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Systematic investigation of protein kinase A substrate proteins using on-chip protein kinase kinetic profiling

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Abstract

Protein kinases play a critical role in a wide variety of cellular processes through post-translational protein phosphorylation and identification of their substrate proteins is important for understanding the enzymes’ mechanism of action and elucidating their functions in physiological and disease processes. However, there have been few systematic investigations of protein kinase substrates due to a lack of high-throughput kinetic assays. Thus, we designed an on-chip protein kinase assay for profiling kinase kinetic parameters by introducing the phosphorylation rate (Vp) under physiological conditions, instead of the maximal velocity (Vmax), in a high-throughput manner. We applied this method to the kinetic analysis of protein kinase A (PKA) for 28 nuclear, cytosolic, plasma membrane, and extracellular target proteins to determine the substrate affinity (Km), Vp (FIATP10/s), and substrate preference (Vp/Km). We then constructed a map of PKA's kinetic parameters with respect to the 28 proteins based on subcellular localization. Thus, the on-chip protein kinase kinetic profiling is an ideal approach for systematically investigating substrate proteins of protein kinases and fully characterizing the enzymes’ physiological functions.

Graphical abstract: Systematic investigation of protein kinase A substrate proteins using on-chip protein kinase kinetic profiling

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Publication details

The article was received on 19 Dec 2016, accepted on 03 May 2017 and first published on 05 May 2017


Article type: Paper
DOI: 10.1039/C6AN02682F
Citation: Analyst, 2017, Advance Article
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    Systematic investigation of protein kinase A substrate proteins using on-chip protein kinase kinetic profiling

    S. Jung, D. Kong, H. Jeon, E. Han, W. S. Park, S. Hong, Y. Kim and K. Ha, Analyst, 2017, Advance Article , DOI: 10.1039/C6AN02682F

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