In-depth mapping of DNA-PKcs signaling uncovers noncanonical features of its kinase specificity
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Updated: Jun 22, 2025

Identification of Kinase-substrate Pairs Using High Throughput Screening
Published on: August 29, 2015
Shannon Marshall1, Marcos V A S Navarro2, Carolline F R Ascenҫão1
1Weill Institute for Cell and Molecular Biology, Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York, USA.
Abstract:
DNA-PKcs is a DNA damage sensor kinase with established roles in DNA double-strand break repair via nonhomologous end joining. Recent studies have revealed additional roles of DNA-PKcs in the regulation of transcription, translation, and DNA replication. However, the substrates through which DNA-PKcs regulates these processes remain largely undefined. Here, we utilized quantitative phosphoproteomics to generate a high coverage map of DNA-PKcs signaling in response to ionizing radiation and mapped its interplay with the ATM kinase. Beyond the detection of the canonical S/T-Q phosphorylation motif, we uncovered a noncanonical mode of DNA-PKcs signaling targeting S/T-ψ-D/E motifs. Sequence and structural analyses of the DNA-PKcs substrate recognition pocket revealed unique features compared to closely related phosphatidylinositol 3-kinase-related kinases that may explain its broader substrate preference. These findings expand the repertoire of DNA-PKcs and ATM substrates while establishing a novel preferential phosphorylation motif for DNA-PKcs.
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