PKA and the potency of PKA inhibitors can be affected by the sequence of the peptide substrate. The technology enables kinetic monitoring of kinase activity in a multiplex setting such as a cell or tissue lysate. Finally, this high-throughput method allows fast identification of peptide substrates for serine/threonine kinases that are still uncharacterized.
Keywords:Peptide microarray  PKA  cAMP-dependent protein kinase A  Substrate identification  Enzymology  Michaelis constant  Kinetics  Mechanism of action  IC50  Staurosporin  PKA inhibitor peptide  AMP-PNP
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