Journal
JOURNAL OF IMMUNOLOGY
Volume 203, Issue 1, Pages 178-187Publisher
AMER ASSOC IMMUNOLOGISTS
DOI: 10.4049/jimmunol.1801657
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Funding
- National Institutes of Health (NIH)/National Cancer Institute (NCI) [R01CA158073, R01CA184187, P01CA174653, R01CA226852]
- Leukemia Lymphoma Society Scholar Award
- NIH/NCI Grant [T32-CA09503]
- NIH/NCI [F31CA183504-01A1]
- Clinical and Translational Science Award/NIH Grant [TL1 TR000082]
- NIH/NCI Cancer Center Support Grant [P30CA013696]
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The classical nonhomologous end-joining (cNHEJ) pathway is a major DNA double-strand break repair pathway in mammalian cells and is required for lymphocyte development and maturation. The DNA-dependent protein kinase (DNA-PK) is a cNHEJ factor that encompasses the Ku70-Ku80 (KU) heterodimer and the large DNA-PK catalytic subunit (DNA-PKcs). In mouse models, loss of DNA-PKcs (DNA-PKcs(-/-)) abrogates end processing (e.g., hairpin opening), but not end-ligation, whereas expression of the kinase-dead DNA-PKcs protein (DNA-PKcs(KD/KD)) abrogates end-ligation, suggesting a kinase-dependent structural function of DNA-PKcs during cNHEJ. Lymphocyte development is abolished in DNA-PKcs(-/-) and DNA-PKcs(KD/KD) mice because of the requirement for both hairpin opening and end-ligation during V(D)J recombination. DNA-PKcs itself is the best-characterized substrate of DNA-PK. The S2056 cluster is the best-characterized autophosphorylation site in human DNAPKcs. In this study, we show that radiation can induce phosphorylation of murine DNA-PKcs at the corresponding S2053. We also generated knockin mouse models with alanine- (DNA-PKcs(PQR)) or phospho-mimetic aspartate (DNA-PKcs(SD)) substitutions at the S2053 cluster. Despite moderate radiation sensitivity in the DNA-PKcs(PQR/PQR) fibroblasts and lymphocytes, both DNA-PKcs(PQR/PQR) and DNA-PKcs(SD/SD) mice retained normal kinase activity and underwent efficient V(D)J recombination and class switch recombination, indicating that phosphorylation at the S2053 cluster of murine DNA-PKcs (corresponding to S2056 of human DNA-PKcs), although important for radiation resistance, is dispensable for the end-ligation and hairpin-opening function of DNA-PK essential for lymphocyte development.
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