Phosphorylation at S2053 in murine (S2056 in human) DNA-PKcs is dispensable for lymphocyte development and class switch recombination

W Jiang, VM Estes, XS Wang, Z Shao… - The Journal of …, 2019 - journals.aai.org
W Jiang, VM Estes, XS Wang, Z Shao, BJ Lee, X Lin, JL Crowe, S Zha
The Journal of Immunology, 2019journals.aai.org
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
(eg, hairpin opening), but not end-ligation, whereas expression of the kinase-dead DNA …
Abstract
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 (eg, 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 DNA-PKcs. 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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