In-depth mapping of DNA-PKcs signaling uncovers noncanonical features of its kinase specificity.

Shannon Marshall, Marcos V A S Navarro, Carolline F R Ascenҫão, Diego Dibitetto, Marcus B Smolka

Journal: The Journal of biological chemistry 2024;300(8):107513

PMID: 38945450

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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.

Address: Weill Institute for Cell and Molecular Biology, Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York, USA.; Weill Institute for Cell and Molecular Biology, Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York, USA; IFSC Institute of Physics of São Carlos, University of São Paulo, São Carlos, São Paulo, Brazil. Electronic address: [email protected].; Weill Institute for Cell and Molecular Biology, Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York, USA; Department of Experimental Oncology, Istituto di Ricerche Farmacologiche Mario Negri IRCCS, Milan, Italy.; Weill Institute for Cell and Molecular Biology, Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York, USA. Electronic address: [email protected].

DOI: 10.1016/j.jbc.2024.107513

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https://assays.cancer.gov/CPTAC-912

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https://data.niaid.nih.gov/resources?id=pxd042258

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https://antibodies.cancer.gov/detail/CPTC-ATM-4

MeSH Terms: DNA-Activated Protein Kinase, Humans, Ataxia Telangiectasia Mutated Proteins, Phosphorylation, Signal Transduction, Substrate Specificity, Amino Acid Motifs